Dissertations / Theses: 'Bacterial virulence factors'

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Prasad, Joni M. "Hemostatic Factors in Bacterial Virulence and Host Defense." University of Cincinnati / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1329495133.

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Johansson, Linda. "Host responses and bacterial virulence factors in Neisseria infections /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-017-6/.

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Tomenius, Henrik. "Bacterial virulence and adaptation mediated by two-component system signalling /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-792-8/.

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Luo, Wenyi. "Identification and characterization of virulence factors of mycoplasmas." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2010p/luo.pdf.

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Hällgren, Anita. "Enterococci in Swedish intensive care units : studies on epidemiology, mechanisms of antibiotic resistance and virulence factors /." Linköping : Linköping University, 2005. http://www.bibl.liu.se/liupubl/disp/disp2005/med880s.pdf.

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Elswaifi, Shaadi Fouad. "The Molecular Characterization of Phosphorylcholine (ChoP) on Histophilus somni Lipooligosaccharide: Contribution of ChoP to Bacterial Virulence and Pathogenesis." Diss., Virginia Tech, 2006. http://hdl.handle.net/10919/30079.

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Histophilus somni virulence factors include expression and antigenic variation of lipooligosaccharide (LOS). Phosphorylcholine (ChoP) is often expressed on H. somni LOS and also undergoes antigenic variation. In this study, five genes that play a role in expression and antigenic variation of ChoP, lic1ABCD and glpQ, were identified in the genome sequence of H. somni through sequence homology with Haemophilus influenzae genes. The open reading frame (ORF) of lic1A contained a variable number of tandem repeats of the tetranucleotide unit 5'-AACC-3'. Slipped strand mispairing in the repeat region during replication leads to shifting the downstream reading frame in and out of frame with the start codon, thus controlling phase variation of lic1A expression. Removal of the repeats from lic1A, cloning the gene in E. coli, and performing a functional assay on the product indicated that lic1A encodes a choline kinase and that the repeats were not required for expression of a functional gene product. Variation in the number of repeats in lic1A correlated with the antigenic variation of ChoP expression in strain 124P, but not in strain 738. This result supported previous findings that antigenic variation of ChoP expression in strain 738 is controlled through extension/truncation of the LOS outer core. Therefore, these results indicated that the lic1ABCD and glpQ genes control expression and antigenic variation of ChoP on the LOS of H. somni and that there are two possible mechanisms for ChoP antigenic variation. The role of H. somni expression of ChoP in colonization of the host respiratory tract was also examined. Experimental infection in the natural host showed that the population of H. somni that expresses ChoP was enriched in the bacteria that colonized the respiratory tract. In addition, bacteria expressing ChoP were able to aggregate bovine platelets through binding to the platelet activating factor receptor (PAF-R), which is also present on epithelial and endothelial cells. These results indicated that ChoP may play a role in the process of colonization and subsequent systemic invasion of host tissues, which may occur through binding of ChoP to PAF-R. Bacteria that did not express ChoP were more prevalent in systemic sites, indicating that ChoP expression may be disadvantageous for the organism during systemic dissemination.
Ph. D.

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Tubby, S. "The effect of light-activated antimicrobial agents on bacterial virulence factors and key modulators of inflammation." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1318137/.

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Photodynamic therapy is a promising new strategy for the treatment of superficial skin infections and periodontitis. A limitation of antibiotic treatment for these diseases is that even after successful killing of the infecting organism, secreted virulence factors may still be present and cause significant damage to host tissues. If light-activated antimicrobial agents can inactivate microbial virulence factors in addition to killing the pathogenic microorganisms, this would represent an advantage of photodynamic therapy over conventional treatment options. The light-activated antimicrobial agents methylene blue and tin chlorin e6 in combination with laser light of 665 and 633 nm respectively, were assessed for their antibacterial activity and ability to reduce the activity of selected virulence factors of Staphylococcus aureus and Porphyromonas gingivalis. In addition to successfully reducing the microbial burden, it was demonstrated that photosensitisation was able to cause significant reductions in the activity of a number of secreted and cell wall-associated virulence factors produced by these species when irradiated with laser light of the appropriate wavelength. Photosensitisation was also shown to reduce the biological activities of the proinflammatory cytokines tumour necrosis factor-alpha and interleukin-6, which are produced in response to infecting bacteria and are associated with damage to host tissues. The results of these studies indicate that light-activated antimicrobial agents may be useful in reducing the pathology associated with bacterial virulence factors and host-mediated inflammation when used as part of an antimicrobial treatment regimen.

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Huish, Sian. "Mechanistic studies on Zymogen-Activator and Adhesion Proteins (ZAAPs) as thrombolytic drugs and bacterial virulence factors." Thesis, Imperial College London, 2015. http://hdl.handle.net/10044/1/33721.

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Streptokinase (SK), expressed by Lancefield Group A, C and G β-haemolytic Streptococci and Staphylocoagulase (SCG), expressed by S. aureus, are bacterial virulence factors which belong to a family of proteins known as Zymogen-activator and adhesion proteins (ZAAPs). SK and SCG are responsible for the non-proteolytic activation of plasminogen and prothrombin, respectively. Understanding of SK activity is exclusively based on the Group C (GCS) S. equisimilis H46a SK, a 'clot buster' or thrombolytic used in the treatment of Myocardial Infarction (MI), which exhibits no fibrin specificity. SK is the most used thrombolytic worldwide. Here, detailed kinetic studies in purified assay systems explored the mechanistic variation between a recombinant H46a SK (rSK H46a) and a Group A Streptococcal SK (M1GAS), most typically isolated in invasive human infection. This work demonstrates a fibrin specific mechanism for M1GAS SK and proposes a kinetic model for M1GAS SK plasminogen activation, to compliment the 'Trigger and Bullet' hypothesis for H46a SK by Bock and colleagues. This work has relevance to the use of SK variants, with enhanced fibrin specificity, for improvement of thrombolytic therapies. Cardiovascular diseases such as myocardial infaraction and ischaemic stroke are significant casues of mortality, particularly in the developing world. Access to Alteplase, an expensive recombinant tPA and the only licensed treatment for stroke, is limited and there is interest in the use of SK for this purpose. Furthermore, microbial resistance is an increasing health burden, as demonstrated by programs such as the Longitude prize. Exploring the mechanisms of bacterial virulence factors at the molecular level such as this could provide rationale for the development of much-needed new antimicrobial technologies.

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Tano, Eva. "Survival of infectious agents and detection of their resistance and virulence factors." Doctoral thesis, Uppsala universitet, Institutionen för medicinska vetenskaper, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-248786.

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In the first study, three different transport systems for bacteria were evaluated. The CLSI M40-A guideline was used to monitor the maintenance of both mono- and polymicrobial samples during a simulated transportation at room temperature that lasted 0-48 h. All systems were able to maintain the viability of all organisms for 24 h, but none of them could support all tested species after 48 h. The most difficult species to recover was Neisseria gonorrhoeae, and in polymicrobial samples overgrowth was an observed problem. The aim of the second study was to study the presence of TSST-1 and three other important toxin genes in invasive isolates of Staphylococcus aureus collected during the years 2000-2012 at two tertiary hospitals. The genes encoding the staphylococcal toxins were detected by PCR, and whole-genome sequencing was used for analyzing the genetic relatedness between isolates. The results showed that the most common toxin was TSST-1, and isolates positive for this toxin exhibited a clear clonality independent of year and hospital. The typical patient was a male aged 55-74 years and with a bone or a joint infection. The third study was a clinical study of the effect of silver-based wound dressings on the bacterial flora in chronic leg ulcers. Phenotypic and genetic silver-resistance were investigated before and after topical silver treatment, by determining the silver nitrate MICs and by detecting sil genes with PCR. The silver-based dressings had a limited effect on primary wound pathogens, and the activity of silver nitrate on S. aureus was mainly bacteriostatic. A silver-resistant Enterobacter cloacae strain was identified after only three weeks of treatment, and cephalosporin-resistant members of the Enterobacteriaceae family were relatively prone to developed silver-resistance after silver exposure in vitro. The last study was undertaken in order to develop an easy-to-use method for simulating the laundering process of hospital textiles, and apply the method when evaluating the decontaminating efficacy of two different washing temperatures. The laundering process took place at professional laundries, and Enterococcus faecium was used as a bioindicator. The results showed that a lowering of the washing temperature from 70°C to 60°C did not affect the decontamination efficacy; the washing cycle alone reduced the number of bacteria with 3-5 log10 CFU, whereas the following tumble drying reduced the bacterial numbers with another 3-4 log10 CFU, yielding the same final result independent of the washing temperature. To ensure that sufficient textile hygiene is maintained, the whole laundering process needs to be monitored. The general conclusion is that all developmental work in the bacterial field requires time and a large strain collection.

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Kanth, Anna. "Studies on global regulators involved in virulence gene expression in Staphylococcus aureus /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-494-1/.

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Arens, Stefan [Verfasser], and Theresia [Akademischer Betreuer] Stradal. "Characterization of type III secreted bacterial virulence factors that interfere with Rho GTPase signalling / Stefan Arens ; Betreuer: Theresia Stradal." Münster : Universitäts- und Landesbibliothek Münster, 2014. http://d-nb.info/1138279897/34.

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Crossman, David K. "Characterization of a novel acetyltransferase found only in pathogenic strains of Mycobacterium tuberculosis." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2007r/crossman.pdf.

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Laabei, Maisem. "Using phospholipid vesicles to assay bacterial lytic agents, examining factors and identifying virulence loci which alter toxin production in Staphylococcus aureus." Thesis, University of Bath, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.636526.

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Burns represent one of the most devastating forms of injury with infection complications representing the highest risk of mortality. The primary objective of the Bacteriosafe project was the development of a smart wound dressing that would respond to the presence of bacteria in burn wounds. The basis of this sensing system employed the use of phospholipid vesicles, containing a self-quenchable fluorescent dye. These vesicles mimic the eukaryotic cell membrane and as such are susceptible to bacterial cytolytic factors which lyse the vesicles, generating an observable and measurable fluorescent response. My primary role in this project was to identify the vesicle lysing agents secreted from the two most frequent burn wound colonisers, Staphylococcus aureus and Pseudomonas aeruginosa. We identified the small amphipathic alpha helical peptide toxins from S. aureus and glycolipid molecules derived from P. aeruginosa as the agents responsible for vesicle lysis. The identification of these molecules led to the development of two novel phenotypic assays designed to measure these important virulence factors, as discussed in chapter 3 and 4. In chapter 5 we examined the role of toxic shock syndrome toxin-1 (TSST-1) in repressing global exoprotein expression. Our results demonstrate that TSST-1 does not repress toxin secretion and strains expressing TSST-1 retain their ability to lyse vesicles. In chapter 6 we explored the use of subinhibitory oxacillin in inducing the alternative penicillin binding protein 2a (PBP2a) in community-acquired methicillin resistant S. aureus (CA-MRSA) strains to down-regulate toxicity. Previous work in the Massey lab demonstrated that the expression of the mecA gene, which encodes PBP2a, resulted in reduced toxicity in hospital-acquired (HA) -MRSA. CA-MRSA strains are considered highly toxic and have a considerably lower level of PBP2a expression. Treatment of CA-MRSA strains with subinhibitory oxacillin did result in a down-regulation of some toxins but also the up-regulation of others, highlighting the pleiotropic effect oxacillin had on virulence regulation. In chapter 7 we developed an approach that uses the genome sequences of a set of related clinical S. aureus strains to identify novel virulence loci by associating genetic polymorphisms with specific virulence phenotypes using a genome wide association study (GWAS). This analysis resulted in the identification of four novel loci which when mutated lead to a reduction in toxicity. We demonstrate that the GWAS approach is an effective method in identifying candidate SNPs which may be important in altering virulence but do highlight limitations of this approach, primarily the generation of false positives.

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Furlanetto, Alessandra. "Síntese de nanoemulsão e nanopartícula de ouro (AuNPs) contendo nisina e seus efeitos sobre os fatores de virulência de Staphylococcus aureus." Botucatu, 2020. http://hdl.handle.net/11449/192336.

