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The mammalian immune system has developed diverse strategies to protect the gastrointestinal tract, as this tissue locale represents a huge absorptive surface and is susceptible to microbial breach. Paradoxically, one key aspect of this protective strategy is the maintenance of selected commensal microorganisms. These commensals serve essential roles in digestion, interfere with pathogenic microbial invasion and stimulate development of the host immune system. Therefore, immune responses which deplete these commensal populations are detrimental to the host. One effective intestinal immune response which selectively promotes the survival of commensals is production of antibodies of the IgA isotype which bind to bacteria without triggering inflammatory cytokines. Proteins of the tumor necrosis factor (TNF) family such as Lymphotoxin and BAFF contribute to the induction of IgA responses. Lymphotoxin is required for generation and organization of most organized lymphoid tissues, where B cell differentiation occurs, while BAFF is necessary for B cell survival and induces B cells to produce IgA. In this thesis, I describe work I have done in examining the roles of the TNF family members Lymphotoxin, BAFF and two related TNF family member cytokines, LIGHT and APRIL, in the regulation of IgA production in mice and in humans. Specifically, LIGHT over-expression drives immense production of IgA, leading to renal deposition of immune complexes in mice. Similar to LIGHT, BAFF over-expression drives increases in IgA production in the intestine, however I have shown that the effects of the BAFF pathway on IgA hyper-production are independent of LIGHT activity. Secondly, examining the phenotype of BAFF-over-expressing mice, I have shown that this phenotype resembles human IgA nephropathy (IgAN) and is dependent on intestinal commensals. Finally, I have described a lymphotoxin-dependent chemokine system in the intestinal lamina propria that could be responsible for organizing cells for the development of IgA responses in this mucosal site.

<p>Resistance to antimicrobials is an increasing problem in the world of today, and develops faster than man can counter. It is therefore of importance to study metabolic pathways in order to develop new antibiotics, but also to understand how resistance spreads and stabilizes in microbial populations.</p><p>The commensal flora could be an important factor in the spread of antimicrobial resistance, as drugs aimed at other targets also hit the harmless commensal bacteria. If stable resistance develops in such a population, it could seriously impair a later treatment with the same drug. After a treatment with the macrolide clarithromycin, resistance to this antibiotic increased markedly in the untargeted throat flora, and resistance levels did not recede until at least one year later. </p><p>Another example of stable resistance can also be seen in sulfonamide resistant <i>Streptococcus pyogenes</i>. Sequence determinations of the dihydropteroate synthase (<i>dhps</i>) gene conferring this resistance revealed a mosaic organisation implying that the it had been brought there by horizontal transfer. Molecular characterization of this gene showed that the sulfonamide resistance was due to mutations of structurally important amino acids in position 65 and 213.</p><p>The folate synthesis pathway has potential for being exploited further as a drug target. One possible new drug target is hydroxymethyl-dihydropterin pyrophosphokinase (<i>hppk</i>). In the malaria parasite <i>Plasmodium falciparum</i> this enzyme is part of a polyfunctional entity, also encoding <i>dhps</i>. The HPPK part can be separated from DHPS, but that the opposite is not possible. The PfHPPK has two insertions: one also present in other plasmodia, and one apparently unique to <i>P. falciparum</i>. Both are crucial for enzyme activity.</p><p>To further characterize HPPK, we developed a spectrophotometric activity assay and a method to measure substrate channelling of hydroxymethyl-dihydropterin diphosphate.</p>

Resistance to antimicrobials is an increasing problem in the world of today, and develops faster than man can counter. It is therefore of importance to study metabolic pathways in order to develop new antibiotics, but also to understand how resistance spreads and stabilizes in microbial populations. The commensal flora could be an important factor in the spread of antimicrobial resistance, as drugs aimed at other targets also hit the harmless commensal bacteria. If stable resistance develops in such a population, it could seriously impair a later treatment with the same drug. After a treatment with the macrolide clarithromycin, resistance to this antibiotic increased markedly in the untargeted throat flora, and resistance levels did not recede until at least one year later. Another example of stable resistance can also be seen in sulfonamide resistant Streptococcus pyogenes. Sequence determinations of the dihydropteroate synthase (dhps) gene conferring this resistance revealed a mosaic organisation implying that the it had been brought there by horizontal transfer. Molecular characterization of this gene showed that the sulfonamide resistance was due to mutations of structurally important amino acids in position 65 and 213. The folate synthesis pathway has potential for being exploited further as a drug target. One possible new drug target is hydroxymethyl-dihydropterin pyrophosphokinase (hppk). In the malaria parasite Plasmodium falciparum this enzyme is part of a polyfunctional entity, also encoding dhps. The HPPK part can be separated from DHPS, but that the opposite is not possible. The PfHPPK has two insertions: one also present in other plasmodia, and one apparently unique to P. falciparum. Both are crucial for enzyme activity. To further characterize HPPK, we developed a spectrophotometric activity assay and a method to measure substrate channelling of hydroxymethyl-dihydropterin diphosphate.

