The emergence and rapid spread of antimicrobial resistance (AMR) is a phenomenon that extends beyond clinical settings. AMR has been detected in multiple environmental compartments, including agricultural soils and water bodies impacted by wastewater discharges. The purpose of this research project was to evaluate what factors could influence the environmental persistence of antibiotic resistance genes (ARGs), as well as to identify potential strategies employed by human pathogens to survive in secondary environment outside the host. The first part of this dissertation describes the incidence of the New Delhi metallobeta lactamase gene (blaNDM-1) – an ARG conferring resistance to last resort antibiotics – in the influent of a wastewater treatment facility processing municipal wastewater from Jeddah, Saudi Arabia. Detection of blaNDM-1 was followed by the isolation of a multi-drug resistant strain of E. coli (denoted as strain PI7) at a frequency of ca. 3 x 104 CFU/m3 in the untreated municipal wastewater. Subsequently, we described the decay kinetics of E. coli PI7 in microcosm experiments simulating biological treatment units of wastewater treatment plants. We identified that transition to dormancy is the main strategy prolonging the persistence of E. coli PI7 in the microcosm experiments. Additionally, we observed slower decay of E. coli PI7 and prolonged stability of extracellular DNA in anoxic/anaerobic conditions. In the last chapter of this thesis, the fate of extracellular DNA is further explored. Using as a model Acinetobacter baylyi ADP1, we describe the stimulation of natural transformation frequencies in the presence of chlorination disinfection byproducts (DBPs). Moreover, we demonstrate the ability of BAA to stimulate transformation is associated with its capacity to cause DNA damage via oxidative stress. Overall, this dissertation addresses important knowledge gaps in our current understanding of ARB and extracellular ARG persistence in the environment. The results from this project highlight the importance of retrofitting the existing water treatment process with advance membrane filtration units, and the need to relook into the current disinfection strategies. Wastewater treatment technologies should be assessed for their efficacies in not only inactivating ARB and ARGs, but also whether unintended consequences such as stimulated horizontal gene transfer would occur.

Le biofilm est défini comme une communauté microbienne adhérant à une surface biotique ou abiotique, et engluée dans une matrice extracellulaire auto-produite. Les biofilms naturels sont composés de plusieurs espèces microbiennes et leurs interactions, qu'elles soient synergiques ou antagonistes, jouent un rôle important dans le développement, la composition et le fonctionnement des consortia impliqués. De plus, ces relations impliquent souvent la production de molécules antagonistes limitant la croissance ou l'adhésion bactérienne. Enfin la composition de la matrice extracellulaire joue un rôle important dans la robustesse du biofilm. Au cours de ce travail, l'étude des interactions au sein de biofilms mixtes constitués par K. pneumoniae et S. epidermidis a permis d'isoler un polysaccharide produit par K. pneumoniae, capable d'inhiber l'adhésion aux surfaces abiotiques de plusieurs autres espèces bactériennes à Gram-négatif et à Gram-positif. La caractérisation physico-chimique de cette molécules de haut poids moléculaire a permis de mettre en évidence qu'elle était composée de galactose, glucose, rhamnose et d'acide glucuronique. Par ailleurs, l'analyse d'extraits de mutants déficients pour la production de capsule a montré que ce polysaccharide correspondait à la capsule de K. pneumoniae. Son mode d'action consisterait à inhiber les interactions initiales entre bactéries et surface. Le suivi dans le temps de la formation de biofilm monospecies par K. pneumoniae avec la technique Biofilm Ring Test® a également permis de mettre en évidence un phénotype original. En effet, la détection initiale d'agrégats bactériens est suivie par une modification apparente de leur structure, qui serait liée à des changements de leur robustesse face aux forces d'aimantation magnétique. La présence d'ADN extracellulaire au sein de la matrice du biofilm pourrait jouer un rôle dans la survenue de ce phénotype comme l'indique les mesures effectuées en présence de l'enzyme DNAse I. En parallèle, l'observation de biofilm formés par K. pneumoniae dans des modèles expérimentaux cinétiques a mis en évidence des décrochements massifs de biomasse au cours de la maturation du biofilm, qui pourraient être corrélées aux modifications internes de robustesse de la matrice. L'ensemble de ces données permet de mieux caractériser les interactions intimes survenant à l'intérieur de biofilms constitués par K. pneumoniae et à terme de mieux caractériser et donc prévenir leur formation et dissémination. / Biofilms are defined as microbial communities adhering to biotic or abiotic surfaces and embedded in a self-produced extracellular matrix. Natural biofilms are composed of several microbial species and their interactions, synergistic or antagonistic, play important roles in development, composition and functioning of the consortia. Furthermore, the relationships often involve the production of antagonist molecules that impair competitors' growth or adhesion. The composition and evolution of the extracellular matrix plays also an important role in the biofilms' robustness. In this work, study of the interactions within biofilms formed by K. pneumoniae and S. epidermidis led to the isolation of a polysaccharide produced by K. pneumoniae able to inhibit the adherence to abiotic surfaces of several Gram-negative and Gram-positive bacterial species. The physico-chemical characterization of this high molecular weight molecule showed it was composed of galactose, glucose, rhamnose and glucuronic acid. This data, together with the analysis of extracts from capsule-deficient mutants, indicated that the capsule of K. pneumoniae was responsible for the biofilm inhibition phenotype, probably by inhibiting the initial interactions between bacteria and surface. By monitoring the formation of monospecies biofilm by K. pneumoniae with the Biofilm Ring Test® technique, we were able to detect an original phenotype. Indeed, detection of bacterial aggregates still occurred after a few hours of incubation but in a different way, probably related to changes of the biofilm robustness towards magnetic forces. The presence of extracellular DNA in the matrix of the biofilm is likely to play a role in the occurrence of this phenotype, as indicated by the assays performed in presence of the enzyme DNase I. At the same time, observations of biofilm formed by K. pneumoniae in kinetic experimental models showed massive detachment events during biofilm maturation, which could be correlated to changes in internal strength of the matrix. All these dtat contribute to a better characterization of the intimate interactions occuring within biofilms formed by K. pneumoniae and will ultimately lead to the development of efficient anti-biofilm strategies.