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Orientador: Ary Fernandes Júnior
Resumo: O aumento no número de bactérias multirresistentes aos fármacos antibacterianos é preocupação de saúde pública e tem motivado pesquisas na buscsa por antimicrobianos alternativos para minimizar este problema, e na obtenção de substâncias com capacidade de matar bactérias e/ou interferir com a sua patogenicidade. O Staphylococcus aureus é uma bactéria altamente virulenta, capaz de causar inúmeras doenças, incluindo intoxicações alimentares. Esta bactéria se tornou resistente aos diversos antimicrobianos ao longo dos anos com destaque para o S. aureus meticilina-resistente (MRSA). O peptídeo antimicrobiano (AMP) nisina, bacteriocina produzida por Lactococcus lactis é um que vem sendo estudado na forma de nanoemulsões e nanopartículas. O objetivo desse estudo foi sintetizar, caracterizar e testar nanoemulsões e nanopartículas de ouro (AuNPs) de nisina, para a verificação da ação antibacteriana, através da Concentração Inibitória Mínima (CIM), atividade antibiofilme, antienterotoxina, atividade hemolítica, ação sobre a membrana bacteriana determinada pelo extravasamento de proteínas, sobre linhagens padrões ATCC de S. aureus, e teste de viabilidade celular em linhagem HCT-116 por citometria de fluxo. Os tratamentos utilizados foram nisina, cinco nanoemulsões com nisina (Nano-Nis), AuNPs com nisina (AuNPs-Nis), AuNPs com Nano-Nis (AuNPs + Nano-Nis) e AuNPs-Nis com nanoemulsão (AuNPs-Nis + Nano). De acordo com os resultados obtidos para CIM, observou-se que para a cepa ATCC 33591 d... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The growth in the number of multiresistant bacteria resistant to traditional antimicrobial drugs is a public health concern which has been motivated researchs worldwide, seeking new antimicrobial drugs to minimize this problem besides getting new substances able to erradicate bacteria and/or interfer with their pathogenicity. Staphylococcus aureus is a highly virulent bacteria, able to cause countless diseases including food poisoning. This bacteria got resistant to many antimicrobials within the years, highlighting the S. aureus metchilin-resistant (MRSA). The antimicrobial peptide (AMP) nisin, bacteriocin synthesized by Lactococcus lactis, is one that has been studied in nanoemulsions and nanoparticles. The objective of this study was to synthesize, characterize and evaluate nanoemulsions and gold nanoparticles (AuNPs) with nisin, to verify their antibacterial action, using the Minimum Inhibitory Concentration (MIC), antibiofilm activity, antienterotoxin activity, hemolytic activity, action on bacterial membrane determined by protein leakage, on MRSA ATCC strains, and cell viability in HCT-116 strain by flow citometry. The treatments used were nisin, five nanoemulsions with nisin (Nano-Nis), AuNPs with nisin (AuNPs-Nis), AuNPs with Nano-Nis (AuNPs + Nano-Nis) and AuNPs-Nis with nanoemulsion (AuNPs-Nis + Nano). According to the results obtained for MIC, it was observed that for the MRSA strain ATCC 33591, the number 1 formulation of nanoemulsions was the more efficient betwe... (Complete abstract click electronic access below)
Mestre

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Palace, Samantha G. "Plague and the Defeat of Mammalian Innate Immunity: Systematic Genetic Analysis of Yersinia pestis Virulence Factors: A Dissertation." eScholarship@UMMS, 2016. http://escholarship.umassmed.edu/gsbs_diss/836.

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Yersinia pestis, the causative agent of plague, specializes in causing dense bacteremia following intradermal deposition of a small number of bacteria by the bite of an infected flea. This robust invasiveness requires the ability to evade containment by the innate immune system. Of the various mechanisms employed by Y. pestis to subvert the innate immune response and to proliferate rapidly in mammalian tissue, only a few are well-characterized. Here, I present two complementary genetic analyses of Y. pestis adaptations to the mammalian environment. In the first, genome-wide fitness profiling for Y. pestis by Tn-seq demonstrates that the bacterium has adapted to overcome limitation of diverse nutrients during mammalian infection. In the second, a series of combinatorial targeted mutations disentangles apparent functional redundancy among the effectors of the Y. pestis type III secretion system, and we report that YpkA, YopT, and YopJ contribute to virulence in mice. We have also begun to investigate a novel relationship between Y. pestis and mammalian platelets, a highly abundant cell type in plasma. I present evidence that Y. pestis has evolved specific mechanisms to interfere with platelet activation, likely in order to evade immune responses and promote maintenance of bacteremia by undermining platelet thrombotic and innate immune functions. The principles guiding this work – systematic genetic analysis of complex systems, coupled with rational modification of in vitro assays to more closely mimic the in vivo environment – are a generalizable approach for increasing the efficiency of discovering new virulence determinants in bacterial pathogens.

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Pouliot, Kimberly Lea. "Surface of Yersinia pestis: LCRV, F1 Production, Invasion and Oxygen: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/358.

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Of the eleven species of bacteria that comprise the genus Yersinia of the family Enterobacteriaceae, three species are pathogenic for humans. Yersinia pseudotuberculosis and Yersinia enterocolitica usually cause a mild, self-limiting mesenteric lymphadenitis or ileitis. Yersinia pestis causes a highly invasive often fatal disease known as plague. All three elaborate a type three secretion system that is essential for virulence and encoded on closely related plasmids. In Y. pestis, all the effectors, structural components and chaperones are encoded on the 70kb plasmid, pCD1. Of these, LcrV from Y. enterocolitica has been implicated in playing an immunosuppressive role through its interaction with host Toll-like receptor 2 (TLR2) and induction of IL-10. Through expression and purification of recombinant LcrV from Escherichia coliwe show that only high molecular weight species of rLcrV are able to stimulate TLR2. In a highly sensitive subcutaneous mouse infection model we demonstrate no difference in the time to death between TLR2-sufficient or deficient mice. Analysis of cytokine levels between these two genotypes also shows no significant difference between splenic IL-10 and IL-6 or levels of bacteria. We conclusively show that this interaction, if it does occur, plays no significant role in vivo. In a separate set of experiments, we also determined that the expression of F1, a peptide shown to be responsible for 37°C-dependent inhibition of invasion by Y. pestis in vitro, was significantly decreased under high oxygen conditions. This led us to re-examine the invasion phenotype both in vitro and in vivo. These results give new insights into virulence gene expression in Y. pestis by environmental cues other than temperature.

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Perrin, Jackie. "Virulence bactérienne et défenses de l’hôte : contribution des cellules phagocytaires dans l’immunité innée chez la drosophile." Université Joseph Fourier (Grenoble ; 1971-2015), 2009. http://www.theses.fr/2009GRE10098.

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Chez les eucaryotes, les cellules phagocytaires participent à la défense contre les pathogènes en assurant la reconnaissance des corps étrangers, leur internalisation et leur élimination. Nous avons développé l’utilisation de la mouche Drosophila melanogaster pour l’étude prospective de la contribution des phagocytes dans l’immunité innée et leur rôle dans les interactions hôte-pathogène. Dans les phagocytes, les GTPases de la famille Rho ont un rôle essentiel dans la dynamique du cytosquelette d’actine au cours de l’adhésion cellulaire et de l’internalisation des pathogènes. Nous avons montré que la RhoGTPase Rac2 est spécifiquement impliquée dans la réponse immunitaire cellulaire chez la drosophile et contribue à la résistance aux infections par la bactérie pathogène Pseudomonas aeruginosa. Cette bactérie pose des problèmes majeurs de santé publique, notamment chez les patients atteints de mucoviscidose. Je me suis intéressée à la toxine ExoS de P. Aeruginosa qui est injectée dans les cellules hôtes où elle cible les RhoGTPases. En particulier, Rac2 est inhibée par cette toxine ce qui a pour conséquence d’affecter la phagocytose et la résistance aux pathogènes. En parallèle, j’ai participé à l’identification de nouveaux facteurs de virulence de P. Aeruginosa en collaboration avec le Pr P. Cosson (Centre Médical Universitaire, Genève). Mon étude a aussi porté sur la recherche de nouveaux acteurs de la réponse immunitaire cellulaire. Je me suis intéressée à une famille de protéines conservées au cours de l’évolution, les nonaspanines ou protéines TM9SF, dont la fonction dans la phagocytose avait été montrée chez l’amibe. Nous avons prouvé que TM9SF4 joue un rôle important dans la résistance aux infections chez la drosophile via son implication dans la phagocytose et l’adhésion cellulaire
In eukaryotes, phagocytic cells are involved in the defense against pathogens by insuring the recognition of foreign bodies, their internalization and elimination. We developed the use of the fly Drosophila melanogaster for the prospective study of the phagocytes contribution in the innate immunity and their role in host-pathogen interactions. In phagocytes, the GTPases of the family Rho have an essential role in the actin cytoskeleton dynamics that take place during cellular adhesion and pathogen internalisation. We proved that the RhoGTPase Rac2 is specifically involved in drosophila cellular immune response and contributes to the resistance in infections by the pathogenic bacteria Pseudomonas aeruginosa. This bacteria raises major public health problems, in particular at the patients affected by cystic fibrosis. I focused in the toxin ExoS of P. Aeruginosa which is injected in host cells targeting RhoGTPases. In particular, Rac2 is inhibited by this toxin resulting in reduced phagocytosis and resistance to infection. In parallel, I participated in the identification of new virulence factors of P. Aeruginosa in association with Pr P. Cosson (Centre Médical Universitaire, Geneva). My work also concerned the research for new players of the cellular immune response. I looked into an evolutionary conserved family of proteins, the nonaspanins also called TM9SF proteins, who’s function in the phagocytosis had been previously shown in amoeba. We proved that TM9SF4 plays an important role in drosophila infection resistance via its implication in phagocytosis and cellular adhesion

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Freitas, Natalia Cristina de. "Fímbrias Pil em Escherichia coli enteropatogênica atípica: Caracterização e investigação do papel de PilS e PilV na adesão bacteriana." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-20092012-095345/.

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Fímbrias do tipo IV estão associadas a diversos fenótipos em bactérias gram-negativas, e o presente estudo consistiu na caracterização da fímbria Pil e investigação de seu papel na adesão bacteriana de isolados de EPEC atípica. Por PCR e RT-PCR foram investigadas a presença e a funcionalidade do operon Pil e os resultados demonstraram que este está sendo transcrito somente nos isolados BA558 e BA956. Os genes pilS e pilV foram clonados em vetor de expressão para obtenção das proteínas Pil recombinantes e produção de anticorpos policlonais. A análise qualitativa dos testes de inibição da adesão utilizando os soros anti-PilS e anti-PilV juntos demonstraram que o isolado BA558 apresentou mudança de fenótipo de adesão. Esses resultados nos permitem concluir que o operon Pil está funcional em BA558 e BA956, e a expressão da fímbria Pil nessas cepas não está relacionada à formação de biofilme e autoagregação, porém a proteína fimbrial PilS juntamente com a adesina PilV parecem exercer uma função acessória importante na interação de BA558 às células HEp-2.
Type IV fimbriae are associated with several phenotypes in gram-negative bacteria. The aim of this study was the characterization of the Pil fimbria and its role in the interaction of atypical EPEC isolates in bacterial adhesion. Using PCR and RT-PCR, we investigated the presence and functionality of the pil operon genes. The results showed that these genes are transcribed only in the BA558 and BA956 isolates. The pilS and pilV genes were cloned into an expression vector for recombinant proteins and polyclonal antibodies production. Qualitative analysis of the adherence inhibition assays using both rabbit sera changed to localized-like the phenotype of BA558 isolate adhesion. Together, these results allow us to conclude that the Pil operon is functional only in the BA558 and BA956 isolates and that the expression of Pil fimbriae in aEPEC is not related to biofilm formation and autoaggregation but, the fimbrial PilS protein together with PilV adhesin seem to play an important accessory function in the interaction between the BA558 and epithelial cells in vitro.

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Mello, Maristela Previato. "Caracterização funcional de fatores de transcrição da família MarR de Chromobacterium violaceum." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/17/17136/tde-13092018-104909/.