The Neisseria genus includes pathogens, Neisseria gonorrhoeae (Ngo) and Neisseria meningitidis, as well as commensals. Ngo, the cause of gonorrhea, induces massive inflammation but a surprising lack of adaptive immune responses. We have observed that Ngo can inhibit both T cell activation and dendritic cell maturation through interaction with the host expressed co-inhibitory receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Therefore, I wondered whether B cells may also be affected in this manner. Herein, I examine primary human B cell responses to infection with Ngo, as well as the other Neisseria species. B cells infected with Ngo show no sign of inhibition, regardless of their ability to bind CEACAM1, instead responding to gonococci with robust activation and proliferation. There are distinct subsets of B cells found in the periphery and, intriguingly, the IgM memory B cell subset expand and produce polyreactive IgM in response to goncoccal infection. These cells are innate in function, producing low affinity, polyclonal IgM that is protective against bacterial and fungal dissemination. This effect was broadly specific for Neisseria sp., as B cell infection with all commensal Neisseria species examined induced innate B cell responses. Curiously, meningococcal strains avoid inducing the innate B cell responses, making it enticing to hypothesize that its avoidance of such an ancient immune response may contribute to its ability to cause disease in humans. Finally, I tested whether gonococcal Opa protein binding to CEACAM1 affects primary human B cell activation, and show that no inhibition was observed. This absence of co-inhibitory function of neisserial-bound CEACAM1 may reflect inherent differences between distinctive cell types. Combined, the results in this thesis contribute new insight regarding the poorly characterized human IgM memory B cells, as well as to the function of CEACAM1 in lymphocytes.

No description available.

The Neisseria genus includes pathogens, Neisseria gonorrhoeae (Ngo) and Neisseria meningitidis, as well as commensals. Ngo, the cause of gonorrhea, induces massive inflammation but a surprising lack of adaptive immune responses. We have observed that Ngo can inhibit both T cell activation and dendritic cell maturation through interaction with the host expressed co-inhibitory receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Therefore, I wondered whether B cells may also be affected in this manner. Herein, I examine primary human B cell responses to infection with Ngo, as well as the other Neisseria species. B cells infected with Ngo show no sign of inhibition, regardless of their ability to bind CEACAM1, instead responding to gonococci with robust activation and proliferation. There are distinct subsets of B cells found in the periphery and, intriguingly, the IgM memory B cell subset expand and produce polyreactive IgM in response to goncoccal infection. These cells are innate in function, producing low affinity, polyclonal IgM that is protective against bacterial and fungal dissemination. This effect was broadly specific for Neisseria sp., as B cell infection with all commensal Neisseria species examined induced innate B cell responses. Curiously, meningococcal strains avoid inducing the innate B cell responses, making it enticing to hypothesize that its avoidance of such an ancient immune response may contribute to its ability to cause disease in humans. Finally, I tested whether gonococcal Opa protein binding to CEACAM1 affects primary human B cell activation, and show that no inhibition was observed. This absence of co-inhibitory function of neisserial-bound CEACAM1 may reflect inherent differences between distinctive cell types. Combined, the results in this thesis contribute new insight regarding the poorly characterized human IgM memory B cells, as well as to the function of CEACAM1 in lymphocytes.

