The first observations of biofilms were made by the “father of microbiology” Antonie van Leeuwenhoek in the 17th century. The number of publications on biofilms has grown exponentially in the last 20 years, highlighting the medical relevance of the field. The complexity of the bacterial biofilm as well as its variability across species provides a continual channel for discovery. While all biofilms differ, there are some components that remain standard such as proteins, polysaccharides and DNA allowing for linkages between seemingly distinct biofilms. Biofilm-associated infections account for more than 65% of all infections implicating the need for understanding the stages of biofilm formation and development. Our lab focuses on the amyloid component of the biofilm and has identified that curli and extracellular DNA (eDNA) complex irreversibly within the biofilms of Salmonella enterica serovar Typhimurium and Escherichia coli. Here, we investigate the formation and pathogenicity of cytotoxic curli intermediates previously unidentified in the in vitro biofilm. The identification of multiple curli conformations within the biofilm biogenesis aides in the understanding of amyloid kinetics in the enteric biofilm. Together, these studies provide a link between biofilm-associated infections and autoimmune responses in the host. In these studies, we planned to isolate curli from different stages in biofilm development to observe their differences both structurally and through their interactions. We identified turbulence has a significant impact on the formation of mature biofilm. We were able to isolate an intermediate form of curli through increased turbulence during biofilm growth. There has never been an intermediate form of curli isolated before our studies due to the high efficiency of the nucleation-precipitation process of curli fiber formation. From this isolation, we characterized these intermediates in comparison to the mature curli complexes. We observed that higher turbulence leads to lesser biofilm formation by sedimentation assay and crystal violet staining. Additionally, we investigated the expression of the curli forming genes csgBA via flow cytometry analysis which indicated that csgBA was preferentially expressed under low turbulence conditions. When investigating the curli conformations isolated from the biofilm, we found that intermediate complexes incorporated less thioflavin T (ThT) indicating lesser amyloid content. We also differentiated the mature and intermediate curli aggregate populations using multiple microscopy techniques. Under confocal microscopy, intermediate fibers seldom measured larger than 100 µm, while mature curli did. Electron dense regions were observed under transmission electron microscopy in the mature curli indicating high fibrillization and compact structure of these aggregates, not seen in the intermediates. Due to known interactions of curli with eDNA in the biofilm, next we investigated the DNA content of the complexes. We hypothesized that the mature structured complexes would have greater DNA content supporting the maturation of the fibrils and the structural compaction. Indeed, we found more DNA could be extracted from the mature curli fibers. Interestingly, we increased the fibrillization of intermediates upon addition of exogenous genomic DNA suggesting DNA incorporation was necessary for the formation of the mature fibrillar aggregates. Intermediates of amyloid β are found to be more cytotoxic than the mature form of the amyloid. For this reason, we hypothesized that curli intermediates could also be cytotoxic. After treating bone marrow-derived macrophages with mature and intermediate curli complexes, we observed that the intermediate aggregates were significantly more cytotoxic to immune cells than mature aggregates. Together, this data implicates a role for the cytotoxic intermediate form of curli in the pathogenesis of Salmonella as well as other enteric bacteria. Curli complexes have been previously described as a novel pathogen associated molecular pattern (PAMP) by their ability to activate numerous receptors in immune cells. The host immune response to curli complexes has been elucidated in our lab. First, binding to Toll-like receptor-2 (TLR2) begins with recognition of the conserved cross-β sheet secondary structure. Mutations disrupting this structure are shown to abrogate immune cell recognition and signaling. For this reason, we next investigated the pathogenicity of the intermediates discovered and characterized above. As cytotoxic oligomers exist for human amyloids, we aimed isolate an earlier form of the intermediate curli and investigate the ability of these conformations to activate host immune responses. As mature curli has been reported to induce anti-dsDNA antibodies in murine models of systemic lupus erythematosus (SLE), we anticipated differences in the autoimmune response to these different curli aggregates as well. First, we isolated and characterized an early form of intermediates isolated at 24 hours which were smaller in size and incorporated less DNA within their complexes than the aforementioned intermediates. Further investigations into the structure of these early intermediates described altered