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Os fatores de transcrição da família MarR atuam como sensores diretos de sinais intracelulares e regulam vários processos em bactérias, incluindo virulência e degradação de compostos aromáticos. Neste trabalho, identificamos de modo global os fatores de transcrição da família MarR envolvidos na virulência do patógeno oportunista de humanos Chromobacterium violaceum. Usando mutagênese por troca alélica, geramos mutantes nulos não polares para doze dos quinze reguladores da família MarR encontrados no genoma de C. violaceum. Em ensaios de virulência, quando injetados por via intraperitoneal em camundongos BALB/c, os mutantes ?CV_0210 (?ohrR), ?CV_0577 e ?CV_2726 foram menos virulentos, enquanto o mutante ?CV_1776 foi mais virulento, quando comparados à linhagem selvagem. Para os demais nove mutantes MarR não houve diferença na virulência. Para definir o regulon de alguns destes reguladores da família MarR, os perfis de expressão gênica foram determinados por ensaios de microarranjo de DNA e Northern blot para as linhagens mutantes ?CV_0210 (?ohrR), ?CV_1776, ?CV_1810 e ?CV_2726, para a linhagem selvagem superexpressando CV_2726 e para a linhagem selvagem em estresse oxidativo com hidroperóxido de cumeno (CHP). O regulon do repressor CV_1810 compreendeu dois operons divergentes, que codificam enzimas que possivelmente metabolizam compostos aromáticos, mas produtos do catabolismo destes compostos não funcionaram como ligantes capazes de antagonizar a repressão de CV_1810 no gene CV_1801. O regulon do ativador CV_2726, definido como quatorze genes comuns diferencialmente expressos em ensaios na ausência e na condição de superexpressão do gene CV_2726, revelou poucos genes (cstA) com potencial de estar envolvidos no fenótipo de menor virulência do mutante ?CV_2726. Os reguladores CV_0577 e CV_1776 foram alocados na subfamília UrtR de resposta a urato e provavelmente influenciam a virulência de C. violaceum com regulons sobrepostos. O regulon de CV_1776 abrangeu dezenas de genes, muitos deles relacionados ao catabolismo de aminoácidos, mas há poucos candidatos a fatores de 10 virulência clássicos (pecM, escU). Alguns genes do catabolismo/utilização de purinas (CV_0578 e CV_3771) foram regulados tanto por CV_1776 quanto por CV_0577 e responderam a presença de urato. O perfil transcricional da resposta adaptativa de C. violaceum a CHP, um ligante que oxida o regulador OhrR, revelou aumento na expressão de genes relacionados à detoxificação de peróxidos (enzimas antioxidantes e sistemas redutores de tiol), degradação da porção aromática do CHP (oxigenases) e proteção contra estresses secundários (reparo de DNA, choque térmico, limitação de ferro e nitrogênio). O regulon de OhrR revelou-se pequeno, incluindo dois genes com expressão aumentada, CV_0209 (ohrA) e CV_0208 (possível diguanilato ciclase), e três genes com expressão diminuída (hemolisina, quitinase e colagenase) no mutante ?ohrR. Assim, a virulência atenuada do mutante ?ohrR deve estar relacionada ao aumento da produção do segundo mensageiro cíclico di-GMP (c-diGMP) e à diminuição da expressão de enzimas degradativas extracelulares. Em conclusão, definimos a resposta transcricional à CHP, identificamos potenciais fatores de virulência, como a diguanilato ciclase, no regulon OhrR, e mostramos que C. violaceum utiliza os fatores de transcrição da família MarR CV_0577, CV_1776, CV_2726 e OhrR para modular sua virulência.
Transcription factors belonging to the MarR family act as direct intracellular sensors of signals and control many processes in bacteria, including virulence and degradation of aromatic compounds. In this work, we identify and characterize MarR family transcription factors controlling virulence in Chromobacterium violaceum, an opportunistic pathogen of humans. Using allelic exchange mutagenesis, we generate non-polar null mutants for twelve of the fifteen MarR family regulators found in the C. violaceum genome. In virulence tests, when introduced by intraperitoneal injection in BALB/c mice, the ?CV_0210 (?ohrR), ?CV_0577 and ?CV_2726 mutant strains were less virulent, while the ?CV_1776 was more virulent, when compared to the wild-type strain. The other nine MarR mutants showed no difference in virulence tests. To define the regulon of some MarR family transcription factors, the gene expression profiles were determined by DNA microarray analysis and Northern blot assays for the ?CV_0210 (?ohrR), ?CV_1776, ?CV_1810 and ?CV_2726 mutant strains, for the wild-type strain overexpressing CV_2726 and for the wild-type strain exposed to oxidative stress generated by cumene hydroperoxide (CHP). The CV_1810 is a repressor of a regulon that comprised two divergent operons encoding enzymes that possibly metabolize aromatic compounds, but catabolic products of these compounds did not function as ligands capable of antagonizing the repression of CV_1810 on the CV_1801 gene. The regulon of the activator CV_2726, defined as fourteen differentially expressed genes commonly found in assays in the absence and overexpression of the CV_2726 gene, revealed few genes (cstA) with potential to be involved in the phenotype of lower virulence of the ?CV_2726 mutant strain. Regulators CV_0577 and CV_1776 were allocated in the urate-responsive UrtR subfamily and probably afect the virulence of C. violaceum with overlapping regulons. The CV_1776 regulon contains dozens of genes, many of them related to amino acid catabolism, but there are few candidates for classical virulence factors (pecM, escU). Some genes related to catabolism/utilization of purine (CV_0578 and CV_3771) were 12 regulated by both CV_1776 and CV_0577 and responded to the presence of urate. The transcriptional profile of the adaptive response of C. violaceum to CHP, a ligand that oxidizes the OhrR regulator, revealed the upregulation of genes related to the detoxification of peroxides (antioxidant enzymes and thiol-reducing systems), degradation of the aromatic moiety of CHP (oxygenases), and protection against other secondary stresses (DNA repair, heat shock, iron limitation, and nitrogen starvation responses). The OhrR regulon was shown to be small, including two upregulated genes, CV_0209 (ohrA) and CV_0208 (putative diguanylate cyclase), and three downregulated genes (hemolysin, chitinase, and collagenase) in the ?ohrR mutant. Thus, the attenuated virulence of the ?ohrR mutant might be related to the increased production of the second messenger cyclic di-GMP (c-di-GMP) and the decreased expression of extracellular enzymes required for tissue dissemination, in this mutant strain. In conclusion, we have defined the transcriptional response to CHP, identified potential virulence factors such as diguanylate cyclase as members of the OhrR regulon, and shown that C. violaceum uses the transcription factors of the MarR family CV_0577, CV_1776, CV_2726 and OhrR to modulate its virulence.

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20

Endo, Marcos Sergio. "Investigação de microrganismos e endotoxinas em infecções intrarradiculares associadas ao insucesso do tratamento endodôntico antes e após o preparo químico-mecânico." [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/290436.

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Orientador: Brenda Paula Figueiredo de Almeida Gomes
Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Introdução: Microrganismos resistentes à terapia endodôntica ou que invadiram o sistema de canais radiculares após os procedimentos clínicos de desinfecção são considerados as principais causas do insucesso endodôntico. Objetivos: 1) investigar a prevalência de Enterococcus faecalis nos casos de retratamento endodôntico e lesão periapical utilizando a técnica de cultura, PCR tradicional e nested PCR, e avaliar a suscetibilidade antimicrobiana e os fatores de virulência dos E. faecalis isolados dos canais radiculares investigados (Capítulo 1); 2) quantificar bactérias viáveis e endotoxinas em dentes com infecções endodônticas secundárias e correlacionar seus níveis com aspectos clínicos e radiográficos, e também avaliar o efeito do preparo químico-mecânico (PQM) com clorexidina 2% gel + EDTA 17% na redução de bactérias e endotoxinas. Também visou investigar determinadas espécies bacterianas Gram-negativas por meio da técnica de PCR (Capítulo 2). Métodos: Amostras microbiológicas foram coletadas de 30 canais radiculares de dentes com tratamento endodôntico prévio e lesão periapical após a remoção da guta-percha (C1) e após o PQM (C2). Técnicas de cultura microbiana, PCR (16S rDNA) e nested PCR foram empregadas para investigação de E. faecalis. Determinadas espécies bacterianas Gram-negativas foram investigadas por meio da técnica de PCR. Níveis de endotoxinas e unidades formadoras de colônias (UFCs) foram monitorados em C1 e C2, utilizando o método LAL e cultura, respectivamente. Cepas clínicas de E. faecalis foram testadas quanto sua suscetibilidade antimicrobiana frente a 12 tipos de antibióticos por meio do E-test. Fatores de virulência (efaA, ace, asa, asa373, gelE, esp e cylA) dos E. faecalis isolados foram investigados pela técnica de PCR. Resultados: E. faecalis foi encontrado pela técnica de cultura (7/30), PCR tradicional (13/30) e nested PCR (23/30). PCR tradicional e nested PCR revelaram maior sensibilidade do que a cultura na detecção de E. faecalis (p<0,05, teste McNemar). P. nigrescens (4/15), P. intermedia (2/15), F. nucleatum (1/15), T. denticola (1/15), T. socranskii (1/15) e T. forsythia (2/15) foram detectadas nos canais radiculares. Endotoxinas foram detectados em todos os casos (C1 e C2). Correlação positiva entre níveis de endotoxinas e destruição óssea periapical foi observada (p<0,05). E. faecalis apresentou-se suscetível frente à Amoxicilina, Amoxicilina + ácido clavulânico, Benzilpenicilina, Moxifloxacina e Vancomicina, entretanto algumas cepas mostraram resistência contra Eritromicina (3/12), Azitromicina (8/12), Rifampicina (4/12), Tetraciclina (2/12) e Doxiciclina (1/12). Foram encontrados os seguintes fatores de virulência nos E. faecalis isolados: ace e efaA (100%), gelE (91,6%), asa (83,3%), esp (25%) e cylA (16,6%). Conclusão: 1) A percentagem de E. faecalis encontrada variou dependendo da técnica empregada. Todos os E. faecalis isolados apresentaram fatores de virulência relacionados à aderência (genes ace e efa). Foi observada resistência frente a alguns antibióticos comumente utilizados na Odontologia (Capítulo 1); 2) Foram encontrados microrganismos e endotoxinas em todos os canais radiculares, antes e após o PQM. Os níveis de endotoxinas presentes inicialmente nos canais apresentaram associação com o tamanho da lesão periapical. O PQM com clorexidina 2% gel + EDTA 17% foi efetivo na redução da carga bacteriana e dos níveis de endotoxinas nos casos de infecção endodôntica secundária. (Capítulo 2)
Abstract: Introduction: Microorganisms resistant to the endodontic therapy or that invaded the root canal system after clinical disinfection procedures are considered the main causes of endodontic failure. Aims: 1) to investigate the prevalence of E. faecalis in cases of endodontic retreatment with periapical lesions using culture technique, traditional PCR and nested PCR; and also to evaluate the antimicrobial susceptibility and virulence factors of E. faecalis isolates (Chapter 1); 2) to quantify cultivable bacteria and endotoxin in root canals with secondary endodontic infection correlating their levels with the presence of clinical features; and also to evaluate the effect of chemomechanical preparation (CMP) with 2% chlorhexidine-gel + 17% EDTA on bacterial and endotoxin removal. Moreover, it was also aimed to investigate the presence of target strict Gram-negative anaerobic bacteria by PCR/ nested PCR (Chapter 2). Methods: Microbial samples were collected from 30 root-filled canals of teeth with secondary endodontic infection after removal of gutta-percha (S1) and after CMP (S2). Microbial culture techniques, PCR (16S rDNA) and nested PCR were used to investigate the presence of E. faecalis. Target Gram-negative bacteria species were investigated by PCR. Levels of endotoxin and CFU were monitored in S1 and S2, using the LAL assay and culture, respectively. Clinical strains of E. faecalis were tested for their antimicrobial susceptibility to 12 types of antibiotics by E-test. The virulence factors (efaA, ace, asa, asa373, gelE, esp and cylA) of E. faecalis isolates were investigated by PCR technique. Results: E. faecalis was found by culture technique (7/30), traditional PCR (13/30) and nested PCR (23/30). The traditional PCR and nested PCR technique revealed higher sensitivity for detection of E. faecalis than culture (p<0.05, McNemar's test). P. nigrescens (4/15), P. intermedia (2/15), F. nucleatum (1/15) T. denticola (1/15) T. socranskii (1/15) and T. forsythia (2/15) were detected in the root canals investigated. Endotoxins were detected in all cases (S1 and S2). Positive correlation between endotoxin levels and periapical bone destruction was observed (p <0.05). All E. faecalis strains were susceptible to Amoxicillin, Amoxicillin + clavulanic acid, Benzylpenicillin, Vancomycin and Moxifloxacin, however some strains showed resistant to Erythromycin (3/12), Azithromycin (8/12), Rifampicin (4/12), Tetracycline (2/12) and Doxycycline (1/12). The virulence factors of the E. faecalis strains were ace and efaA (100%), gelE (91.6%), asa (83.3%), esp (25%) and cylA (16.6%). Conclusion: 1) The percentage of E. faecalis found varied according to the technique employed. All E. faecalis isolated showed virulence factors related to adherence (genes ace and efa). They also showed resistance to some antibiotics commonly used in dentistry (Chapter 1); 2) Microorganisms and endotoxins were found in all root canals investigated, before and after CMP. The endotoxin levels initially found in the infected root canals were associated with a larger size of periapical radiolucent area. CMP with 2% chlorhexidine-gel + 17% EDTA was effective in reducing both bacterial load and endotoxin contents in the post-treatment apical periodontitis (Chapter 2)
Doutorado
Endodontia
Doutor em Clínica Odontológica

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Fischer, Joshua Richard. "Mechanisms of Host Cell Attachment by the Lyme Disease Spirochete: A Dissertation." eScholarship@UMMS, 2005. http://escholarship.umassmed.edu/gsbs_diss/194.

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Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent, Borrelia burgdorferi, which colonizes multiple tissues, is capable of attachment to diverse cell types. Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple B. burgdorferi surface proteins. We previously showed that B. burgdorferi strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind. The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen. Complementation of a high-passage non-adherent B. burgdorferi strain reveals that the expression of DbpA, DbpB, or BBK32, is sufficient to confer efficient spirochete attachment to 293 epithelial cells. Epithelial cell attachment by DbpA and B was mediated by dermatan sulfate, while BBK32 recognized dermatan and heparan sulfate. The GAG binding properties of bacteria expressing DbpB or DbpA were distinguishable in that DbpB, but not DbpA, promoted spirochetal attachment to C6 glial cells. Furthermore, DbpA alleles from diverse Lyme disease spirochetes exhibit allelic variation with respect to binding decorin, dermatan sulfate, and epithelial cells. Targeted disruption of bbk32 resulted in decreased spirochete binding to fibronectin, GAGs, and mammalian cells. Thus, DbpA, DbpB, and BBK32 may play central but distinct roles in cell type-specific binding by Lyme disease spirochetes. This study illustrates that transformation of high-passage B. burgdorferi strains and targeted gene disruption provide a comprehensive genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors.

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Sanford, Amy. "The Characterization of a Putative Virulence Factor Expressed By Sneathia amnii." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3997.

