Tesi sul tema "Flagella (Microbiology)"

Orientador: Domingos da Silva Leite
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Escherichia coli é um membro comensal da microbiota de animais, porém podem causar doenças desde diarréias até sepses. A caracterização dos seus antígenos de superfície O (somático) e H (flagelar) auxilia na determinação de linhagens patogênicas dentro da espécie. Contudo, algumas bactérias não expressam flagelo in vitro, demonstrado que a amplificação do gene fliC, a análise dos fragmentos de polimorfismo (PCR-RFLP) e sequenciamento podem ser utilizadas para identificação dos antígenos H, em substituição à sorologia convencional. Até meados de 1980, pensava-se que, diferentemente da Salmonella, E. coli possui um único gene para expressão de flagelina (fliC), mas algumas amostras podem conter genes para expressão de flagelina flkA, fllA, flmA, flnA e fljA (repressor de fliC). Em nosso trabalho, analisamos 31 amostras de E. coli isolados de animais e alimentos que apresentavam o fenótipo HNT em ensaios de sorologia. Utilizamos PCR-RFLP e sequenciamento para descrever novos genes para flagelina, da qual foram obtidos antissoros. Identificamos por PCR e sequenciamento os genes responsáveis pela variação de fase fljA, flkA e flmA, realizamos experimentos de motilidade para determinar a variação de fase flagelar e detectar a expressão dos genes através de RT-PCR. Dezessete amostras tiveram seus antígenos H caracterizados, sendo nove caracterizadas por PCR-RFLP: H2 (duas amostras) H16 (duas amostras), H34 (três amostras), H33 (uma amostra) e H38 (uma amostra). Na análise de sequenciamento identificamos duas amostras portadoras do gene fliCh25, duas amostras fliCh7 e uma amostra apresentando fliCh32. Três novos genes para flagelina foram descritos: fliCh2', fliC4c, fliC40c. Identificamos o gene fljA em duas amostras HNT (3C e 4C) e na amostra padrão H35. O gene das amostras HNT apresentaram homologia ao fljA de Salmonella enterica, cuja variação de fase é bem estabelecida. As amostras padrão H11, H35, H40 e H47, bem como as amostras HNT 3C e 4C foram positivas para o gene flmA. As amostras padrão H3 e H53 são portadoras do gene flkA, contudo apenas a amostra H53 apresentou fljA. A amostra H54 é portadora de fljA e flmA. Nenhuma amostra H padrão mostrou variação de fase, diferentemente da literatura, sugerindo a perda da capacidade de variar a fase flagelar. A amostra 4C mostrou variação de fase positiva quando induzida em meios de cultura contendo antissoros anti-H48, anti-H54 e anti-H4C. Do mesmo modo, a detecção dos RNAm em diferentes condições de cultura confirmou a variação de fase. Como resultado um esquema de identificação para detecção de grupos de antígenos H e identificação de fliC foi testado. A técnica de fliC-RFLP provou ser eficiente e rápida, auxiliando a sorologia clássica para detecção de antígenos H de E. coli. Um modelo geral de variação de fase da amostra 4C é expresso por fliCoff + flmAon ? fliCon + flmAoff. Além disso, nós verificamos que a amostra 4C apresenta um gene novo para expressão de flagelina. Este trabalho é pioneiro em relação à variação de fase flagelar, demonstrando uma nova associação entre os antígenos H48 e H54
Abstract: Escherichia coli are a species of microflora, and characterization of the cell surface lipopolysaccharide O antigen and the flagellar H antigen allow the grouping of pathogenic clones within this species. Moreover, some bacteria in vitro do not obtain to express its flagella, demonstrated that PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing analysis has been used for the identification of these antigens, in substitution of traditional serology. Moreover, until middle of years 80, are believed, differently of the Salmonella, E. coli possesss an only gene for flagelin expression (fliC), but some s/strains can contain genes for flagellin expression flkA, fllA, flmA, flnA and fljA (repressor of fliC). In this work, we analyzed 31 strains of E. coli isolated from animals and foods that presented HNT phenotype in serology assays. We use PCR-RFLP and sequencing to describe new genes for flagellin, of which antiserum were obtained. We identify for PCR and sequencing the genes for phase variation fljA, flkA and flmA, we carry through motility experiments to determine the flagellar phase variation and to detect the expression of the genes (RNAm) through RT-PCR. Seventeen strains had had its H antigen characterized and nine of then were characterized for PCR-RFLP: H2 (two strains) H16 (two strains), H34 (three strains), H33 (one strain) and H38 (one strain). Through sequencing analysis we identify to two carrying strains of the gene fliCh25, two strains fliCh7 and one strain presenting fliCh32. Three new genes for flagellin had been described: fliCh2', fliC4c, fliC40c. Using PCR and sequencing, we identify fljA gene in two strains HNT (3C and 4C) and in the H35 control strain. The HNT genes showed homology to fljA of Salmonella enterica, whose variation of phase well is established. The control strains H11, H35, H40 and H47, as well as HNT 3C and 4C strains were positive for flmA gene. The control strains H3 and H53 are carrying of flkA gene, however only the H53 strain presented fljA. The H54 control strain is carrying of fljA and flmA. No H control strain showed phase variation, differently of literature, suggesting the loss of the capacity to flagellar phase variation. The 4C strain showed positive phase variation when cultured with antiserum anti-H48, anti-H54 and anti-H4C. In a similar way, the detention of RNAm in different conditions of culture confirmed the phase variation. As a result, an identification scheme was tested to deduce H antigen groups and new genes of fliC. The fliCRFLP technique proved to be faster than classic serotyping for the deduction of the E. coli H antigen, characterizing the antigens with few days and indicating new putative genes. Thus, a general model for flagellar phase variation in 4C strain can be expressed as fliCoff + flmAon ? fliCon + flmAoff. In addition, we found that strains 3C and 4C express unidentified flagellin antigens. This is the first report of flagellar phase variation in wild E. coli strains. We have also provided evidence that strain 4C, identified here for the first time, expresses three flagellar antigens, H48, H54 and a previously unidentified flagellin
Doutorado
Microbiologia
Doutor em Genetica e Biologia Molecular

