Dissertations / Theses on the topic 'Bordetella'

The present work confirmed that B. pertussis infection or pertussis toxin produce hypoglycaemia in mice. The hypoglycaemia was associated with hyperinsulinaemia, and both were abolished by destruction of the pancreatic β cells with alloxan. Impaired glucose counterregulatory mechanisms may also contribute to pertussis-induced hypoglycaemia, as the hypoglycaemic action of insulin was prolonged in pertussis infected mice. On the other hand, impaired responsiveness to lower doses of insulin was found. Pertussis-induced hyperinsulinaemia had two components. First, the increase in serum insulin in response to food intake was both greater and more prolonged in pertussis-infected mice. Second, infected or pertussis toxin-treated animals, unlike controls, showed a marked increase in serum insulin in response to certain stresses, such as ether, histamine, anoxia and 2-deoxyglucose. However, other stresses (LPS, cold and hypoxia) did not cause hyperinsulinaemia in pertussis infected mice. Stress-induced hyperinsulinaemia was also seen in normal mice receiving the a2- adrenoceptor blocking drug idazoxan. Stress-induced hyperinsulinaemia in a2 adrenoceptor blocked mice, but not in pertussis-treated mice, was prevented by β adrenoceptor blockade using propranolol. Adrenal demedullation or ganglionic blockade (using hexamethonium) in normal mice also allowed stress induced hyperinsulinaemia. Thus, adrenal medullary catecholamines may normally serve to prevent stress induced hyperinsulinaemia, which becomes unmasked when they are absent or when their action is prevented. Stress-induced hyperinsulinaemia in pertussis treated mice was unlikely to involve autonomic, cholinergic oropioid mechanisms as it was not blocked by hexamethonium, atropine or naloxone. Human infants with pertussis showed no hypoglycaemia compared with non-pertussis controls, although their plasma insulin concentrations were slightly but significantly raised. It remains possible that hyperinsulinaemia with resultant profound hypoglycaemia might occur in susceptible patients following exposure to pertussis-toxin (either during the disease or following vaccination).

Bordetella pertussis, l'agent de la coqueluche secrete une adenyl cyclase qui constitue l'un des principaux facteurs de virulence de cet organisme. Cet enzyme bacterien presente l'originalite d'etre active par la calmoduline, une proteine regulatrice eucaryote. Nous avons purifie l'adenyl cyclase a l'homogeneite; elle possede l'activite specifique la plus elevee connue pour cette classe d'enzymes. Elle est constituee d'une chaine polypeptidique de 43-50 kilodaltons qui fixe une molecule de calmoduline avec une grande affinite. Par proteolyse limitee du complexe adenyl cyclase/calmoduline, nous avons identifie, dans la molecule d'adenyl cyclase, deux domaines d'interaction avec la calmoduline. Le clonage du gene de l'adenyl cyclase de b. Pertussis nous a permis d'etudier et de caracteriser la proteine exprimee chez e. Coli. La determination de la sequence nucleotidique du gene a revele l'existence d'une phase ouverte de 1706 acides amines: l'adenyl cyclase se situe dans la partie n-terminale; la partie c-terminale presente une homologie de sequence avec l'hemolysine d'e. Coli. Ces donnees suggerent que l'adenyl cyclase pourrait etre synthetisee sous forme d'un precurseur commun, adenyl cyclase-hemolysine. Un tel precurseur, d'un poids moleculaire apparent de 200 kilodaltons a ete identifie dans les souches virulentes de b. Pertussis; nous avons montre que, dans certaines conditions de culture, cette forme d'adenyl cyclase de 200 kilodaltons peut etre secretee par b. Pertussis sans proteolyse. Enfin, nous avons mis en evidence une parente antigenique entre les adenyl cyclases calmoduline-dependantes de b. Pertussis et de cerveau de rat. L'hypothese d'une origine evolutive commune pour ces deux enzymes est ainsi serieusement etayee

B. Pertussis, agent pathogene de la coqueluche, produit de nombreux facteurs de virulence dont la toxine adenylcyclase. Cet enzyme est active par une proteine eucaryote, la calmoduline ; particularite partagee avec une autre adenylcyclase bacterienne, celle de b. Anthracis, agent du charbon. Ces deux adenylcyclases comportent un motif peptidique hautement conserve que nous avons caracterise comme faisant partie du site catalytique de ces deux enzymes. Des anticorps diriges contre ce motif conserve nous ont permis de preciser une parente immunologique entre les deux adenylcyclases bacteriennes calmoduline-dependantes et la sous-unite catalytique de l'adenylcyclase de cerveau de rat. Chez b. Pertussis, l'expression des genes codant pour les facteurs de virulence est regulee de facon coordonnee en reponse a des signaux de l'environnement par les produits du locus bvg. Nous avons etudie la regulation du gene de l'adenylcyclase, cyaa chez e. Coli et chez b. Pertussis. Nous avons montre que le promoteur du gene cyaa n'est pas active chez e. Coli par les produits du locus bvg introduit en trans. Afin de mieux comprendre la regulation de ce promoteur, nous avons entrepris sa caracterisation et determine les regions impliquees dans son activation soit par deletion, soit par mutagenese. Ceci nous a egalement permis d'isoler des mutations conduisant a l'expression constitutive du gene cyaa, aussi bien chez e. Coli que chez b. Pertussis. L'ensemble des nos resultats suggere l'existence de sites multiples et d'une regulation complexe necessitant la presence de differentes proteines

Dermonecrotic toxin (DNT) is produced by all the Bordetella species, and DNT from B. bronchiseptica is considered to be an important virulence factor in turbinate atrophy of pigs.;Recombinant DNT (rDNT) was purified by sonication, ion-exchange and hydroxylapatite chromatography. Other methods for the purification of wild-type DNT and rDNT, including preparative isoelectric focusing and hydrophobic chromatography, were investigated in detail.;Partially pure preparations of rDNT contained a 145 kDa protein band and were cytotoxic to embryonic bovine lung (EBL) cells. Partially pure rDNT induced the formation of actin stress fibres and focal adhesions in Swiss 3T3 cells. In addition, rDNT stimulated DNA synthesis in quiescent Swiss 3T3 cells but prevented cell proliferation, resulting in binucleated cells. Recombinant DNT has been shown to directly modify the small GTP-binding protein, Rho, (Pullinger, unpublished), which regulates the cell cytoskeleton. Results from this thesis indicate that rDNT causes the assembly of actin stress fibres and focal adhesion possibly by direct activation of the Rho protein.;Partially purified rDNT with a site-directed mutation in a putative nucleotide-binding motif did not induce cytoskeletal rearrangements and did not stimulate DNA synthesis in Swiss 3T3 cells. This suggests that the nucleotide-binding motif is essential for activity.;Two lines of evidence indicate that the toxin is internalised in the endosomal/lysosomal compartment: i) stimulation of DNA synthesis by transient exposure of Swiss 3T3 cells to rDNT, and ii) blocking of rDNT-induced DNA synthesis with methylamine.;Three monoclonal antibodies (mAbs) were produced against B. bronchiseptica DNT. These mAbs recognised rDNT and B. pertussis DNT, but none neutralised the cytotoxic activity of DNT on EBL cells.;The partial purification of rDNT and characterisation of its biological effects provide valuable information for further studies of the toxin, including analysis of its enzymatic mode of action and its role in infection. Also, DNT may prove to be a useful tool for analysis of cell responses involving the important signalling molecule, Rho.

