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Haemophilus influenzae encounters niches within the human host that are predicted to differ in availability of oxygen and reactive nitrogen species (RNS: nitrite and nitric oxide), which influence the environmental redox state. Previously reported data has indicated that an altered redox condition could serve as a signal recognized by H. influenzae to optimize its survival within host microenvironments. To elucidate the role of redox signaling in virulence, we examined regulation by the FNR homolog of H. influenzae, whose counterpart in E. coli has been reported to be a direct oxygen sensor and a regulator of genes responsible for RNS metabolism and resistance. Many members of the FNR regulon are subject to coordinated transcriptional control by NarP, a regulator in E. coli that is activated by cognate sensor NarQ in response to environmental nitrite. To study the regulatory activities of FNR and NarQ-NarP in H. influenzae, I targeted a gene predicted to be FNR-regulated, nrfA, which encodes nitrite reductase, a periplasmic cytochrome-c involved in anaerobic respiration. The fnr, narP and nrfA mutants were assayed for nitrite reduction, which implicated the roles of FNR, NarP and NrfA in RNS metabolism. Using Western blot detection of an epitope-tagged reporter protein fused to the endogenous nrf promoter (Pnrf-HA), I demonstrate that FNR and NarP, but not NarQ, are required for full activation of the nrf promoter. Additionally, Pnrf-HA expression increases as oxygen becomes depleted and decreases when exposed to high concentrations of nitrite, implying that the nrfpromoter is modulated by environmental redox signals. FNR of E. coli has been implicated in regulation of resistance mechanisms to a reactive nitrogen species, nitric oxide (NO), which is produced by innate immune cells during infection as a host defense mechanism. A mutant lacking FNR is more sensitive to NO exposure and killing by activated macrophages than wild type H. influenzae after anaerobic pre-growth. Mutants of nrfA and narP have been tested and initial experiments have shown both mutants have a lesser NO sensitivity phenotype as compared to the fnr mutant, suggesting that other factors could be involved in FNR-mediated NO resistance in H. influenzae. Upon examination of potential factors that might be involved to this phenotype, we discovered FNR-regulated gene, ytfE, which contributes to defense against nitrosative stress. The fnr and ytfE mutants are more susceptible to killing by activated macrophages indicating that FNR regulation of ytfE might be important for in vivo infection.

DNA repair processes are crucial for mutation avoidance and the maintenance of genetic integrity in all organisms. Organisms rely on repair processes to combat genotoxic stress imposed by hostile host environment, and sometimes by therapeutic agents. Most pathogens rapidly generate genetic variability to acquire increased virulence and evade host immune response. Therefore, there needs to exist a fine balance between mutation avoidance and fixation, which is perhaps regulated by repair processes. Haemophilus influenzae and Helicobacter pylori contribute significantly to morbidity and mortality caused by bacteria worldwide. H. influenzae is an obligate commensal of upper respiratory tract with the potential to cause a variety of diseases in humans like meningitis and respiratory infections. H. pylori, which inhabits the human stomach, is associated with gastric and duodenal ulcers and cancerous gastric lesions. One of the striking differences between these two genetically diverse bacterial species is the absence of recognized DNA mismatch repair (MMR) pathway homologs in H. pylori. MMR is a highly conserved post-replicative process, which corrects base pairing mismatches and small loops arising during DNA replication and recombination due to misincorporated nucleotides, insertions, and deletions. Defective MMR results in increased mutation frequency that can alter the pathogenic potential and antibiotic resistance of pathogens. MMR has been extensively studied in Escherichia coli, and requires an orchestrated function of different proteins like MutS, MutL, MutH, UvrD, SSB, RecJ, ExoVII, ExoI, ExoX, beta-clamp, DNA polymerase III and DNA ligase. A growing body of evidence suggests that bacteria other than the well-characterized E. coli paradigm differ in basic DNA repair machinery. MMR proteins involved in mismatch recognition and strand discrimination like MutS, MutL and MutH from H. influenzae have been characterized, but other downstream repair genes like UvrD helicase and exonucleases like RecJ have not been studied functionally in detail. H. pylori harbors a UvrD homolog, which shares limited homology with other UvrD proteins (29% identity with E. coli UvrD and 31 % with H. influenzae UvrD) and its cellular functions are not clear. Moreover, it is not well-understood how the activities of UvrD and RecJ proteins are regulated within these pathogens. It was, therefore, envisaged that biochemical characterization of UvrD and RecJ would lead to a better