Orientador: Domingos da Silva Leite
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Escherichia coli tem sido isolada, com certa freqüência, apresentando antígenos flagelares (H) que não são reconhecidos por nenhum dos anti-soros disponibilizado pelo mais importante centro de referência de E. coli, The International Escherichia and Klebsiella Centre (WHO) do Statens Serum Institut, Copenhague, Dinamarca. Atualmente são reconhecidos 53 antígenos "H" e, nos últimos 29 anos, nenhuma modificação ocorreu na lista dos antígenos flagelares associados à Escherichia coli. Isto posto, os objetivos deste trabalho foram identificar os antígenos flagelares das cepas de E. coli que expressam H não tipável (HNT) e que apresentam fatores de virulência associados à diferentes enteropatias. Esta identificação foi realizada inicialmente, pela reação em cadeia da polimerase (PCR) do gene fliC, responsável pela proteína flagelina, das 53 amostras padrões para os antígenos H e das 20 amostras HNT (H não-tipável). Em seguida, os amplicons foram digeridos por enzimas de restrição e daquelas amostras que apresentaram perfis de restrições distintos daqueles observados para as amostras padrões de antígeno H, foram produzidos soros em coelhos. Foram realizados testes de titulação frente aos 53 antígenos padrões, frente ao antígeno homólogo e frente aos antígenos das amostras HNT. As seqüência gênicas das amostras HNT, obtidas na reação de sequenciamento, foram comparadas aos diferentes genes de fliC armazenados no banco de dados do "National Center for Biotecnology Information" (NCBI) através do sistema BLAST, e o programa ClustalW foi utilizado para alinhamento das seqüências. Os resultados demonstraram que estas amostras apresentaram similaridade com antígenos padrões, entretanto, elas não possuem a mesma seqüência nucleotídica e também não reagiram fenotipicamente com o anti-soro esperado. Os dados obtidos permitem concluir que no conjunto de amostras estudado, treze amostras apresentaram antígeno flagelar diferente daqueles já descritos na literatura, quando utilizado as técnicas de PCR e/ou sorologia.
Abstract: Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Centre (WHO) of the Statens Serum Institute, Copenhagen, Denmark. Are currently recognized 53 H antigens and in the last 29 years, no change occurred in the list of flagellar antigens associated with Escherichia coli. The objectives of this study were to identify the flagellar antigens of E. coli that do not express non-typeable H antigens and presenting the virulence factors associated with different diseases. This identification was performed initially by gene amplification of the fliC, (flagellin protein) by the polymerase chain reaction (PCR) in all 53 standards E. coli strains for the H antigens and 20 non-typeable H-antigens E. coli strains, being then, the amplicons were digested by restriction enzymes. Anti-sera were produced in rabbits, those strains that showed different restriction profiles of these patterns observed for the nontypeable H antigens E. coli strains. Agglutination testes were carried out against the 53 antigens standards, against the homologous antigen and H antigens of the non-typeable strains. DNA sequences were compared to different fliC genes stored in the database of the National Center for Biotecnology Information (NCBI) through the BLAST, and ClustalW program was used to align the sequences. The results showed that although these strains have homology with a standard H-antigen, they do not have the same nucleotide sequence and did not phenotypically reacted with the antiserum expected. The data obtained showed that thirteen strains had a different H antigen those already described in the literature when used the techniques of PCR-RFLP and/or serology.
Doutorado
Microbiologia
Doutor em Genetica e Biologia Molecular

Uma das estratégias mais promissoras para a biotecnologia de vacinas é o desenvolvimento de linhagens precisamente atenuadas, e que possam ser usadas como carregadoras de antígenos heterólogos. Mutantes de Salmonella Typhimurium têm sido extensivamente utilizados com essa fmalidade. A flagelina, monômero constituinte do filamento flagelar, vem sendo empregada como carregadora de antígenos heterólogos, inseridos na região central, hipervariável (região IV). Inserções nessa região são freqüentemente funcionais, e levam à exposição do epitopo na superfície do filamento. O presente trabalho explora o potencial de outras regiões da molécula para a inserção de epitopos. Nós inserimos a mesma seqüência usada anteriomente (epitopo da proteína M de S. pyogenes, Tipo 5) em regiões com diferentes níveis de homologia (III e VI), e em região totalmente conservada (VIII). Também foram feitas inserções duplas em regiões que se mostraram toleráveis (III e IV; IV e VI). Todas as proteínas híbridas foram sintetizadas pela Salmonella, como demonstrado em imunoblots, usando anticorpo contra a flagelina e contra o peptídeo. Todas as regiões, exceto a VIII, aceitaram a inserção sem perda de motilidade, apesar de, em alguns casos, ela ter sido extremamente reduzida. A imunogenicidade foi avaliada pela imunização de camundongos com bactérias vivas, inativadas ou, quando possível, flagelina purificada. Os resultados foram similares aos descritos na literatura para inserções envolvendo a região IV, obtendo-se um elevado título de anticorpos contra flagelina. Um baixo nível de anticorpo contra o peptídeo também foi detectado para todas as novas linhagens testadas. Nossos resultados com imunização de bactérias vivas sugerem uma resposta levemente melhor ao peptídeo quando duas cópias estão presentes, mas os dados não são conclusivos.
One of the most promising strategies for the biotechnology of vaccines is the development of precisely attenuated strains, which could be used as carriers of heterologous antigens. Mutants of Salmonella Typhimurium have been extensively explored to this effect, since the infection ofmice by S. Typhimurium mimics the infection of humans by S. Typhi, and the genetics of the species is extremely well known, making it easy the obtention of defined mutants with reduced pathogenicity. Mutants with auxotrofy in genes of the aromatic pathway are particularly attractive, since they need PABA and DHB to grow, and these compounds are unavailable in mammalian tissues. Flagellin, the monomer which constitutes the flagellar filament, has been used as a carrier for heterologous epitopes, inserted in a central, hypervariable region (region IV). Insertions in this region are often functional, and lead to exposition of the epitope at the filament\' s surface. The present work explored the potential of the other regions ofthe molecule for the insertion of epitopes. We inserted the same reporter sequence (MS epitope from S. pyogenes M protein) in regions with different levels of homology (III and VI), and totally conserved (VIII). We also made double insertions in regions shown to be permissive (III and IV; IV and VI). All hybrid proteins were synthesized by Salmonella, as demonstrated by immunoblots using antibody against flagellin and against the synthetic peptide. All regions, except the highly conserved region VIII, accepted the insertions without loss of motility, albeit, in some cases, motility was seriously reduced. Immunogenicity of the hydrids was evaluated by immunization with live bacteria, killed bacteria, and purified flagellin (when possible). Results obtained with the new constructs were similar to the ones published for insertions involving region IV, in the sense that antibody titers to the carrier protein were very high. A low level of antibody to the inserted peptide was also detected in all groups of animals. Our results with live immunization suggest a slightly better response to the peptide when two copies are present, but the data are not conclusive.