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Preterm birth, defined at birth before 37 weeks gestation, affects millions of newborns worldwide every year. Preterm birth is a leading cause of infant morbidity and mortality. One major cause of preterm birth is preterm premature rupture of membranes (PPROM), which can be triggered by bacterial infection and inflammation. A bacterial species that has been implicated in preterm birth and other obstetric complications is Sneathia amnii. The goals of this study were to observe cytopathogenic effects caused by S. amnii strain Sn35 and identify putative virulence factors causing those effects. Sn35 was able to adhere to, invade, and damage/kill various host cell lines. We characterized these virulence attributes. A putative virulence determinant was identified, and a fragment of the protein was expressed for polyclonal antiserum production. Antiserum was used to characterize the expression and subcellular localization of the protein in Sn35. However, antiserum was unable to prevent cytopathogenic effects.

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Thanabalasuriar, Ajitha. "Functional characterization of the bacterial virulence factor NleA." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=119381.

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Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that cause severe diarrheal illnesses and death in humans. Citrobacter rodentium is a mouse pathogen that serves as a small animal model for EPEC and EHEC infections of humans. EPEC, EHEC, and C. rodentium translocate bacterial virulence proteins directly into host cells via a type III secretion system (T3SS). NleA (non-LEE-encoded effector A) is a T3SS effector that is common to these pathogens and is required for bacterial virulence. NleA localizes to the host cell secretory pathway and inhibits vesicle trafficking by interacting with the Sec24 subunit of mammalian COPII (coatamer protein II complex). Mammalian cells express four paralogues of Sec24 (Sec24A-D), that mediate selection of cargo proteins for transport and possess distinct, but overlapping cargo specificities. We have recently shown that NleA binds Sec24A-D with two distinct mechanisms. Furthermore, a mutant NleA protein with greatly diminished interaction with all Sec24 paralogues does not properly localize in the host cell, does not inhibit vesicle trafficking, and does not confer virulence in the mouse infection model. The inhibition of COPII results in NleA playing a role in intestinal epithelial barrier tight-junction disruption during EPEC infections. EPEC mediated tight-junction disruption occurs in a two-tier process. While the bacterial effectors EspF and Map are proposed to play a direct role in the breakdown of existing tight-junctions, NleA actively inhibits the translocation of newly synthesized tight junctions to the sites of tight-junctions. This results in tight-junctions being irreparably disrupted during infection. Together, this work provides strong evidence that the interaction and inhibition of COPII by NleA is an important aspect of EPEC and EHEC-mediated disease.
Les bactéries entéropathogènes et entérohémorragiques Escherichia coli (EPEC et EHEC) sont des pathogènes d'origine alimentaire pouvant causer de sévères diarrhées et même la mort chez l'humain. Citrobacter rodentium est un pathogène de la souris qui sert de modèle animal pour les infections causées par EPEC et EHEC chez l'homme. EPEC, EHEC et Citrobacter rodentium transportent les protéines de virulence bactériennes directement dans la cellule hôte via un système de sécrétion de type III (T3SS). Le NleA (non-LEE-encoded effector A ou effecteur A non encodé par LEE) est un effecteur T3SS commun chez ces bactéries et est requis pour conférer la virulence bactérienne. Le NleA repère la voie de sécrétion de la cellule hôte et inhibe le transport vésiculaire en interagissant avec la sous-unité Sec24 du COPII (coatamer protein II complex ou complexe de protéines coatamer II) des mammifères. Les cellules de mammifères expriment quatre paralogues de Sec24 (Sec24A-D), qui interviennent dans la sélection des protéines transporteuses cargo et possèdent des spécificités cargo distinctes mais se chevauchant. Nous avons récemment démontré que NleA lie Sec24A-D selon deux mécanismes distincts. De plus, une protéine mutante ayant une interaction grandement diminuée avec tous les paralogues Sec24 ne se localise pas adéquatement dans la cellule hôte, n'inhibe pas le transport des vésicules et ne confère pas de virulence dans le modèle d'infection de la souris. L'inhibition de COPII résulte en une rupture des jonctions serrées de la barrière épithéliale impliquant NleA durant l'infection à EPEC. La rupture des jonctions serrées associée à EPEC survient en deux phases. Tandis que les effecteurs bactériens EspF et Map démantèlent les protéines existantes de la jonction serrée, NleA inhibe activement la translocation vers leur site des protéines de la jonction serrée nouvellement synthétisées par l'épithélium de l'hôte. Ceci résulte en une rupture irréparable de la jonction serrée durant l'infection. Ces travaux suggèrent fortement que l'intéraction et l'inhibition de COPII par NleA est un aspect important de la maladie médiée par EPEC et EHEC.

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Rodrigues, Marjory Xavier. "Molecular characterization of bacterial isolates and microbiome: study of mastitic milk, bulk tank milk, and cheese processing plants." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/11/11141/tde-30092016-185025/.

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The present study aimed to evaluate bacterial isolates and the microbiome of dairies. The specific aims were: to characterize Staphylococcus spp. isolated from mastitic milk, to evaluate the presence of Lactococcus in mastitic milk as a potential causative agent of mastitis, to evaluate the association between microbiome and milk quality parameters, and to characterize Staphylococcus spp. isolated from production lines of Minas Frescal cheese. The detection of genes encoding virulence factors (enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, and selx), hemolysins (hla, hlb, hld, hlg, and hlgv), exfoliative toxins (eta, etb, and etd), Panton-Valentine leukocidin (pvl), and toxic shock syndrome toxin (tst)), genes encoding antibiotic resistance (resistance to tetracycline (tetK, tetL, and tetM), erythromycin (ermA, ermB, and ermC), methicillin (mecA and mecC), and tobramycin (ant(4\')-Ia)), molecular typing (spa, SCCmec, and agr types), and phenotyping regarding antibiotic resistance were performed in staphylococci isolates from mastitic milk, and from cheese processing plant samples. Staphylococcus aureus was identified in the majority of isolates from both origins. Several virulence factor genes were detected. The distribution of genes encoding staphylococcal enterotoxins (85.0% - 85.7% of isolates were positive for one or more enterotoxin gene) was highlighted and the gene related to H toxin was the most prevalent. Methicillin-resistant Staphylococcus aureus were identified in isolates from mastitic milk (4.1%) and cheese processing (6.0%); the genotyping and phenotyping of these isolates were described. t605 had the highest frequency in the S. aureus population studied. In mastitic milk, Lactococcus was suggested as the causative agent of an outbreak of mastitis in a dairy farm. Using next generation sequencing, the abundance of Lactococcus was observed in microbiome samples. Bacterial isolation and DNA sequencing confirmed the presence of Lactococcus lactis and Lactococcus garvieae. The microbiome of environmental samples and bulk tank milk from the dairy farm showed the Lactococcus genus among the most common bacterial taxa, suggesting other sources of this genus. Regarding milk quality parameters, the microbiome of bulk tank milk from several dairy farms was associated with somatic cell count and bacterial count. The core microbiome was described and many genera of importance were identified. Among the associations performed between microbiome and milk quality parameters, the identification of Streptococcus in samples classified with high somatic cell count and high bacterial count was highlighted. Several bacterial taxa with relative abundance significantly higher in samples classified as high and low cell count and bacterial count were shown. Real-time polymerase chain reaction was also performed associated with bacterial diversity, bacterial taxa, and bacterial count. These findings highlight the need to control and prevent bacterial contamination in the dairy industry, from herd to consumers.
O presente estudo apresentou como objetivo avaliar isolados bacterianos e microbioma de lácteos. Os objetivos específicos foram: caracterizar Staphylococcus spp. isolados de leite de vacas com mastite, avaliar a presença de Lactococcus em leite de vacas com mastite como um potencial agente causador de mastite, avaliar a associação entre microbioma de leite de tanque e parâmetros da qualidade de leite, e caracterizar Staphylococcus spp. isolados de linhas de processamento de queijo Minas frescal. A detecção de genes codificadores de fatores de virulência (enterotoxinas (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, e selx), hemolisinas (hla, hlb, hld, hlg, e hlgv), toxinas exfoliativas (eta, etb e etd), leucocidina de Panton-Valentine (pvl), toxina da síndrome do choque tóxico (tst)), genes codificadores de resistência a antibióticos (resistência a tetraciclina (tetK, tetL e tetM), eritromicina (ermA, ermB e ermC), meticilina (mecA e mecC) e tobramicina (ant(4\')-Ia)), tipagem molecular (spa, SCCmec e agr types), e fenotipagem quanto à resistência a antibióticos foram realizadas em estafilococos isolados de leite de vacas com mastite e de amostras de planta de processamento de queijo. Staphylococcus aureus foi identificado na maioria dos isolados de ambas as origens. Diversos genes de fatores de virulência foram detectados, com destaque para a distribuição de genes codificadores de enterotoxinas estafilocócicas (85,0%-85,7% dos isolados foram positivos para um ou mais genes codificadores de enterotoxinas), sendo o gene relacionado com a toxina H o mais frequente. Staphylococcus aureus meticilina resistente foram identificados em isolados de leite de vacas com mastite (4.1%) e em processamento de queijo (6.0%); o perfil genotípico e fenotípico destes isolados foram descritos. t605 foi o mais freqüente na população de S. aureus estudada. Em leite de vacas com mastite, Lactococcus foi sugerido como o agente causador de um surto de mastite numa fazenda leiteira. Usando sequenciamento de nova geração, a abundância de Lactococcus foi observada no microbioma das amostras. O isolamento e sequenciamento de DNA confirmaram a presença de Lactococcus lactis e Lactococcus garvieae. O microbioma de amostras ambientais e de leite de tanque da fazenda mostrou o gênero Lactococcus entre os mais comuns, sugerindo outras fontes deste gênero. Contemplando parâmetros da qualidade de leite, o microbioma de leite de tanque de várias fazendas leiteiras foi relacionado com contagem de células somáticas e contagem bacteriana. O core microbiome foi descrito e muitos gêneros bacterianos de importância foram identificados. Dentre as análises realizadas associando microbioma com parâmetros da qualidade de leite, foi destacada a identificação de Streptococcus em amostras classificadas com alta contagem de células somáticas e alta contagem bacteriana. Diversos táxons bacterianos com abundância relativa significativamente maior em amostras classificadas com alta e baixa contagem de células somáticas e contagem bacteriana foram mostrados. Reação em cadeia da polimerase em tempo real também foi realizada e associada com diversidade bacteriana, táxons bacterianos e contagem bacteriana. Estes levantamentos confirmam a necessidade de controlar e prevenir a contaminação bacteriana na indústria de lácteos, do rebanho leiteiro até os consumidores.

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Da, Silva Livia Pilatti Mendes 1989. "Construção de mutantes para os genes ychO, luxS e qseC presentes em uma linhagem de Escherichia coli patogênica para aves (APEC) : análises in vivo e in vitro." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317366.

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Orientador: Wanderley Dias da Silveira
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Linhagens de Escherichia coli patogênica para aves causam doenças extraintestinais em aves, levando a perdas econômicas consideráveis para a indústria avícola em todo o mundo. Fatores de virulência ainda não conhecidos podem apresentar papéis importantes na patogenicidade, os quais podem ser significativos para o desenvolvimento de medidas de controle de processos infecciosos dessas linhagens. Invasinas permitem que o patógeno invada células hospedeiras, sobrepujando as defesas do hospedeiro, por interagir com receptores específicos na superfície celular. Baseando-se em análises do genoma da linhagem APEC SEPT362, detectou-se a presença de uma provável invasina, homóloga ao gene ychO, ainda não caracterizado, da linhagem E. coli str. K-12 substr. MG1655, sendo seu efeito na patogenicidade estudado. Neste trabalho, a linhagem SEPT362 deficiente para ychO reduziu a taxa de mortalidade em pintos, a adesão a células de fibroblasto de embrião de galinha (FEG), a invasão em células fibroblasto de embrião de galinha (CEC-32), a sobrevivência em macrófagos de aves (HD11), a formação de biofilme e a motilidade. Esses resultados sugerem, pela primeira vez, que o gene ychO codifica para uma invasina e que tem papel na patogenicidade da linhagem APEC SEPT362. Além dos diversos fatores de virulência, o estudo de diferentes tipos de sinais extracelulares que medeiam a comunicação entre bactérias e regulam a expressão gênica abre grandes possibilidades no estudo de mecanismos de patogenicidade e possíveis meios de interferir nos mesmos, transformando organismos patogênicos em organismos não virulentos. Quorum sensing (QS) é um mecanismo dependente da densidade celular bacteriana que as permitem agir como complexos multicelulares, aumentando suas chances de sobrevivência no meio ambiente. O sistema QS autoindutor 2 (AI-2) é um sistema de comunicação universal de bactérias mediado pelo AI-2, produzido pela ação da enzima LuxS. O sistema QS autoindutor 3 (AI-3) é um sistema que pode também mediar a comunicação entre reinos, sendo regulado pela hisitidina quinase QseC. Neste contexto, mutantes para os genes luxS e qseC foram construídos na linhagem SEPT362. A linhagem deficiente para luxS apresentou uma redução significativa na adesão a células FEG, devido a um papel do gene na regulação da fímbria do tipo 1. A linhagem deficiente para qseC teve uma menor taxa de mortalidade em pintos e uma redução significativa na adesão a células FEG, pela falta da fímbria do tipo 1 no mutante
Abstract: Avian pathogenic Escherichia coli strains cause extraintestinal diseases in poultry, leading to substantial economic losses to the poultry industry worldwide. Virulence factors not yet known may play important roles in pathogenicity, which may be significant for measures control development of infectious processes of these strains. Invasins allow the pathogen to invade host cells, overcoming host defenses by interacting with specific cell surface receptors. Based on analysis of APEC SEPT362 strain genome detected the presence of a putative invasin gene homologous to ychO, not yet characterized, of E. coli str. K-12 substr. MG1655, and its effect on pathogenicity was studied. In this work, the SEPT362 strain deficient for ychO reduced the mortality rate in chicks, the adherence to chicken embryo fibroblast (CEF) cells, the invasion of chicken embryo cells (CEC-32), the survival in poultry macrophages (HD11), the motility and biofilm formation. These results suggest, for the first time, that ychO gene encodes for an invasin and plays a role in APEC SEPT362 pathogenicity. In addition to the many virulence factors, the study of different types of extracellular signals that mediate communication between bacteria and regulate gene expression opens up great possibilities to study pathogenic mechanisms and possible ways to interfere in it, transforming pathogenic organisms into non-virulent organisms. Quorum sensing (QS) is a mechanism dependent on the bacterial cell density that enables them to act as multicellular complexes, increasing their chances of survival in the environment. The QS system autoinducer 2 (AI- 2) is a universal communication system of bacteria mediated by AI- 2 produced by the action of the enzyme LuxS. The QS system autoinducer 3 (AI- 3) is a system that can also mediate communication between realms, being regulated by kinase QseC. In this context, mutants for the luxS and qseC genes were built in SEPT362 strain. The strain deficient for luxS showed a significant reduction in CEF cell adhesion due to a role in the regulation of gene type 1 fimbriae. The deficient strain for qseC had a lower rate of mortality in chicks and a significant reduction in cell adhesion to CEF, due to a lack of type 1 fimbriae in the mutant
Mestrado
Genetica de Microorganismos
Mestra em Genética e Biologia Molecular