understanding of the mechanistic aspects of repair processes within these pathogens. The following sections summarize the results presented in this investigation. Functional characterization of UvrD from H. influenzae UvrD or DNA helicase II is a member of superfamily I of DNA helicases with well-documented roles in nucleotide excision repair (NER) and MMR, in addition to roles in replication and recombination. The 727-amino acid H. influenzae Rd KW20 UvrD (HiUvrD) protein was purified as an N-terminal (His)6-tagged protein to near homogeneity, and its authenticity was confirmed by peptide mass fingerprint analysis. HiUvrD displayed robust binding with single-stranded (ss) DNA as compared to double-stranded (ds) DNA. HiUvrD was found exhibit ~ 1000-fold higher affinity for ssDNA as compared to dsDNA as determined by surface plasmon resonance (SPR). In addition, to gain insights into the role of HiUvrD in replication, repair, recombination and transcription, the ability of HiUvrD to bind different DNA structures resembling intermediates of these processes was investigated using electrophoretic mobility shift assays. HiUvrD exhibited relatively high affinities for a number of branched DNA substrates and the order of affinity observed was; splayed-duplex ≥3’-flap ≥ ssDNA > 3’-overhang > four-way junction > three-way junction > nicked duplex > looped duplex ≥ duplex. Concurrent with its high affinity for ssDNA, HiUvrD exhibited a robust ssDNA-specific and Mg2+ - dependent ATPase activity. HiUvrD was able to unwind different DNA structures with varying efficiencies (3’ flap ≥ 3’-overhang > three-way junction > splayed-duplex > four-way junction > nicked > loop = duplex >>> 5’-overhang) and with a 3’-5’ polarity, which underpins its role in replication fork reversal, recombination and different DNA repair pathways. Multiple sequence alignment of HiUvrD with other helicases showed the presence highly conserved helicase motifs of which motif I and II are essential for ATP binding and hydrolysis. Mutation of an invariant glutamate residue (E226Q) in motif II of HiUvrD resulted in a dominant negative growth phenotype since, it was not possible to recover transformants when wild-type E. coli expression strains BL21(DE3)plysS or BL21(DE3)plysE were transformed with expression vector carrying hiuvrDE226Q. Mutation of a conserved arginine residue to alanine (R288A) in motif IV resulted in approximately 80 % reduction in ATP hydrolysis, and abrogation of helicase activity as compared to the wild-type protein. This can be attributed to ~ 70 % reduced ATP binding by HiUvrDR288A as determined by UV-crosslinking of radioactive ATP without change in affinity for ssDNA. HiUvrD was found to exist predominantly as a monomer with small amounts (~ 2-3 %) of higher oligomers like dimers and tetramers in solution. Deletion of 48 amino acid residues from distal C-terminus of HiUvrD resulted in abrogation of the oligomeric species implicating C-terminus to be involved in protein oligomerization. Interplay of UvrD with MutL and MutS in H. influenzae, and its modulation by ATP To investigate the effects of H. influenzae MutS (HiMutS) and MutL (HiMutL) on the helicase activity of HiUvrD, two different nicked DNA substrates were generated- a homoduplex and a heteroduplex DNA with a GT mismatch. HiMutL and HiMutS did not exhibit any helicase activity on either homoduplex or heteroduplex DNA, and unwinding of these substrates was observed only in presence of HiUvrD. In the presence of HiMutL the helicase activity of HiUvrD was stimulated on both homoduplex and heteroduplex nicked substrates whereas no significant modulation of HiUvrD ATPase activity in presence of HiMutL was observed. A much higher stimulation of unwinding of heteroduplex DNA was obtained, in presence of increasing concentrations of HiMutS. With increasing concentrations of HiMutL a progressive increase in HiUvrD mediated unwinding of the radiolabeled DNA strand was observed, which was ~ 15-fold higher than unwinding by HiUvrD alone. To investigate the effect of ATP in the stimulation of HiUvrD by HiMutL, two mutants of HiMutL–E29A (E29 is involved in ATP hydrolysis in E. coli UvrD), and D58A (D58 is essential for ATP binding in E. coli UvrD) were generated. HiMutLE29A retained only ~ 30 % of the wild-type ATPase activity, which was completely abolished in HiMutLD58A. Similar to wild-type protein, HiMutLE29A was able to stimulate HiUvrD helicase activity whereas HiMutLD58A failed to stimulate this activity. This indicated that ATP-bound form of MutL was essential for stimulation and perhaps interaction with UvrD. SPR analysis was carried out to validate and quantitate the direct protein-protein interaction between HiUvrD and HiMutL in absence or in presence of ATP, AMPPNP, and ADP. In the presence of ATP as well as AMPPNP, almost ~ 10,000-fold increase in the affinity between HiMutL and HiUvrD was observed but the same was not the case in presence of ADP. This clearly suggested that ATP binding rather than its hydrolysis promotes the interaction of MutL with UvrD. The effect of HiMutS on MutL-stimulated DNA unwinding by HiUvrD was