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Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil
A coqueluche, ou pertússis, é uma doença do trato respiratório causada principalmente pela bactéria Bordetella pertussis. Após 50 anos de vacinação, pertussis reemergiu, passando a ser a doença imunoprevinível mais frequente mesmo em países desenvolvidos. Várias são as hipóteses para a reemergência de pertússis, uma delas é a adaptação do patógeno frente à vacinação. Linhagens contemporâneas de B. pertussis diferem de linhagens do período pré-vacinal, especialmente em genes codificadores de proteínas usadas na produção de vacinas acelular. Esta re-emergência também tem sido observada no Brasil, assim, realizamos a caracterização genética por MLST baseado nesses genes, de 26 isolados B. pertussis de surtos de três regiões brasileiras (Norte, Sul e Nordeste). Foram identificados dois perfis alélicos, em 24 isolados: prn2-ptxS1A-fim3B-ptxP3, de surtos (2008-2013) de Alagoas, Pernambuco e Rio Grande do Sul - e o perfil prn2-ptxS1A-fim3A-ptxP3 , em dois isolados de Pará/2004. Análises filogenéticas agruparam esses perfis com isolados do período pós vacinal de outras partes do globo. Deste conjunto, três do perfil mais frequente e um do perfil menos frequente, tiveram seus genomas sequenciados na plataforma GS 454 Junior. A comparação desses genomas com outros genomas de B. pertussis disponíveis em dados públicos não identificou SNPs ou genes únicos que caracterizassem os isolados do Brasil Este estudo desenvolveu uma metodologia que permitiu definir a posição da IS481 nos genomas, e uma delas corresponde a um gene relacionado a regulação da transcrição da família MarR, Análise filogenômica, baseada em 826 SNPs, demonstrou que os isolados recentes do Brasil da linhagem pandêmica que presente em todos os continentes, exceto a África. Foi observado também que as relações filogenéticas inferidas pelo MLST são semelhantes àquelas inferidas quando se utiliza o genoma completo, isso denota a pressão seletiva sobre esses genes. Sendo assim, a cepa utilizada na produção da vacina no Brasil, que apresenta o perfil alélico prn1-ptxS1D - fim3A-ptxP2, pode não ser capaz de gerar uma resposta imune protetora frente às linhagens circulantes no país. Este estudo traz, pela primeira vez, informações genéticas e genômicas de isolados de B. pertussis do Brasil, país que apresenta cobertura vacinal bastante heterogênea, que utiliza, oficialmente, a vacina celular, mas que, também, aplica a vacina acelular. As informações reveladas neste estudo podem auxiliar a tomada de ações para o controle de pertússis no Brasil, além do conhecimento sobre epidemiologia e evolução de B. pertussis
Pertussis more commonly referred as whooping cough is respiratory tract disease mainly caused by the bacteria B. pertussis. After 50 years of vaccination pert ussis remerged, becoming the most frequent vaccine preventable disease in developed countries. Many hypotheses have been proposed for the re - emergence of pertussis, one being the pathogen adaptation in a vaccinated environment. Current pertussis strains ar e different than those from the prevaccination era, especially in genes that code for proteins used in acelluar pertussis vaccines. This re - emergence is also observed in Brazil, therefore we characterized 26 isolates from 3 regions of Brazil (North,South,N ortheast) using an MLST approach based on these genes. We identified two allelic profiles, 24 isolates from the states of Rio Grande do Sul (2008 - 2009), Alagoas (2008 - 2009), Pernambuco (2013) and Pará (2004) presented the prn2 - ptxS1A - fim3B - ptxP3 allelic pr ofile, while 2 isolates from Pará (2004) presented the prn2 - ptxS1A - fim3A - ptxP3 allelic profile. Phylogenetic analysis branch these two allelic profiles along with other post vaccination isolates around the globe. Four isolates, three from the dominant prof ile and one from the less frequent profile, had their genomes completed sequenced on the GS 454 Junior Platform. We compared these genomes with others available in public databases and no SNP or unique genes were identified in the Brazilian genomes. This s tudy also developed a methodology that identifies the location of the repetitive region IS481, and what genes it interrupted. One of them was the MarR transcriptional regulator gene. Phylogenomic analysis based on 826 SNPs revealed that Brazilian B. pertus sis lineages are part of the current pandemic linage present in all continents, except Africa. We also observed that phylogenomic relationships are similar to MLST’s. Therefore, strain used for pertussis vaccine in Brazil, that presents the prn1 - ptxS1D - f im3A - ptxP2 allelic profile, might not be able to induce immune response to the current linage circulating in the country. This is the first study with genetic and genomic informations of B. pertussis isolates in Brazil, which is a country with heterogeneou s vaccine coverage and mixed and has both cellular and acellular vaccine administrated to the population. Information brought with this study can help the decision making on the control of pertussis in Brazil and gives new insights on the epidemiology and evolution of B. pertussis

Bordetella pertussis, o agente etiológico da coqueluche ou tosse comprida, que estabelece a infecção através da fixação bacteriana no epitélio do trato respiratório superior. Os principais mediadores de adesão da bactéria são a toxina pertússica (PT) e a hemaglutinina filamentosa (FHA). A FHA é a adesina majoritária e contém pelo menos 4 domínios: porção Nterminal, domínio de reconhecimento de carboidrato (CRD) (FHA1141-1279), trinca de aminoácidos Arginina-Glicina-Ácido aspártico (RGD) (FHA1097-1099) e o sítio de ligação a heparina (domínio Hep) (FHA442-863). Neste trabalho, foi realizado a amplificação de duas regiões do domínio de ligação à heparina, as regiões MAL80 (FHA299-873) e HEP (FHA430-873). As regiões foram amplificadas, clonadas no vetor de expressão pAE, expressas utilizando a cepa BL21 SI de E. coli e purificadas. A proteína HEP purificada de E. coli possui baixa afinidade por heparina e não é capaz de aglutinar hemácias. A proteína recombinante HEP purificada foi utilizada para a produção de anticorpos. Através do experimento de ELISA foi demonstrado que o anti-soro anti-HEP é capaz de reconhecer além da região HEP, a região MAL80 e a proteína FHA. Estes resultados foram confirmados por experimentos de Western. Neste período também foi realizada a amplificação do domínio HEP de FHA e da subunidade S1 da toxina pertússica (PT) de B. pertussis através do método de TAP Express. Este método envolve duas reações de PCR e no final do processo é obtido um fragmento que contém uma região promotora (CMV), uma seqüência codificadora e uma região terminadora (SV40), que está pronto para ser introduzido e expresso em animais. De posse deste material e da proteína recombinante HEP, foi realizado o desafio intracerebral com Bordetella pertussis e através do monitoramento dos camundongos foi observado que nenhum dos candidatos testados foi capaz de proteger os animais contra B. pertussis. Foi realizado também a expressão do domínio Hep em lactobacilos, visando um possível sistema de imunizações de mucosas. Os anticorpos produzidos nos camundongos imunizados com a proteína HEP expressa em E. coli, lactobacilos e por vacina de DNA foram capazes de inibir a hemaglutinação promovida pela proteína FHA.
Bordetelfa pertussis, the agent of whopping cough, establishes infection by attaching to the ciliated epithelial cells of the respiratory tract. The bacterial adherence is mediated by pertussis toxin and filamentous hemagglutinin (FHA). FHA is the major adhesin of B. pertussis and displays multipie adherence activities. FHA contains four distin\'ct domains that exhibit specific affinities for different ligands or receptor, the amino-terminal end, the RGD triplet (FHA1097-1099), the lectin domain (FHA1141-1279) and the heparin-binding domain (FHA442-863). In this study, two overlapping regions of the heparin-binding domain, Mal80 (FHA299-873) and Hep (FHA442-873), were amplified by peR and subcloned in pAE expression vectors for E. coli. The fusion proteins in pAE were transformed in E. coli BL21 SI, induced with NaCI 0,3 M and purified using a nickel-charged metal chelating resin. The purified protein has low heparin affinity and does not have hemagglutination activity. The purified protein HEP was used to produce polyclonal antibodies in mouse. The anti-HEP antibodies are able to recognize the HEP, MAL80 and FHA proteins in ELISA and western assays, but anti-FHA only recognized the FHA protein. The genetically detoxified S1 subunit of pertussis toxin and Hep domain were amplified by the TAP Express method. There are two PCR reactions involved in the TAP processo At the end of the process the fragment of interest will carry a CMV promoter and a SV40 terminator and is ready to be introduced into animals or cell by transfection. Groups were immunized with proteins and/or DNA, challenged i.c. with a lethal dose of live Bordetelfa pertussis and the survival was monitored. No groups were protected against the challenge. The recombinant protein HEP were also expressed in Lactobacilfus aiming the development of potential mucosal vaccines. The polyclonal antibodies produce in mouse immunized with DNA and protein Hep expressed in E. coli and Lactobacillus were able to inhibition the FHA hemagglutination activity.

La coqueluche est une maladie respiratoire dangereuse pour les nouveaux nés non vaccinés. Celle-ci est causée par Bordetella pertussis et Bordetella parapertussis, deux bactéries à gram négatif dont le seul hôte connu est l'homme. L'introduction de la vaccination contre B. Pertussis, le pathogène majoritairement responsable de cette maladie, à grandement impacté l'épidémiologie de la coqueluche ainsi que les populations de B. Pertussis circulant dans la population humaine. Les premier vaccins introduit dans les 1950 et utilisés pour la primo vaccination et le rappel enfant, dit à germes entier, ont permit de contrôler les isolats circulants semblables aux souches vaccinales. L'introduction des vaccines acellulaire, ne ciblant que quelques antigènes bactériens, pour le rappel adolescent puis pour la primo-vaccination et le rappel adulte a changé la donne. En plus d'un changement de l'immunité induite par le vaccin on a assisté à une augmentation de l'immunité vaccinale au sein de la population. Quelques années après l'introduction des vaccins acellulaires nous avons mis en évidence l'augmentation de la prévalence d'isolat n'exprimant plus un des antigènes vaccinaux alors que la population restait inchangée par rapport à l'ère post-germe entier au niveau génétique. La caractérisation phénotypique des ces isolats nous a permit de montrer qu'ils n'étaient pas plus virulent dans différents modèles in vitro ou in vivo mais qu'ils pouvaient présenter un avantage sélectif dans population immunisée par des vaccins acellulaires ciblant cet antigène en question. Bien que les vaccins restent efficaces, la surveillance doit continuer
Whooping cough is an acute respiratory disease life threatening for unvaccinated young children. It is caused by Bordetella pertussis and Bordetella parapertussis, two gram-negative bacteria restricted to humans. The introduction of vaccination against B. Pertussis in the 1950s greatly changed the epidemiology of pertussis as well as the bacterial population itself. Whole cell vaccines were first introduced for children immunization and first booster and enabled to control isolates similar to strains included in the vaccines. Acellular pertussis vaccines, only targeting few bacterial antigens, were later introduced for adolescent booster vaccination before being generalized to the whole population, including adults and new-borns. In addition to a change in the type of vaccine-induced immunity, B. Pertussis also had to face increased herd immunity. Few years alter acellular pertussis vaccine introduction we demonstrated a temporal increase in the prevalence of isolates lacking the production of one vaccine antigen, pertactin, while the population remained quite monomorphic at the genetic level as compare to the post-whole cell vaccine era (allelic stability of known virulence factors). We characterized these isolates at the phenotypic level and demonstrated that they remain as virulent in different in vitro and in vivo models of host pathogen interaction while they might present a selective advantage in an acellular immunized background targeting pertactin. Although vaccines remain effective, surveillance must continue to follow the evolution of these isolate prevalence and the possible impact of pertactin loss on vaccine effectiveness