Campylobacter jejuni is a major cause of bacterial gastroenteritis in the developed world; however, many aspects of its biology remain poorly understood, including its colonization of the mucus layer lining the gastrointestinal tract. In this study, we utilized microarray transposon tracking to compile a list of 195 genes essential for the growth of C. jejuni in vitro under microaerophilic conditions. Then we characterized C. jejuni growing in an extracted intestinal mucus medium. We found that C. jejuni will grow efficiently in a medium comprised of either chick and piglet intestinal mucus, and that these media have a dramatic impact on its transcriptome. Within the genes identified as differentially expressed during growth in a mucus medium, we identified a single operon, (cj0481-cj0490), which we have subsequently characterized as being responsible for both the uptake and metabolism of L-fucose. This represents the first observation of carbohydrate metabolism by the otherwise asaccharolytic C. jejuni. We further found that the inability to utilize L-fucose puts C. jejuni at a competitive disadvantage when colonizing the piglet intestine, but not the chick cecum. Finally, we examined C. jejuni’s ability to utilize mucins as a carbon source while growing within the mucus layer. We found that despite mucins being a major source of L-fucose and amino acids within the intestine, C. jejuni has a minimal ability to degrade and utilize mucins on its own. However, close proximity to mucolytic bacteria within the microbiota of the intestine, allows for increased C. jejuni growth. Together, this paints the picture of an organism that is well adapted to survival within the mucus lining of the intestine and establishing itself as part of the intestinal microbiota.

The mammalian immune system has developed diverse strategies to protect the gastrointestinal tract, as this tissue locale represents a huge absorptive surface and is susceptible to microbial breach. Paradoxically, one key aspect of this protective strategy is the maintenance of selected commensal microorganisms. These commensals serve essential roles in digestion, interfere with pathogenic microbial invasion and stimulate development of the host immune system. Therefore, immune responses which deplete these commensal populations are detrimental to the host. One effective intestinal immune response which selectively promotes the survival of commensals is production of antibodies of the IgA isotype which bind to bacteria without triggering inflammatory cytokines. Proteins of the tumor necrosis factor (TNF) family such as Lymphotoxin and BAFF contribute to the induction of IgA responses. Lymphotoxin is required for generation and organization of most organized lymphoid tissues, where B cell differentiation occurs, while BAFF is necessary for B cell survival and induces B cells to produce IgA. In this thesis, I describe work I have done in examining the roles of the TNF family members Lymphotoxin, BAFF and two related TNF family member cytokines, LIGHT and APRIL, in the regulation of IgA production in mice and in humans. Specifically, LIGHT over-expression drives immense production of IgA, leading to renal deposition of immune complexes in mice. Similar to LIGHT, BAFF over-expression drives increases in IgA production in the intestine, however I have shown that the effects of the BAFF pathway on IgA hyper-production are independent of LIGHT activity. Secondly, examining the phenotype of BAFF-over-expressing mice, I have shown that this phenotype resembles human IgA nephropathy (IgAN) and is dependent on intestinal commensals. Finally, I have described a lymphotoxin-dependent chemokine system in the intestinal lamina propria that could be responsible for organizing cells for the development of IgA responses in this mucosal site.

Campylobacter jejuni is a major cause of bacterial gastroenteritis in the developed world; however, many aspects of its biology remain poorly understood, including its colonization of the mucus layer lining the gastrointestinal tract. In this study, we utilized microarray transposon tracking to compile a list of 195 genes essential for the growth of C. jejuni in vitro under microaerophilic conditions. Then we characterized C. jejuni growing in an extracted intestinal mucus medium. We found that C. jejuni will grow efficiently in a medium comprised of either chick and piglet intestinal mucus, and that these media have a dramatic impact on its transcriptome. Within the genes identified as differentially expressed during growth in a mucus medium, we identified a single operon, (cj0481-cj0490), which we have subsequently characterized as being responsible for both the uptake and metabolism of L-fucose. This represents the first observation of carbohydrate metabolism by the otherwise asaccharolytic C. jejuni. We further found that the inability to utilize L-fucose puts C. jejuni at a competitive disadvantage when colonizing the piglet intestine, but not the chick cecum. Finally, we examined C. jejuni’s ability to utilize mucins as a carbon source while growing within the mucus layer. We found that despite mucins being a major source of L-fucose and amino acids within the intestine, C. jejuni has a minimal ability to degrade and utilize mucins on its own. However, close proximity to mucolytic bacteria within the microbiota of the intestine, allows for increased C. jejuni growth. Together, this paints the picture of an organism that is well adapted to survival within the mucus lining of the intestine and establishing itself as part of the intestinal microbiota.