determined using a heteroduplex nicked DNA with a GT mismatch. Interestingly, in the presence of HiMutS ~ 20-fold activation of DNA unwinding was observed, which is higher than the stimulation by HiMutL alone. The role of ATP-hydrolysis by MutS in regulation of UvrD helicase was studied by replacing wild-type protein with HiMutSE696A in the helicase assays. HiMutSE696A failed to hydrolyze ATP but was able to bind ATP with the same affinity as the wild-type protein and interacted with heteroduplex DNA with ~ 8-fold reduced affinity as compared to wild-type MutS. Intriguingly, increasing concentrations of HiMutSE696A failed to stimulate HiUvrD helicase activity in presence of HiMutL indicating that ATP hydrolysis by HiMutS is essential for stimulation of HiUvrD helicase activity post MutH-nicking during MMR. SSB, an essential component of all DNA metabolism pathways, possibly functions to stabilize the ssDNA tract generated by UvrD and exonucleases during MMR. ATPase and helicase activities of HiUvrD were inhibited by the cognate SSB protein. This inhibition could be overcome by increasing the concentration of HiUvrD helicases thus, pointing out the fact that SSB and UvrD perhaps compete with each other for ssDNA substrate. Noticeably, MutL and MutS proteins could alleviate the inhibition of HiUvrD by HiSSB. Functional characterization of UvrD from H. pylori In H. pylori, UvrD has been reported to limit homologous recombination and DNA-damage induced genomic recombinations but the protein has not been functionally studied. UvrD from H. pylori strain 26695 (HpUvrD) was over-expressed and purified as an N-terminal (His)6-tagged protein, and its authenticity was confirmed by peptide mass fingerprint analysis. HpUvrD exhibited high affinity for ssDNA as compared to dsDNA as determined by electrophoretic mobility shift assays and SPR. In addition, HpUvrD was able to bind a number of branched DNA structures (splayed duplex > ssDNA > 3’-flap > 3’overhang > three-way junction = four-way junction > loop >>> nicked ≥ duplex) suggesting its role in different DNA processing pathways. HpUvrD exhibited a Mg2+ - dependent ssDNA-specific ATPase activity, and a 3’-5’ helicase activity. HpUvrD was able to unwind different branched DNA structures with 3’-ssDNA regions like splayed duplex, 3’-overhang and 3’-flap. Blunt-ended duplex, duplexes with nick and loop as well as three-way and four-way junctions were unwound with less efficiency. Interestingly, the helicase activity of HpUvrD was supported by GTP and dGTP to almost the same level as ATP and dATP, which is in stark contrast to other characterized UvrD proteins. Moreover, HpUvrD was able to hydrolyze GTP albeit with ~ 1.5-fold reduced rate as compared to ATP. However, motifs associated with GTP binding and hydrolysis were not found in HpUvrD and it is possible that GTP binds in the same site as ATP. To investigate this possibility, helicase assay was done in the presence of ATP together with different concentrations of GMP-PNP, which is a non-hydrolysable analog of GTP, and did not support HpUvrD helicase activity. With increasing concentrations of GMP-PNP, a progressive inhibition of DNA unwinding by HpUvrD was observed suggesting that GMP-PNP could compete with ATP for a common binding site within HpUvrD. Replacement of a highly conserved glutamate residue with gluatamine (E206Q) in Walker B motif of HpUvrD resulted in ~17-fold reduced ATPase activity, and abrogation of helicase activity as compared to the wild-type protein. HpUvrDE206Q was able to bind ssDNA and ATP with comparable affinities as the wild-type protein suggesting the role of E206 in ATP hydrolysis. Like HiUvrD, HpUvrD was found to exist predominantly as a monomer in solution together with the presence of small amounts of higher oligomeric species. However, unlike HiUvrD, deletion of distal C-terminal 63 amino acids in HpUvD did not abrogate the oligomeric species suggesting that additional regions of the protein may be involved in protein oligomerization. The ATPase and helicase activities of HpUvrD were inhibited by the cognate SSB protein, and this inhibition could be overcome by increasing HpUvrD concentrations again suggesting that both UvrD and SSB proteins compete for ssDNA substrate. To investigate the role of UvrD in the physiology of H. pylori, a knock-out of hpuvrD was constructed in H. pylori strain 26695 by insertion of chloramphenicol cassette in its open reading frame. The mutant H. pylori strain 26695 obtained after disruption of hpuvrD was extremely slow growing under the normal microaerophilic conditions compared to the wild-type strain. Growth defect of H. pylori strain 26695ΔhpuvrD highlights the importance of UvrD in H. pylori cellular processes and in vitro fitness. Characterization of H. influenzae RecJ and its interaction with SSB Among the four exonucleases involved in MMR pathway, RecJ is the only known nuclease that degrades single-stranded DNA with 5’ to 3’ polarity. RecJ exonuclease plays additional important roles in base-excision repair, repair of stalled replication forks, and recombination. RecJ exonuclease from H. influenzae (HiRecJ) is