O câncer cervical é o segundo maior responsável por mortes atribuídas a câncer em mulheres e dados epidemiológicos tem demonstrado a associação entre a infecção do HPV e o desenvolvimento da neoplasia. Sabe-se que num dado momento da infecção pelo HPV, ocorre integração do genoma viral ao genoma da célula hospedeira e consequente hiperexpressão de dois oncogenes virais, E6 e E7, o que contribui fortemente para a transformação celular. O presente trabalho propõe o uso de vacinas terapêuticas expressando a oncoproteína E7 do HPV-16 geneticamente fusionada à porção amino terminal da flagelina FliCd de Salmonella enterica sv müenchen; e verificação de seu possível papel adjuvante. Vacinas de DNA foram construídas de modo a direcionar as proteínas hibridas ao espaço intracitoplasmático. A verificação da expressão in vitro foi feita utilizando transfecções de culturas celulares, seguida de imunofluorescência e imunodetecção. Em seguida, essas construções vacinais foram administradas em camundongos C57BL6. Ensaios de ELISPOT foram feitos para mensuração o nível de linfócitos T secretores de IFN-g. Os dados de imunofluorescência e imunodetecção demonstraram a correta expressão da proteína. Ensaios de proteção realizados com 5 x 106 células TC-1 administrada por via subcutânea mostraram 80% de proteção em camundongos que haviam recebido previamente 4 doses das vacinas de DNA por biobalística. Ensaios de ELISPOT mostraram em média 9 células do baço secretoras de IFN-g por 106 células do baço (INF-g+/106) responsivas ao peptídeo CD8 / E7 de forma específica. Nossos dados sugerem que a formulação vacinal possui um efeito terapêutico significativo frente ao desafio com as células tumorais TC-1. Em paralelo, foi construída a cepa de salmonela vacinal SL FlaE7 que não mostrou efeito protetor frente ao desafio com células TC-1.
The cervical cancer is the second major responsible for deaths attributed to cancer in women and epidemiologic data have been demonstrating the association between the infection of HPV and the development of this illness. It is known that in a certain moment of the infection with HPV, occurs the integration of the viral genome in the genome of the host cell and consequent over expression of two oncogenes, E6 and E7, it strongly contributes to the transformation of that cell. The present work proposes the use of DNA vaccines expressing the oncoprotein E7 of HPV-16 genetically fused to the portion A-terminal of the flagellin FliCd of Salmonella enterica sv müencheun and verification of your possible performance as adjuvant. The DNA vaccines were constructed in expression vector for eukaryotic to address the hybrid proteins to the intracitoplasmatic space. The verification of the expression in vitro was made using transfections of cellular cultures (COS-7) followed by immunofluorescence and western blot analyses. Soon after, those vaccines were injected in mice C57BL6 that were challenged then with 5^105 tumor cells TC-1 subcutaneous. ELISPOT assays were performed for measure the level of spleen cells secreting interferon g. The immunofluorescence and Western blot data are complementary demonstrating the correct expression of the protein. Protection assays showed 80% of protection in mice that had previously received 4 doses of the DNA vaccines in gene gun form. Assays of ELISPOT show 9 cells of the spleen secreting of IFN-g (average) for 106 cells of the spleen (INF-g /106). Our data suggest that the formulation of vaccines possesses a significant therapeutic effect front to the challenge with the tumor cells TC-1 accompanist splenocyte IFN-g secretory.