secondary structure by circular dichroism. The lack of fully formed secondary structure in the early intermediates we hypothesized to decrease the ability of these complexes to interact with immune receptors as mature curli. Indeed, we saw decreased response in pro-inflammatory cytokine production as well as type I IFN production. This lack of type I IFN production, lead us to investigate the autoantibody response to the early intermediates. When treating both wild-type and autoimmune-prone mice with curli complexes, early autoantibodies responses were dependent on the DNA content of the complex. However, after continual treatment for 10 weeks, intermediate complexes produced levels of anti-dsDNA similar to that of the mature curli treatment. In addition to anti-dsDNA antibodies, for the first time, other anti-nuclear antibodies, such as anti-C1q and anti-nucleosome, were produced in response to these treatments as well. Finally, chronic exposure to curli complexes led to significant histopathological changes including synovial proliferation and periosteal resorption, in the joints of mice autoimmune-prone mice. Together, this data identified that chronic exposure to curli induced autoimmune sequelae which is thought to be transient in genetically healthy individuals, but leads to joint inflammation in individuals whom are genetically predisposed to autoimmunity. In summary, these data significantly broaden the knowledge of curli amyloid formation during biofilm biogenesis in vitro through the identification of previously unidentified cytotoxic intermediate conformations of curli. Additionally, our work forwards the fields of both autoimmunity and biofilm-associated infection research by providing evidence of the direct impact that chronic exposure to the biofilm has on the host, both transient and long-lasting in those whom are pre-disposed to autoimmunity. / Biomedical Sciences

Verocytotoxin-producing Escherichia coli (VTEC) are important human pathogens that may cause diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome (HUS). Worldwide, non-sorbitol-fermenting (NSF) VTEC O157:H7 is the most common serogroup associated with HUS but several non-O157:H7 serogroups have emerged as causes of this disease. This research investigated the pathogenic potential of two non-O157:H7 serogroups: O26 and sorbitol-fermenting (SF) O157:NM. While VTEC O26 have emerged as a significant cause of HUS in continental Europe, human infections associated with this pathogen are uncommon in Scotland and generally only result in simple diarrhoea. The study characterised E. coli O26 isolates recovered from human infections in Europe and Scotland and isolates collected from Scottish cattle with the objectives to identify factors which may allow strains to cause more serious clinical disease and to investigate the potential of bovine VTEC O26 in Scotland to cause human infection. MLST analysis of housekeeping genes found little genetic variation in the genomic ‘backbone’ among the vast majority of E. coli O26 isolates. The gene for verocytotoxin 2 (vtx2) alone was carried by VTEC O26 isolates recovered from patients in continental Europe but was found in no Scottish human isolate, where the majority of isolates did not harbour a vtx gene. It was demonstrated that among the European VTEC O26 human isolates, 67% carried a specific allele within the promoter region for LEE1 and 87% harboured the tccP2 gene. In contrast, no Scottish VTEC O26 human isolate carried this allele or the tccP2 gene. The impact these genotypic characteristics have on the pathogenic potential of a strain remains uncertain. There were no clear differences in verocytotoxin titres, levels of LEEencoded protein secretion or levels of adherence to Caco-2 cells between VTEC O26 isolates recovered from human infections of varying severity. However, levels of LEE-encoded protein secretion from cattle isolates were generally higher than those from many of the human isolates. The differences in pathogenic potential between isolates are likely to be due to horizontally acquired DNA, including vtx2 carriage and the O-island-phage-associated effector protein repertoire. Further work is required to determine if the differences identified may also impact on shedding levels from cattle and therefore the likelihood of transmission to humans. Since 1988, SF VTEC O157:NM strains have emerged and have been associated with a higher incidence of progression to HUS than NSF VTEC O157:H7. This study investigated bacterial factors that may account for the increased pathogenic potential of SF VTEC O157:NM. While no evidence of toxin or toxin expression differences between the two VTEC O157 groups was found, the SF VTEC O157:NM strains adhered at significantly higher levels to a human colonic cell line. Under the conditions tested, curli were shown to be the main factor responsible for the increased adherence to Caco-2 cells. The capacity of SF VTEC O157:NM strains to express curli at 37C may have relevance to the epidemiology of human infections as curliated strains could promote higher levels of colonization and inflammation in the human intestine. In turn this could lead to increased toxin exposure and an increased likelihood of progression to HUS.