Autotransporters are a superfamily of Gram-negative secreted proteins, composed of a Cterminal domain which forms a β-barrel in the outer membrane and plays a significant role in translocation of the N-terminal passenger domain to the cell surface. A subfamily of autotransporters is N-terminally acylated during their biogenesis, a post-translational modification demonstrated to be essential for their function. Considering the autotransporter passenger domain is secreted beyond the outer membrane, how translocation can occur in the presence of N-terminal acyl groups is undetermined. The work in this thesis describes the biogenesis of the Bordetella autotransporter F (BapF) from B. bronchiseptica RB50, which is Nterminally acylated during the initial stages of its secretion to the cell surface. The acyl groups attached to BapF at the Cys28 residue were shown to be subsequently removed by signal peptidase 1 cleavage between residues Ala34 and Ala35, indicating that BapF forms an acylated intermediate in its secretion pathway. Studying the secretion of BapF mutants in which Nterminal acylation was ablated revealed that the passenger domain was still capable of reaching the cell surface; the surface-expressed passenger domain mediated phenotypes of cellular aggregation and an increased rate of culture sedimentation, similar to that observed in E. coli cultures expressing the wild-type BapF protein. However, cells expressing these mutants appear to have damaged outer membranes, potentially due to the observed increase in fulllength protein accumulating in the periplasm. Alternatively, E. coli cells expressing a BapF mutant in which signal peptidase 1 cleavage is blocked do not exhibit obvious aggregation and sedimentation phenotypes. Yet, an independent passenger domain is clearly produced. Based on the results presented in this thesis, it can be hypothesized that sequential processing of the BapF signal peptide, producing an acylated intermediate in the secretion pathway, helps to regulate the passage of BapF through the periplasm ultimately permitting surface expression. In addition, bioinformatic and molecular analysis strongly suggest the BapF passenger domain folds into a β-propeller structure, and if proven to do so, BapF will be the first autotransporter reported with this conformation

Bordella pertussis, agent etiologique de la coqueluche, synthetise, entre autres toxines, une adenylate cyclase dont l'activite depend d'une proteine de l'hote, la calmoduline. Clonage et structure du geni de l'adenylate cyclase (gene cya) ont ete realises par complementation d'une souche cya d escherichia coli, en reconstituant la bacterie l'activation par la calmoduline. L'adenylate cyclase et synthetisee sous forme d'un precurseur bifonctionnel de haut poids moleculaires

The resurgence of pertussis in several highly vaccinated countries has stimulated this study of the genotypic diversity and epidemiological typing of Bordetella pertussis. The genotypic variation of B. pertussis was investigated in 318 UK clinical isolates from 1920-2002, vising pertactin (prnA) and pertussis toxin SI subunit (ptxA) gene typing. To date, the evaluation of typing methods used for B. pertussis has not been performed extensively. Therefore, in this thesis, the recognised methods, namely serotyping, pulsed-field gel electrophoresis (PFGE) using Xbal, and IS1002-RFLP analysis, were evaluated. It was found that, PFGE typing gave a good discrimination index value, but gave a low score for reproducibility, and further work is required to optimise this method. Furthermore, if prnA and ptxA gene typing were included in the evaluation, combined with serotyping, this combination would equal the discrimination of PFGE. Other typing methods attempted for B. pertussis included direct sequencing of adenylate kinase (adk) and filamentous haemagglutinin genes (fhaB), and single-enzyme amplified fragment length polymorphism (AFLP) analysis with a selection of enzymes and selective primers. The type strain and a clinical strain, generated one and six single nucleotide polymorphisms (SNPs) in adk and fhoB, respectively. The discriminatory ability of the single-enzyme AFLP analysis was not satisfactory, as only a few different profiles were seen in the nine isolates tested. However, at least four AFLP profiles were generated using PstI enzyme, and the selective primer Pst-C. The direct detection and epidemiological typing of B. pertussis in 20 previously untypable clinical samples was attempted using prnA, ptxA as targets. Six clinical extracts generated prnA and ptxA (5/6) sequence data, therefore confirming that these typing procedures on B. pertussis PCR-positive clinical specimens is worthwhile in order to generate the prnA and ptxA distributions from babies or adults presenting atypical symptoms. This strategy should also be encouraged in other country that have studied prnA and ptxA allele distributions, in order to update the representation of the genetic diversity of B. pertussis.

An aspartate transcarbamoylase (ATCase) gene from Bordetella pertussis was amplified by PCR and ligated into pT-ADV for expression in Escherichia coli. This particular ATCase (pyrB) was an inactive gene found adjacent to an inactive dihydroorotase (DHOase) gene (pyrC'). This experiment was undertaken to determine whether this pyrB gene was capable of expression alone or if it was capable of expression only when cotransformed with a functional pyrC'. When transformed into E. coli TB2 pyrB-, the gene did not produce any ATCase activity. The gene was then co-transformed into E. coli TB2 pyrB- along with a plasmid containing the pyrC' gene from Pseudomonas aeruginosa and assayed for ATCase activity. Negative results were again recorded.

Le cuivre est devenu un élément essentiel à la plupart des organismes vivants depuis l’apparition de l’oxygène dans l’atmosphère terrestre il y a 2,7 milliards d’années. Ce métal est utilisé dans de nombreux processus biologiques, mais les propriétés chimiques qui le rendent indispensable sont également à l’origine de sa toxicité. Cette ambivalence du cuivre a fait que tous les organismes ont développé divers systèmes permettant un maintien extrêmement strict de l’homéostasie de ce métal. Certains organismes comme les amibes ont également mis à profit ses propriétés toxiques pour la prédation d’autres organismes unicellulaires par la phagocytose. Un des mécanismes qui leur permet ensuite de tuer leurs proies est l’empoisonnement par les métaux, et en particulier par le cuivre. La phagocytose a perduré au cours de l’évolution jusqu’à constituer un acteur essentiel de l’immunité des métazoaires, dont l’humain fait partie. Les systèmes de détoxification du cuivre mis en place par les organismes procaryotes ont donc été sélectionnés par la pression exercée lors de la phagocytose. C’est ainsi que les bactéries pathogènes ont acquis des systèmes de survie à la phagocytose, dont des mécanismes de l’homéostasie du cuivre. Ces mécanismes sont variés et dépendent de la niche écologique de chaque bactérie. Les connaissances sur ce sujet se concentrent essentiellement sur les bactéries environnementales ainsi que sur quelques bactéries pathogènes, principalement intracellulaires. Ici, c’est l’homéostasie du cuivre chez Bordetella pertussis qui a été au centre de ces travaux de thèse. Cette bactérie est l’agent étiologique de la coqueluche et présente la particularité d’être un pathogène totalement dépendant de son hôte. De plus, sa niche écologique est extrêmement restreinte, se limitant à l’épithélium de l’arbre respiratoire supérieur de l’humain. Cette bactérie constitue donc un modèle nouveau pour l'étude du rôle du cuivre dans la relation hôte-pathogène. Ces travaux ont mis en évidence la perte de la plupart des systèmes de l’homéostasie du cuivre chez B. pertussis. Néanmoins, un système de détoxification original avec une régulation double a été découvert, composé de la métallochaperonne cytoplasmique CopZ et de deux enzymes impliquées dans la détoxification du stress oxydant. Les travaux de cette thèse ont permis d’émettre l’hypothèse que ce système propre aux Bordetella est impliqué dans la survie à la phagocytose par les cellules immunitaires, seule source d’excès de cuivre dans la niche écologique de B. pertussis. La métallochaperonne CopZ détoxifie le cuivre libre par complexation et les deux enzymes permettent de réduire la concentration de peroxyde d’hydrogène à l’origine du stress oxydant causé par les réactions de Fenton et d’Haber-Weiss. La co-régulation de ce système par le cuivre et le peroxyde d’hydrogène permet son activation sélective et très dynamique en réponse aux mécanismes liés à la phagocytose. Le rapport entre coût énergétique de ce système pour la bactérie et la protection qu’il lui assure doit être suffisamment favorable pour qu’il ait été spécifiquement sélectionné dans l’évolution de ce pathogène très spécialisé.Ces travaux ont permis de déterminer les mécanismes de tolérance au cuivre présents chez B. pertussis, ainsi que la sélection évolutive que ces systèmes ont subie chez Bordetella
Copper has become an essential element for most living organisms since the adventof oxygen in the Earth’s atmosphere, 2.7 billion years ago. This metal is used in manybiological processes. However, the electronic properties that make it crucial for life are alsowhat makes it poisonous. This ambivalent biological impact has forced organisms to acquirean increasing number of mechanisms dedicated to maintaining a strict control over copperhomeostasis. In addition, a few organisms such as amoebae have taken advantage of its toxicproperties. Those lower eukaryotes have developed a complex predation mechanism to feedon other unicellular organisms, phagocytosis, and make use of metal poisoning, in particularcopper, to kill their preys. Phagocytosis has persisted over the course of evolution to becomea key actor of innate immunity in metazoans, including humans. Copper detoxificationmechanisms developed by prokaryotic organisms have likely been selected through thepressure exerted by phagocytosis. In particular, copper homeostasis regulation mechanismsare part of the strategies adopted by pathogenic bacteria to survive phagocytosis. Thesemechanisms vary depending on the ecological niche of each species. Studies on this topichave mainly focused on environmental bacteria and on pathogenic bacteria, especially thosewith an intracellular lifestyle. Copper homeostasis in Bordetella pertussis is at the centre ofthis thesis. This bacterium is the etiologic agent of whooping cough whose peculiarity is to bestrictly dependent on its host. Furthermore, its ecological niche is very limited, as it onlyinfects the human upper respiratory epithelium. This bacterium represents a new model tostudy the role of copper in host-pathogen relationships. This work has revealed the loss ofmost copper homeostasis systems in B. pertussis. An original detoxification mechanism witha composite regulation has been identified that includes the cytoplasmic metallochaperoneCopZ and two enzymes catalyzing detoxification of peroxides. This thesis work has led to thefollowing hypothesis: this system, which is a distinctive feature of the Bordetella genus, isinvolved in survival to phagocytosis, the only situation where the bacterium experiencescopper excess in its ecological niche. The metallo-chaperone CopZ mediates thedetoxification of free copper by complexation, and together the two enzymes decrease theconcentration of hydrogen peroxide, which causes oxidative stress through the Fenton andHaber-Weiss reactions. The co-regulation of this system by copper and hydrogen peroxideresults in a very dynamic and selective activation, specific to phagocytosis-relatedmechanisms. This system offers a sufficiently favorable cost-benefit balance to have beenselected over the course of the evolution of this host-restricted pathogen.This work has led to the identification of copper tolerance mechanisms in B. pertussisand to the description of their evolutionary selection among Bordetellae