Escherichia coli is the best characterized bacterium; it grows rapidly, and it is easy to manipulate genetically. An increased knowledge about the physiology of this model organism will facilitate the development of engineered E.coli strains for applications such as production of biofuels and biofilm control. The aims of this work were the application of protein engineering to increase E. coli hydrogen production, the identification of the proteins regulating extracellular DNA production (eDNA), and the evaluation of the effect of the proteins synthesizing the signal 3'-5'-cyclic diguanylic acid (c-di-GMP) on biofilm formation. The Escherichia coli hydrogen production rate was increased 9 fold through random mutagenesis of fhlA. Variant FhlA133 (Q11H, L14V, Y177F, K245R, M288K, and I342F) enhances hydrogen production by increasing transcription of the four transcriptional units regulated by FhlA. The amino acid replacements E363G and L14G in FhlA increased hydrogen production 6 fold and 4 fold, respectively. The complete E. coli genome was screened to identify proteins that affect eDNA production. The nlpI, yfeC, and rna mutants increased eDNA production and the hns and rfaD mutants decreased eDNA production. Deletion of nlpI increases eDNA 3 fold while overexpression of nlpI decreases eDNA 16 fold. Global regulator H-NS is required for eDNA with E. coli since deletion of hns abolished eDNA production while overexpression of hns restored eDNA to 70 percent of the wild-type levels. Our results suggest that eDNA production in E. coli is related to direct secretion. Deletions of the genes encoding the diguanylate cyclases YeaI, YedQ, and YfiN increased swimming motility and eDNA as expected for low c-di-GMP levels. However, contrary to the current paradigm, early biofilm formation increased dramatically for the yeaI (30 fold), yedQ (12 fold), and yfiN (18 fold) mutants. Hence, our results suggest that c-di-GMP levels should be reduced for initial biofilm formation because motility is important for initial attachment to a surface.