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. / Nickel is known for its use in alloy preparation, which is renowned for not being very sensitive to bacterial contamination, and thus widely used in the food-processing and medical sectors. However, bacteria appeared to be able to adhere even on this type of material, and to colonize it in the form of biofilms. Biofilms are microorganisms’ communities, adherent between themselves and to a surface, thanks to the secretion of an adhesive matrix, which offers them protection against desiccation, host defences and a great number of antimicrobial agents. Biofilms express biocidal multiresistance properties, which causes major sanitary problems in hospitals and industries. Biofilm life comes with important modifications in the genetic expression, which induces specific characteristics like increased biocides resistances and virulence factors production. In the latter, curli are a major proteic component of the extracellular matrix in the bacteria Escherichia coli and Salmonella spp., and curli play a key role in biofilm formation on inert and biologic surfaces. This work had consisted of the exploration of the biofilm life-style contribution in the survival of E. coli K-12 bacteria that produces curli in the presence of nickel. In that aim, physiological effect of soluble ions of nickel on the bacterial survival has been tested on inert supports such as polystyrene and stainless steel. We have shown that sub-inhibitory concentrations of nickel induce an increase in the biofilm thickness and density. This effect does not rely on physicochemical modifications on the bacterial surface, nor on RcnA activity, which is the only known efflux pump dedicated to nickel in E. coli. On the other hand, low concentrations of nickel induce curli expression and their production. Hence, the nickel-induced biofilm increase occurs through transcriptional activation of curli genes. This work also tried to find the nature of the relay that governs curli setting up in response to the presence of nickel. None of the curli principal regulators seem to be implicated in this curli production increase. Thus, our results suggest that nickel effect lies on a more global response phenomenon like oxidative stress, but the involved mechanism needs to be determined.

Les bactéries dans la nature existent principalement en biofilm, qui est une communauté structurée et adhérente de microbes enveloppés dans des matrices polymériques. Dans le corps humain, la plupart de biofilms sont composés de microorganismes commensaux et le tractus gastro-intestinal est le site le plus fortement colonisé. L’attachement bactérien à la couche de gel de mucus couvrant l’épithélium intestinal est fondamental à l’établissement d’une microflore commensale stable. Cependant, les interactions entre les bactéries et le gel de mucus restent mal décrites. En plus, la complexité et la diversité du microbiote intestinal lui-même est un obstacle pour les analyses de son fonctionnement biologique. Les fonctions du microbiote sont le produit de communautés bactériennes complexes, et des interactions entres les différentes espèces qui les composent. De nouvelles approches sont nécessaires pour étudier la génétique de l’espèce la plus étudiée du microbiote de l’intestin humain, Escherichia coli. Cette thèse est consacrée à l’exploration de la réponse transcriptionnelle d’E. coli à différents facteurs présents dans l’intestin humain à travers la réalisation de 3 objectifs principaux. La première partie de mon travail concerne la conception et l’optimisation d’outils génétiques permettant de détecter E. coli au sein de biofilms multi-espèces tout en mesurant simultanément l’activité d’un gène d’intérêt. L’utilisation du gène codant la protéine fluorescente verte (GFP) et de ses dérivés a permis d’importantes avancées sur le marquage des cellules entières ainsi que le suivi d’activité transcriptionnelle. Par contre, l’utilisation de marqueurs fluorescents rouges s’est révélée décevante. Dans un deuxième temps, grâce aux outils mis au point dans la première partie de mon travail, l’influence de la mucine sur la capacité d’E. coli à former des biofilm a pu être étudiée. J’ai montré que la mucine augmente la formation du biofilm d’E. coli par modulation transcriptionnelle de structures d’adhérences telles que les curli et les pili de type 1. Enfin, l’influence de la culture en biofilms multi-espèces constitués d’E. coli et de bactéries commensales (K. pneumoniae and E. faecalis) sur la croissance de chacun des partenaires a été analysée, en focalisant notre attention sur l’influence possible de structures d’adhérence telles que les curli. Les résultats indiquent que la production de curli en biofilm augmente le développement d’E. coli en co-culture avec K. pneumoniae alors qu’elle favorise l’interaction synergique entre E. coli et E. faecalis. Les implications basées sur ces données ont été examinées. Ce travail contribue à l’amélioration des connaissances sur la réponse d’E. coli à l’environnement intestinal et apporte les fondations pour construire des outils plus puissants pour la poursuite des investigations sur les biofilms multi-espèces. / 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.

