The genetic requirements for Influenza virus to infect and adapt to new species is largely unknown. To understand the evolutionary steps required by a virus to become virulent, a human virus (A/HK/1/68) (HK), avirulent in mice, was subjected to 20 and 21 serial lung-to-lung passages in mouse. Sequence analysis revealed the emergence of eleven mutations within the NS1 gene of the new virulent strains, many of which occurred in binding sites for transcriptional and translational cellular factors. In the present study we have rescued viruses containing each of the NS1 mouse adapted mutations onto A/PR/8/34 (PR8) backbone. We found 9 of 16 NS1 mutants were adaptive by inducing mortality, body weight loss in BALB/c mice and enhanced virus replication in MDCK cells with properties of host cell interferon transcription inhibition. Sequence comparisons with the highly pathogenic A/Hong Kong/156/1997 (H5N1) and the most severe pandemic A/Brevig Mission/1/1918 (H1N1) NS1 genes showed convergent evolution with some of the mouse adapted viruses for F103L plus M106I and V226I plus R227K mutations respectively. The F103L and M106I mutations in the HK NS1 gene were shown to be adaptive by assessment with respect to replication, early viral protein synthesis, interferon-β antagonism and tropism in the mouse lung. We extended the study and proved increased virulence associated with F103L+M106I mutations in their respective H5N1 NS1 gene on the PR8 and HK backbones, as well as the PR8 NS1 gene and the H9N2 (A/Ck/Bj/1/95) gene in the PR8 and A/WSN/33 backbones respectively. However the V226I and R227K mutations in their respective HK and 1918 NS1 genes slightly enhanced virulence and viral growth at later stages of infection. This study demonstrates that NS1 is a virulence factor; involved in multiple viral processes including interferon antagonism and viral protein synthesis. Furthermore, NS1 mutations acquired during mouse adaptation are proven to be adaptive in human, mouse and avian NS1 genes.

Les Formes Actives de l’Oxygène (FAO), molécules dérivées de l’oxygène, sont capables d’oxyder et d’endommager les macromolécules biologiques. Au cours de son cycle de vie, Salmonella typhimurium est exposée à des FAO provenant de deux sources : soit de son métabolisme aérobie, soit du macrophage, sa cellule hôte au cours de l’infection. Parmi les FAO existantes, l’H2O2 est l’une des plus néfastes. Au cours de ma thèse, j’ai étudié la contribution des catalases et des peroxyrédoxines dans le métabolisme et la virulence de S.typhimurium. Cinq enzymes ont ainsi été identifiées pour leur capacité à éliminer l’H2O2 : les catalases KatG, KatE, KatN et les peroxyrédoxines AhpCF et TsaA. Des tests de virulence ont également permis de montrer que ces enzymes participaient à l’établissement de la virulence.A l’aide d’une sonde moléculaire capable de détecter et de signaler l’H2O2, nous avons montré que S. typhimurium percevait cette FAO au cours de l’infection dans des macrophages murins. Ces résultats ont souligné l’importance des catalases et des peroxyrédoxines au cours de la vie intracellulaire de S. typhimurium. L’analyse du mutant DahpCF DtsaA Dtpx a également révélé que les peroxyrédoxines AhpCF, TsaA et Tpx contribuaient à la capacité de prolifération de la bactérie dans le macrophage. Enfin, l’étude des méthionine sulfoxyderéductases a montré que les caractéristiques d’un mutant DmsrA DmsrB étaient proches de celles de la souche sauvage. Les gènes msrA et msrB ont également été inactivés dans une souche dépourvue de katG, katE et ahpCF. Dans cette souche accumulant de l’H2O2endogène, la contribution de MsrA et MsrB devient évidente pour lutter contre les effets liés au stress oxydant. L’ensemble de ces travaux a permis d’identifier et de caractériser l’implication de systèmes antioxydants dans la virulence et le métabolisme de S. typhimurium. / Reactive Oxygen Species (ROS), produced from molecular oxygen, can oxidize and damagebiological macromolecules. During its lifestyle, Salmonella typhimurium is submitted to ROScoming from two sources: its aerobic metabolism and its host cell upon infection, themacrophage. Among the ROS, H2O2 is one of the most toxic. In this work, the contribution ofcatalases and peroxiredoxins in the metabolism and the virulence of S. typhimurium wasstudied. Five enzymes are implied in H2O2 degradation, the catalases KatG, KatE, KatN andthe peroxiredoxins, AhpCF and TsaA. Virulence tests showed that these enzymes wereinvolved in virulence. Using a molecular probe able to detect and quantify H2O2, we showedthat S. typhimurium sensed H2O2 during infection in murine macrophages. These resultsunderlined the importance of catalases and peroxoxyredoxines for the intracellular life of S.typhimurium. Analysis of the mutant DahpCF DtsaA Dtpx revealed that the peroxiredoxinsAhpCF, TsaA and Tpx contributed to the bacterial proliferation inside macrophage. Finally,the study of the methionine sulfoxyde reductases showed that the phenotype of the mutantDmsrA DmsrB was related to the wild type strain. Then, msrA and msrB were inactivated in astrain deleted of katG, katE and ahpCF. In this strain impaired in H2O2 degradation, thecontribution of MsrA and MsrB to fight against oxidative stress effect is stronger. Altogether,these results allowed the identification and the contribution of antioxidant systems in S.typhimurium virulence and metabolism.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