a 575 amino acid protein, which harbors the characteristic motifs conserved among RecJ homologs. Due to limited solubility of HiRecJ, the protein was purified as a fusion protein with maltose binding protein (MBP). The purified protein exhibited a Mg2+ or Mn2+- dependent, and a highly processive 5’ to 3’ exonuclease activity, which is specific for ssDNA. MBP did not affect the exonuclease activity of HiRecJ. The processivity of HiRecJ was determined as ~ 700 nucleotides per binding event, using a ssDNA substrate labelled internally with 3H and at its 5’-terminus with 32P. Cd2+ inhibited the Mg2+ - dependent exonuclease activity of RecJ, which could not be overcome by increasing Mg2+ concentration. Site-directed mutagenesis of highly conserved residues in HiRecJ- D77A, D156A and H157A abolished the enzymatic activity. Interestingly, HiRecJD77A was found to interact with ssDNA with a 10-fold higher affinity than wild-type protein suggesting that this conserved aspartate residue may function to coordinate the binding of metal ion or DNA to hydrolysis of DNA. E. coli HU protein inhibited the HiRecJ exonuclease activity in a concentration-dependent manner possibly due to sequestration of ssDNA, thus making it unavailable for HiRecJ. During MMR, ssDNA tracts generated by UvrD helicase activity are most probably stabilized by SSB and hence, the in vivo substrate for RecJ would be SSB-ssDNA complex. The exonuclease activity of HiRecJ was stimulated approximately 3-fold by H. influenzae SSB (HiSSB) protein. HiSSB was able to stimulate HiRecJ exonuclease activity on a ssDNA substrate, which formed either a very strong secondary structure or on a homopolymeric ssDNA substrate, which did not form any secondary structure, suggesting that HiRecJ exonuclease was stimulated independent of the ability to HiSSB to melt secondary structures and stabilize ssDNA. Significantly, steady-state-kinetic analysis clearly showed that HiSSB increases the affinity of HiRecJ for ssDNA. H. influenzae SSBΔC and T4 gene 32 protein, a SSB homolog from bacteriophage T4, failed to enhance the HiRecJ exonuclease activity suggesting a specific functional interaction between HiSSB and HiRecJ mediated by C-terminus tail of HiSSB. More importantly, HiRecJ was found to directly associate with its cognate SSB. The C-terminus of HiSSB protein was found to be essential for this interaction. To delineate the regions of HiRecJ that interact with HiSSB, different truncated forms of HiRecJ were generated in which regions external to conserved motifs required for exonuclease activity were deleted. Different deletion mutants of HiRecJ- RecJ∆N34, RecJ∆C76 and the core catalytic domain (which contains amino acid residues 35-498) were purified as fusion proteins with MBP. HiSSB was found to interact with all the truncated forms of HiRecJ suggesting that its core-catalytic domain harbors a site for interaction with SSB. Taken together, the results presented in this study lead to a better understanding of the structure-function relationships of the UvrD helicase and RecJ exonuclease. Importantly, they provide insights into the interplay between various proteins in DNA MMR pathway. Characterization of repair proteins that are involved in multiple genome fidelity pathways is of fundamental importance to understand repair processes, more so in pathogenic bacteria wherein they regulate mutation rates, which can alter the fitness and virulence of the pathogens. Publication Sharma R., and Rao, D.N. (2009). Orchestration of Haemophilus influenzae RecJ exonuclease by interaction with single-stranded DNA-binding protein. J. Mol. Biol., 385, 1375-1396.

La Broncho-Pneumopathie Chronique Obstructive (BPCO) est une maladie pulmonaire inflammatoire consécutive à l'exposition chronique à la pollution atmosphérique et surtout au tabagisme dans environ 90% des cas. Cette maladie se caractérise par une obstruction des bronches due à une hypersécrétion de mucus, une hypertrophie des muscles lisses, ainsi qu’une destruction de la paroi des alvéoles respiratoires amenant le patient à l’emphysème. Le stress induit par la fumée de cigarette provoque une activation de la barrière épithéliale pulmonaire associée à une altération de la réponse immunitaire responsable d’une susceptibilité accrue aux infections pulmonaires. De ce fait, les patients atteints de cette maladie développent des exacerbations principalement liées à ces infections bactériennes en particulier à Non-Typable Haemophilus influenza (NTHi) et Streptoccocus pneumoniae (Sp).La cytokine IL-22 est un acteur très important des défenses antibactériennes et du maintien de la barrière épithéliale. Cette cytokine appartient à la grande famille de l’IL-10, et à la sous-famille des cytokines IL-20 composée de l’IL-19, l’IL-20 et l’IL-24. L’IL-22 se lie au récepteur formé par les sous-unités IL-10Rb et IL-22Ra, tandis que les cytokines IL-19, IL-20 et IL-24 utilisent deux récepteurs associant l’IL-20Rb avec l’IL-20Ra ou l’IL-22Ra. Il a été démontré que les cytokines de la famille IL-20 (IL-19, IL-20, IL-24) agissent sur la clairance bactérienne au cours d’une infection cutanée par