Antibiotics have been extensively used to treat bacterial infections since Alexander Fleming’s discovery of penicillin 1928. Disease causing microbes that have become resistant to antibiotic drug therapy are an increasing public health problem. According to the world health organization (WHO) there are about 440 000 new cases of multidrug-resistant tuberculosis emerging annually, causing at least 150 000 deaths. Consequently there is an immense need to develop new types of compounds with new modes of action for the treatment of bacterial infections. Presented herein is a class of antibacterial ring-fused 2-pyridones, which exhibit inhibitory effects against both the pili assembly system in uropathogenic Escherichia coli (UPEC), named the chaperone usher pathway, as well as polymerization of the major curli subunit protein CsgA, into a functional amyloid fibre. A pilus is an organelle that is vital for the bacteria to adhere to and infect host cells, as well as establish biofilms. Inhibition of the chaperone usher pathway disables the pili assembly machinery, and consequently renders the bacteria avirulent. The focus of this work has been to develop synthetic strategies to more efficiently alter the substitution pattern of the aforementioned ring-fused 2-pyridones. In addition, asymmetric routes to enantiomerically enriched key compounds and routes to compounds containing BODIPY and coumarin fluorophores as tools to study bacterial virulence mechanisms have been developed. Several of the new compounds have successfully been evaluated as antibacterial agents. In parallel with this research, manipulations of the core structure to create new heterocycle based central fragments for applications in medicinal chemistry have also been performed.

Hospital Acquired Infections and equipment contamination are some of the biggest issues faced by the healthcare industry worldwide. These infections generally range from mild to life threatening human infections which lead to increased costs and prolonged hospitalization time. The primary factor which caused these issues were biofilm forming bacteria which are able to withstand medications and defend themselves from various cleaning procedures. Another aspect which make these bacteria troublesome is that they are able to hide inside the biofilm, thus evading a lot of diagnostic tests. The methods used to detect these biofilms are unfortunately toxic to cells and cannot be used in vivo. Though there is enough data on the formation of biofilm on abiotic surfaces, the data present on the biophysical properties, structural organizations within the biofilm or their viscoelastic properties is very limited. In this master’s degree project, a dynamic monitoring platform is made for 2 different strains of the Salmonella Enteritidis bacteria where their structural and biophysical properties was investigated. Each strain lacks either curli or cellulose which are major components responsible for proper biofilm formation so performing these experiments on them gave us important information on how their properties get affected over time. Bacterial growth monitoring for all the strains were performed by measuring the absorbance every hour over a period of 5h and it was observed that all the strains had a very similar growth pattern until the end of the 4th hour after which they showed very mild differences. The next set of experiments involved using an eQCM to monitor the formation of biofilm on the surface of a quartz chip over 48h. There were differences observed in the biofilm formation pattern and mass deposition which provided clues as to how the biofilm formation and their viscoelastic properties are affected due to the mutations. This led to finding further clues regarding which aspect of biofilm formation is targeted by a specific mutant.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies directed against ubiquitous self-antigens, many of which are nuclear autoantigens like dsDNA and chromatin (Pisetsky, 2016), and by elevated type I interferons (IFN) (Hooks et al., 1979; Weckerle et al., 2011), a family of pro-inflammatory cytokines that have antiviral activity (Pestka et al., 2004). Microarray analysis of peripheral blood mononuclear cells (PBMC) from SLE patients discovered the increased expression of IFN-responsive genes that was named the IFN Signature (Baechler et al., 2003a; Bennett et al., 2003b; Crow et al., 2003). Genome wide association studies indicate a clear genetic component in lupus pathogenesis (Chung et al., 2011; SLEGEN et al., 2008) and murine models of SLE confirm genetic drivers of the disease (Morel, 2010; Morel et al., 2000). However, the concordance of SLE in monozygotic twins is only 30-40% (Connolly and Hakonarson, 2012), while the inc / Microbiology and Immunology