Aeromonas spp. são bactérias Gram negativas capazes de causar doenças intestinais e extra-intestinais. A virulência de algumas espécies do gênero já foi associada a diversos fatores, como a expressão de flagelos e conseqüente mobilidade bacteriana. Aeromonas caviae apresenta dois distintos sistemas flagelares, um deles responsável pela natação em meio líquido (swimming) onde está envolvido o flagelo polar, e outro associado ao deslizamento em superfícies sólidas ou viscosas (swarming), o qual participam flagelos laterais. A fim de avaliar a contribuição de ambos os flagelos no contexto da formação de biofilme em superfície plástica e aderência à linhagem celular HEp-2, foram estudadas 76 cepas de A. caviae isoladas de diferentes origens. Os resultados obtidos em nosso estudo demonstraram que a detecção do gene flaA (flagelina polar) foi mais freqüente (94%) que a do gene lafA (flagelina lateral) (71%) e, aparentemente, a presença deste último parece estar condicionada à do primeiro. Todas as cepas que apresentaram o genótipo flaA-lafA-, foram incapazes de formar biofilme, bem como 76% das cepas flaA+lafA-. As cepas provenientes de água foram as que exibiram menor percentual de positividade para o gene da flagelina lateral (57%) e aquelas onde se observou maior incapacidade de formação de biofilme (71%). Além disso, 95% das cepas flaA+lafA+ formaram biofilme e todas as cepas que apresentaram, quantitativamente, um biofilme forte, albergavam ambos os genes. A aderência à HEp-2 se mostrou eficiente em todos os casos onde ao menos um dos flagelos estava presente e em dois casos onde ambos estavam ausentes, com prevalência do padrão agregativo. Em contrapartida, não foi notado uma interferência exclusiva dos flagelos laterais nesse processo. Todas as três cepas que não aderiram ao modelo celular utilizado, ou o fizeram muito discretamente, não exibiram nem o gene flaA, tampouco lafA. Em conclusão aos dados observados, apontamos que uma plena atividade de ambos os flagelos, geralmente, é requerida nos processos de aderência às superfícies abióticas e bióticas. Contudo, trabalhos adicionais sobre a contribuição dessas estruturas na patogênese de A. caviae necessitam ser realizados.
Aeromonas spp. are bacteria Gram-negative able to cause intestinal and extraintestinal diseases. The virulence of some species of this genus has already been associated to several aspects, such as the expression of flagellum and following bacterium mobility. Aeromonas caviae presents two distinct flagellar systems, one of which is responsible for mobility in liquid environments (swimming) at where the polar flagellum is involved, and the other is associated to sliding in solid or viscous surfaces (swarming), in which lateral flagella take part. In order to evaluate the contribution of both flagella in the biofilm formation in plastic surfaces and its adherence to cell line HEp-2, 76 strains of isolated A. caviae from distinct sources were analyzed. The results from our studies showed that detection of gene flaA (polar flagellin) was more frequent (94%) than gene lafA (lateral flagellin) (71%), and apparently, the presence of the last one seems to be conditioned to the first one (the gene flaA). All the strains that presented the genotype flaA-lafA- were unable to form biofilm, such as 76% of strains flaA+lafA-. The aquatic strains were those who showed lesser percentage of positivity for the lateral flagellin gene (57%), and higher incapacity of biofilm formation (71%). Moreover, 95% of the flaA+lafA+ strains formed biofilm, and all strains that quantitatively showed a strong biofilm, contained both genes. The adherence to HEp-2 proved to be efficient in all cases where at least one of the two flagella was present at, and in two cases where both were absent, with prevalence of the aggregative pattern. However, an exclusive interference from the lateral flagella was not perceived in this process. All of the three strains that did not adhere to the cell model used, or that adhered in a very discrete way, did not show neither gene flaA nor gene lafA. In conclusion, we suggest that the complete function of both flagella usually is a requirement in the adherence processes to biotic and abiotic surfaces. However, additional studies on the contribution of these structures in the pathogenesis of A. caviae need to be carried out.

Campylobacter jejuni is one the leading causes of food poisoning in the UK. Motility in C. jejuni is key to its survival within the gut and other environments. Without the two flagella, one at either end of the cell, it is highly attenuated to cause disease. As the flagellar structure is imperative to Campylobacters lifestyle, studying the regulation of flagella synthesis is important. Unlike other bacteria, Campylobacter lacks a master regulator atop the flagella synthesis pathway. It does however, have an FlhF protein with a signal recognition particle (SRP) domain and GTPase activity whose role is still enigmatic. Previous studies and observations have found that in a C. jejuni strain lacking FlhF, loss of motility and an aflagellate phenotype are observed. However under selective pressure, C. jejuni ΔflhF is able to revert to have partial motility. These pseudorevertants have an increasing unipolar phenotype when transitioning from exponential to stationary growth phase and have 50-70% of the wild-type motility. These pseudorevertant strains have provided a novel way of studying the FlhF protein. Sequencing of the pseudorevertants identified mutations in the fliF and fliG genes. FliF forms an oligomeric structure called the MS ring, which sits at the base of the flagella structure and surrounds the T3SS. FliG interacts with FliF and forms part of the C ring, placed below the MS ring. Analyses of these mutations suggest that they cause changes that make the structure more rigid, potentially forcing an interaction between the rings and T3SS. In the pseudorevertants, unlike the ΔflhF strain, transcription levels of σ54 and σ28 genes reach wild-type levels. The data in this thesis suggest that the role of FlhF is to activate a conformational change within the flagella base, locking it into the correct position and thereby creating the signal for the FlgSR cascade and σ54 transcription. The global analysis conducted in this thesis also provides many other insights into the C. jejuni flagellar cascade.