Staphylococcus aureus (Myles et al., 2013), en inhibant la production des cytokines IL-17 et IL-22. De plus, des précédents travaux au laboratoire, ont montré un défaut de l’expression des cytokines IL-17 et IL-22 qui participaient à la susceptibilité à l’infection chez les souris atteintes de BPCO (Pichavant et al., 2015). Enfin, nos données actuelles montrent que l'exposition à la fumée de cigarette augmente l'expression des cytokines de la famille IL-20 et que l'inhibition de cette voie permet de bloquer le développement d'épisodes d'exacerbation chez des souris BPCO.L'objectif de cette thèse est de préciser le rôle des cytokines IL-20 dans la réponse à l'infection bactérienne (Sp, NTHi) tant dans un contexte physiologique qu'au cours d’un contexte mimant la BPCO. Pour cela, nous nous focaliserons sur le rôle de l’épithélium pulmonaire tant dans la production que dans la fonction de ces cytokines en contexte infectieux.Pour répondre à ces questions, nous avons analysé l’expression des cytokines IL-20 par l’épithélium pulmonaire in vitro et ex vivo dans un modèle murin mimant l’exacerbation de la BPCO ainsi que dans des biopsies pulmonaires de patients fumeurs atteints ou non de BPCO. Dans un second temps nous avons évalué la modulation par un anticorps bloquant le récepteur des cytokines IL-20 (anti-IL-20Rb) au cours de la réponse anti-infectieuse de l'épithélium dans nos modèles in vivo (souris infectées par Sp) et in vitro (cellules épithéliales de trachées murines). Nous avons en parallèle évalué l'implication des cytokines IL-20 dans la réparation épithéliale.L’ensemble des résultats acquis au cours de la thèse nous a permis de démontrer l'implication des cytokines IL-20 et de préciser leur rôle sur l’épithélium pulmonaire au cours de l'infection bactérienne ainsi que dans la pathologie de la BPCO. De plus, les résultats obtenus avec l’anticorps neutralisant anti-IL-20Rb dans ces contextes d’infections et de BPCO, font de celui-ci une potentielle piste thérapeutique pour le traitement des lésions dues à l’infection. / Chronic Obstructive Pulmonary Disease (COPD) is an inflammatory lung disease due to chronic exposure to air pollution and especially to cigarette smoke exposure in approximately 90% of the cases. This disease is characterized by obstruction of the bronchi due to hypersecretion of mucus, hypertrophy of the smooth muscles, and destruction of the alveolar wall leading the patient to emphysema. The stress induced by cigarette smoke exposure causes activation of resident cells including pulmonary epithelial cells and an alteration of the immune system responsible for an increased susceptibility to pulmonary infections. As a result, patients with this disease develop exacerbations especially du to Non-Typable Haemophilus influenza (NTHi) and Streptoccocus pneumoniae (Sp).The IL-22 cytokine plays a key role in antibacterial defenses and maintenance of the epithelial barrier. This cytokine belongs to the large IL-10 family, and to the IL-20 cytokine subfamily also including IL-19, IL-20 and IL-24. IL-22 binds to the receptor formed by the IL-10Rb and IL-22Ra subunits, while the IL-19, IL-20 and IL-24 cytokines binds to IL-20Rb associated with either IL-20Ra or IL-22Ra subunits. IL-20 cytokines (IL-19, IL-20, IL-24) have been shown to impair bacterial clearance during cutaneous infection with Staphylococcus aureus (Myles et al., 2013), by inhibiting the production of IL-17 and IL-22 cytokines. In addition, previous work in the laboratory showed a defect in the expression of IL-17 and IL-22 cytokines contributing to the susceptibility to infection in COPD mice (Pichavant et al., 2015). In fact, our current data show that exposure to cigarette smoke increases cytokine expression of the IL-20 family and that inhibition of this pathway blocks the development of exacerbation episodes in COPD mice.The aim of this thesis is to clarify the role of IL-20 cytokines in the response to bacterial infections (Sp, NTHi) both in a physiological context and in a context mimicking COPD. To do so, we will focus on the role of pulmonary epithelium both in the production and function of these cytokines in infectious context.To answer these questions, we analyzed the expression of IL-20 cytokines by pulmonary epithelium in vitro and ex vivo in a mouse model mimicking the COPD exacerbation as well as in pulmonary biopsies of smokers and non-smokers patients and of COPD patients. In a second step we evaluated the modulation by an IL-20 receptor blocking antibody (anti-IL-20Rb) of the anti-infectious response in our in vitro (murine tracheal epithelial cells) and in vivo models (Sp-infected mice). In parallel, we evaluated the involvement of IL-20 cytokines in the epithelial repair.All the results acquired during the thesis allowed us to demonstrate the expression of IL-20 cytokines and to demonstrate their role on the pulmonary epithelium during bacterial infection as well as in COPD. In addition, the results obtained with the anti-IL-20Rb neutralizing antibody in these contexts of infections and COPD, suggests a potential therapeutic application for respiratory infection.