No description available.

A virulent strain of Legionella pneumophila serogroup 1 was established in continuous culture under defined iron-replete conditions at pH 6.9. Iron-limitation and extremes of pH (6.0 and 7.8) influenced the growth and metabolism of L. pneumophila, as manifested by increased metabolic activity, impaired energy coupling, and altered metabolic fluxes. In particular, the physiological versatility of L. pneumophila was demonstrated by a significant decrease in the iron content of biomass (6-fold increase in Yiron), coupled with reduced metabolic efficiency (Y, on), in response to iron-limited growth. Iron limitation promoted the accumulation of significant intracellular reserves of poly- ß-hydroxybutyrate (16 % cell dry wt.), which supported long-term survival of L. pneumophila under starvation conditions. Expression of the important pathogenicity factor, the zinc metalloprotease, was regulated by iron availability. Common iron acquisition mechanisms, such as siderophores and transferrin receptors, were not elaborated by iron-limited cells. However, human transferrin was identified as a potential iron source for L. pneumophila, with the zinc metalloprotease mediating transferrin digestion and possibly iron acquisition. Iron-limitation and extremes of pH also influenced cellular morphology and the surface properties of L. pneumophila, promoting the formation of uniform cultures of short rod-shaped bacteria, with altered fatty acid, phospholipid and protein composition. In addition to morphological and physiological adaptation, iron limitation had a significant effect on the virulence of L. pneumophila. Iron-replete cells grown at pH 6.9 and 6.0 were highly virulent in a guinea pig model. However, the virulence of L. pneumophila was significantly attenuated (P < 0.05) in response to iron-limited growth. This phenomenon was reversible, and correlated with reduced phagocytosis and / or reduced intracellular survival following infection. Decreasing the pH of iron-limited cultures to 6.0 did not stimulate recovery of culture virulence. Therefore, this study clearly demonstrates that environmental stresses, including iron limitation and extremes of pH, play an important role in modulating the physiology and virulence of L. pneumophila, inducing the expression of distinct phenotypes differing in their ability to persist in nature and cause infection.

Bacteria interact with one another in many ways, through helpful behaviours like producing fitness-enhancing secretions and signals as well as harmful ones like the release of anti-competitor toxins. These interactions are essential for bacterial growth and survival and can have substantial impacts on the virulence of bacterial pathogens. This thesis explores the theory of social interactions among bacteria, focusing on both the mechanisms that underlie them as well the consequences for pathogens coinfecting a host. I first propose a hypothesis for the regulation of competitive traits in bacteria. By analysing published literature on anti-competitor toxin regulation I suggest that one of the principal mediators of antagonistic behaviour in bacteria is sensing harm from competitors. In particular, I argue that certain types of stress responses, known to protect bacteria from environmental assault, are fundamental in allowing bacteria to sense competitive threats. Next I focus on another mechanism of sensing social partners, quorum sensing, which has been argued alternatively to either sense bacterial cell density or the mass transfer properties of an environment. I propose a hypothesis on how the use of multiple quorum sensing signals molecules, a common feature across many bacteria, can potentially help resolve ambiguity between social and physical aspects of a cell’s environment. The rest of the thesis focuses on the epidemiology of coinfection, bacterial and otherwise. In some parasites, high coinfection rates lead to an increased level of evolved virulence due to competition between lineages inside the host. In contrast, when cooperative secretions contribute to virulence, the opposite can occur because high producing virulent strains are out-competed by parasites that do not produce public goods. I develop a mathematical model to show that the structure of parasites inside the host largely determines the fate of virulence when there is social interaction at a local level within the host. This analysis shows that multiplicity of infection can have either a positive or negative effect on virulence depending on structuring within the host. Lastly I explore how host contact structure influences coinfection rates and show that when hosts have very heterogeneous numbers of contacts, a small fraction of individuals in the population has a disproportionate effect on coinfection, which in turn shapes pathogen evolution.