A espécie Escherichia coli constitui um grupo de bactérias tipicamente não patogênicas e que fazem parte do trato intestinal de humanos e animais. As amostras são sorotipadas com base em seus antígenos de superfície O (somático), H (flagelar) e K (capsular). O antígeno flagelar correspondente ao filamento é formado pela polimerização da flagelina, codificada pelo gene fliC. O presente trabalho empregou a técnica de PCR-RFLP para analisar os padrões de antígenos flagelares de 112 amostras de EPEC e STEC. Quatorze amostras não amplificaram o gene fliC, 17 tiveram seu antígeno flagelar determinado apenas por PCR-RFLP e 75 amostras tiveram seus antígenos flagelares confirmados por esta técnica. Três antígenos H com padrões irregulares foram clonados e sequenciados. Após o sequenciamento, inserções e remoções de nucleotídeos foram encontradas. Até o momento, poucos estudos utilizam um número abrangente de amostras de STEC e EPEC provenientes de diferentes animais para a determinação do antígeno H empregando a técnica de PCR-RFLP do gene fliC. De acordo com os resultados encontrados neste estudo, podemos concluir que a técnica de PCR-RFLP do gene fliC é mais rápida, menos trabalhosa e mais eficiente que a metodologia de sorotipagem clássica.
The Escherichia coli species consists of a group of typically non-pathogenic bacteria present in the intestinal tract of humans and animals. Strains are serotyped according to their O (somatic), H (flagellar) and K (capsular) surface antigens, in order to distinguish these microorganisms from the non-pathogenic members of the intestinal microbiota. The flagellar antigen corresponding to the filament is formed by the polymerization of the flagellin, codified by the fliC gene. This study employed the PCR-RFLP technique to analyze flagellar antigen patterns from 112 EPEC and STEC strains. Fourteen strains have not amplified the fliC gene, 17 had their flagellar antigen determined only by the PCR-RFLP and 75 strains had their flagellar antigen confirmed by this technique. Three H antigens with irregular patterns were cloned and sequenced. After sequencing, insertions and deletions of nucleotides were discovered. So far, few studies used a significant number of STEC and EPEC strains originated from different animals to determine H antigens employing the PCR-RFLP technique of the fliC gene. According to the findings of this study, we assumed that PCR-RFLP of the fliC gene is faster, less laborious and more efficient than classic serotyping methodology.

Coinfection with bacteria and viruses is an understudied area of microbiology, despite its potential to modulate pathogen abundance and host survival. We investigated the effect of bacteria on virus infection and developed an in vitro system to study the first step: viral internalization. Our studies show that multiple bacterial species promote the entry of a diverse panel of viruses into lung and gut epithelial cells. Bacteria expressing the toll-like receptor (TLR)5 agonist, flagellin, are most efficient at inducing viral uptake and studies using recombinant flagellin or aflagellate bacterial strains confirm that flagellin has pro-viral activity. Flagellin promotes epithelial cells to support virus entry via TLR5-dependent activation of NF-KB. To extend these observations and study the role of flagellin in the complete viral replicative lifecycle, we studied human immunodeficiency virus (HIV)-1 replication in T cells. Flagellin augments HIV-1 entry and promoter activity and increases the production of extracellular virus. The data presented in this thesis highlight a new role for bacterial flagellin to promote diverse virus infection of epithelial barriers and enhance the spread of HIV-1. This has significant implications for understanding how exposure to multiple pathogens can alter susceptibility to infection and its associated pathogenesis.

El género Aeromonas está constituido por bacilos gram negativos y comprende patógenos de animales poiquilotermos y oportunistas en humanos. La virulencia de Aeromonas hydrophila AH-1 es multifactorial, siendo el lipopolisacárido (LPS), el flagelo (polar y lateral) y la lámina S importantes factores de patogenicidad. La mutación del gen wbpL, implicado en la biosíntesis del antígeno O del LPS, da lugar a la pérdida de antígeno O, una bajada en el peso molecular de las flagelinas polares y la aparición de fimbrias en la superficie bacteriana. Se procedió a estudiar si las flagelinas polares y laterales de la cepa AH-1 se hallaban glicosiladas. Los análisis mediante LC-MS mostraron que las flagelinas laterales de la cepa AH-1 no se hallaban glicosiladas, mientras que las polares se hallaban glicosiladas con un glicano de 403 Da, derivado del ácido pseudamínico y otro glicano de 1060 Da, que no se encontraba en el mutante del gen wbpL. Este dato, junto con el hecho de que el mutante del gen rmlB que codifica para la biosíntesis de ramnosa (azúcar que compone el antígeno O) también pierde el glicano de 1060 Da, muestra la relación entre dicho glicano y el antígeno O del LPS. Asimismo se procedió a localizar los genes implicados en la síntesis de pseudamínico, adyacentes a la región 2 de flagelo polar; mutaciones tanto en alguno de estos genes (pseI, pseB) como en la glicosiltransferasa maf-1 abolía la presencia de flagelo polar pero no del lateral, demostrando así una regulación post-traduccional de las flagelinas polares mediante la glicosilación. También se observó la aparición de fimbrias. Se procedió a analizar las fimbrias que se inducían en los mutantes wbpL, pseI, pseB y maf-1. Se trata de fimbrias de tipo IV, denominadas MSHA por su homología a las descritas en Vibrio spp y ampliamente distribuidas en el género Aeromonas. La secuenciación de la agrupación mostró 22 ORF agrupadas en dos transcritos. Para analizar la inducción de las fimbrias en los mutantes se realizaron análisis de promotores, RT-PCR y detección mediante anticuerpos, que mostraron que la cepa salvaje AH-1 presenta las fimbrias pero no es capaz de ensamblarlas correctamente y las libera al exterior. La acumulación de azúcares activos en los mutantes parece ser el detonante para la expresión de estas fimbrias que juegan un papel fundamental en la formación de biofilms, favoreciendo la persistencia de la bacteria en el medio ambiente.
The genus Aeromonas is composed of Gram-negative rod-shaped bacteria and comprises opportunistic and primary pathogens of poikilothermic and homeothermic animals, including humans. The lipopolysaccharide (LPS) and S layer are important virulence factor of Aeromonas hydrophila AH-1. Mutation of the gene wbpL, which is involved in the O antigen synthesis of the LPS, leads to loss of the O antigen, a lesser molecular weight of the polar flagella and the appearance of fimbriae on the bacterial surface. It was studied whether the polar and lateral flagella of the strain AH-1 appear glycosylated. The LC-MS analysis showed that the lateral flagella were not glycosylated, while polar flagella were carrying a 403 Da glycan, derived from pseudaminic acid, as well as a 1060 Da glycan that was not found in the wbpL mutant strain. This finding, together with the fact that a mutant of the rmlB-gene, which plays a role on ramnose-biosynthesis (a sugar of the O antigen), also loses the glycan of 1060 Da, shows the involvement of this glycan in the o antigen of the LPS. It was proceeded to identify the genes in pseudaminic-synthesis adjacent to region 2 of the polar flagellum genes. Mutations in some of these genes (pseI, pseB) as well as in glycosyltranferase maf-1 abolished the presence of polar but nor of lateral flagella and therefore showed a posttranslational regulation of the polar flagellins by glycosylation. As also fimbria formation was observed it was proceeded to analyse the fimbria that were induced in the mutants of wbpL, pseI, pseB and maf-1. They were identified as type IV fimbria, also called MSHA due to their homology to those observed in Vibrio ssp and broadly distributed in the genus Aeromonas. DNA-sequencing showed 22 ORF that were organized in 2 transcripts. To analyze the induction of the fimbria in above mentioned mutants, promoter analysis, RT-PCR and immunodetection was performed. This showed that wildtype AH-1 also expresses fimbria, but is not capable of assembling them correctly and therefore expulses them to the exterior. The accumulation of active sugars in the mutants seems to trigger the correct assembly of the fimbria that play an fundamental role in biofilm formation and therefore favor the persistence of the bacteria in the environment.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2007.
Title from dissertation home page (viewed Sept. 29, 2008). Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0818. Adviser: Clay Fuqua.