L’asthme équin, une maladie inflammatoire considérée non infectieuse, est néanmoins associé avec la présence de certains streptocoques et Pasteurellaceae. La similarité entre les espèces de Pasteurellaceae complexifie toutefois leur différenciation. L’objectif de cette étude est d’évaluer la présence de différents Pasteurellaceae dans les voies respiratoires de chevaux atteints d’asthme. Des amorces qPCR ont été optimisées pour [Pasteurella] caballi, Nicoletella semolina, Pasteurella multocida et Haemophilus influenzae, mais plusieurs espèces d'intérêt (notamment Actinobacillus spp.) n'ont pas pu être étudiées faute d'amorces spécifiques. Des lavages nasaux, oraux et bronchoalvéolaires de douze chevaux (six sains, six asthmatiques) gardés dans différents environnements ont été analysés. Pour N. semolina, les analyses ont été approfondies chez dix chevaux asthmatiques (phase II : lavages nasaux et bronchoalvéolaires), et chez dix chevaux sains (phase III : lavages nasaux). N. semolina a été identifié dans 0 à 66 % des échantillons et était retrouvé en plus forte quantité dans le nez de chevaux gardés à l’intérieur et nourris avec du foin. Dans les phases II et III, N. semolina a été identifié dans 90 à 100 % des échantillons nasaux, et dans six lavages bronchoalvéolaires. [P.] caballi a été identifié dans la cavité orale de 83 % des chevaux, indépendamment du statut de santé. H. influenzae et P. multocida n’ont pas été détectés. Aucun Pasteurellaceae analysé n’était associé à l’asthme équin sévère. N. semolina était fréquemment retrouvé dans les voies respiratoires et sa charge nasale était influencée par l’environnement. Les amorces synthétisées faciliteront de futures études sur les Pasteurellaceae. / Equine asthma, an inflammatory disease considered non-infectious, is associated with the presence of certain streptococci and Pasteurellaceae. Strong similarities between Pasteurellaceae species prevent specific differentiation. The objective of the study was to evaluate the presence of different Pasteurellaceae in the airways of horses with severe asthma. Quantitative PCR primers were optimized for [Pasteurella] caballi, Nicoletella semolina, Pasteurella multocida and Haemophilus influenzae. Several species of interest (in particular Actinobacillus spp.) could not be studied due to a lack in primer specificity. Nasal, oral and bronchoalveolar lavages from twelve horses (six healthy, six asthmatics) kept in different environments were analyzed. For N. semolina, further investigation was pursued through inclusion of ten asthmatic horses (phase II: nasal washes and bronchoalveolar lavages), and ten healthy horses (phase III: nasal washes). N. semolina was identified in 0 to 66 % of samples and was found in larger loads in the nose of horses kept inside and fed hay. In phases II and III, N. semolina was identified in 90 to 100 % of nasal samples and in six bronchoalveolar lavages. [P.] caballi was only identified in the oral cavity, with 83 % of positive horses regardless of health status. H. influenzae and P. multocida were not detected. No specific Pasteurellaceae studied here were associated with severe equine asthma. N. semolina was identified frequently in respiratory samples and its nasal load was influenced by the horse’s environment. The optimized primers will facilitate future studies on Pasteurellaceae.

Haemophilus parainfluenzae (Hp) and H. influenzae (Hi) are closely related members of the Pasteurellaceae family and are common commensal bacteria of the human nasopharynx. Whilst Hi is frequently implicated in meningitis, otitis media and respiratory tract infections, reports of pathogenic behaviour by Hp are very rare. Lipopolysaccharide (LPS) is a key component of the Gram negative cell wall, and its structure influences the ability of Haemophilus to interact with the host and evade immune clearance. A better understanding of the differences in LPS structure between Hi and Hp could help to ascertain which parts of the molecule are important for commensal and pathogenic behaviour. Hi LPS comprises lipid A, a conserved oligosaccharide inner core, and an oligosaccharide outer core that differs between strains. The latter is partly phase variable by the slipped strand mispairing during replication of DNA repeat tracts within several LPS biosynthesis genes. Very little was known about LPS in Hp so we investigated its biosynthesis and structure in a panel of 20 Hp carriage isolates. Using PCR, DNA sequencing and Southern analysis we demonstrated that Hp possesses homologues of the Hi lipid A and inner core LPS synthesis genes and a few of the genes for outer core synthesis; however, homologues of the Hi phase variable outer core genes were largely absent and did not contain repeat tracts. The results of immunoblotting and collaborative structural analysis were consistent with this data. Phosphocholine, a phase variable Hi LPS epitope that has been implicated in otitis media, was found to be absent in Hp LPS due to the lack of four genes required for its biosynthesis and incorporation. The introduction of these genes into Hp led to the phase variable addition of phosphocholine to the LPS, indicating that there is no fundamental reason why Hp could not use a similar mechanism of variation to Hi if it was advantageous to do so. SDS-PAGE data suggested the presence of O-antigens (repeated chains of sugars) in many of the Hp strains, an unusual feature for Haemophilus, and all of the strains were found to contain a potential O-antigen synthesis locus. Each locus encodes homologues of several glycosyltransferases in addition to either the Wzy polymerase- or ABC transporter-dependent mechanisms of O-antigen synthesis and transport. Comparisons of wild type and isogenic mutant strains showed that the O-antigen enhances resistance to complement-mediated killing and appears to affect adhesion to epithelial cells in vitro. Hp is a successful commensal organism but lacks the flexibility of adapting its LPS using repeat-mediated phase variation, potentially limiting its range of host niches.