The obligate intracellular protozoan, Leishmania infantum chagasi (Lic) is the causative agent of visceral leishmaniasis in South America. The flagellated promastigote life stage of the parasite undergoes receptor-mediated phagocytosis by macrophages. This process is followed by a transient delay in phagolysosome maturation that allows for conversion into the amastigotes, a stage that is resistant to degradation inside host cells. We hypothesized that events occurring early during parasite-host interaction influence whether the pathogen ultimately survives or is eliminated in the intracellular environment, and that these processes are facilitated by determinants from both the macrophage and the incoming Leishmania. We found differences in the pathway through which virulent Lic metacyclic promastigotes or avirulent logarithmic promastigotes are phagocytosed by human monocyte-derived macrophages (MDMs). The macrophage surface receptors that ligated the two forms of promastigotes differed, guiding metacyclic promastigotes into a compartment that supported their replication and logarithmic promastigotes into a vacuole that rapidly assembled its microbicidal machinery. Survival of metacyclic promastigotes following their phagocytosis also varied greatly on characteristics of the host macrophage. U937 cells, a model monocytic cell line lacking the third complement receptor (CR3) on their surface, took up parasites via a unique "coiling" mode of pseudopod extension, leading to a formation of a phagosome that did not fully mature. Since the parasites never demonstrated escape into the macrophage cytosol, it is logical to predict that they synthesize and release virulence factors that localize within the parasitophorous vacuole (PV) in order to establish communication with the host cell. Using a previously assembled bioinformatic catalogue of putatively secreted or excreted (E/S) proteins encoded in the Leishmania infantum genome, we chose four candidate proteins for further analysis. Two of these, serine carboxypeptidase (CBP) and a flavodoxin domain-containing protein (HP) coding sequences, were overexpressed or removed in Lic. Parasites lacking one allele of either CBP or HP were defective in survival within MDMs. Furthermore, recombinant overexpressed HP was detected from parasite lysate in a stage-specific manner, paralleling expression in wild type Lic. This implies that the regulatory elements within the protein coding sequence remain functional outside of their native locus. Taken together, our study shows that quiescent entry of virulent Leishmania spp. into macrophages is accounted for by i) the ability of metacyclic promastigotes to selectively bypass macrophage components leading to deleterious pathways, as well as ii) tightly regulated parasite virulence factors for deliberately enhancing intracellular survival.

Equine infectious anemia virus (EIAV) causes rapid development of acute disease followed by recurring episodes of fever, thrombocytopenia and viremia, termed chronic EIA. Most infected horses control the virus by immune mechanisms and become inapparent carriers. To further our understanding of the equine immune response to EIAV, a multi-probe ribonuclease protection assay (RPA) was developed to quantitate equine-specific cytokine mRNAs. Eleven template plasmids specific to ten equine cytokine genes and the ?-actin gene were generated, from which radiolabeled anti-sense RNA probes were produced. The RPA simultaneously quantitated mRNA levels of interleukin (IL)-1, IL-1, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, interferon (IFN)-, transforming growth factor (TGF)-1 and tumor necrosis factor (TNF)- in equine peripheral blood mononuclear cells and equine monocyte-derived macrophages (EMDM). The assay detected as few as 5105 RNA molecules and displayed coefficients of variation of 0.03-0.08 when normalized to -actin expression. Using this RPA, cytokine expression in EMDM infected with 2 molecularly cloned viruses (EIAV17 and EIAV19) was determined. EIAV17 varies from EIAV19 only in env, rev and LTR and causes fatal disease in Shetland ponies. When added to EMDM cultures, virulent EIAV17 stimulated expression of IL-1, IL-1, IL-6, IL-10 and TNF-. These cytokine mRNAs were significantly elevated by 0.5 to 1 hr post infection (hpi) and returned to basal levels by 12 to 24 hpi, indicating modulation by early event(s), such as receptor binding. In contrast to EIAV17, EIAV19 is avirulent in vivo and failed to induce any of the tested cytokines in EMDM. These data show a direct correlation between the virulence of the EIAV clone and the induction of cytokines. The cytokines stimulated by EIAV17 may contribute to EIA-associated symptoms, enhance viral replication in the host, and regulate the host immune response. To determine whether cytokine induction requires EIAV17 replication, EMDM cultures were exposed to UV-inactivated EIAV17 and cytokine induction was monitored. UV-inactivation did not block cytokine induction by EIAV17, suggesting dispensability of viral replication. Given that EIAV17 induces cytokines in a rapid and replication-independent manner, the activation of cytokine expression is likely mediated by binding of EIAV17 to equine macrophage receptor(s).