There is a strong need to develop a mechanical method to apply external torque to the bacterial flagellar motor. Such a method will allow us to probe the behaviour of the motor at a range of different speeds under different external conditions. In this thesis, I explored various methods to deliver torque at the single-molecule level, in particular the use of angular optical trapping and magnetic tweezers. I have identified rutile particles as suitable handles for use in angular optical trapping due to their high birefringence. Further progress was not achieved using angular optical trapping due to the lack of a suitable method to attach birefringent particles to the bacterial flagellar motor. On the other hand, I was able to make further progress using magnetic tweezers. A highly-reproducible and high-yielding magnetic bead assay was developed along with electromagnets capable of generating fast-rotating magnetic fields at magnitudes on the order of tens of mT. Using the system of delivering magnetic torque developed, I was able to stall and rotate the motor forward at speeds up to 220 Hz and in the reverse direction. Stalling experiments carried out on the motor revealed the stator mechanosensing depends on torque and not rotation. Signatures of stators dropping out at low load experiments further confirm the load dependence of stators.

Campylobacter jejuni is one of the leading causes of food borne illness worldwide; the main cause of infection is the consumption of undercooked, contaminated poultry. Motility, mediated by single polar flagella, is necessary for Campylobacter virulence. The flagellin proteins are extensively glycosylated in C. jejuni with pseudaminic acid and related derivatives; glycosylation is required for flagella filament formation. The flagellin glycosylation locus in C. jejuni NCTC11168 comprises 50 genes, 50% are potentially involved in glycan biosynthesis and others are hypothetical; the regulatory mechanism(s) of flagellin glycosylation remain unclear. The response to nitric oxide stress in C. jejuni is regulated by NssR, which also regulates a small number of genes in the flagellin glycosylation locus; an nssR mutant has defective motility. This work aims to elucidate the mechanisms by which NssR regulates these genes and investigate the consequences of nssR mutation. Transcriptional organisation of the affected genes was deduced and chromatin immunoprecipitation (ChIP) used to map NssR binding to six potential locations in the region. Methods to measure bacterial motility were assessed and used to document the defective motility of the nssR mutant, which was found to be due to truncated, sometimes asymmetric, flagella. Finally, mass spectrometry was used to examine flagellin glycosylation in all strains.

Pseudomonas syringae est une bactérie phytopathogène qui provoque des dégâts de gravité variable sur une large gamme d'hôtes. Les 57 pathovars qui la subdivisent viennent d'être redistribués en cinq espèces génomiques qu'aucun critère phénotypique ne permet de caractériser. Nous avons étudié les protéines de surface présentes chez ces bactéries et tente d'évaluer leur potentiel taxonomique. Deux sérotypes flagellaires, h1 et h2, identifiables en immunofluorescence et en immunotransfert, sont distingués pour les souches de p. Syringae. H1 et h2 montrent une distribution remarquablement homogène dans les cinq espèces génomiques. Certaines souches de p. Viridiflava et p. Cichorii, appartiennent également à ces deux sérotypes. L'analyse des poids moléculaires des flagellines souligne l'homogénéité des pseudomonas (30 à 35 kda), sauf pour le groupe des p. Fluorescens/putida. L'analyse en sds-page des profils des protéines de l'enveloppe, extraites dans une solution de lic1 a 48c, a permis de mettre en évidence deux protéines, de 60 à 65 kda, chez les souches du pathovar pisi (bactérie pathogène du pois). Elles ne sont pas détectées chez les souches du pathovar syringae (bactérie saprophyte présente sur le pois). Des anticorps polyclonaux et monoclonaux sont produits contre ces deux protéines. Elles ne semblent pas situées au niveau de la membrane externe mais leur localisation exacte reste incertaine. Un protocole d'identification rapide des pseudomonas du pois utilisant ces deux protéines est proposé. L'analyse en sds-page des profils électrophorétiques des protéines de la membrane externe extraites par solubilisation de la membrane interne à l'aide de sarcosyl, révèle l'intérêt de cette méthode pour la taxonomie : similitude des profils de certaines espèces génomiques et discrimination de certains pathovars. En fonction de la présence de certaines protéines dans des conditions particulières de culture, et par comparaison avec les protéines de la membrane externe de p. Aeruginosa, quelques hypothèses sur leur rôle possible sont proposées.