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26

Holmes, Ashleigh. "Characterising virulence factors from pathogenic bacteria using fluorescent reporters." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/3317/.

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Protein translocation systems are invaluable to pathogenic bacteria, facilitating the display of virulence factors on their surface or their release into the extracellular environment. Some protein export systems are ubiquitous and essential to cell survival whereas others are horizontally acquired on prophages or pathogenicity islands (PAI), in many cases providing the bacterium with pathogenic advantages. For the majority of the known protein export systems, their structure, function and secreted substrates have been characterised, yet some proteins have been identified that are secreted via unknown mechanisms. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of human foodborne disease worldwide. The pathogenesis of this bacterium is mainly attributed to the secretion of toxins and the presence of a Type III Secretion System (T3SS). The T3SS can translocate bacterial proteins, known as effectors, into the host cell which mediate an effect culminating in the formation of a characteristic attaching and effacing (A/E) lesion. This system is encoded on a horizontally acquired PAI termed the locus of enterocyte effacement (LEE). The LEE not only encodes the T3SS apparatus but also several effectors secreted by the system and transcription factors which regulate its expression. However, it was recently found that T3SS not only secretes LEE encoded effectors but can also secrete proteins encoded on other prophages present in the EHEC genome. Characterisation of these non-LEE encoded effectors is ongoing and this study investigates the expression, regulation and function of non-LEE encoded effector H1 (NleH1) and H2 (NleH2). NleH1 and NleH2 are secreted by the T3SS but are encoded on different prophages. This study demonstrates that expression of NleH1 and NleH2 is induced in the same in vitro conditions which stimulate the expression of the LEE but is diminished upon initial host cell contact in vitro. Transcription of nleH1 and nleH2 is dependant upon factors specific to E. coli O157:H7 and these factors are regulated by LEE encoded regulators Ler and GrlA, as they have a positive effect on nleH transcription. NleH1 and H2 are predicted serine/threonine protein kinases and are able to autophosphorylate. Yeast two hybrid screening and 2D differential gel electrophoresis did not elucidate a eukaryotic protein binding partner of NleH1 or NleH2. Transfection assays show that they do not have a significant effect upon NF-κB activation in vitro. Determining the expression, regulation and function of non-LEE encoded effectors contributes towards further understanding of how this pathogen causes disease. Streptococcus pneumoniae, also known as the pneumococcus, is another globally important human pathogen. It is a very diverse pathogen, with over 90 capsular serotypes and is naturally competent for DNA uptake. Pneumococcal pathogenesis is facilitated by the production of a pore-forming toxin, pneumolysin. Pneumolysin’s activities in pneumococcal pathogenesis extend beyond its cytolytic function as it can also activate the complement pathway and modulate the host cytoskeleton. Pneumolysin is a member of a conserved family of toxins known as the cholesterol dependant cytolysins but differs due to the lack of a secretion signal peptide within its sequence. This indicates that it is not secreted from the bacterium however it has been reported that some strains can release pneumolysin in a cell lysis-independent manner. Additional to this, pneumolysin can also localise to the cell wall, and this localisation is not strain dependent. This study characterised codon-optimised N-terminally labelled pneumolysin constructs and applied them to assess the localisation of pneumolysin. In addition, the importance of autolysin and genes which are co-transcribed with Ply upon the localisation/secretion of pneumolysin was investigated by construction of a pneumococcal strain carrying an autolysin-pneumolysin fusion which naturally occurs in equine strains. These genes were not required for the translocation of pneumolysin or its association with the cell wall. Growth of this strain, and its isogenic parent, in vitro at a low density and low temperature resulted in the pneumolysin being detected in the broth culture. This indicates that pneumolysin can be released from the cell wall and that this action is not dependant upon the genes which were deleted in the mutant. The distribution of pneumolysin on the pneumococcal surface was assessed with immunofluorescence, and LumioTM substrate fluorescence, microscopy and found to have a general distribution. As a contribution to future pneumococcal research, codon-optimised fluorescent protein reagents were developed and can be used as reporters for gene expression and protein localisation.

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Botham, Crystal Marie. "Molecular and genetic dissection of host pathways disrupted by Helicobacter pylori's virulence factor, cytotoxin associated gene A /." view abstract or download file of text, 2007. http://proquest.umi.com/pqdweb?did=1417800931&sid=1&Fmt=2&clientId=11238&RQT=309&VName=PQD.

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Thesis (Ph. D.)--University of Oregon, 2007.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 60-66). Also available for download via the World Wide Web; free to University of Oregon users.

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Rompikuntal, Pramod Kumar. "Outer membrane vesicle-mediated export of virulence factors from Gram-negative bacteria." Doctoral thesis, Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten), 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-57475.

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The Gram-negative, motile bacterium Campylobacter jejuni is a causative agent of food-borne gastroenteritis. Cytolethal distending toxin (CDT) is one of the important virulence factors for C. jejuni pathogenesis. It was not previously known how CDT is released from C. jejuni into the surrounding environment. In our study, CDT proteins were observed in the periplasmic fraction and all CDT subunits from C. jejuni were released from the bacterial cells in association with OMVs. The OMV-associated toxin caused cytolethal distending effects on tissue culture cells. Our results strongly suggest that the release of OMV-associated CDT is a route by which C. jejuni delivers all CDT toxin subunits (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.The Gram-negative, motile bacterium Vibrio cholerae is primarily known as the causal organism of the severe dehydrating diarrheal disease cholera. OMVs released from non-O1 non-O139 V. cholerae (NOVC) strain V:5/04 induced an inflammatory response in human host cells. The inflammatory potential is mediated by the nucleotide-binding domain, leucine-rich repeat containing family members NOD1 and NOD2. Physiochemical analysis in conjunction with NOD1/2 reporter assays in HEK293T cells confirmed the presence of the NOD1/2 active peptidoglycan (PGN) in OMVs. Deletion of the quorum sensing master regulator HapR specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. These findings suggest that OMVs from a NOVC strain delivered PGN to the host cells, where they elicited an immune response mediated by NOD1 and NOD2.The Gram-negative, non-motile coccobacillus Aggregatibacter actinomycetemcomitans is a natural inhabitant of the oral cavity, but the bacterium can translocate from the oral cavity into the bloodstream and thereby be transported to other regions of the body. A. actinomycetemcomitans is implicated in aggressive forms of periodontitis. The mechanism behind this aggressive periodontitis was not fully known. In addition to several virulence factors, this organism also produces CDT. We have demonstrated that OMVs released by A. actinomycetemcomitans contain several virulence factors, including CDT. We showed that OMVs delivered CDT to the host cells and that CDT was localized inside the nucleus, which led to a cytolethal distending effect on two different cell lines tested: HeLa cells and human gingival fibroblasts (HGF). These results suggest that A. actinomycetemcomitans OMVs could deliver biologically active CDT toxin into the periodontal tissue and may contribute to periodontitis.In our earlier studies, we discovered that an M6 family metalloprotease PrtV was an essential factor for V. cholerae survival from predator grazing. Pure PrtV protein effectively degraded human blood plasma components. In addition, it also showed a dose-dependent cytotoxic effect in the human intestinal HCT8 cell line. V. cholerae produces a large amount of outer membrane vesicles (OMVs) during the normal course of cell growth. OMVs are composed of periplasmic proteins, membrane lipids, lipopolysaccharides and outer membrane proteins. We showed that OMVs can transport several biologically active toxins and enzymes to the surrounding environment and ultimately into the host cells. We have initiated analysis of OMV-associated secretion of virulence factors in V. cholerae. It was observed that PrtV is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs and OMV-associated PrtV protein is biologically active and more stable than the free, soluble PrtV protease.

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Martinez, Castillo Alexandre. "Movilidad de factores de virulencia del patógeno E. Coli O157:H7 y otros serotipos." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/327313.

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Los elementos genéticos móviles (EGMs) tienen la capacidad de transportar el ADN bacteriano y hacer que prácticamente cualquier gen presente en una bacteria pueda ser movilizado a otra. Los bacteriófagos son un tipo de EGM que permiten la movilización de genes de una cepa bacteriana a otra, pudiendo generar nuevas cepas patógenas si el gen que transfieren codifica para una proteína que provoca virulencia. En esta tesis doctoral nos hemos centrado en el estudio de los bacteriófagos portadores de genes de virulencia como EGMs. Para ello se estudió, en primera instancia, la presencia de bacteriófagos Stx2 en heces de individuos sanos, dado que se ha demostrado su presencia y capacidad de infección en alimentos aptos para el consumo humano y en aguas residuales. La presencia de fagos Stx en heces humanas aparece como la conexión entre su prevalencia en los alimentos, los cuales al ser ingeridos incorporan los fagos Stx, y su detección en agua residual, como resultado de su excreción. Se detectó la presencia de fagos Stx en las heces de individuos sanos y se evaluó su capacidad infectiva. La evolución de las cepas Shiga toxin Escherichia coli (STEC) se determinó a partir de ancestros stx-negativos y que inicialmente no poseían el resto de genes necesarios para producir la enfermedad. Por ello se estudiaron los genes de virulencia en cepas STEC ambientales, permitiendo observar los diferentes estadios intermedios en las evoluciones de las cepas STEC. Las variantes de stx encontradas fueron stx2a y stx2c, que corresponden a las variantes asociadas a mayor virulencia. El estudio de la distribución de los genes de virulencia según su origen reveló claramente que las cepas que contenían un mayor número de factores de virulencia eran cepas de origen vacuno, seguidas de las de origen humano, siendo el serotipo O157:H7 el que contenía mayor número de genes de virulencia. En general, los genes de virulencia que se detectaron con mayor frecuencia fueron aquellos que no estaban codificados en el denominado locus for enterocyte effacement (LEE). El gen más predominante fue hlyA que codifica para una enterohemolisina seguido por el gen nleC y el gen saa El desarrollo de una matriz para visualizar la distribución de los genes entre sí permitió la detección de dos agrupaciones: el grupo formado por genes codificados en LEE y otros genes como espJ, espM, nleB2, nleC, nleD, nleE, nleF, nleA, nleH1 y tccP; y los genes saa, hlya, stx1, cdt y cif que raramente se encontraban en combinación con otros genes, y probablemente se movilicen independientemente mediante algún tipo de EGM. A continuación, nos centramos en el estudio de los genes no descritos en fagos que parecía que se movilizaban de forma independiente y, más en concreto, en el gen saa. El gen saa codifica para una adhesina que se encuentra en las cepas STEC que no codifican para LEE. Se analizaron las diferentes variantes del gen en las cepas STEC saa+ aisladas y se observó y cuantificó la adherencia a células Hep2. En contacto con un agente inductor como la Mitomicina C, el número de copias del gen saa aumentaba hasta 4 unidades logarítmicas y se observó hibridación positiva con una sonda saa en calvas de lisis. Estos resultados sugieren que saa puede ser un gen movilizado por un bacteriófago o, al menos, movilizado mediante una cápside proteica. La banda de CsCl en la que se detectaba el gen saa se observó por microscopia electrónica revelando una morfología tipo Podoviridae. Se confirmó por proteómica la presencia de proteínas fágicas en la fracción de CsCl donde se detectaba saa, siendo una de ellas homóloga a proteínas tipo gene transfer agents (GTA). A sí mismo, se detectó la presencia de saa en la fracción viral de diferentes muestras de origen animal y humano. Finalmente, dado que el primer paso para que una cepa llegue a ser infectada por un fago es la unión de éste a los receptores de membrana de la bacteria provocando la activación de las respuestas de estrés de membrana, se estudiaron la influencia de los sistemas de estrés de membrana en la conversión lisogénica de la bacteria. Los resultados obtenidos indican que la vía BaeSR está implicada en la formación de lisógenos. A pesar de que los fagos Stx son un grupo muy heterogéneo, este resultado se observó con distintos fagos Stx y se determinó que este efecto se debía a los receptores de membrana BamA.
In this thesis we focused on studying bacteriophages mobilization of virulence genes as MGE. To achieve this goal we studied the presence of Stx2 bacteriophages in feces of healthy individuals since it has been shown their presence and infectivity in food for human consumption and in wastewater. We were able to detect Stx phages in feces of healthy individuals and their infectivity was evaluated. Virulence genes in environmental STEC strains were studied, allowing the observation of the different intermediate stages in the evolution of STEC strains. Strains from bovine origin showed the largest number of virulence factors, followed by those from human origin, with serotype O157: H7 containing the largest number of virulence genes. In general, the most frequently virulence genes detected were those not encoded in LEE. The most predominant virulence gene was hlyA, which encodes for enterohemolysin, followed by the nleC gene and the saa gene. The development of a matrix to visualize the gene distribution allowed the detection of two different groups: LEE encoded genes and other genes such as espJ, espM, nleB2, nleC, nleD, nleE, nleF, nleA, nleH1 and tccP; and the saa, hlya, stx1, cdt and cif genes rarely found in combination with other genes, which probably are mobilized independently by a MGE. Then, we focused on the study of genes not described in phages that seemed to be mobilized independently, particularly, the saa gene. saa encodes for an adhesine found in LEE-negative STEC strains. The variants of the gene were analyzed in isolated saa+ STEC strains and the adherence to Hep2 cells was evaluated and quantified. In the presence of an inducing agent such as Mitomycin C, the number of saa copies increased 4 log units and positive hybridization plaques were detected by using a saa probe, suggesting that saa could be mobilized by a bacteriophage or, at least, by a protein capsid. The CsCl band in which saa was detected was analyzed by electron microscopy revealing a Podoviridae morphology and by proteomic studies it was confirmed the presence of phage proteins, one of them showing homology to gene transfer agents (GTA) proteins. saa was also detected in the viral fraction of human and animal origin samples. Finally, since the first step for a strain to be infected by a phage is the attachment of the phage to specific receptors on the surface of bacteria triggering membrane stress responses, the influence of membrane stress systems in bacteria lysogenic conversion were studied. We demonstrated that BaeSR is involved in the formation of lysogens and that this effect was due to BamA membrane receptors.