Soilborne plant pathogens are responsible for many of the major crop diseases worldwide. However, plant root tips are generally resistant to pathogen infections. The goal of this dissertation research is to understand the mechanism of this natural resistance by testing the hypothesis that root caps and root border cells control the rhizosphere community through the biological products which they deliver to the soil. Specific objectives of this dissertation project are 1) identifying, isolating, and characterizing the genes important for border cell development and for root exudates delivery, and 2) analyzing the function of extracellular macromolecules in root exudates in root tip-fungal pathogen interaction. The expression of a primary cell wall synthesis gene, PsFut1, encoding Pisum sativum fucosyltransferase, was characterized during border cell production, and the impact of silencing this gene on border cell development was examined. Another gene, BRDgal1, encoding β-galactosidase, was identified and characterized in Pisum sativum during this study. It was shown that this β-galactosidase is specifically produced in and secreted from root border cells. The microarray transcriptional profiling in M. truncatula and mRNA differential display analysis in pea plants were carried out following the induction of border cell production to gain a broader understanding of the genes which potentially influence border cell development. In order to study the commonality of border cell production across different plant species, the expression of rcpme1, the marker gene for border cell production, was compared between the garden pea and a gymnosperm species, the Norway spruce (Picea abies). To accomplish the second objective, the focus of this study was shifted from border cell development to mucilaginous root exudates excreted by border cells and root cap cells. This resulted in a breakthrough in the understanding of the mechanisms of root tip resistance. The presence of extracellular DNA in the root mucilage was discovered and its requirement for root tip resistance to fungal infection was demonstrated. Extracellular proteins in the root mucilage were identified and they were shown to be also required for the root tip resistance to fungal infection. This work provided new insights into understanding plant defense mechanisms.

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

Neutrophil extracellular traps (NETs) are a link between infection and coagulation in sepsis. The major structural component of NETs is nucleosomes, consisting of DNA and histones. NETs not only act as a scaffold to trap platelets, but NET components also promote coagulation and impair fibrinolysis. Thus, removal of extracellular DNA by DNases may be a potential therapeutic strategy for sepsis. Since heparin is used for thromboprophylaxis in sepsis and may also be a potential anti-sepsis therapy, we investigated the mechanisms by which various forms of heparins modulate DNase function. There are two types of DNases in vivo: DNase I (produced by exocrine and endocrine glands) and DNase1L3 (secreted by immune cells). DNase I cleaves free DNA, whereas DNase1L3 preferentially cleaves DNA in complex with proteins such as histones. In this study, we investigated how DNase I and DNase1L3 activities are modulated by the following heparins: unfractionated heparin (UFH), enoxaparin (a low-molecular-weight heparin), Vasoflux (a low-molecular-weight, non-anticoagulant heparin), and fondaparinux (the pentasaccharide unit). Using agarose gel experiments, we showed that UFH, enoxaparin, and Vasoflux enhance the ability of DNase I to digest DNA-histone complexes (presumably by displacing DNA from histones), whereas fondaparinux does not. These findings are consistent with the KD values of the binding of heparin variants to histones, with fondaparinux having >1000-fold lower affinity for histones compared to the other heparins. Taken together, our data suggests that the ability of heparin to enhance DNase I-mediated digestion of DNA-histone complexes is size-dependent and independent of the pentasaccharide region of heparin. With respect to DNase1L3, we observed that it is able to digest histone-bound DNA, and that all heparins, except fondaparinux, inhibited DNase1L3-mediated digestion of histone-bound DNA. Next, we visualized the degradation of NETs by fluorescence microscopy. DNase I (± heparin variants) completely degraded NETs, presumably by digesting extracellular chromatin at histone-free linker regions, thereby releasing nucleosome units. DNase1L3 also degraded NETs, but not as effectively as DNase I, and was inhibited by all heparins except fondaparinux. Finally, we showed that DNase I levels are decreased and DNase1L3 levels are elevated in septic patients. Taken together, our findings demonstrate that heparin modulates the function of DNases, and that endogenous DNase levels are altered in sepsis pathophysiology. / Thesis / Master of Science (MSc) / Sepsis, a life-threatening condition due to hyperactivation of the immune system in response to infection, results in widespread inflammation and blood clotting. During sepsis, immune cells release sticky strands of DNA that block blood vessels and damage organs. Two different enzymes in the blood (DNase I and DNase1L3) can digest these DNA strands, and may represent a new class of anti-sepsis drugs. Our goal was to determine how heparins, commonly used blood thinners, alter the function of these enzymes. We found that (a) larger-sized heparins improved the activity of DNase I towards DNA-histone complexes and do not require any specific portion of heparin, (b) DNase I is more efficient than DNase1L3 in digesting DNA strands released from immune cells, and (c) levels of DNase I and DNase1L3 are altered in septic patients. Taken together, our studies provide new insights into how these enzymes function.

