Les bactéries pathogènes Escherichia coli entérohémorrhagiques (EHEC) O157:H7 causent des toxi-infections sévères chez l’humain. Les biofilms des EHEC rendent difficile leur contrôle dans les environnements favorisant leur persistance. Certaines souches O157:H7 dont celle de référence Sakai, possèdent une capacité accrue à s'autoagréger et former des biofilms. L’étude a visé à identifier les gènes impliqués dans l’autoagrégation et la formation de biofilm chez la souche Sakai, et vérifier l’association des facteurs identifiés avec d’autres EHEC O157:H7 du même phénotype. Avec une banque de mutants Tn10 de la souche Sakai, des mutants non-autoagglutinants et non-formateurs de biofilms des gènes csgB et csgG furent sélectionnés et caractérisés. Ces mutants formaient significativement moins de biofilms et d´autoagrégats que Sakai, et ne produisaient plus curli. La complémentation des mutants restaurait le phénotype sauvage. De plus, des gènes responsables de la biogenèse de curli csgA, csgB et csgG étaient significativement surexprimés chez Sakai, comparativement avec la souche EDL933 non-autoagglutinant, qui forme moins de biofilm. Parmi les souches d’E. coli 0157:H7 on distingue deux groupes d’isolats: Sakai-like et EDL933-like selon leur production de curli, et leur capacité à former des autoagrégats et biofilms. Nos résultats suggèrent qu’une surproduction de fibres de curli dans un sous-ensemble de souches O157:H7 pourrait être responsable de leur phénotype particulier d'autoagrégation et de formation de biofilms forts. Le projet permet de mieux cerner le mécanisme de formation de biofilm dans EHEC et renforce l’hypothèse que le curli est une cible intéressante pour contrer la persistance des EHEC en environnements naturels et industriels. / Enterohemorrhagic E. coli (EHEC) O157:H7 is an important foodborne pathogen that causes severe toxi-infections in humans. These bacteria have a higher capacity to form biofilm, impeding the control of the contamination in different environments and allowing their persistence. Some E. coli O157:H7 strains, including the Sakai reference strain, display a distinctive ability to autoaggregate and form strong biofilms. The aim of this work was to identify the genes involved in autoaggregation and biofilm formation in Sakai strain, and to verify the association between identified factors and the same phenotype in other EHEC O157: H7. We found that csgA, csgB and csgG curli genes were significantly overexpressed in strain Sakai compared to strain EDL933, a low biofilm-former and non-autoaglutinating strain. Sakai csgB and csgG Tn10 mutants formed significantly less biofilm and autoaggregation than the wild-type strain, and lost the curli-producing phenotype. Complementation restored the strong autoaggregation and biofilm formation phenotype, and the curliated morphotype of Sakai. In addition, E. coli O157: H7 isolates tested for curli formation, Sakai-like strains were curli-producing, whereas EDL933-like strains were non-curliated. These results suggest that overproduction of extracellular curli fibers in a subset of E. coli O157: H7 strains may be responsible for their particular phenotype of autoaggregation and strong biofilm formation. The project provides a better understanding of the mechanism of biofilm formation in EHEC, as well as reinforcing the hypothesis that curli fibers are an attractive target to counter the persistence of these bacteria in natural and industrial environments.

Le nickel est connu pour son utilisation dans la préparation d'alliages réputés peu sensibles à la contamination bactérienne, très utilisés dans les secteurs agro-alimentaires et médicaux. Cependant, les bactéries apparaissent capables d'adhérer même à ce type de matériau et de le coloniser sous forme de biofilms. Les biofilms sont des communautés de micro-organismes, adhérant entre eux et à une surface, grâce à la sécrétion d'une matrice adhésive offrant une protection contre la dessiccation, les défenses de l'hôte et un grand nombre d'agents antimicrobiens. Ces biofilms manifestent des propriétés de multirésistances aux biocides qui causent des problèmes sanitaires majeurs dans les installations hospitalières et industrielles. D'importantes modifications de l'expression génétique accompagnent la vie en biofilm et induisent des caractères spécifiques dont des résistances accrues aux biocides et la production de facteurs de virulence. Parmi ces derniers, les curli, qui sont un composé protéique majeur de la matrice extracellulaire chez les bactéries Escherichia coli et Salmonella spp., jouent un rôle clé dans la formation de biofilms sur surfaces inertes et biologiques. Ce travail a consisté à explorer la contribution de la vie en biofilm à la survie des bactéries Escherichia coli K-12 productrices de curli en présence de nickel. Pour cela, l'effet physiologique d'ions solubles de nickel sur la survie des bactéries a été testé sur des supports inertes en polystyrène ou en acier. Nous avons montré que des concentrations sub-inhibitrices de nickel induisent une augmentation de l'épaisseur et de la densité du biofilm. Cet effet ne dépend pas des modifications physico-chimiques de la surface cellulaire par le nickel, ni de l'activité de la seule pompe d'efflux à nickel connue d'E. coli, RcnA. Par contre, le nickel à faible concentration induit l'expression des curli, ainsi que leur production. C'est donc via l'activation transcriptionnelle des gènes codant les curli que l'augmentation du biofilm par le nickel se produit. Ce travail s'est également appliqué à rechercher la nature du relais gouvernant la mise en place des curli en réponse à la présence de nickel. Aucun des régulateurs principaux de l'expression des curli ne joue un rôle décisif. Nos résultats nous conduisent à suggérer que l'effet du nickel repose sur un phénomène global de réponse au stress oxydant dont le mécanisme reste à déterminer.