There are three human pathogenic species of the genus Yersiniae: Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis. To cause disease, these strains inhibit several key innate defense mechanisms, including phagocytosis, the critical process for bacterial clearance. The ability of Yersinia to evade the immune defense is dependent on delivery of virulence effectors, Yersinia outer proteins (Yops), into the interacting cell by a mechanism involving the type III secretion machinery. We have shown that the virulence protein YopK plays an important role in the control of Yop effector translocation via a feedback mechanism involving another virulence protein, YopE. We also found that YopK participated in regulation of Yop effector translocation by modulating level and ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, using a yeast two-hybrid screen with YopK as a bait, the eukaryotic protein RACK1 was identified as a target for this virulence protein. We found that RACK1 was engaged upon Y. pseudotuberculosis-mediated β1-integrin activation, where it was recruited to phagocytic cups. Downregulation of RACK1 by RNAi resulted in a reduced ability of Y. pseudotuberculosis to block phagocytosis, indicating that RACK1 is required for efficient Yersinia-mediated antiphagocytosis. Based on our data, we suggest a model where Yersinia, via YopK, targets RACK1 to ensure a directed delivery of the Yop effectors to the “right place” where they bind to and inactivate their targets, resulting in efficient inhibition of phagocytosis.   A yopK mutant strain over-delivers Yop effectors, but is still avirulent in mice, indicating that YopK is important for the fine-tuning of effector protein delivery during infection. To analyse this, we investigated the importance of YopK during in vivo infection. We found that a yopK mutant colonized Peyer’s patches and the mesenteric lymph node more rapidly compared to wild-type Y. pseudotuberculosis, but was unable to spread systemically to liver and spleen and cause full disease in mice. Further, we showed that a yopK mutant was able to colonize liver and spleen and cause full disease in mice lacking the main phagocytes, polymorphonuclear leukocytes (PMNs). We also showed that YopK was important for Yersinia-mediated silencing of the PMN response. To summarize, we suggest that YopK is important for Yersinia to evade the PMN defense and thereby spread systemically and cause disease. YopK is proposed to do this by allowing a controlled, directed Yop effector delivery that is just sufficient to inhibit host immune defense mechanisms. The controlled and precise delivery of virulence effectors avoids inappropriate triggering of PMNs and thereby an enhanced immune response favoring the host.

The genetic requirements for Influenza virus to infect and adapt to new species is largely unknown. To understand the evolutionary steps required by a virus to become virulent, a human virus (A/HK/1/68) (HK), avirulent in mice, was subjected to 20 and 21 serial lung-to-lung passages in mouse. Sequence analysis revealed the emergence of eleven mutations within the NS1 gene of the new virulent strains, many of which occurred in binding sites for transcriptional and translational cellular factors. In the present study we have rescued viruses containing each of the NS1 mouse adapted mutations onto A/PR/8/34 (PR8) backbone. We found 9 of 16 NS1 mutants were adaptive by inducing mortality, body weight loss in BALB/c mice and enhanced virus replication in MDCK cells with properties of host cell interferon transcription inhibition. Sequence comparisons with the highly pathogenic A/Hong Kong/156/1997 (H5N1) and the most severe pandemic A/Brevig Mission/1/1918 (H1N1) NS1 genes showed convergent evolution with some of the mouse adapted viruses for F103L plus M106I and V226I plus R227K mutations respectively. The F103L and M106I mutations in the HK NS1 gene were shown to be adaptive by assessment with respect to replication, early viral protein synthesis, interferon-β antagonism and tropism in the mouse lung. We extended the study and proved increased virulence associated with F103L+M106I mutations in their respective H5N1 NS1 gene on the PR8 and HK backbones, as well as the PR8 NS1 gene and the H9N2 (A/Ck/Bj/1/95) gene in the PR8 and A/WSN/33 backbones respectively. However the V226I and R227K mutations in their respective HK and 1918 NS1 genes slightly enhanced virulence and viral growth at later stages of infection. This study demonstrates that NS1 is a virulence factor; involved in multiple viral processes including interferon antagonism and viral protein synthesis. Furthermore, NS1 mutations acquired during mouse adaptation are proven to be adaptive in human, mouse and avian NS1 genes.