Bordetella pertussis is a gram-negative, human pathogen and etiologic agent of whooping cough (pertussis), a highly contagious, acute respiratory illness. In this study, the analysis of whole immunproteome and subproteomes of this microorganism was performed. The soluble cytoplasmic proteomes of B. pertussis Tohama I strain and a local isolate Saadet were separated by 2DE. By Western blot analysis, we identified 25 immunogenic proteins of three categories. In the first group, there were well-known proteins of the pathogen The second group comprised proteins which were already shown antigenic in certain pathogenic bacteria, but not in B. pertussis before. The third group of proteins were those which have not been shown to be immunogenic in any pathogen till the present study such as putative chromosome partition protein, preprotein translocase SecA subunit, carbamoyl-phosphate synthase large chain, PRP synthase, putative substrate-CoA ligase, lysyl-tRNA synthetase, fumaryl acetoacetase, putative peptidyl-prolyl cis-trans isomerase, aspartate-semialdehyde dehydrogenase, putative DNA-binding protein and a putative outer membrane protein. In our surfaceome study, surface proteins of two strains were identified by 2DE followed by MALDI-TOF-MS/MS analysis and also geLC-MS/MS. With these techniques 45 proteins were identified by 2DE and 226 proteins by geLC-MS/MS. The immunogenicity of surface proteins on 2DE gels were analyzed by Western blotting and among 11 identified immunogenic proteins glutamine-binding periplasmic protein, leu/ile/val-binding protein, one putative exported protein, and iron-superoxide dismutase were found to be immunogenic for the first time in Bordetella. It was also found that 16 proteins were differentially expressed in B. pertussis Saadet and Tohama I. Five proteins were expressed only in Saadet (adhesin, chaperone protein DnaJ, fimbrial protein FimX, putative secreted protein Bsp22 and putative universal stress protein), and two (ABC transporter substrate-binding protein and a putative binding protein-dependent transport periplasmic protein) only in Tohama I. In the secretome study, we identified 40 proteins by 2DE and 357 proteins by geLC-MS/MS. It was found that 12 proteins were immunogenic by Western blot analysis and the immunogenicity of putative secreted protein (BP1047) was shown for the first time in this study. In our study, PT subunit 2 and putative outer protein D (BopD) were more abundant in Saadet while one protein, glutamate synthase subunit beta was expressed at a higher level in Tohama I. Four proteins were expressed only in Saadet (two capsular polysaccharide biosynthesis protein, protein FimX and putative outer membrane permeability protein). The present study comprehensively covered almost the entire proteome of a crucial pathogen, demonstrated many novel antigens and identified hundreds of membrane-bound proteins, cell surface-associated and extracellular proteins. Thus, it is anticipated to greatly aid in a better understanding of pathogen-host relations, rational design of novel drugs and developing new generation vaccines against B. pertussis.

The identification and characterisation of new virulence determinants of 5. pertussis is providing important information for understanding the colonisation and survival strategies of the microorganism. B. pertussis deploys a range of surface-associated components to enable its successful colonisation of the host. Bap-5 has been identified as a new member of the B. pertussis autotransporter family of proteins that includes PRN, BrkA, TCF and Vag-8, largely due to its homology at the C-terminus and some other similar regions such as the RGD (integrin-binding) and SGXG (glycosaminoglycan-binding) motifs. The bap-5 gene also exists in B. bronchiseptica and B. parapertussis. Characteristic upstream regulatory sequences such as a ribosome-binding site were not seen in bap-5, but a potential heptameric BvgA-binding motif was identified. The expression of Bap-5 was confirmed by RT-PCR and Western blotting and was shown to be bvg dependent. Although Bap-5 does not possess a typical signal sequence like pertactin (PRN), its surface localisation was confirmed by agglutination and immunofluorescence assays. A potential role for Bap-5 in infection was studied by generating Bap-5 deficient mutants in two strains of B. pertussis. An allelic exchange procedure with the suicide vector pSS1129 carrying the bap-5 gene disrupted with a kanamycin-resistance cassette was used. PCR and Southern blotting confirmed the replacement of the wild-type bap-5 gene with the mutated version. Moreover, SDS-PAGE and Western blotting of outer-membrane preparations of B. pertussis Taberman wild-type and its Bap-5-deficient mutant showed a clear difference in their outer-membrane profile at ~79.9kDa presumably representing the unprocessed form and bands at ~65 kDa and ~16 kDa may represent the processed forms of the protein. The Bap-5 characterisation studies showed that the Taberman Bap-5-deficient strain was less able than the parent strain to colonise the lower respiratory tract of mice and adhesion studies (in vitro) showed that the Taberman parent was better in adhering to certain cell types than the Bap-5-deficient mutant.

A 1kb HinfI DNA fragment, containing a repeat DNA sequence element was isolated from a Bordetella pertussis BP348 cosmid gene bank. This repeat DNA sequence has subsequently been named IS148. The insertion sequence was demonstrated by have a high copy number only in B.pertissus and to be absent in Bordetella parapertussis and Bordetella bronchiseptica. Using a restriction fragment of IS148 labelled with radioactivity as a probe in DNA or whole cell dot blots between 10-100 cells could be detected or from 100pb-1ng DNA. Furthermore, a pair of oligonucleotide primers was synthesised corresponding to a central region of IS148 that could generate a 242bp fragment upon PCR amplification. The use of PCR amplification with specific primers allows the detection of 0.5pg of B.pertussis chromosomal DNA and as little as one B.pertussis cell. The prn genes encoding the outer membrane P.70 and P.68 pertactin from B.parapertussis and B.bronchiseptica have been cloned, sequenced and expressed in Escherichia coli. Analysis of the DNA sequences reveal that the genes have open reading frames capable of encoding proteins with molecular weights of 95,177 (P.95, B.parapertussis) and 93,996 (P.94, B.bronchiseptica). These precursors molecular are processed to form the P.70 and P.68 antigens on the surface of B.parapertussis and B.bronchiseptica respectively. Heterologous expression of the full-length gene encoding P.95 and P.94 in E.coli result in similar processing, with the P.70 and P.68 antigens targetted to the bacterial outer membrane. Comparison of P.95, P.94 and P.93, encoding homologous proteins from B.parapertussis, B.bronchiseptica and B.pertussis, shows a high degree (> 90%) of homology. The major differences between all three proteins occur in the number of repeat of the two families (Gly-Gly-Xaa-Xaa-Pro)n and (Pro-Gln-Pro)n of reiterated sequence motifs.