Bacteria in nature mostly exist in biofilms, which are structured adherent communities encased in polymeric matrices. In the human body, most biofilms are composed of commensal microorganisms with the gastrointestinal tract being the most heavily colonized site. Bacterial attachment to the overlying mucus gel layer of the intestinal epithelium is fundamental to the establishment of a stable commensal microflora. However the interaction of bacteria with the complex mucus gel is poorly described. Moreover, the complexity and diversity of the gut microbiota is itself an obstacle to studying its biology. Microbiota functions are the product of communities of bacteria and interactions between multiple species. New approaches are needed to study this aspect of even the most well-studied member of the human gut microbiota, Escherichia coli. This thesis was devoted to the exploration of the transcriptional response of E. coli facing different elements of human gut following 3 main objectives. First, the initial part of my work was related to the conception and optimization of appropriate genetic tools to both track E. coli within the multispecies context that constitute human gut commensals, and survey the expression of genes of interest. Use of the Green Fluorescent Protein (GFP) genes allowing enhanced fluorescence and shortened half-life has permitted significant progress both in whole cell tagging as well as transcriptional reporting, while the red fluorescent counterparts were disappointing. Second, using the subset of tools that has been validated to be reliable, influence of mucin on the biofilm formation ability of E. coli has subsequently been studied. I have shown that mucin promotes E. coli biofilm formation through transcriptional modulation of surface adhesion structures such as curli and type 1 pili. Third, concurrently, E. coli's population relationship to commensal bacteria (K. pneumoniae and E. faecalis) was investigated and demonstrated, with the possible influence of surface adhesion structures such as curli as the biological focus. The results suggest that curli production in biofilm increases the fitness of E. coli when co-cultured with K. pneumoniae while promoting synergistic interaction between E. coli and E. faecalis. The implication based on the data is discussed. This work improves the understanding of E. coli response to the gut environment, and provides foundations to build more powerful tools for further investigations.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that affects at least five million people worldwide. An increased expression of type I interferon (IFN) regulated genes is a hallmark of SLE, but the precise etiology of SLE initiation and flares is poorly understood. Because plasmacytoid dendritic cells (pDCs) are the primary type I IFN producers, their role in SLE has long been suspected, with murine pDC depletion models successfully delaying the progression of murine lupus-like disease. However, the mechanism behind how exactly how pDCs contribute to lupus autoimmunity is unknown, contributing to the current dearth lack of disease modifying treatments; current treatments only succeed in suppressing symptoms, and do not halt disease progression. In this study, we take a multifactorial approach to understanding the biology of pDCs in the context of lupus autoimmunity. Although the exact etiology of lupus is unknown, infections are an important environmental trigger for / Infectious Disease & Immunity

The first recorded observation of bacterial biofilms dates back to the 17th century by Antoine Van Leeuwenhoek. Today, biofilms are known as bacteria encapsulated within a self-produced extracellular matrix adherent to biotic or abiotic surfaces. Since the initial discovery of biofilms, research has explored the structure and function of biofilms. Only until recently has the role of biofilms within the medical setting become apparent. Here, we investigate the role of curli-extracellular DNA (eDNA) complexes in disease pathogenicity and explore the ability to target bacterial amyloid curli as a novel anti-biofilm therapeutic target. Biofilms of enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, are composed of various components that act in consortium to fortify the extracellular matrix. One of the main components of enteric biofilms is amyloid curli. Curli, one of the best characterized bacterial amyloids, is a protein with a conserved cross beta sheet structure that forms basket like structures encapsulating the bacteria. Within the biofilm, curli serves to fortify the extracellular matrix, aids in bacterial attachment and protects bacteria from harsh environmental conditions. Extracellular DNA (eDNA) is another integral component of enteric biofilms. Recent reports from our lab has suggested that curli forms irreversible complexes with eDNA. Even with exposure to DNases, co-localized curli and eDNA can be observed. Other components of enteric biofilms include cellulose and Biofilm Associated Protein A. Biofilms of S. Typhimurium have been associated with