Made available in DSpace on 2016-08-17T18:39:34Z (GMT). No. of bitstreams: 1 3277.pdf: 7015864 bytes, checksum: 9834075e819d2bb62213cf25d21ce5e5 (MD5) Previous issue date: 2010-08-27 / Haemophilus influenzae type b (Hib) is a Gram negative bacterium responsible for causing meningitis worldwide. The capsular polysaccharide b, a polymer composed by repeating units of ribosyl-ribitol phosphate (PRP), is the major virulence factor and it is used in the formulation of the vaccine against this microorganism. Despite their high efficiency, the conjugated vaccine against Hib is a product of high production cost, which involves fermentation, purification and conjugation processes to obtain a final product within the specifications of the World Health Organization (WHO). The improvement of the culture medium and cultivation conditions can contribute to reduce the cost of this vaccine in order to facilitate its dissemination in developing countries. The main objective of this work was to identify culture conditions that result in higher production of capsular polysaccharide, helping to reduce costs in the steps of purification and conjugation. The experiments were carried out in shake flasks or in bioreactors with 7-13 liters of capacity. The temperature was maintained at 37 °C, pH controlled at 7.5 by adding NaOH 5M and the concentration of dissolved oxygen (CDO) maintained at 30% of air saturation. The specific flow rate of air ranged between 0.2 and 1 VVM. Samples were collected at regular time intervals to measure optical density (DO540nm), biomass concentration, capsular polysaccharide (PRP) production and concentrations of glucose and metabolites. Two possibilities for increasing polysaccharide production were studied: 1) different strategies of fed-batch cultivation consisting of: a) intermittent addition of glucose (FBIG), b) constant feeding (FBCF), c) exponential feeding (FBEF), e d) exponential feeding with cell recycle and perfusion (FBER + P); 2) improvement of culture media composition regarding the carbon/nitrogen ratio through the use of central composite rotational design (CCRD) methodology, having as independent variables: Soy Peptone (S), Yeast Extract (YE) and Glucose (G). Quality parameters were also evaluated to assess the molecular weight profile of the product (PRP) as well as morphological aspects of the microorganism. Economic analysis of different cultivation strategies was used to identify the more economically viable process. The results of the different cultivation strategies together with the outputs of the studied processes cost analysis showed that FBCF, with a cost of U.S.$ 425.50/g PRP and productivity of 88 mg/L.h, showed to be the best alternative among PRP production processes due to its lower cost with a good productivity. In the study of the culture media composition through the statistical analysis of the CCRD results showed that the best culture media composition (BCM) consisted of S 5 g/L; YE 5.5g /L and G of 15.25 g/L. DO540nm and PRP volumetric production values of 8.4 and 410 mg/L, respectively, were attained in validation experiments carried out in shake flasks at the BCM condition. For the bioreactor BCM validation experiment, biomass concentration of 3 g DW/L and polysaccharide production of 600 mg PRP/L were observed. Similar values were reached at validation runs performed in shake flasks and bioreactor for the central point CP condition, showing that both BCM and CP conditions belong to the optimum region. The analysis of quality parameters showed that the cultivation time influences strongly the size of the polysaccharide molecule. The longer the cultivation time, the lower molecular weight was found. The analysis by transmission electron microscopy (TEM) of H. influenzae cells revealed a predominance of round cells in the sixth hour of cultivation, whereas in the twelfth hour of cultivation the cells exhibited a more elongated morphology with the presence of cytoplasmic inclusions in the shape of granules, possibly due to the accumulation of some reserve material. Based on these results, the new composition of the culture medium resulted in an increased of cell growth and capsular polysaccharide production with half of the sources nitrogen (soybean peptone and yeast extract) concentrations, which reduces the production cost. The cultivations that resulted in higher production and productivity of polysaccharide were FBCF (1600 mg PRP/L and 88 mg PRP/(L.h)) and FBER+ P (1800 mg PRP/L and 129 mg PRP/(L.h)). The FBER+P reached 30% higher productivity of polysaccharide than the best result described in the literature (90 mg PRP/(L.h)). However, the FBCF cultivation was economically