La coqueluche, infection des voies aériennes inférieures par Boedetella pertussis (Bp), est en recrudescence chez les adultes qui contaminent les nouveaux-nés exposés aux formes sévères de la maladie. Nous rapportons une épidémie de coqueluche parmi les membres du personnel soigant (MPS) d'un hôpital avec contamination de 2 patients adultes. Ces données font discuter une vaccination des MPS. Les interactions de l'hépithélium respiratoire humain avec Bp ont été étudiées dans le but initial de rechercher un réservoir bactérien chez l'adulte. Nous avons montré que Bp pénètre dans des cellules épithéliales bronchiques humaines en lignée HTE. L'invasion est inhibée par une toxine, l'adényl cyclase-hémolysine (AC-Hly). Les cellules HTE en contact avec Bp sont capables de sécréter des cytokines inflammatoires dont l'IL-6. L'AC-Hly est responsable de cette sécrétion d'IL-6 confirmant le rôle de cette toxine dans l'induction de la réponse inflammatoire
Whooping cough is a respiratory disease caused by Bordetella pertussis (Bp). Because of waning immunity after vaccination, adult are thought to have been acting as a reservoir for infections in infants, for whom the disease can be serious. We report a pertussis out break among health care workers (HCW) in an hospital with the contamination of 2 adults immunosuppressed patients. Such observations could support the vaccination of HCW. We also studied interactions between human respiratory epithelium and Bp as these cells could constitute a bacterial reservoir in adults. We showed that Bp invades human HTE tracheal apithelial cells line. The invasive capacity of the bacterium is inhibited by the expression of a toxin, the adenylate cyclase-hemolysin (AC-Hly). Interaction of Bp with HTE cells induces the secretion of inflammatory cytokines such as IL-6 by the cells. AC-Hly is responsible of this secretion confirming that this toxin plays an important role in the inflammatory response induced by Bp

Thèse de doctorat : Sciences de la vie et de la santé. Biologie cellulaire et moléculaires des épithéliums : Paris 12 : 2004.
Version électronique uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 332 réf.

Pertussis is a vaccine preventable disease, primarily caused by the bacterium Bordetella pertussis (and occasionally B. holmesii and B. parapertussis). The disease is highly contagious, and results in extended periods of coughing. Prior to vaccination, pertussis was the foremost cause of infantile death by infection globally. Global vaccination efforts against pertussis were introduced in the 1950s and this resulted in the rate of pertussis infections to decrease tremendously. In recent decades, public health authorities around the world have reported a resurgence of the number of pertussis cases, despite high vaccination coverage. The reason behind this resurgence is unknown, however, there are multiple theories. A major contributing factor is the reduced molecular surveillance of B. pertussis, a consequence of the change in diagnostic methods from culture isolates to direct PCR on clinical samples. Culture isolates enable molecular surveillance by providing information on the strain, such as sequence type and antibiotic resistance information. Without molecular surveillance we know little about the currently circulating strains of B. pertussis. The aims of this thesis were to the provide characterise the genomes of the Bordetella genus. Ultimately, it aimed to attempt explaining the driving forces behind B. pertussis resurgence by identifying their properties of relevance for laboratory diagnosis and surveillance. The thesis also aimed to develop new laboratory protocols to circumvent the lack of isolates and achieve high resolution data and information from clinical samples. It also aimed to determine the differences in the expression of B. pertussis vaccine antigens using metatranscriptomics. Furthermore, the thesis aimed to determine the potential for antimicrobial resistance within the Bordetella spp., and their mechanisms behind resistance. This thesis confirmed through the generation of high-quality closed reference sequences, that Bordetella spp. isolates from NSW carried the same genomic rearrangement characteristics as global isolates. Thus, demonstrating evidence of genomic evolution of the Bordetella spp. in the Southern hemisphere. These reference sequences can serve as a representative of the prevalent strains in the Australian community and be utilised in future public health surveillance. This thesis also describes the development of a protocol to circumvent culture isolates, by combining Saponin depletion with an improved library preparation method, Nextera Flex kit (Illumina) and sequencing so that more reads per sample are generated (i.e. deep sequencing). It demonstrated that with high bacterial load samples, this method can effectively produce the entire genome of B. pertussis with an average 20 X coverage. Therefore, through the utilisation of pre-extraction depletion methods, in conjunction with the proper library preparation kit, and increased sequencing depths, sufficient information can be obtained for molecular surveillance. Culture-independent RNA sequencing was also determined as a feasible method in determining high-resolution strain typing information for public health investigations. Metatranscriptomics was able to show that assemblies could reliably identify segments of the genome coding the vaccine antigens, primarily fhaB and prn. In addition, the thesis identified 10 new genomic markers for further investigation, especially as potential targets for CI-WGS enrichment. The study showed that two of the three Bordetella spp. developed resistance (>256 μg /mL) within 15 weeks upon consistent macrolide exposure. WGS was able to confirm the resistance mutation in B. holmesii – a non-synonymous SNP mutation in the 23S ribosomal RNA (C2585T, G2031A, and A2032G). Thus, the study proved that B. parapertussis and B. holmesii carried a higher capability in developing macrolide resistance under antibiotic pressure in vitro. The mechanism of resistance in these two species was identified in B. holmesii, however remains unclear in B. parapertussis. Overall, the findings of this thesis will facilitate the genomic surveillance of Bordetella spp. in the era of culture-independent diagnostic testing.

Adenylate cyclase toxin (CyaA) was produced and purified in the toxic CyaC-modified form and the unmodified non-toxic form from both B. pertussis and recombinant E. coli strains in sufficient quantity to allow large scale experimentation. Immunoblot analysis of crude and purified CyaA preparations revealed that the toxins were prepared as full length 200 kDa proteins with a small amount of degradation products of lower molecular weight protein in both toxic and non-toxic forms. The in vivo CyaC-modified CyaA toxin produced from recombinant E. coli showed comparable levels of cytotoxic and invasive activity to that produced from B. pertussis but its haemolytic activity was weaker. In addition, the leukotoxin (LktA) from Pasteurella haemolytica was also produced by expression from recombinant plasmids in E. coli, in both the LktC-modified toxic form and unmodified non-toxic form. The A and C proteins of both toxins were produced separately in E. coli and each could be co-expressed on compatible plasmids. This allowed heterologous activation of CyaA by LktC and LktA by CyaC. The LktC- or CyaC-modified LktA of 105 kDa protein was produced and partially purified from recombinant E. coli strains, but the yield of LktA production was low compared to that of CyaA in the same T7 expression system. The LktC-modified CyaA toxin was also produced but showed no cytotoxic or haemolytic activity. Heterologous activation of LktA by CyaC was successful and the toxin was almost as active against bovine lymphoma (BL3) cells as the LktC-modified toxin. However, the heterologous activation by CyaC did not change its specificity for ruminant cells, because no cytotoxic activity against mouse J774.2 cells or haemolytic activity against horse red blood cells was detected. The CyaC-modified LktA showed a greater haemolytic to cytotoxic ratio than LktC-modified LktA. Thus, LktA modified by CyaC was more haemolytic than the LktC-modified form, but nevertheless retained the specificity for ruminant cells which is a feature of the native toxin. Two hybrid toxins derived from CyaA and LktA were also produced and purified. Hybl contained the N-terminal enzymic domain and the pore forming domain from CyaA (amino acids [aa] 1-687), with the remainder of the protein derived from the C-terminal end of LktA (aa 379-953). Hyb2 was created from Hybl by replacement of the LktA C-terminal domain of Hybl with the C-terminal domain of CyaA (aa 919-1706 ). Part of the region concerned with C-modification site of CyaA (aa 688-918) was therefore replaced with the equivalent region from LktA. The Hybl toxin of 150 kDa protein had normal AC enzymic activity, but showed no toxic activity when modified in vivo by CyaC or LktC. In contrast to CyaA, the 200 kDa Hyb2 protein was activated more efficiently by LktC than by CyaC, although the cytotoxic and haemolytic activity of Hyb2 modified with LktC or CyaC was lower compared to recombinant active CyaA modified by CyaC. However, LktC-modified Hyb2 showed more toxic activity against ruminant than against murine nucleated cells, whereas CyaC-activated Hyb2 displayed a similar, but lower, activity against both cell types, indicating that LktC and the region with which it interacted had an influence on the target cell specificity of the activated toxin.