significant disease pathologies. In addition to identifying the existence of curli-eDNA complexes within S. Typhimurium biofilms, our lab has also reported that curli-eDNA complexes of S. Typhimurium potentiate the autoimmune disease Systemic Lupus Erythematosus (SLE). SLE is an autoimmune disease characterized by the production of type I interferons and autoantibodies, although the etiology remains unknown. Systemically, curli binds to and activates the Toll like Receptor (TLR)1/2 complex leading to a pro-inflammatory response. In these studies we aimed to identify the innate immune mechanisms leading to the autoimmune phenotype following stimulation with curli-eDNA complexes. As TLR9 is activated by unmethylated bacterial DNA CpG DNA sequences leading to the production of type I interferons we hypothesized a potential role for TLR9 in recognizing eDNA of the curli-eDNA complex leading to the generation of the hallmarks of SLE. To investigate this hypothesis, we stimulated wild-type, TLR2 knockout, TLR9 knockout and TLR2-9 double knockout immortalized macrophages with curli-eDNA complexes purified from S. Typhimurium biofilms. We observed a significant reduction in the transcript level of type I interferons (IFN), Ifnβ, Isg15 and Cxcl10, upon stimulation of TLR2 knockout, TLR9 knockout and TLR2-9 double knockout immortalized macrophages implicating a role in TLR9 recognition of the curli-eDNA complex. As there was a significant reduction of type I interferon levels upon stimulation of TLR2 knockout macrophages, we hypothesized that TLR2 may serve as a carrier to bring the curli-eDNA complex into the endosome containing TLR9. To inhibit phagocytosis, we pretreated cells with endocytosis inhibitors and stimulated wild-type macrophages with curli-eDNA complexes. We observed a reduction in the transcript level of Ifnβ suggesting that curli-eDNA complexes gain access to endosomal TLR9 via TLR2 engagement. Finally, to explore the role of TLR2 and TLR9 in the production of autoantibodies, curli-eDNA complexes were intraperitoneal injected twice weekly for six weeks into C57BL/6 wild-type, TLR2 knockout, TLR9 mutant and TLR2 knockout-TLR9 mutant mice. We observed a robust generation of anti-double stranded autoantibodies within the first three weeks, however the production of autoantibodies was significantly decreased and delayed in the TLR2 knockout, TLR9 mutant and TLR2 knockout-TLR9 mutant mice. Overall, these data suggest that curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9. Within biofilms of S. Typhimurium, curli is the main proteinaceous component. Biofilms lacking curli destabilize and fail to form mature biofilms. Recent research has shown that in response to the production of host amyloids, the body will generate anti-amyloid antibodies in the serum. Monoclonal antibodies (mAb) generated from serum antibodies have been shown to have pan anti-amyloid properties in vitro and in vivo due to the β-sheet conformational epitope. As amyloids from both human and bacterial origin share a β-sheet conformational structure, we hypothesized as to if the anti-amyloid mAbs can eradicate S. Typhimurium biofilms by targeting curli. We incubated S. Typhimurium biofilms in the presence of various mAbs (ALZ.4A6, ALZ.4GI, ALZ.2C10 and ALZ.3H3) and observed a significant reduction of biofilm thickness and curli content within the biofilm. We deduced that ALZ.3H3 conferred the greatest anti-biofilm response. When we visualized the three-dimensional architecture of biofilms incubated with ALZ.3H3, we observed that ALZ.3H3 induced the formation of a loose architecture compared to untreated biofilms that were dense and compact. The resulting loose biofilm architecture induced by incubation with ALZ.3H3 enhanced the susceptibility of the biofilms to antibiotic exposure and macrophage clearance. We also observed enhanced biofilm eradication in vivo when catheters precoated with S. Typhimurium biofilms were inserted into the back flanks of mice that were percutaneously injected with ALZ.3H3. Both in vitro and in vivo, combination therapy of ALZ.3H3 and antibiotic enhanced biofilm clearance. In summary, we propose a novel anti-biofilm strategy by targeting the amyloid component of the biofilm, thus satisfying an unmet need in the art of biofilm prevention. Overall, these data in summation significantly broadens our understanding of disease pathogenicity and the role of curli-eDNA complexes in S. Typhimurium biofilms. As amyloid-eDNA complexes may be found in other biofilms, these results may extend beyond enteric bacteria proving novel insight into host-microbe interactions and the generation of novel anti-biofilm therapeutics. / Microbiology and Immunology