The fate of DNA from dead cells is an important issue when interpreting results from root canal infections analysed by the PCR technique. DNA from dead bacterial cells is known to be detectable long time after cell death and its stability is dependent on many different factors. This work investigated factors found in the root canal that could affect the recovery of microbial DNA. In an ex vivo experiment, DNA from non-viable gram-positive Enterococcus faecalis was inoculated in instrumented root canals and recovery of DNA was assessed by PCR over a two-year period. DNA was still recoverable two years after cell death in 21/25 teeth. The fate of DNA from the gram-negative bacteria Fusobacterium nucleatum and the gram-positive Peptostreptococcus anaerobius was assessed in vitro. DNA from dead F. nucleatum and P. anaerobius could be detected by PCR six months post cell death even though it was clear that the DNA was released from the cells due to lost of cell wall integrity during the experimental period. The decomposition rate of extracellular DNA was compared to cell-bound and it was evident that DNA still located inside the bacterium was much less prone to decay than extracellular DNA. Free (extracellular) DNA is very prone to decay in a naked form. Binding to minerals is known to protect DNA from degradation. The fate of extracellular DNA was assessed after binding to ceramic hydroxyapatite and dentine. The data showed that free DNA, bound to these materials, was protected from spontaneous decay and from enzymatic decomposition by nucleases. The main conclusions from this thesis were: i) DNA from dead bacteria can be detected by PCR years after cell death ex vivo and in vitro. ii) Cell-bound DNA is less prone to decomposition than extracellular DNA. iii) DNA is released from the bacterium some time after cell death. iv) Extracellular DNA bound to hydroxyapatite or dentine is protected from spontaneous decomposition and enzymatic degradation.

Introduction: Extracellular DNA (ecDNA) is a common component of blood plasma. Increased levels of ecDNA in plasma can be found in some autoimmune diseases like systemic lupus erythematosus (SLE), rheumatoid arthritis or celiac disease which are associated with inflammatory processes. These diseases are also associated with an increased risk of osteoporosis. Bone is a dynamic structure undergoing constant modelling caused by osteoblasts, osteocytes and osteoclasts. Shifting their equilibrium can lead to pathological conditions such as osteoporosis. In this thesis we focused on elucidating whether ecDNA, an inflammatory agent with proven immunoregulatory effects can alter differentiation potential of monocytes and alternatively lead to osteoclastogenesis via TLR9. Material and methods: We obtained monocytes from peripheral blood of healthy donors and cultivated them with four types of ODNs control (CO), stimulatory (ST), inhibitory (INH, telomeric (TLM) with phosphodiester (-pO) or phosphorothioate (-pS) backbone for two weeks to establish their effect on differentiation potential of monocytes into osteoclasts. Osteoclastogenesis was evaluated by number of yielded osteoclasts observed on a light microscope. To establish the effect of ODNs on osteoclast activity samples were analysed by qPCR for...

Histones are chromatin-associated proteins localized to the nucleus. However, extracellular histones are present in biofluids from healthy individuals and become elevated under disease conditions, such as neurodegeneration and cancer. Hence, extracellular histones may have important biological functions in healthy and diseased states, which are not understood. Histones have been reported in the proteomes of extracellular vesicles (EVs), including microvesicles and exosomes. The main aim of this thesis was to determine whether or not extracellular histones are secreted via EVs/exosomes. In an initial study (Paper I), I optimized methods for human embryonic kidney (HEK293) cell culture, transfection and protein detection using western blotting. In the main study (Paper II), I used oligodendrocyte cell lines (rat OLN-93 and mouse Oli-neu) to investigate the localization of histones to EVs. Western blotting of EVs purified from OLN-93 cell-conditioned media confirmed the presence of linker and core histones in them. Immunolocalization and transmission electron microscopy confirmed that histones are localized to EVs, as well as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). This suggests that histones are secreted via the MVB/exosome pathway. Localization of histones in EVs was investigated by biochemical/proteolytic degradation and purification followed by western blotting. Surprisingly, histones were associated with the membrane but not the luminal fraction. Overexpression of tagged histones in HEK293 cells confirmed their conserved, membrane localization. OLN-93 cell EVs contained both double stranded and single stranded DNA but nuclease and protease digestion showed that the association of histones and DNA with EVs was not interdependent. The abundance of histones in EVs was not affected by differentiation in Oli-neu cells. However, histone release was upregulated as an early response to cellular stress in OLN-93 cells and occurred before the release of markers of stress including heat shock proteins. Interestingly, a notable upregulation in secretion of small diameter (50-100 nm) EVs was observed following heat stress, suggesting that a sub-population of vesicles may be involved specifically in histone secretion in response to stress. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) as a possible mechanism underlying increased histone secretion. In Paper III, I developed methods to quantify extracellular histone proteins in human ascites samples from ovarian cancer patients.   In summary, we show for the first time that membrane-associated histones are secreted via the MVB/exosome pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.