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Sinha, Sarmistha Gates Kent S. "Oxidative DNA damage by 1-hydroxyphenazine, virulence factor of Pseudomonas aeruginosa towards a molecular understanding of the bacterial virulence factor 1-hydroxyphenazine /." Diss., Columbia, Mo. : University of Missouri-Columbia, 2008. http://hdl.handle.net/10355/7119.

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The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed April 27, 2010). Thesis advisor: Dr. Kent S. Gates. Vita. Includes bibliographical references.

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Distelhorst, Steven Lindau. "Understanding virulence factors of Mycoplasma penetrans: attachment organelle organization and gene expression." Miami University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=miami149060747354379.

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Felek, Arif. "Discovery of antimicrobial peptides active against antibiotic resistant bacterial pathogens." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/discovery-of-antimicrobial-peptides-active-against-antibiotic-resistant-bacterial-pathogens(cc408f16-e24a-4f49-ac8e-e7a5fe7185d0).html.

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Rapid development of antimicrobial resistance (AMR) among bacteria, combined with diminished new antibiotic discovery rates, is an increasing threat to human health. Bacterially derived antimicrobial peptides (AMP) hold excellent potential as potent novel therapeutics. This study embraces traditional natural AMP discovery methods and the newer in silico genome mining tool BAGEL 3 to facilitate identification of novel antimicrobial agents. The traditional screening efforts led to the discovery of two promising antimicrobial producer strains; Bacillus pumilus J1 producing two AMPs, peptides NI03 and NI04, and Klebsiella pneumoniae A7, which produced peptide NI05. In silico mining of the B. pumilus J1 and K. pneumoniae A7 genomes and those from under exploited anaerobic bacteria using BAGEL 3 yielded 18 putative bacteriocin structures that were associated with multiple known and relevant bacteriocin accessory genes and/or carried significant homologies to known bacteriocins. Peptide NI04 proved to be active against Gram positive species only, including meticillin resistant Staphylococcus aureus and vancomycin resistant enterococci and peptide NI03, in addition to these pathogens, showed activity against E. coli. Peptide NI05 was active against Gram-negative pathogens including extended spectrum β-lactamase producing E. coli. All isolated peptides were observed to be proteinaceous in nature and highly heat stable. Peptides were purified or partially purified using solid phase extraction followed by RP-HPLC. The mass of the peptides was determined using ESI or MALDI-TOF mass spectrometry. Tandem MS fragmentation of peptide NI04 generated several sequence tags. Draft genome sequences of the B. pumilus J1 and K. pneumoniae A7 producer strains were obtained using the Illumina MiSeq platform. This allowed identification of the genes encoding peptide NI04, which was confirmed to be novel and was named pumicin NI04. Further characterisation of pumicin NI04 demonstrated it was non-toxic to keratinocytes, Vero cells and non-haemolytic up to at least 18x the minimum inhibitory concentration. The discovery revealed that pumicin NI04 belongs to the WXG-100 peptide superfamily, having homology with the mycobacterial and staphylococcal virulence factor EsxA. This represents the first report of antimicrobial activity in a WXG-100 peptide and has intriguing evolutionary implications. Although it was not possible to fully characterise peptides NI03 and NI05, when BAGEL 3 was used to mine the B. pumilus J1 genome, a promising putative bacteriocin candidate was identified that was homologous to Enterocin AS-45, which also confers anti Gram-negative activity and may be related to the activity observed for NI03, however more evidence is required. Investigations of the K. pneumoniae A7 bacteriocin on the other hand helped establish that the K. pneumoniae microcin E492 pathway was present and highly conserved in strain A7, and is likely to be responsible for the activity observed indicating that NI05 was not a novel peptide.

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Georgiades, Kalliopi. "Phylogénomique des bactéries pathogènes." Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX20690/document.

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La pathogénicité des bactéries a toujours été attribuée à des facteurs de virulence et les bactéries pathogènes sont considérées comme étant mieux armées, comparé à des bactéries ne provoquant pas de maladies. Selon les premières études génomiques, le fait de supprimer un certain nombre de gènes des bactéries pathogènes, limiterait leur capacité à infecter leurs hôtes. Au contraire, des études de génomique comparatives récentes, démontrent que la spécialisation des bactéries dans les cellules eucaryotes est associée à une perte de gènes massive, en particulier pour les endosymbiontes allopatriques qui sont isolés depuis longtemps dans une niche intracellulaire. En effet, les bactéries sympatriques, extracellulaires, ont souvent des génomes plus grands et présentent une résistance et une plasticité plus importante. Ces bactéries constituent, de fait, plutôt des complexes d’espèces que de vraies espèces. Certaines bactéries spécialistes, comme les bactéries pathogènes, arrivent à s’échapper de ces complexes et à coloniser une niche, bénéficiant alors d’un nom d’espèce. Leur spécialisation leur permet de devenir allopatriques et leurs pertes de gènes favorisent une évolution réductive. Ces observations nous ont conduits à réaliser une étude afin de quantifier le taux de perte de gènes lors de l’évolution de ces bactéries extracellulaires vers celle de bactéries spécialistes intracellulaires. Notre objectif était de vérifier que ce qui caractérise l’évolution des bactéries intracellulaires est bien la réduction génomique, en prenant en compte tous les événements possibles de gains de gènes. Par ailleurs, dans une étude neutre comparant les 12 espèces pandémiques les plus dangereuses pour l’homme avec les espèces non-épidémiques les plus proches, nous avons voulu identifier des spécificités génomiques associées à la capacité virulente de bactéries pathogènes et démontrer que, à part les toxines et les modules toxine-antitoxine, ce qui caractérise ces espèces ce ne sont pas les facteurs de virulence, mais la perte des gènes de régulation. Au final, les bactéries pathogènes ont un répertoire virulent dans lequel les gènes absents sont aussi importants que les gènes présents
The virulence of pathogenic bacteria has been attributed to virulence factors and pathogenic bacteria are considered to have more genes compared to bacteria that do not cause disease. According to the first genomic studies, removing a certain number of genes from pathogenic bacteria impairs their capacity to infect hosts. However, more recent studies have demonstrated that the specialization of bacteria in eukaryotic cells is associated with massive gene loss, especially for allopatric endosymbionts that have been isolated for a long time in an intracellular niche. Indeed, bacteria living in sympatry often have bigger genomes and exhibit greater resistance and plasticity and constitute species complexes rather than true species. Specialists, including specific pathogenic bacteria, escape these bacterial complexes and colonize a niche; thereby gaining a species name. Their specialization allows them to adopt allopatric lifestyle and experience reductive genome evolution. These observations led us to design a study to quantify the rate of gene losses during the evolution of free-living bacteria to intracellular specialists. Our objective was to verify that what characterizes the evolution of intracellular bacteria is genomic reduction, taking under consideration all possible gene gain events. Furthermore, in another neutral study comparing the 12 most dangerous pandemic bacteria to Humans to their closest non-epidemic species, we wished to identify any genomic specificities associated to the virulent capacity of pathogenic bacteria and demonstrate that, besides toxins and surprisingly, toxin-antitoxin modules, pathogenic bacteria are not characterized by more virulence factors, but rather by a loss of regulatory genes. Finally, virulent bacteria exhibit a genomic repertoire in which absent genes are as important as present ones

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Cole, Jason Nicklaus. "Characterisation of cell wall proteins, virulence factor maturation and invasive disease trigger of Group A streptococcus." Access electronically, 2006. http://www.library.uow.edu.au/adt-NWU/public/adt-NWU20070130.144214/index.html.

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Guzmán-Verri, Caterina. "Virulence mechanisms of two Gram negative bacteria : studies on Escherichia coli hemolysin HlyA and on the interaction of Brucella abortus with non-phagocytic cells /." Stockholm : Karolinska Univ. Press, 2002. http://diss.kib.ki.se/2002/91-7349-114-4.

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Monnanda, Ponnappa Bopanna [Verfasser]. "Host Responses to Presentation of Particulate Virulence Factors of Bacteria and Parasites / Bopanna Monnanda Ponnappa." Berlin : Freie Universität Berlin, 2016. http://d-nb.info/111702847X/34.

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Liu, Lulu. "Characterization of a new virulence factor secreted by the plant pathogenic bacteria Dickeya dadantii." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSEI020.

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Peu de protéines nécessaires à l’infection des plantes communes aux bactéries, aux champignons et aux oomycètes ont été identifiées à part les enzymes dégradant les parois des cellules végétales. Nous avons caractérisé la structure et les propriétés d’une protéine, IbpS, sécrétée par la bactérie phytopathogène nécrotrophe Dickeya dadantii. Des homologues de cette protéine sont présents non seulement chez des bactéries à Gram+ mais aussi chez des champignons, des oomycètes et quelques animaux. Ce gène d’origine bactérienne a été transféré une fois chez les oomycètes et probablement plusieurs fois chez les champignons. IbpS peut fixer le fer et le cuivre, des métaux à activité redox. Elle a une structure en Venus Fly Trap classique mais avec des caractéristiques originales : elle forme des dimères en solution et possède un nouveau mode de fixation du métal. IbpS est impliquée dans le processus infectieux de D. dadantii et de Botrytis cinerea, un champignon phytopathogène nécrotrophe. Nous proposons qu’IbpS, une fois sécrétée, fixe le fer et le cuivre exogène, réduisant ainsi leur concentration intracellulaire et la formation de ROS dans le microorganisme. La sécrétion de cette protéine fixant les métaux semble être un mécanisme de protection contre l’oxydation requis pendant l’infection partagé par des phytopathogènes nécrotrophes
Few secreted proteins involved in plant infection common to necrotrophic bacteria, fungi and oomycetes have been identified except for plant cell wall-degrading enzymes. Herein, we have characterized the structure and properties of a protein (IbpS) secreted by the plant pathogenic bacterial necrotroph Dickeya dadantii. Homologs of this protein are present in not only Gram+ bacteria but also in fungi, oomycetes, most phytopathogens, and some animals. The gene originating from bacteria was transferred once in oomycetes and most likely several times in fungi. IbpS is capable of binding the redox-active metals iron and copper and has a classical Venus Fly trap fold with some original characteristics: it forms dimers in solution and has a novel metal binding site. IbpS is involved in D. dadantii and of the Botrytis cinerea, a fungal necrotroph, infection process. We propose that secreted IbpS binds exogenous iron and copper, reducing their intracellular concentrations of these metals and ROS formation in the microorganisms. Secretion of this metal scavenging protein appears to be a common antioxidant protection mechanism shared by necrotrophic phytopathogens and required during infection

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Rosadini, Charles V. "Roles of Secreted Virulence Factors in Pathogenicity of Haemophilus Influenzae: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/541.