Fresh produce is increasingly associated with foodborne outbreaks. In order to develop effective intervention and measures to reduce microbial risks, it is essential to attain a better understand the mechanisms of attachment of foodborne pathogens to fruits and vegetables. Using lettuce as a model, the attachment of Escherichia coli O157:H7 to produce surfaces was studied. Strains expressing various extracellular proteins (curli, O157-antigen, and intimin) known to influence attachment of E. coli to intestinal cells were evaluated for their physicochemical properties and ability to adhere to cut edge and whole leaf lettuce. Escherichia coli O157:H7 strains included: 0018, 43894 and 43895 (curli producing and non-producing); 86-24 (WT), F-12 (O157-antigen negative), pRFBE (O-antigen replaced on plasmid); and 86-24, 86-24Ã eae10 (intimin negative). The eleven strains were surveyed for their hydrophobicity and cell charge using hydrophobic interaction chromatography (HIC) and electrostatic interaction chromatography (ESIC) techniques. Iceberg lettuce squares (2 x 2 cm) were inoculated with E. coli O157:H7 strains separately (7.0 log CFU/square) and dried in a laminar flow hood. Lettuce was sampled before (unrinsed) and after being rinsed twice with sterile de-ionized water (rinsed). Strips (2 mm wide) of each cut edge of the lettuce were aseptically removed. Cut-edge and whole-leaf samples were homogenized and spiral plated onto Luria-Bertani agar, supplemented with nalidixic acid (50ppm), to assess levels of bacteria remaining on the lettuce leaf after rinsing. The rinse steps were not effective in significantly removing bacteria from lettuce (p>0.05). Curli-producing and non-producing strains preferentially attached to cut edge versus the whole leaf portions of lettuce (p<0.05); however the 86-24 strains showed no preference for attachment. With the exception of 0018 curli-producing and non-producing strains, presence/absence of extracellular proteins surveyed did not influence attachment of E. coli O157:H7 to either cut edge or whole leaf lettuce. There was significantly greater attachment of the curli-producing 0018 strain over the curli non-producing 0018 strain to cut and whole lettuce surfaces (p<0.05). Production of curli and O-polysaccharide significantly increased (p<0.05) the cell's overall hydrophobicity of the cell; however this did not affect attachment (p<0.05). The overall cell charge of all strains was negative; however, charge did not affect attachment of E. coli O157:H7 to lettuce. The presence of extracellular appendages (curli, O157-antigen, intimin) as well as hydrophobicity and cell charge properties had no affect on attachment of the cell to lettuce. / Ph. D.

Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability. By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.

New strategies are needed to counter the growing problem of bacterial resistance to antibiotics. One such strategy is to design compounds that target bacterial virulence, which could work separately or in concert with conventional bacteriostatic or bactericidal antibiotics. Pilicides are a class of compounds based on a ring-fused 2-pyridone scaffold that target bacterial virulence by blocking the chaperone/usher pathway in E. coli and thereby inhibit the assembly of pili. This thesis describes the design, synthesis, and biological evaluation of compounds based on the pilicide scaffold with the goal of improving the pilicides and expanding their utility. Synthetic pathways have been developed to enable the introduction of substituents at the C-2 position of the pilicide scaffold. Biological evaluation of these compounds demonstrated that some C-2 substituents give rise to significant increases in potency. X-ray crystallography was used to elucidate the structural basis of this improved biological activity. Furthermore, improved methods for the preparation of oxygen-analogues and C-7 substituted derivatives of the pilicide scaffold have been developed. These new methods were used in combination with existing strategies to decorate the pilicide scaffold as part of a multivariate design approach to improve the pilicides and generate structure activity relationships (SARs). Fluorescent pilicides were prepared using a strategy where selected substituents were replaced with fluorophores having similar physicochemical properties as the original substituents. Many of the synthesized fluorescent compounds displayed potent pilicide activities and can thus be used to study the complex interactions between pilicide and bacteria. For example, when E. coli was treated with fluorescent pilicides, it was found that the compounds were not uniformly distributed throughout the bacterial population, suggesting that the compounds are primarily associated to bacteria with specific properties. Finally, by studying compounds designed to inhibit the aggregation of Aβ, it was found that some compounds based on the pilicide scaffold inhibit the formation of the functional bacterial amyloid fibers known as curli; these compounds are referred to as 'curlicides'. Some of the curlicides also prevent the formation of pili and thus exhibit dual pilicide-curlicide activity. The potential utility of such 'dual-action' compounds was highlighted by a study of one of the more potent dual pilicide-curlicides in a murine UTI model were the compound was found to significantly attenuate virulence in vivo.