ASTMH 64th Annual Meeting. October 25-29, 2015 Philadelphia Marriott Downtown Philadelphia, Pennsylvania USA
Acute respiratory infections (ARI) are the main cause of morbidity and mortality in children under 5 years worldwide. Bordetella pertussis is a highly contagious bacterium that can cause serious illness, and approximately half of infected infants less than 1 year old are hospitalized. Also, pertussis immunization series is not completed until six months of age, leaving young infants vulnerable to pertussis. In Peru, pertussis is an increasing health problem despite immunization efforts, and the role of B. pertussis in ARI is unknown. We determined the prevalence of B. pertussis among children under 5 years old admitted to Hospital Nacional Cayetano Heredia in Lima with diagnosis of ARI between Jan-2009 and Dec 2010. Epidemiological and clinical features were collected, and presence of B. pertussis was determined by PCR (pertussis toxin and IS481 gene). A total of 596 nasopharyngeal samples among children under 5 years were analyzed. In 114 (19.1%) samples were positive for B. pertussis. 32.5% of sample positive to B. pertussis were diagnosed as viral pneumonia at diagnosis. Importantly, 71.9% of cases were under 12 months of age and 58.8% have been contact with other ARI infected people. Significant differences in clinical symptoms between the total ARI cases and B. pertussis cases were not found. The most frequent symptoms in B. pertussis cases were fever (100%), rhinorrhea 78%, cough 71.9% and respiratory distress 60.5%. One child died due to the infection. B. pertussis cases showed a seasonal distribution with peaks during the months March June and November. This study shows the high prevalence of B. pertussis in infants who were hospitalized due to severe acute respiratory infections in Lima, Peru. Epidemiologic surveillance programs for B. pertussis are essential in the future in Peru

La tosferina és una malaltia infecciosa aguda causada generalment per Bordetella pertussis, un cocbacil gramnegatiu, de reservori exclusivament humà i amb una elevada transmissibilitat per via aèria. Es caracteritza per presentar un quadre respiratori de tos intensa que, sovint, pot perllongar-se durant setmanes o mesos. Entre les possibles causes implicades en el ressorgiment de la tosferina, destaca l’adaptació de B. pertussis a la immunitat conferida per les vacunes acel·lulars (DTPa), les quals es van introduir en el nostre entorn a finals dels anys noranta, així com l’emergència de noves espècies del gènere Bordetella contra les quals les vacunes utilitzades no confereixen protecció. Per una banda, el procés d’adaptació de B. pertussis a la immunitat induïda per les vacunes DTPa ha evidenciat com, coincidint amb la introducció d’aquestes, han aparegut noves poblacions de B. pertussis que han anat reemplaçant de forma progressiva les variants antigèniques presents a les composicions vacunals utilitzades. En aquest sentit, s’ha observat un viratge en l’estructura de les poblacions de B. pertussis circulants, reflectida pel recanvi dels perfils electroforètics que han circulat entre els períodes d’ús de les vacunes de cèl·lules completes (DTPw) i les vacunes DTPa, així com de l’emergència i posterior predomini del genotip MT27, el qual s’ha observat en el 79,3% dels aïllats de B. pertussis circulants després de la introducció de les vacunes DTPa. Addicionalment, en aquestes poblacions, s’han observat polimorfismes en els gens que codifiquen la toxina pertussis (ptxA) i en el seu promotor (ptxP), la pertactina (prn) i la fimbria de tipus 3 (fim3). Així, la combinació al·lèlica ptxA1/prn2/fim3-2, la qual presenta formes antigèniques diferents a les que es troben contingudes en les vacunes DTPa, s’ha observat com a majoritària (52,7%) entre els aïllats que han circulat en el nostre medi després de la introducció d’aquest tipus de vacuna. Addicionalment, la presència majoritària (97,7%) del promotor de la toxina pertussis de tipus 3 (ptxP3) condiciona un increment de la producció d’aquest determinant de virulència en aquests aïllats de B. pertussis que el posseeixen. D’altra banda, s’ha posat de manifest la circulació d’aïllats de B. pertussis que deixen d’expressar l’antigen vacunal de la pertactina (PRN), mantenint la seva virulència i capacitat per produir malaltia. Aquests han suposat 27,2% del total d’aïllats, identificant-se únicament en el període d’ús de les vacunes DTPa. En aquest sentit, l’aparició i disseminació exitosa, d’un clúster d’aïllats que han presentat una deleció en el promotor i part del gen que codifica aquest determinant de virulència (58,1%, prn::del(-292, 1340)), ha evidenciat l’expansió dels aïllats de B. pertussis que no expressen PRN en el nostre medi. D’aquesta manera, la disseminació d’aïllats de B. pertussis que expressen antígens vacunals que posseeixen epítops no reconeguts pels anticossos generats mitjançant l’ús de les vacunes DTPa, així com la pèrdua de l’expressió d’aquests antígens com a importants mecanismes d’adaptació i evasió de la immunitat conferida per les vacunes antipertússiques utilitzades al llarg dels últims anys, sembla ser un mecanisme que pot comprometre la seva eficàcia. Finalment, la descripció de l’emergència de B. holmesii com a agent causal de la tosferina, observant-se una prevalença d’aquest microorganisme del 3,9% l’any 2015 i del 8,8% l’any 2016, demostra que la seva circulació ha pogut contribuir en el ressorgiment de la tosferina en el nostre medi. En relació amb aquests, no s’han observat diferències en les característiques clíniques-epidemiològiques dels casos de B. holmesii en comparació amb aquells causats per B. pertussis, ni s’han evidenciat complicacions o recidives després del seu tractament amb azitromicina. Així, el conjunt d’observacions reforça la necessitat d’adaptar les eines diagnòstiques actuals per la identificació correcta de les etiologies de la tosferina.
La tosferina es una enfermedad infecciosa aguda causada generalmente por Bordetella pertussis, un cocobacilo gramnegativo, de reservorio exclusivamente humano y con una elevada transmisibilidad por vía aérea. Se caracteriza por presentar un cuadro respiratorio de tos intensa que, a menudo, puede prolongarse durante semanas o meses. Entre las posibles causas implicadas en el resurgimiento de la tosferina, destaca la adaptación de B. pertussis a la inmunidad conferida por las vacunas acelulares (DTPa), las cuales se introdujeron a finales de los años noventa, así como la emergencia de nuevas especies del género Bordetella contra las que las vacunas utilizadas no confieren protección. Por un lado, el proceso de adaptación de B. pertussis a la inmunidad inducida por las DTPa ha evidenciado como, coincidiendo con la introducción de éstas, han aparecido nuevas poblaciones de B. pertussis que han ido reemplazando progresivamente las variantes antigénicas presentes en las composiciones vacunales utilizadas. En este sentido, se ha observado un viraje en la estructura de las poblaciones de B. pertussis circulantes, reflejada por el recambio de los perfiles electroforéticos que han circulado entre los períodos de uso de las vacunas de células completas (DTPw) y las DTPa, así como de la emergencia y posterior predominio del genotipo MT27, el que se ha observado en el 79,3% de los aislados de B. pertussis circulantes tras la introducción de las DTPa. Adicionalmente, en estas poblaciones, se han observado polimorfismos en los genes que codifican la toxina pertusis (ptxA) y en su promotor (ptxP), la pertactina (prn) y la fimbria de tipo 3 (fim3). Así, la combinación alélica ptxA1/prn2/fim3-2, la cual presenta formas antigénicas diferentes a las contenidas en las DTPa, se ha observado como mayoritaria (52,7%) entre los aislados que han circulado en nuestro medio después de la introducción de estas vacunas. Adicionalmente, la presencia mayoritaria (97,7%) del promotor de la toxina pertusis de tipo 3 (ptxP3) condiciona un incremento de la producción de este determinante de virulencia entre los aislados de B. pertussis que lo poseen. Por otra parte, se ha puesto de manifiesto la circulación de aislados de B. pertussis que dejan de expresar el antígeno vacunal de la pertactina (PRN), manteniendo su virulencia y capacidad para producir enfermedad. Éstos han supuesto 27,2% del total, identificándose únicamente en el período de uso de las DTPa. En este sentido, la aparición y diseminación exitosa, de un clúster de aislados que han presentado una deleción en el promotor y parte del gen que codifica este determinante de virulencia (58,1%, prn::del(-292, 1340)), ha evidenciado la expansión de los aislados de B. pertussis que no expresan PRN. De este modo, la diseminación de aislados de B. pertussis que expresan antígenos vacunales que poseen epítopos no reconocidos por los anticuerpos generados mediante el uso de las DTPa, así como la pérdida de la expresión de estos antígenos como importantes mecanismos de adaptación y evasión de la inmunidad conferida por las vacunas utilizadas, parece ser un mecanismo que puede comprometer su eficacia. Finalmente, la descripción de la emergencia de B. holmesii como agente causal de la tosferina, observándose una prevalencia de este microorganismo del 3,9% en 2015 y del 8,8% en 2016, demuestra que su circulación ha podido contribuir en el resurgimiento de la tosferina. En relación con éstos, no se han observado diferencias en las características clínicas-epidemiológicas de los casos de B. holmesii en comparación con aquellos causados por B. pertussis, ni se han evidenciado complicaciones o recidivas después de su tratamiento con azitromicina. Así, el conjunto de observaciones refuerza la necesidad de adaptar las herramientas diagnósticas actuales para la identificación correcta de las etiologías de la tosferina.
Whooping cough is an acute infectious disease usually caused by Bordetella pertussis, a gram-negative coccobacillus, with an exclusively human reservoir and high airborne transmissibility. It is characterized by presenting a respiratory picture of intense cough that can often last for weeks or months. Among the possible causes that are involved in the pertussis resurgence, the adaptation of B. pertussis to the immunity conferred by acellular vaccines (DTPa), which were introduced in the late 1990s, as well as the emergence of new species of the genus Bordetella, against which the vaccines used do not confer protection, are some of the most important ones that have be considered. On the one hand, the process of adaptation of B. pertussis to the immunity induced by DTPa has been evidenced by the new populations of B. pertussis that have appeared and have gradually replaced the antigenic variants present in the vaccine, concurrently with its introduction. In this sense, a change in the structure of circulating populations has been observed, reflected by the replacement of the electrophoretic profiles that have circulated within the different vaccine periods. Is remarkable the emergence and subsequent predominance of the MT27 genotype, which has been observed in 79.3% of circulating B. pertussis isolates after the introduction of DTPa. Additionally, in these populations, polymorphisms in the genes encoding the pertussis toxin (ptxA) and its promoter (ptxP), pertactin (prn) and fimbria type 3 (fim3). Thus, the allelic combination ptxA1/prn2/fim3-2, which include different antigenic forms than those contained in DTPa, has been observed as the most prevalent (52.7%) among the isolates that have circulated in our environment after the introduction of this type of vaccine. Additionally, a high prevalence (97.7%) of the type 3 pertussis toxin promoter (ptxP3), which induces an increase in the production of this virulence determinant in these B. pertussis isolates, is found in isolates obtained after the DTPa introduction. Other factor that has evidenced the B. pertussis adaptation is the emergence of pertactin (PRN) negative isolates maintaining its virulence and ability to produce disease. In our study, PRN-negative isolates have accounted for the 27.2% of the total studied isolates. They have been identified only during the period of use of DTPa. In this sense, the emergence and successful dissemination of a cluster of isolates that possess a deletion in the promoter and part of the gene encoding this determinant of virulence (58.1%, prn::del(-292 , 1340)), has evidenced the expansion of most of the B. pertussis isolates that do not express PRN. Thus, the spread of B. pertussis isolates expressing vaccine antigens possessing epitopes not recognized by antibodies generated by the use of DTPa, as well as the loss of expression of these antigens as an important mechanism of adaptation and evasion of immunity conferred by pertussis vaccines, seems to be a mechanism contributing to compromise the vaccine effectiveness. Finally, the description of the emergence of B. holmesii as the causative agent of pertussis, observing a prevalence of this microorganism of 3.9% in 2015 and 8.8% in 2016, shows that its circulation has been able to contribute to the pertussis resurgence. In relation to these, no differences were observed in the clinical-epidemiological characteristics of the cases by B. holmesii and those caused by B. pertussis, nor were there any complications or relapses after its treatment with azithromycin. Accurate diagnosis of the causative agent is crucial to determine the incidence and prevalence of the microbial species involved, to assess its contribution to the epidemiology of whooping cough, to evaluate whether specific antimicrobial drug treatments should be implemented and, in terms of public health, to assess the efficacy of the pertussis vaccine.
Universitat Autònoma de Barcelona. Programa de Doctorat en Microbiologia