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Haemophilus influenzae is a pathogenic Gram-negative bacterium that colonizes the upper respiratory tract of humans and can cause otitis media, upper and lower respiratory infections, and meningitis. Factors important for H. influenzae to colonize humans and cause disease are not fully understood. Different bacterial pathogens are armed with virulence mechanisms unique to their specific strategies for interacting with their hosts. Many of the proteins mediating these interactions are secreted and contain disulfide bonds required for function or stability. I postulated that identifying the set of secreted proteins in H. influenzae that require periplasmic disulfide bonds would provide better understanding of this bacterium's pathogenic mechanisms. In this thesis, the periplasmic disulfide bond oxidoreductase protein, DsbA, was found to be essential for colonization and virulence of H. influenzae. Mutants of dsbA were also found to be sensitive to the bactericidal effects of serum. However, the DsbA-dependent proteins important for pathogenesis of this organism have not been previously identified. To find them, putative targets of the periplasmic disulfide bond pathway were identified and examined for factors which might be important for mediating critical virulence aspects. By doing so, novel virulence factors were discovered including those important for heme and zinc acquisition, as well as resistance to complement. Overall, the work presented here provides insight into requirements for H. influenzae to survive within various host environments.

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Fernandes, Matilde Costa. "Antibiotic resistance and virulence profiles of gram-negative bacteria isolated from loggerhead sea turtles (Caretta caretta) of the Island of Maio, Cape Verde." Master's thesis, Universidade de Lisboa, Faculdade de Medicina Veterinária, 2021. http://hdl.handle.net/10400.5/21241.

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Dissertação de Mestrado Integrado em Medicina Veterinária
Loggerhead sea turtles (Caretta caretta) have been suggested as carriers of potential zoonotic pathogens and prime reservoirs of antibiotic-resistant and virulent bacteria. In the present study, the isolation of Gram-negative bacteria of the Cape Verdean loggerhead subpopulation, currently believed to be the largest subpopulation of this species worldwide, is described for the first time. This study aimed to characterize the aerobic and facultative anaerobic Gram-negative bacteria of the loggerhead colony of the Island of Maio, to evaluate their pathogenic potential and to unveil both the impact on sea turtles’ conservation and the underlying public health risks resulting from interactions with these animals and the consumption of turtle-derived products. Cloacal, oral and egg content swab samples from 33 nesting loggerheads (n = 99) were analysed regarding the presence of Gram-negative bacteria and the respective antibiotic resistance and virulence profiles. Conventional bacteriological techniques were applied. Shewanella putrefaciens (26.32%), Vibrio alginolyticus (21.05%) and Morganella morganii (21.05%) were the most prevalent species. A low prevalence of antibiotic-resistant bacteria (15.79%) was detected, and no multidrug-resistant isolates were identified. The identified bacterial species revealed the ability to produce numerous virulence factors, including hemolysins (100.0%), DNases (89.47%), lipases (78.95%), biofilms (73.68%), proteases (52.63%), lecithinases (21.05%), and gelatinases (15.79%). These findings suggest that due to the low anthropogenic impact observed in both their nesting (the Island of Maio) and foraging sites, this loggerhead subpopulation may be less exposed to antimicrobial compounds. Furthermore, Gram-negative bacteria isolated from these turtles may be less susceptible to acquiring resistance genes from environmental bacteria via horizontal gene transfer. Nevertheless, the presence of potentially pathogenic bacteria expressing virulence factors may threat both sea turtles’ and human’s health.
RESUMO - PERFIS DE RESISTÊNCIA A ANTIBIÓTICOS E VIRULÊNCIA DE BACTÉRIAS GRAM-NEGATIVAS ISOLADAS DE TARTARUGAS MARINHAS COMUNS (CARETTA CARETTA) DA ILHA DO MAIO, CABO VERDE - A tartaruga-marinha-comum (Caretta caretta) é conhecida por ser portadora de agentes potencialmente patogénicos e zoonóticos, e um relevante reservatório de bactérias virulentas e resistentes aos antibióticos. O presente estudo descreve, pela primeira vez, o isolamento de bactérias Gram-negativas da subpopulação de tartarugas-comuns de Cabo Verde, a qual se estima ser a maior população mundial desta espécie. Este estudo teve como objectivo a caracterização de bactérias Gram-negativas aeróbias e anaeróbias facultativas da colónia de tartarugas-comuns da Ilha do Maio, a avaliação do seu potencial patogénico e o respectivo impacto na conservação de tartarugas marinhas e do potencial risco para saúde pública, resultante de interações com estes animais e do consumo de productos derivados de tartarugas. Neste trabalho foram analizadas amostras de zaragatoas da cloaca, cavidade oral e ovos de 33 tartarugas comuns fêmeas (n = 99), de modo a isolar bactérias Gram-negativas e os respectivos perfis de resistência a antibióticos e virulência. Para o efeito, foram usados métodos de bacteriologia convencionais. As espécies isoladas mais prevalentes foram Shewanella putrefaciens (26,32%), Vibrio alginolyticus (21.05%) e Morganella morganii (21.05%). Foi detetada uma baixa prevalência de bactérias resistentes (15.79%), e não foram identificados isolados multirresistentes. As espécies bacterianas identificadas revelaram a capacidade de produzir vários factores de virulência, incluindo hemolisinas (100.0%), DNases (89.47%), lipases (78.95%), biofilmes (73.68%), proteases (52.63%), lecitinases (21.05%), gelatinases (15.79%). Os resultados deste estudo sugerem que, devido ao reduzido impacto antropogénico observado nos locais de nidificação (Ilha do Maio) e de alimentação da subpopulação em estudo, esta estará menos exposta a compostos antimicrobianos. Além disso, bactérias Gram-negativas isoladas neste estudo, poderão estar menos suscetíveis à aquisição de genes de resistência provenientes de bactérias ambientais, via transferência horizontal de genes. Contudo, a presença de bactérias potencialmente patogénicas e com a capacidade de expressar diversos factores de virulência representa uma ameaça tanto à saúde destes animais como à saúde humana.
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Albarral, Ávila Vicenta. "Diversidad intraespecífica y factores de virulencia en el “complejo de especies de Aeromonas hydrophila” (A. Hydrophila, A. Salmonicida, A. Bestiarum)." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/125472.

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The genus Aeromonas comprises 26 currently recognized species, some of them divided in to several subspecies. They are ubiquitous in fresh water, but found in chlorinated, brackish and marine water. Aeromonas strains are obtained from a wide variety of foods, as well as clinical samples. Aeromonas species are the cause of intestinal and extra-intestinal infections although the mechanisms that cause bacterial diarrhoea are not yet clearly established. The “A. hydrophila complex” (A. hydrophila, A. salmonicida, A. bestiarum, A. piscicola and A. popoffii), includes A. salmonicida, an important pathogen of fish, and A. hydrophila, an opportunistic pathogen in humans. The virulence of these species is multifactorial and involves complex pathogenic mechanisms associated with toxins (cytotoxic and cytotonic), proteases, haemolysins, lipases, adhesins, agglutinins, pili, etc. A phylogenetic study was performed from a collection of 128 strains belonging to the “A. hydrophila complex", determining partial sequences of dnaJ, cpn60, gyrB and rpoD genes, this analysis allowed us to clarify the taxonomy of this group of Aeromonas species, showing a polyphyletic origin that challenges the existence of the group as such. The population analysis of the studied strains revealed as strong linkage disequilibrium, typical of a clonal population, and a high genetic diversity. We also studied the prevalence and distribution of different virulence factors in the population to establish its pathogenic potential. For this purpose, we determined several enzymatic activities and the antibiotic sensitivity profile of these strains. We also detected the presence of act (aerolysin), alt (cytotonic toxin) and ast (cytotonic toxin) genes by PCR as well as the adhesion capacity and cytopathic effect of the strains on the Caco-2 cell line. The results obtained revealed a high pathogenic of potential the studied Aeromonas strains, regardless of their origin.
El género Aeromonas está constituido actualmente por 26 especies reconocidas, algunas de las cuales incluyen varias subespecies. Son habitantes ubicuos del agua dulce, pero también de aguas cloradas, salobres y marinas. Se han obtenido cepas de Aeromonas de una amplia variedad de alimentos, y se han aislado de muestras clínicas. Algunas especies de Aeromonas son causantes de infecciones intestinales y extraintestinales aunque los mecanismos por los que causan diarreas bacterianas no están todavía establecidos de manera clara. Dentro del “complejo A. hydrophila” (A. hydrophila, A. salmonicida, A. bestiarum, A. piscicola y A. popoffii), hay que destacar A. salmonicida como patógeno importante de peces y A. hydrophila como patógeno oportunista de humanos. La virulencia de estas especies es multifactorial e implica mecanismos de patogenicidad complejos asociados a toxinas (citotóxicas y citotónicas), proteasas, hemolisinas, lipasas, adhesinas, aglutininas, pili, etc. A partir de una colección de 128 de cepas del “complejo A. hydrophila”, se ha realizado un estudio filogenético, poblacional y de factores de patogenicidad. El estudio filogenético con las secuencias parciales de los genes cpn60, dnaJ, gyrB, y rpoD, ha permitido clarificar la taxonomía de las especies del “complejo A. hydrophila”, demostrándose que posee un origen polifilético que cuestionaría la posible existencia del grupo como tal. El análisis de la población de las cepas estudiadas revela un fuerte desequilibrio de ligamiento, típico de una población clonal, y una elevada diversidad genética. También se ha estudiado la prevalencia y distribución de diversos factores de virulencia en la población estudiada para poder establecer su potencial patogénico. Para ello se han determinado actividades enzimáticas, el perfil de sensibilidad a antibióticos, la detección por PCR de los genes act (aerolisina/hemolisina), alt (toxina citotónica) y ast (toxina citotónica), y la capacidad de adherencia y efecto citopático de las cepas en la línea celular Caco-2. Los resultados obtenidos revelan un elevado potencial patogénico de las cepas de Aeromonas estudiadas, independientemente de su origen.

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Masadeh, Majed. "Studies on the effects of sub-minimal inhibitory concentrations of antibiotics on the virulence factors of biofilm bacteria." Thesis, Abertay University, 2005. https://rke.abertay.ac.uk/en/studentTheses/ee318b1c-d3f5-4357-8165-2bf1caed633d.

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Pseudomonas aeruginosa is a notorious nosocomial opportunist. Planktonic forms of this pathogen have been traditionally studied for its pathogenicity. Such studies have shown that sub-minimal inhibitory concentrations (sub-MICs) of antibiotics are able to negatively modulate pathogenicity. However, more recent findings suggest a biofilm basis of infection. In this study, monospecies and binary biofilms of Pseudomonas aeruginosa ATCC 15692 (PAOl) and Escherichia coli ATCC 10000 were investigated for their pathogenic potential using resistance and virulence as key pathogenic determinants, in the presence of sub-MICs of selected antibiotics (Ampicillin, Nalidixic acid and Streptomycin). MICs of biofilms were observed to be at least 7-fold greater than those of the corresponding planktonic form of the same species (as judged from results obtained from MIC experiments). SDS-PAGE and 2D-PAGE analysis indicate alteration of outer membrane proteins (OMPs) within the envelope of the pathogen in sub-MIC antibiotic treated samples. The observed rearrangement of lipopolysaccharide (LPS; as observed in LPS gel experiments) may also contribute to the pathogens increased tolerance to antibiotics within the biofilm state. While LPS changes may possibly help the biofilm bacteria escape host immune system in vivo, more direct evidence of increases in virulence of the pathogen comes from investigation of its secreted proteases and cytotoxins (leucocidin). Virulence-specific azocasein and micro-culture tetrazolium (MTT) assays against both monospecies and binary biofilms of Pseudomonas aeruginosa indicate significant increases in virulence potential of proteases and cytotoxins, respectively. These results were further substantiated in phase contrast microscopy images showing advanced stages of oncosis in tissue cultured mouse spleen myeloma (Sp2) cells treated with leucocidin isolated from Ps. aeruginosa treated with sub-MIC of ampicillin (8 pg mL'1). The results reported in this thesis provide evidence of observed increases in virulence and pathogenicity in biofilm cells of Pseudomonas aeruginosa in the presence of sub-MICs of selected antibiotics, in vitro. Although these findings are those of in vitro experiments, they may have significant implications regarding the usage and therapeutic control of antibiotics in clinical situations.

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Yang, Tao [Verfasser], and Thomas [Gutachter] Rudel. "Functional insights into the role of a bacterial virulence factor and a host factor in Neisseria gonorrhoeae infection / Tao Yang ; Gutachter: Thomas Rudel." Würzburg : Universität Würzburg, 2021. http://d-nb.info/1236503406/34.

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Nassif, Xavier. "Etude genetique des facteurs de virulence des bacteries a multiplication extracellulaire : le modele klebsiella pneumoniae." Paris 6, 1989. http://www.theses.fr/1989PA066370.