Salmonella enterica serotype Typhimurium is a rod-shaped, motile, Gram-negative bacterium that causes gastroenteritis in immunocompetent individuals. S. Typhimurium produces an extracellular protein termed curli, a bacterial amyloid with a cross beta-sheet tertiary structure that is common across all amyloids. Curli formation is critical for biofilm formation by enteric pathogens such as S. Typhimurium and E. coli. Curli expression requires the production of multiple proteins, which are encoded by two operons known as csgBAC and csgDEFG. Curli production can be induced in vitro by low temperature and low osmolarity, which is evident by growth on T-medium plates for 72 hours at 28oC. Earlier studies have shown that curli is expressed in sepsis patients with E. coli, as well as in mice after S. Typhimurium infection. This is evidenced by the production of antibodies to CsgA, the major subunit of curli. Our lab has shown that curli fibers are recognized by the TLR2/TLR1 complex of the innate immune system during infection. Infection with curli expressing bacteria causes elevated levels of proinflammatory cytokines, nitric oxide, and autoantibodies. Nonetheless, the details of curli expression in vivo during bacterial infection remain unknown. The focus of these studies was to elucidate the location where bacteria expresses curli in vivo during infection. Initially, we used S. Typhimurium strains carrying plasmids with csgB and csgD promoter regions fused to the gfp gene to study curli expression in vivo by use of flow cytometry. Unfortunately, we were unable to determine curli expression with this model, due to the diminished fluorescence intensity of GFP under anaerobic conditions in the gastrointestinal tract. As the question of curli expression in vivo was left unanswered, we next used a long-term infection model of S. Typhimurium with the goal of determining seroconversion to curli as well as the location and timing of curli expression. Using CBA/J mice infected with wild-type S. Typhimurium or a curli mutant strain, we were able to identify seroconversion to CsgA in the mice infected with the wild-type strain through ELISA and western blot analysis. We were also able to identify autoantibody production in mice infected with the wild-type strain through ELISA. However, we were unable to determine curli expression in the feces of mice either by western blot or qPCR data. We were also able to identify autoantibody production in mice infected with the wild-type strain through an anti-double stranded DNA ELISA. Preliminary findings lead us to hypothesize that curli expression may occur very early on in infection, and may be expressed inside cells such as macrophages. Overall, our results partially elucidate curli expression in vivo, although more research is needed in order to answer our remaining questions regarding location and timing of expression. / Biomedical Sciences

Many bacterial species exist in nature as part of highly structured multicellular communities known as biofilms. Amyloids, proteins with a conserved β-sheet quarternary structure, show high resistance to many chemical and enzymatic processes including proteinase K and SDS treatments and are produced as essential adhesins during biofilm formation. Curli fibers expressed by Enterobacteriaceae family members including E. coli and S. Typhimurium are the most studied amyloids to date. Curli-like fibers are also produced by members of the predominant phyla found in the host gastrointestinal microbiota in environmental biofilms. Curli fibers are the predominant microbial-associated molecular pattern (MAMP) on enteric bacteria recognized by the Toll-like receptor (TLR) 2/1-heterodimer complex. Interestingly, the TLR2/1 complex has been implicated as a key player in modulating gastrointestinal homeostasis. The focus of the current