Bordetella pertussis is the causative agent of whooping cough which is a worldwide acute respiratory disease that predominantly involves infants. Whooping cough is one of the ten most common causes of death from infectious diseases worldwide. The increased coverage of the primary pertussis vaccination (DaBT-IPA-Hib) decreased the incidence of disease in Turkey dramatically. However, in spite of the incidence decline, the circulation of B. pertussis has not yet been eliminated, and a change in the clinical spectrum and age-related incidence of the disease has been observed. On the other hand, in view of the moderate changes that have been observed in the genomic sequences of certain virulance factors over time, there are concerns about the gradual loss of the efficacy of the current pertussis vaccines as a result of antigenic drift and continuous selection of the least vaccine-sensitive clones. Proteomics deals with whole protein content (proteome) of cells as a function of space and time. Gel-based approach in proteomics involves two dimensional gel electrophoresis (2-DE) followed by peptide mass fingerprinting (PMF) employing matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). Immunoproteomics which is a combination of gel based proteomics and Western blot analysis determines tumor-specific antigens as well as immunoreactive proteins of pathogens by combining proteomics with Western blot technique. Although immunoproteomics is a rather new research tool, it has been quite effective to determine the virulence factors of various pathogenic microorganisms. The present study aims at comparing immunoproteome of the standard B. pertussis strain &ldquo
Tahoma I&rdquo
with those of two other strains, namely &ldquo
Saadet&rdquo
and &ldquo
Nursel&rdquo
, which are the local isolates that have been preferred as the vaccine strains for many years in our country for their ability to provide a better protection. Of a total of 38 immunogenic proteins identified, 14 were shown to be the novel antigens for B. pertussis. Among 14 proteins, one was detected as immunogenic in only Tohama I strain where two proteins were specific for Nursel strain. Among the strains compared, Saadet strain had the highest antigenic variety, than the others.

Les endotoxines sont des constituants heterogenes majeurs de la paroi des bacteries a gram negatif. Elles sont composees de trois regions principales dont la structure generale et les voies de biosynthese sont differentes : (i) le lipide a generalement, constitue d'un disaccharide de glucosamines reliees par une liaison 1-6, contient des groupes phosphates et des acides gras ; (ii) le noyau oligosaccharidique compose d'une dizaine de residus osidiques et (iii) les chaines-o specifiques formees par des unites repetitives portant les determinants antigeniques. L'importance de ces lipopolysaccharides (lps) reside dans le fait qu'ils sont responsables d'un nombre impressionnant d'activites aussi bien nefastes que benefiques pour l'homme et l'animal. La structure de l'endotoxine de bordetella pertussis, agent responsable de la coqueluche, a ete elucidee au sein de notre laboratoire. D'autres especes bacteriennes du meme genre : bordetella parapertussis et bordetella bronchiseptica sont impliquees dans la coqueluche ou responsable d'autres infections respiratoires. Une troisieme espece est pathogene chez les oiseaux : bordetella avium. Le but de notre travail etait de determiner la structure des endotoxines de ces trois bacteries dans le cadre d'une etude globale de relation entre structures et activites. Les resultats obtenus devant aider a la mise au point de tests de detection comme a la prevention des infections causees par ces pathogenes. En premier lieu, nous avons compare les endotoxines de b. Bronchiseptica, de b. Parapertussis et de b. Avium a la souche de reference b. Pertussis qui est de type rough constituee seulement de la structure i et ii. Les trois souches etudiees, sont de type smooth constituees des trois structures. Nous avons pu separer les lps avec et sans chaine-o specifiques presents dans une meme preparation. L'analyse par pdms des noyaux de trois souches de b. Bronchiseptica qui presentent des structures apparentees a celle de b. Pertussis, revele une variabilite due a la presence de groupes phosphates et dans le cas d'une souche de b. Bronchiseptica, la variabilite est due a l'absence d'un heptose. Les trois souches presentent une espece moleculaire commune de masse 1710 correspondant a l'espece nonasccharidique de b. Pertussis plus un phosphate. La spectrometrie de masse nous a permis de montrer que les noyaux lies au chaines-o n'etaient pas toujours identiques a ceux qui sont libres, ce qui est effectivement le cas de noyaux libres de b. Parapertussis et montre qu'il est injustifie d'extrapoler leur structure a celle des noyaux lies comme cela etait systematiquement realise dans la litterature. L'analyse de la structure fine des lipides a isoles des endotoxines de b. Bronchiseptica, de b. Parapertussis et partiellement celle de b. Avium a ete realisee. Des analyses chimiques, biochimiques et physiques (spectrometrie de masse de desorption par plasma et resonnance magnetique nucleaire) de ces lipides a, ont permis de montrer des similitudes importantes avec l'espece de reference b. Pertussis. Cependant chaque lipide a presente une structure fine differente et deux originalites de subsitutions, concernant les acides gras, ont pu etre mise en evidence pour la premiere fois.

Detection of Bordetella Infection in a Paediatric Population at CHW Hettiarachchige ANP1*, Kesson AM1, Leung KC2 Department of Infectious Diseases and Microbiology. The Children’s Hospital at Westmead. Discipline of Paediatrics and Child health, University of Sydney, Sydney, Australia. Introduction The Bordetella bacteria, such as B. pertussis, B. parapertussis and B. holmesii, are causative agents of whooping cough in humans. While B. pertussis is well documented to be an important agent of this disease in Australia, the roles of B. parapertussis and B. holmesii are unknown. To address this question the present project aims: To develop specific and sensitive PCR tests for B. parapertussis and B. holmesii, and to investigate the incidence of infection of B. parapertussis and B. holmesii compared to that of B. pertussis in patients of The Children’s Hospital at Westmead Methods PCR assays for B. parapertussis and B. holmesii were developed and combined with the existing in-house PCR test for B. pertussis to form a multiplex assay. The insertion sequences IS481, pIS1001 and hIS1001 were used as targets for B. pertussis, B. parapertussis and B. holmesii, respectively. Inhibitor control for each target was also synthesized and included in the multiplex PCR assay to detect false negative results. In this project, mainly nasopharyngeal aspirates and swabs were tested using the multiplex PCR during the year of 2013. Results The specificities and sensitivities of the multiplex PCR assay for the three Bordetella species were confirmed. A total of 464 specimens were tested by multiplex PCR assay, in the period of January to December 2013. Among 25 positive cases of Bordetella, 12 (48%) were positive for B. pertussis, 12 (48%) for B. parapertussis and none for B. holmesii. One (4%) case of co-infection with B. pertussis and B. parapertussis was detected in these specimens. Conclusion We have developed a highly specific and sensitive multiplex PCR assay for the three Bordetella species causing whooping cough in humans. Using this assay, we show that the infection rates of B. pertussis and B. parapertussis to be similar in children with respiratory disease. B. holmesii infection is rare in this study group.