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Parmi les facteurs de virulence des bacteries a multiplication extracellulaire, les plus anciennement connus sont la capsule et le lipopolyoside. Cependant, au sein d'une espece bacterienne donnee, voire meme a l'interieur d'un serotype, certaines souches ont un pouvoir invasif plus marque. Le but de ce travail a ete, en utilisant klebsiella pneumoniae comme agent pathogene a multiplication extracellulaire, d'expliquer la virulence plus importante des souches de serotype k1 et k2 lorsqu'elles sont injectees par voie intraperitoneale chez la souris. Un plasmide de 180 kilobases present dans les souches virulentes a ete identifie. Des experiences de cure suivies de transfert de ce plasmide ont permis de demontrer son role dans la virulence des souches de k. Pneumoniae de serotype k1 et k2. Deux phenotypes codes par ce plasmide ont ete associes a la virulence: un systeme de captation du fer, l'aerobactine et l'aspect muqueux des colonies. Il a ete demontre que ce dernier phenotype n'etait pas en rapport avec une hyperproduction de polyoside capsulaire mature. Chez e. Coli, le plasmide recombinant contenant le gene plasmidique (rmpa pour regulator of mucoid phenotype) de k. Pneumoniae necessaire a l'induction du phenotype muqueux induit, a 30#oc, la production d'acide colanique responsable d'un phenotype muqueux. Aucune production d'acide colanique n'a ete detectee dans les souches muqueuses de k. Pneumoniae. L'etude du mecanisme de regulation par lequel rpma controle la production d'acide colanique chez z. Coli a ete realisee. Dans une derniere partie, nous avons etudie le role de la production de siderophore, dans la virulence de shigella flexneri, une bacterie a multiplication intra- et extracellulaire. Un mutant defectif dans la production de ce siderophore, l'aerobactine, a ete construit et teste sur les differents modeles disponibles in vitro et in vivo qui permettent l'etude des fac

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Baker, Patrick Ericson. "Genetic regulation of virulence factors contributing to colonization and pathogenesis of helicobacter pylori." The Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1061414659.

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Paquette, Sara Montminy. "Evasion of LPS-TLR4 Signaling as a Virulence Determinate for Yersinia pestis." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/458.

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Yersinia pestis, the gram-negative causative agent of plague, is a master of immune evasion. The bacterium possesses a type three secretion system which translocates Yop effector proteins into host immune cells to inhibit a number of immune and cell signaling cascades. Interestingly, this apparatus is not expressed at low temperatures such as those found within the flea vector and is therefore neither in place nor functional when the bacteria are first transmitted into a mammalian host. However, the bacterium is still able to avoid activating the immune system, even very early during infection. When grown at 37°C (human body temperature) Y. pestis produces a tetra-acyl lipid A molecule, which is antagonistic to the human Toll like receptor 4/MD2, the major lipopolysaccharide recognition receptor. Although tetra-acyl lipid A binds this receptor complex, it does not induce signaling, and in fact inhibits the receptors interaction with other stimulatory forms of lipid A. The work undertaken in this thesis seeks to determine if the production of tetra-acyl lipid A by Y. pestis is a key virulence determinant and was a critical factor in the evolution of Y. pestis from its ancestral parent Yersinia pseudotuberculosis. By examining the enzymes involved in the lipid A biosynthesis pathway, it has been determined that Y. pestis lacks LpxL, a key enzyme that adds a secondary acyl chain on to the tetra acyl lipid A molecule. In the absence of this enzyme, Y. pestis cannot produce a TLR4 stimulating form of lipid A, whereas Y. pseudotuberculosis does contain the gene for LpxL and produces a stimulatory hexa acyl lipid A. To determine if the absence of LpxL in Y. pestis is important for virulence, LpxL from E. coli and Y. pseudotuberculosis were introduced into Y. pestis. In both cases the addition of LpxL led to bacterium which produced a hexa-acylated lipid A molecule and TLR4/MD2 stimulatory LPS. To verify the LpxL phenotype, lpxL was deleted from Y. pseudotuberculosis, resulting in bacteria which produce tetra-acylated lipid A and nonstimulatory LPS. Mice challenged with LpxL expressing Y. pestis were found to be completely resistant to infection. This profound attenuation in virulence is TLR4 dependent, as mice deficient for this receptor rapidly succumb to disease. These altered strains of the bacterium also act as vaccines, as mice infected with Y. pestis expressing LpxL then challenged with wild type Y. pestis do not become ill. These data demonstrate that the production of tetra-acyl lipid A is a critical virulence determinant for Y. pestis, and that the loss of LpxL formed a major step in the evolution of Y. pestis from Y. pseudotuberculosis. These bacterial strains were also used as tools to determine the contributions of different innate immune receptors and adaptor molecules to the host response during Y. pestis infection. The use of LpxL expressing Y. pestis allowed identification of the innate immune pathways critical for protection during Y. pestis infection. This model also established that CD14 recognition of rough LPS is critical for protection from Y. pestisexpressing LpxL, and activation of the IL-1 receptor and the induction of IL-1β plays a major role in this infection as well. The lipid A acylation profile of gram negative bacteria can have a direct and profound effect on the pathogenesis of the organism. This work illustrates a previously unknown and critical aspect of Y. pestis pathogenesis, which can be extended to other gram-negative pathogens. The greater detail of the contributions which different host adaptor and receptor molecules make to the overall innate immune signaling pathway will allow a better insight into how gram negative infections progress and how they are counteracted by the immune system. Alterations of the lipid A profile of Y. pestis have important implications for the production of vaccines to Y. pestis and other gram negative pathogens. Taken together, this work describes a novel mechanism for immune evasion by gram negative bacteria with consequences for understanding the immune response and the creation of more effective vaccines, both of which will decrease the danger posed by this virulent pathogen.

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Saadeh, Bashir. "Characterization and search for virulence-related factors in “Classical” and “New” Brucella species." Thesis, Montpellier 1, 2013. http://www.theses.fr/2013MON1T010/document.

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L'étude qu'on a entreprise a pour but d'analyser les facteurs de virulence des espèces "Classiques" et "nouvelles" de Brucella. Dans cette perspective, on a analysé les génomes des espèces récemment découvertes : Brucella inopinata BO1 et Brucella inopinata-like BO2, isolés pour la première fois de patients humains sans réservoir animal connu. On a découvert que ces deux espèces possèdent des profils de restriction uniques. De plus, BO2 possède deux chromosomes de taille identique, un profil jamais décrit pour une autre espèce de Brucella. L'analyse de la réplication intracellulaire de ces deux espèces révèle que BO2 ne se réplique pas dans les macrophages humains et murins alors que BO1 se réplique d'une façon similaire à Brucella suis 1330, ce qui confirme la potentielle implication de BO1 dans la pathogenèse chez l'homme. Sur un autre niveau d'analyse, on a été à la recherche de facteurs de virulence potentiels dans d'autres espèces de Brucella notamment Brucella microti et Brucella suis sur les niveaux génomique et post-transcriptionnel. Sur le niveau génomique, on a découvert que le système GAD (glutamate decarboxylase) confère une résistance à l'acidité à Brucella microti lors de son passage dans l'estomac. Sur le niveau post-transcriptionnel, on a isolé, séquencé et identifié les petits ARNs noncodant associés à la protéine chaperone Hfq, qui joue un rôle important dans la virulence de Brucella
We have undertaken in this study a multidimensional analysis of the virulence factors of "Classical" and new "Brucella species". In this objective, we have analysed the genomes of newly described species Brucella inopinata BO1 and Brucella inopinata-like BO2 isolated for the first time from human patients with no known animal reservoir. We found that these two species have unique restriction profiles. In addition, BO2 has a unique chromosomal distribution with two chromosomes of the same size, never seen before in Brucella. Analysis of the intracellular replication of these strains reveals that BO2 is unable to replicate in neither human nor mouse macrophages while BO1 successfully entered and replicated as efficiently as Brucella suis 1330 confirming the potential virulence of this species for humans. On an other level of analysis, we looked for potential virulence factors in other Brucella species including Brucella microti and Brucella suis at the genomic and post-transcriptional level. At the genomic level we discovered that the glutamate decarboxylase system confers resistance to acidity to Brucella miroti during its transit in the stomach. On the post-transcriptional level, we isolated, sequenced and identified small noncoding RNAs associated to the chaperone protein Hfq, known to play a role in the virulence of Brucella

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Vdovikova, Svitlana. "Roles of membrane vesicles in bacterial pathogenesis." Doctoral thesis, Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-138714.

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The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the MVs and released from the surface of bacteria. MV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37, thereby, enhancing bacterial survival by avoiding this innate immune defense of the host. We also studied another virulence factor of V. cholerae, the pore-forming toxin VCC, which was found to be transported by MVs. MV-associated VCC is biologically active and triggers an autophagic response in the target cells. We suggested that autophagy serves as a cellular defense mechanism against the MV-associated bacterial virulence factor of V. cholerae. Listeria monocytogenes is a Gram-positive intracellular and facultative anaerobic food-borne pathogen causing listeriosis. It causes only sporadic outbreaks in healthy individuals, however, it is dangerous for a fetus or newborn child, and for pregnant and immunocompromised people, leading to a deadly infection in one third of the cases. We have analyzed MVs produced by L. monocytogenes and their interaction with eukaryotic cells. Confocal microscopy analysis showed that MVs are internalized into HeLa and HEK293 cells and are accumulated in lysosomes. Moreover, L. monocytogenes produces MVs inside the host cells and even inside the phagosomes. We found that the major virulence factor of L. monocytogenes, the cholesterol-dependent pore-forming protein listeriolysin O (LLO), is entrapped inside the MVs and resides there in an oxidized inactive state. LLO is known to induce autophagy by making pores in the phagosomal membrane of targeted eukaryotic cells. In our studies, we have shown that MVs effectively abrogated autophagy induced by Torin1, by purified LLO or by another pore-forming toxin from V. cholerae. We also found that MVs promote bacterial intracellular survival inside mouse embryonic fibroblasts. In addition, MVs have been shown to have a strong protective activity against host cell necrosis initiated by pore-forming toxin. Taken together, these findings suggested that in vivo MVs production from L. monocytogenes might be a relevant strategy of bacteria to manipulate host responses and to promote bacterial survival inside the host cells.

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Srimanote, Potjanee. "Analysis of putative virulence factors of a locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli O113:H21 strain." Title page, contents and abstract only, 2003. http://web4.library.adelaide.edu.au/theses/09PH/09php863.pdf.

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"February 2003." Addendum and corrigenda inserted at back Includes bibliographical references (leaves 249-272) Aims to identify and characterise potential virulence-associated factors from the locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli O113:H21 strain 98NK2 which was responsible for an outbreak of haemolytic uremic syndrome. Particular attention was focused on putative virulence genes encoded on the megaplasmid of this strain.

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Salazar, Salvatierra Maria Elena. "Prevalencia de genotipos de pili tipo IV y factores de virulencia asociados en aislados clínicos de Pseudomonas aeruginosa." Doctoral thesis, Universidad Nacional Mayor de San Marcos, 2014. https://hdl.handle.net/20.500.12672/3797.

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Pseudomonas aeruginosa es un importante patógeno oportunista del ser humano y tiene amplia versatilidad metabólica por lo que se puede adaptar a vivir en ambientes ubicuos, pudiendo persistir en ciertas superficies como tejidos formando comunidades especializadas llamadas biopelículas, además de elaborar otros factores de virulencia, como el pili tipo IV (T4P) vinculado no sólo a la motilidad llamada “contracción” sino también pareciera estar relacionada con las etapas tempranas en la formación de las biopelículas. La presente tesis tuvo como principal objetivo determinar la presencia de los diferentes genotipos del T4P así como los factores de virulencia asociados en cepas clínicas de diferentes fuentes, encontrando que la distribución de genotipos fue 25,9 % para el Grupo II, 19 % para el Grupo I, 24,1 % para el Grupo III, 10,3 % el Grupo IV y 20,7 % para el Grupo V, de tal manera que en nuestro medio existe distribución homogénea de los diversos genotipos. Palabras clave: Pseudomonas aeruginosa, Genotipos de pilA, Motilidad
Tesis

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Fine, Lindy. "Genetic Manipulation of the Relapsing Fever Spirochete Borrelia hermsii Permits Novel Investigation into the Role of Factor H Binding in Borrelial Virulence." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/2506.

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Abstract:

Borrelia hermsii, an etiologic agent of tick-borne relapsing fever, binds negative complement regulator factor H (FH) via its FhbA protein. Direct demonstration of the role of FhbA in the disease process has been hindered by the lack of genetic manipulation systems for the relapsing fever Borrelia. Here, we demonstrate successful generation of a B. hermsii strain YOR fhbA deletion mutant (Bh YORΔfhbA) that constitutively produces green fluorescent protein (GFP). Genetic manipulation did not affect growth rate or plasmid composition. Bh YORΔfhbA lost factor H-binding and C3b-inactivation capabilities, but retained resistance to killing in human serum and infectivity in mice. Stable production of GFP was demonstrated in vitro and in vivo. Collectively, these results suggest that B. hermsii employs an unidentified mechanism of complement evasion that is FH-independent and sufficient for persistence within the host. Additionally, this study represents a significant methodological advancement in the molecular characterization of relapsing fever spirochetes.

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Dissertations / Theses on the topic 'Bacterial virulence factors'

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