studies centered on the innate immune recognition of curli fibers by cells of the gastrointestinal tract and how that contributes to gastrointestinal homeostasis. In the first phase of our studies, utilizing intestinal epithelial cells polarized on semi-permeable tissue culture inserts (Transwells®), we observed that the recognition of curli fibers on Salmonella enterica serovar Typhimurium by intestinal epithelial cells led to the augmentation of the intestinal epithelial barrier in a PI3K-dependent manner. We also observed that bacterial translocation of S. Typhimurium from the apical side to the basolateral side of the Transwell system was limited when curli fibers were present. Furthermore, infection of mice with S. Typhimurium showed that translocation of bacteria from the intestinal lumen into the cecal tissue and mesenteric lymph nodes was limited in C57BL/6 mice as compared to TLR2 knockout mice. In the second phase of our studies, we sought to further investigate the effect that curli fibers exert on gastrointestinal homeostasis through the induction of immunomodulatory cytokines such as Interleukin 10 (IL10) from subepithelial lamina propria cells. IL10 has been shown to contribute to the maintenance of the intestinal epithelial barrier and IL10-deficient mice develop lethal colitis within the first 2-3 months of life. 6-8 week-old female C57BL/6 and TLR2-/- mice were given 5mg/kg of curli fibers via intraperitoneal injection. Subsequent RT-PCR analysis of the small intestine showed a significant expression of Il10 in C57BL/6 that was absent in TLR2-/- mice. Interestingly, no changes in Ifnγ or Tgfβ mRNA were observed in these mice. This response was gut-specific, as Il10 was not detected at all in the spleen. Furthermore, in a chemically-induced colitis model, we observed that the administration of curli fibers to 8-week old Balb/c mice ameliorated disease severity as compared to colitic mice that received mock treatments. Interestingly, Il10 was also induced in the colons of colitic mice that received curli and which were euthanized 6 days after colitis was induced. Our results suggest that curli fibers induce IL10 production via a TLR2-dependent manner to dampen inflammation in the gastrointestinal tract. Overall, our results partially describe a novel role for curli amyloid fibers produced by commensal bacteria in modulating gastrointestinal health and homeostasis. We propose that the induction of immunomodulatory cytokine such as IL10 by amyloid fibers is an important mechanism utilized by commensal bacterial to confer beneficial effects that benefit both the host and microbe. We also propose curli fibers as a potential alternative in the treatment of inflammatory bowel disease. / Microbiology and Immunology

Mastitis caused by Escherichia coli is among the most common and severe forms of environmental mastitis. Currently, the severity of E. coli mastitis is thought to be more related to cow factors than bacterial virulence. Some strains of E. coli, however, may be adapted to colonizing mammary tissue, increasing clinical severity, and impairing recovery. Curli are adhesive surface structures produced by some E. coli and Salmonella strains that bind a number of host proteins and have recently been found to play a role in the pathogenesis of bacterial sepsis. Sixty-one E. coli isolates from 36 clinical cases of bovine mastitis were characterized using ERIC-PCR and screened for their ability to produce curli by binding Congo-red dye. The effect of curli production on case recovery, based on a return to milk production, was investigated for a subset of 43 isolates from 20 quarters of 19 cows. Fifty-eight of the 61 isolates were clustered into two clonal groups at 52% genetic similarity. Thirty-five of all 61 isolates (57%) were curli-positive. Twenty-three isolates from 13 cows clustered in clonal group I, of which 5 cases (38%) were curli-positive; 35 isolates from 22 cows were clustered in clonal group II, 15 of which were curli-positive cases (68%). No association was found between genetic similarity and phenotypic curli expression of isolates from cows with clinical E. coli mastitis cases (p=0.16). Phenotypic curli expression in isolates did not affect the recovery of cows' milk yield to pre-mastitis production levels (p=0.18). / Master of Science