Le protozoaire flagellé Histomonas meleagridis est responsable d'une maladie chez les oiseaux galliformes, nommée histomonose. La culture in vitro de ce parasite anaérobie est qualifiée d'agnobiotique car il y a présence d'une flore bactérienne non déterminée. L'identification des bactéries dans cette flore montre l'existence d'une flore très diversifiée. Des essais de culture axénique et monoxénique suggèrent que les bactéries sont essentielles au développement in vitro du parasite. Par des stratégies de PCR, RT-PCR et RACE-PCR, nous avons les gènes complets codant trois protéines intervenant dans le métabolisme énergétique chez H. meleagridis : une enzyme malique, la sous-unité alpha de la succinyl coenzyme A synthétase et une hydrogénase à fer. Ces trois protéines sont localisées dans des organites particuliers à double membrane, produisant de l'énergie sous forme d'ATP, appelés hydrogénosomes. Une partie du travail a porté sur le suivi d'élevages de dindes pour surveiller l'apparition éventuelle d'histomonose et sur la réalisation de tests anti-parasitaire à base de produits naturels

Bacillus cereus est fréquemment associé à des toxi-infections alimentaires et peut être responsable de pathologies opportunistes sévères. La présence de B. Cereus dans les industries agro-alimentaires compromet la qualité et surtout la sécurité des produits finis. La capacité de B. Cereus à former des biofilms sur différentes surfaces, renforce sa persistance sur les équipements de production industrielle. Les travaux présentés dans cette thèse ont permis d'une part de caractériser la dynamique de formation du biofilm par B. Cereus et d'autre part, de mettre en évidence et de clarifier le rôle joué par les flagelles et par la mobilité flagellaire dans la formation de ce biofilm dans différentes conditions de culture. Nous avons montré que, en condition statique de culture, en tubes de verre ou en microplaques, la mobilité flagellaire est indispensable pour la formation du biofilm. La mobilité flagellaire est également impliquée dans le recrutement de bactéries planctoniques par le biofilm en développement et permet l'expansion du biofilm sur la surface colonisée. Nous avons montré que B. Cereus, est aussi capable de former des biofilms immergés dans les conditions de flux continu en flow-cell. La mobilité flagellaire n'est pas requise pour la formation du biofilm dans ces conditions. L'observation de ces biofilms en microscopie confocale montre la présence d'une sous-population de bactéries mobiles capables de traverser les différentes couches du biofilm. Enfin, nous avons montré que la mobilité flagellaire est impliquée dans le pouvoir pathogène de la bactérie chez la larve du lépidoptère Galleria mellonella infectée par voie orale. La mobilité, favorise l'adhésion des bactéries sur des cellules épithéliales HeLa
Bacillus cereus is an opportunistic pathogen frequently associated with food poisoning and involved in rare but severe local or systemic infections. Contamination of food industry products by B. Cereus can result in economical injuries and might raise safety concerns. The capacity of B. Cereus to form biofilm on different surfaces increases its persistence in the food industry equipments. Here, we have determined the dynamics of biofilm formation by B. Cereus and the roles played by flagella and flagellar motility in B. Ce reus biofilm formation in different growth conditions. We have shown that, in static cultures runned in glass tubes or in microtiter plates, flagellar motility is required for biofilm formation. Motility was necessary for the bacteria to have access to the air-liquid interface where the biofilm develops. Flagellar motility was also involved in the recruitment of planktonic bacteria by the biofilm and promoted biofilm expansion on the colonized surface. We found that B. Cereus is able to form immersed biofilms in continuous flow conditions in flow­cells. However, in these growth conditions, flagellar motility was not required for biofilm formation. Observation of biofilms by confocal microscopy have shown the presence of a subpopulation of bacteria able to move through the different biofilm. The speed of these mobile bacteria could reach values up to approximately 16 μm/s. This speed decreased when the biofilm became older and mature, probably as a consequence of an increase in the exopolysaccharide matrix density. Finally, we demonstrated that flagellar mobility was involved in the bacterium pathogenicity in an isect model. In this model, larvae of the wax moth Galleria mellonela was infected by the oral route. Motility, but not the simple presence of flagella, promoted adhesion of bacteria on epithelial HeLa cells