Bordetella pertussis produces a number of virulence determinants believed to contribute to its survival in the host as well as to the pathogenesis of disease. One of these factors, adenylate cyclase toxin (ACT), has been implicated to penetrate human neutrophils and macrophages and abrogate their function by virtue of unregulated production of intracellular cAMP. In order to adequately study the nature of ACT and its role in pathogenesis, it is necessary to isolate the toxin from other virulence factors produced by the organism. Attempts by other investigators to purify ACT and maintain both its invasive and catalytic properties have not been successful. B. pertussis produces a cell associated ACT during mid-log phase of growth in Stainer-Scholte medium. Purification of ACT with both activities from urea extracted whole cells has been achieved by hydroxylapatite and calmodulin-sepharose chromatography. ACT is a single protein of 220 kd molecular weight with an isoelectric point of 7.0. The protein probably contains regions which are strongly hydrophobic. ACT has a specific activity of nearly 17,000 μM cAMP formed/min. An 850 ng sample of ACT induced over 1,400 pmoles cAMP/10⁶ S49 mouse lymphoma cells while 660 ng of ACT inhibited human neutrophil chemiluminescence by 65%.

Little is known regarding the interaction of Bordetella pertussis with polymorphonuclear leukocytes (PMNL) or the role PMNL play as an initial line of defense against B. pertussis infection. An in vitro system was developed to establish optimum conditions for the study of phagocytosis and killing of three virulent strains of B. pertussis and a series of derivative mutants using strictly human PMNL and sera. Optimum phagocytosis of B. pertussis occurred by opsonization with human anti-B. pertussis antibody (HAPA), while autologous normal sera (NS) did not induce significant phagocytosis. An antiserum made in rabbits against an avirulent strain was required as an opsonin for significant phagocytosis of several avirulent strains. Over 50% of all B. pertussis strains and mutants tested survived PMNL bactericidal activities while Escherichia coli controls were readily killed. Therefore, if internalized, B. pertussis has an innate ability to survive within human PMNL. No one virulence factor appears to be superior to another in determining survivability. Electron microscopic studies using acid phosphatase as a lysosomal marker demonstrated that virulent B. pertussis strains are capable of surviving intracellularly within PMNL phagosomes and that such survival is due to inhibition of phagosome-lysosome fusion. PMNL respiratory burst activity is unaffected by internalized B. pertussis. This strongly suggests that inhibition of phagosome-lysosome fusion is the key mechanism of B. pertussis intracellular survival within PMNL.

The Bordetella genus contain primary colonizing bacteria of the mammalian respiratory tract. Bordetella bind specifically to cilia and colonize the healthy conducting airway epithelium. In this dissertation, I examine the following aspects of initial Bordetella/ciliary binding: 1) the contribution of individual Bordetella virulence factors to binding, 2) the identity of ciliary receptors for Bordetella, and 3) the contribution of airway epithelial innate immune effectors in defense against Bordetella colonization. Bordetella/host cell interactions are modeled using Bordetella bronchiseptica and ciliated rabbit tracheal epithelial cells (RTEC) in a 240 second binding assay. B. bronchiseptica expressing the full complement of virulence factors adhere directly to RTEC cilia. Using single knockout strains of B. bronchiseptica in the ciliary binding assay. I show that the virulence factors filamentous hemagglutinin (FHA), fimbriae, pertactin, and adenylate cyclase-hemolysin (CyaA) contributed to ciliary binding, whereas dermonecrotic toxin, FhaS and the type III secretion system did not. Additional B. bronchiseptica adherence factors exist, as a strain that does not express FHA, fimbriae, pertactin or CyaA retained ciliary binding activity. In an attempt to identify ciliary components for binding, B. bronchiseptica bound cilia with greater affinity following enzymatic removal of terminal sialic acid residues from RTEC cilia. Complementary experiments using the immobilized glycocongugates GM1 and asialoGM1 suggest roles for FHA, and possibly fimbriae and pertactin, in ciliary attachment to asialylated compounds. In attempts to identify airway epithelial innate immune responses that limited ciliary attachment, I show that surfactant protein A (SP-A) blocked B. bronchiseptica adherence to RTEC cilia and nitric oxide (NO) inhibited B. bronchiseptica growth. To determine if B. bronchiseptica might encounter NO during colonization, I developed a novel method to isolate ciliated cells from the tracheal mucosa, and showed constitutive expression of iNOS mRNA in these cells. In summary, I present data that further the understanding of initial Bordetella/ciliary interaction and identify potential pathogen and host targets that may be manipulated to alter Bordetella colonization of the airway.

Bordetella bronchiseptica é um dos agentes etiológicos da tosse dos canis, infecções do trato respiratório superior em gatos, rinite atrófica, broncopneumonia em suínos, sendo freqüentemente isolada em coelhos e animais de laboratório. O impacto deste agente em saúde humana ainda é subestimado, o risco da infecção de pessoas imunodeprimidas que tem contato com animais domésticos existe e é relatado na literatura. No Brasil não há estudos caracterizando o agente isolado a partir de espécies de animais de companhia. O conhecimento dos aspectos epidemiológicos da infecção por Bordetella bronchiseptica é de fundamental importância para a adoção de medidas efetivas de controle e prevenção da infecção em seres humanos e animais domésticos, neste sentido, estudos de epidemiologia molecular são de grande relevância. O presente estudo tem como objetivos caracterizar amostras de Bordetella bronchiseptica isoladas de cães e gatos através da eletroforese em campo pulsado (PFGE) e do perfil de resistência a antimicrobianos. Foram isoladas 45 amostras provenientes de três cães e onze gatos de diferentes gatis, canis e clínicas veterinárias e as mesmas foram discriminadas em sete perfis através dos padrões de resistência e em quatorze perfis genotípicos através da PFGE.
Bordetella bronchiseptica is one of the etiologic agents of kennel cough, cat superior respiratory tract infections, swine bronchopneumonia, being frequently isolated from rabbits and laboratory animals. The impact of this agent in human health is still underestimated; the risk of immunosuppressed persons who have contact with domestic animals exists and is reported in literature. In Brasil, there are no studies characterizing the agent isolated from pet species. The knowledge of epidemiological aspects of Bordetella bronchiseptica is of fundamental importance to adopt effective control measures and prevention of human and domestic animals, in that sense, studies of molecular epidemiology are very relevant. This study aims to characterize strains of Bordetella bronchiseptica from dogs and cats through pulsed field gel electrophoresis (PFGE) and antimicrobial resistance profile. Forty five strains from three dogs and eleven cats from different catteries, shelters, and veterinary clinics were separated in seven profiles based in resistance patterns and fourteen genotypic profiles through PFGE.

La régulation de la virulence chez Bordetella pertussis, l'agent de la coqueluche, est sous la dépendance d'un système à deux composants complexe appelé BvgA/S. Ce système est composé d'une protéine senseur membranaire appelée BvgS et d'un régulateur transcriptionnel cytoplasmique, BvgA. En présence d'activateurs de la virulence (non encore définis), BvgS s'autophosphoryle, l'accepteur terminal du phosphate étant BvgA qui est alors actif et va réguler la transcription des gènes impliqués dans la virulence. Par contre, lorsque la bactérie est en présence d'inhibiteurs de la virulence, comme l'acide nicotinique ou le MgSO4, il y a absence d'autophosphorylation de BvgS, BvgA n'est plus activé et les gènes réprimés lors de la virulence sont actifs. Malgré toutes ces informations, nous ne connaissons pas encore les domaines de BvgS impliqués dans la perception des signaux environnementaux activateurs ou inhibiteurs de la virulence. Nous nous sommes donc intéressés à deux domaines de BvgS qui pourraient être impliqués dans ce mécanisme. Le premier domaine est le domaine périplasmique de BvgS homologue à des PBP (Periplasmic Binding Protein). Il pourrait intervenir dans la fixation de ligands activateurs ou inhibiteurs de la virulence. Le deuxième domaine, cytoplasmique, est appelé domaine PAS. Il est très répandu chez les eucaryotes et procaryotes et est impliqué dans l'adaptation du métabolisme de la cellule à des variations de conditions environnementales (comme des variations de potentiel redox, de luminosité ou à la en présence d'oxygène ou de fer. . . ). Ce domaine pourrait intervenir dans la fixation d'inhibiteur ou d'activateur de la virulence. Ce travail permettra donc d'avoir une meilleure compréhension du fonctionnement du complexe BvgAS dans la régulation de la virulence, d'identifier les domaines impliqués dans la perception des signaux positifs ou négatifs de la virulence, et de connaître l'identité de ces différents signaux

joruiz@clinic.ub.es
Objective: To characterize two Achromobacter xylosoxidans recovered from 2 patients diagnosed with pertussis during a Bordetella pertussis surveillance program. Methods: Nasopharyngeal swabs from 2 children under 1 year of age with clinical suspicion of pertussis were analyzed by culture and PCR. Results: Two Achromobacter xylosoxidans A8, closely related to Bordetella spp. were recovered from 2 patients diagnosed of pertussis, both carrying the ptxA gene and IS418 the pertussis toxin encoding gene. Subsequently, antibiotic susceptibility was evaluated by disk-diffusion method and by PCR. Conclusions: Although more detailed studies are needed, the present data highlight the possibility that Achromobacter xylosoxidans, closely related Bordetella pertussis microorganisms and not covered under the vaccine umbrella, might also result in cases of whooping cough. Thereby further surveillance is necessary to determine the extension and relevance of their pathogenic role in order to discriminate their real public health implication.