more viable. / Haemophilus influenzae tipo b (Hib) é uma bactéria Gram negativa responsável por causar meningite em todo o mundo. O polissacarídeo capsular b, um polímero composto por unidades repetidas de ribosil-ribitol-fosfato (PRP) e o principal fator de virulência, sendo utilizado na formulação da vacina contra este microrganismo. Apesar de sua elevada eficiência, a vacina conjugada contra Hib é um produto de alto custo de produção por envolver processos de fermentação, purificação e conjugação para obtenção de um produto final dentro das especificações da Organização Mundial da Saúde (OMS). O melhoramento do meio de cultura e das condições de cultivo pode contribuir para redução do custo desta vacina de forma a facilitar sua difusão nos países subdesenvolvidos. Assim, o principal objetivo deste trabalho foi identificar condições de cultivo que resultem em maior produção de polissacarídeo capsular, contribuindo para reduzir os custos nas etapas de purificação e conjugação. Os ensaios foram conduzidos em frascos agitados ou em biorreatores com 7 a 13 litros de capacidade nominal. A temperatura foi mantida em 37oC, o pH controlado em 7,5 através da adição de NaOH 5M e a concentração de oxigênio dissolvido (COD) mantida em 30 % da saturação do ar. A vazão especifica de ar variou entre 0,2 e 1 VVM. Amostras foram coletadas em intervalos regulares de tempo para mensurar densidade óptica (DO540nm), concentração celular, produção de polissacarídeo capsular (PRP) e concentrações de glicose e metabolitos. Duas possibilidades para aumentar a produção de polissacarídeo foram estudadas: 1) diferentes estratégias de cultivo descontinuo alimentado consistindo por: a) adição intermitente de glicose (CDAIG), b) vazão constante (CDAVC), c) vazão exponencial (CDAVE), e d) reciclo de células seguido de perfusão (CDAVE+P)); e 2) o melhoramento da composição do meio de cultura quanto à relação carbono/nitrogênio através do uso da metodologia de delineamento composto central rotacional (DCCR), tendo como variáveis independentes: Peptona de Soja (S), Extrato de Levedura (EL) e Glicose (G). Parâmetros de qualidade também foram avaliados para verificar o perfil da massa molecular do produto (PRP) e os aspectos morfológicos do microrganismo. Os resultados das diferentes estratégias de cultivo juntamente com os resultados da analise econômica do custo dos processos estudados mostraram que o CDAVC, com custo de US$ 425.50/g PRP por ano e produtividade de 88 mg/(L.h), demonstrou ser a melhor alternativa para o processo de produção de PRP por apresentar menor custo com boa produtividade. Já o estudo da composição do meio de cultura através da analise estatística dos resultados do DCCR mostraram que a melhor composição do meio de cultivo (MMC) consistia em S de 5 g/L, EL de 5,5 g/L e G de 15,25 g/L. Valores de DO540nm de 8,4 e de produção volumétrica de polissacarídeo de 410 mg PRP/L foram alcançados em experimentos de validação na condição MMC realizados em shaker. Para o ensaio de validação da condição MMC em reator, a concentração de biomassa de 3,0 g MS/L e a produção de polissacarídeo capsular de 600 mg PRP/L foram observadas. Valores semelhantes foram obtidos em experimentos de validação realizados em shaker e biorreator na condição CPC, mostrando que tanto a condição CPC como a MMC pertencem a região de ótimo. A analise dos parâmetros de qualidade mostrou que o tempo de cultivo influencia fortemente no tamanho da molécula do polissacarídeo, sendo que quanto maior o tempo de cultivo, menor a sua massa molecular. A analise por microscopia eletrônica de transmissão (MET) das células de H. influenzae mostrou a predominância de células com morfologia arredondada na sexta hora de cultivo, enquanto que, na décima segunda hora de cultivo, as células apresentaram morfologia mais alongada e exibiram a presença de inclusões citoplasmáticas na forma de grânulos, possivelmente devido ao acumulo de algum material de reserva. Diante dos resultados obtidos, a nova composição de meio resultou em maior crescimento celular e produção de polissacarídeo capsular, com metade das concentrações das fontes de nitrogênio (peptona de soja e extrato de levedura), o que reduz o custo de produção. Os cultivos que resultaram em maior produção e produtividade foram o CDAVC (1600 mg PRP/L e 88 mg PRP/L.h) e o CDAVE+P (1800 mg PRP/L e 129 mg PRP/L.h), sendo que este ultimo (CDAVE+P) atingiu produtividade 30 % maior que o melhor resultado ate então descrito na literatura (90 mg PRP/L.h). No entanto, o cultivo CDAVC apresentou maior viabilidade do ponto de vista econômico e de execução e passível de escalonamento.

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