A HP1076 null mutant has been constructed to provide a better understanding of the biological significance of HP1076 in H. pylori . The DeltaHP1076 mutant displays impaired motility and resistance to the antibiotic drug metronidazole. Using a proteomic study, an overall of 40 differentially expressing proteins involved in metabolism and pH homeostasis for bacterial survival, adhesion for colonization, virulence factor to gastric epithelial cells and antigenic proteins have been identified. The virulence factor, Cag pathogenicity island protein (Cag 26) and urease UreA and UreB are confirmed to have enhanced and reduced expression in null mutants. These findings may provide new insight into the infection of H. pylori.
FliS is an export chaperone that binds to flagellin molecules in cytosol in order to prevent pre-mature polymerization. Disruption of FliS would result in formation of shorter flagella and impaired adhesion ability to epithelial cells. Previous yeast two-hybrid study has identified various FliS associated proteins in H. pylori, but with no known implications. Here, we have demonstrated the interaction of FliS and a hypothetical protein HP1076 by biochemical and biophysical methods. Moreover, HP1076 possesses anti-aggregation ability on insoluble FliS-mutants and chaperone activity. Thus, HP1076 is proposed to be a co-chaperone that promotes the folding and chaperone activity of FliS. FliS is demonstrated to have a broad range of substrate specificity that binds to flagellin and flagellar related proteins which may play a key role in flagellar export system different from other flagellated bacteria.
Helicobacter pylori is a pathogenic bacterium and adheres to the gastric mucosal cells. Chronic infection would lead to gastritis or peptic ulceration and is one of the leading causes of gastric cancer. Formation of functional flagella is essential for infection, that it aids in motility of bacteria and colonization on gastric epithelial cells. The process is complex and involves more than 50 proteins in assembly of structural proteins, regulatory proteins, an export apparatus, a motor and a sensory system. Cytosolic chaperones are required to bind to exported proteins in order to facilitate the export or prevent the aggregation of proteins in cytosol. Divergence is found in flagellar system H. pylori that may account for survival inside gastric environment.
The crystal structures of FliS, HP1076 fragment and FliS/HP1076 complex are determined at 2.7A, 1.8A and 2.7A resolution respectively to provide better understanding of their molecular interactions. FliS consists of four helices and HP1076 consists of helical rich bundle structure with three helices and three beta strands that share similar fold to that of a flagellin homologue, hook-associated protein and FliS, suggesting HP1076 is involved in flagellar system. The FliS/HP1076 complex reveals an extensive electrostatic and hydrophobic binding interface which is distinct from the flagellin binding pocket on FliS. HP1076 stabilizes two alpha helices of FliS and therefore the overall bundle structure. Our findings provide new insights into the flagellar export chaperones and other secretion chaperones in Type III secretion system.
Lam, Wai Ling.
Adviser: An Wing-Ngor.
Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 223-243).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Vibrio parahaemolyticus is a Gram-negative, halophilic, human pathogenic bacterium ubiquitous in the marine environment. Like many Vibrio species, V. parahaemolyticus commonly associates with shellfish, particularly oysters. Ingestion of a raw or under cooked oysters contaminated with V. parahaemolyticus can cause gastroenteritis, which is typically self-limiting and rarely causes death. Globally, oyster production is highly lucrative, especially on the West Coast of the United States where approximately 60% of oyster production occurs each year. Outbreaks of V. parahaemolyticus can result in a significant public health problem as well as an economic burden for the oyster farms implicated in the outbreak. With the increase in overall V. parahaemolyticus outbreaks, improved post-harvest processing strategies have been developed to reduce this natural contaminant. Depuration was developed to allow shellfish to purge contaminants from their tissues into the clean, flowing seawater where they are held. This post-harvest processing technique can typically reduce fecal contaminants from the oyster tissues but is relatively ineffective at eliminating V. parahaemolyticus and other Vibrio species.. Thus, improved methods for reducing this and other human pathogenic Vibrio are needed to effectively produce safer oysters for the consumer. To develop more effective and novel V. parahaemolyticus intervention strategies, first we must identify the factors that are involved in V. parahaemolyticus colonization of the oyster, allowing them toresist depuration. This study sought to investigate specific factors utilized by V. parahaemolyticus and, in the process, determined that various strains of V. parahaemolyticus have different alleles of the Type IV pili, mannose-sensitive hemagglutinin (MSHA)and chitin-regulated pilus (PilA). In addition, we expanded our investigations into the allelic diversity of MSHA and PilA from Vibrio cholerae and Vibrio vulnificus and found that V. cholerae strains that possess the Type IV toxin co-regulated pilus (TCP) maintained highly conserved MSHA and PilA sequences while strains of V. cholerae without TCP, and all of the V. vulnificus and V. parahaemolyticus strains examined, had highly divergent sequences with no discernable connection to isolation source or observed phenotype. Following that discovery, we determined that Type I, and Type IV pili, as well as polar and lateral flagellar systems contribute to V. parahaemolyticus persistence in the Pacific oyster during depuration, while Type III secretion systems and phase variation do not. Overall, we have identified factors involved in colonization of the Pacific oyster by V. parahaemolyticus. Future studies investigating conditions that affect pili and flagella production in V. parahaemolyticus may provide novel depuration conditions that could easily and effectively increase the efficiency of oyster depuration, ultimately reducing the risk of seafood-borne illness by V. parahaemolyticus associated with oysters.
Graduation date: 2013

Cell-envelope associated and secreted proteins of Salmonella are integral for host-pathogen interactions, and for the induction of protective immune responses. An array of exported proteins of S. Brandenburg was identified through constructing an expression library using alkaline phosphatase gene technology. A partial digest of S. Brandenburg strain S59 was cloned into the vector pJEM11, and expressed in E. coli. The DNA inserts from randomly selected alkaline phosphatase positive clones were sequenced, and the sequences were analysed using public databases to find the ones that may play a role in host immune cell activation. The phase-1 flagellin (fliC) gene identified from an alkaline phosphatase positive phenotype was chosen for further studies. The complete nucleic acid sequence of the fliC gene was obtained by PCR amplification. The complete ORF, part of the variable region (V456) and region IV (V4) of the fliC gene were cloned into the pET14b vector for the expression of N-terminal histidine-tagged fusion proteins. The proteins were purified through metal affinity chromatography, and were evaluated for their humoral immunogenic properties by Western blotting with sera collected from 81 sheep naturally infected with S. Brandenburg. All 81 naturally infected sheep had IgG antibodies against recombinant FliC, V456, and V4 proteins. Furthermore, Western blotting of sera from 6 salvexinTM+B-vaccinated sheep (Trial 2004) had IgG antibodies against the 3 recombinant proteins. Whole blood cells of vaccinated sheep did not show interferon-gamma production upon stimulation with recombinant FliC and V456 proteins. Western blotting of sera from sheep vaccinated with salvexinTM and salvexinTM+B (Trial 1999), and those from rabbits vaccinated with S. Brandenburg, S. Hindmarsh and S. Typhimurium suggested that recombinant V4 contains epitopes specific for S. Brandenburg. Therefore, V4 was used to develop a novel indirect enzyme-linked immunosorbent assay (ELISA) for the detection of serum IgG antibodies in S. Brandenburg infected sheep. The ELISA showed a specificity of 100%, and a sensitivity of 93.8%. Furthermore, a new PCR assay was developed targeting rfbJ(B) gene in a single reaction, and genes invA, fliC and fljB in a multiplex reaction for the identification of S. Brandenburg from pure cultures. The sensitivity and specificity of the PCR assay was calculated to be 100%.