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Thesis advisor: Babak Momeni / Rothia mucilaginosa is a gram-positive bacterium that is found in the human oral microbiota. Under normal circumstances, it exhibits a commensal relationship with humans. However, in immunocompromised patients and those with medical implants, these bacteria can pose a serious health risk. In this thesis I outline experiments performed to reveal more about the normal role of R. mucilaginosa within the oral microbiota in the hopes of better understanding its transition to virulence. R. mucilaginosa was grown alongside several of its major co-inhabitants in the oral microbiota in pairwise fashion on solid growth media to search for signs of interspecies interactions. After initial screening, those pairs that showed consistent patterns of interaction were selected for further analysis via fluorescence microscopy. A consistent interaction pattern was confirmed between R. mucilaginosa and Streptococcus gordonii. Interactions were also noted between Corynebacterium durum and R. mucilaginosa as well as between Corynebacterium durum and Neisseria elongata. / Thesis (BS) — Boston College, 2018. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Scholar of the College. / Discipline: Biology.

The mammalian intestinal tract is highly colonised with a diverse bacterial microbiota. The importance of this bacterial presence is now recognised; these bacteria contribute both to the nutritional status of their hosts and are required for the development of a competent immune system. In addition, the composition of the microbiota is likely important in influencing how the immune system reacts to antigens, as the presence of specific bacterial species can promote differentiation of T cells towards specific effector or regulatory fates. Though the ability of the microbiota to influence infections with bacterial and viral agents has been reported, whether the microbiota can affect a parasitic infection has not yet been described. It is likely, due to millions of years of co-evolution within mammalian hosts, that helminths have co-opted mechanisms of the microbiota to manipulate the host’s immune system, in order to promote their own survival. In this thesis, the immune parameters required for expulsion of a primary infection with the murine gastrointestinal helminth parasite Heligmosomoides polygyrus are examined, and whether the microflora influence these parameters in order to modulate susceptibility is explored. Firstly, a multiparameter analysis of H. polygyrus infection was performed in two mouse strains which differ in susceptibility to a primary infection, to identify both immune factors and microbial populations which correlate with susceptibility to infection. BALB/c mice exhibited a stronger T helper (Th)2-type response to H. polygyrus excretory-secretory antigen (HES), produced high numbers of intestinal granulomas following infection and were better able to expel H. polygyrus, whereas the more susceptible C57BL/6 strain produced higher levels of inflammatory Th1 cytokines in response to HES. High levels of duodenal Lactobacillus/Lactococcus species positively correlated with H. polygyrus persistence within the BALB/c host, as did high levels of Enterobacteriaceae in the C57BL/6 host. Furthermore, the abundance of both of these bacterial groups was elevated in H. polygyrus-infected C57BL/6 mice compared to naïve controls, and mice given antibiotic treatment to diminish these groups were rendered more resistant to H. polygyrus. Infection persistence was prolonged in BALB/c mice which were administered the single species Lactobacillus taiwanensis, a normal component of the microbiota. Next, the impact of a loss of microbiota signalling by immune cells during H. polygyrus infection was examined, through the use of Toll-like receptor (TLR)- and TLR adaptor protein-deficient mice. MyD88-/- mice were more resistant to H. polygyrus than wildtype (Wt) C57BL/6 mice and exhibited increased granuloma formation: phenotypes which were not recapitulated by individual deficiencies in TLR2, TLR4, TLR5 or TLR9, and not seen in TRIF-/- mice. When MyD88-/- mice were additionally deficient in TRIF, the increased granuloma formation phenotype of MyD88-/- mice was lost. Whether MyD88 controls susceptibility to H. polygyrus infection via a TLR-independent mechanism, and how MyD88 and TRIF antagonistically contribute to granuloma formation remains to be resolved. Finally, the importance of TGF-β signalling during H. polygyrus infection was examined, using mice deficient in TGF-β signalling specifically in T cells (TGF-βRII DN mice). These mice were more susceptible to H. polygyrus than Wt C57BL/6 mice, which was explained by an attenuated Th2 response to infection accompanied by exuberant IFN-γ production. The increased susceptibility to H. polygyrus was lost in TGF-βRII DN IFN-γ-/-mice, in which Th2 responsiveness was partly restored. These data highlight the importance of both immune components, particularly IFN-γ, which promotes susceptibility, and the presence of specific intestinal bacterial populations in controlling the persistence of a primary H. polygyrus infection.

Horizontal gene transfer (HGT) is one of the man driving forces of evolution in prokaryotes, and can also promote within-strain variation of bacterial species. The genomes of three previously sequenced Bacteroides fragilis strains, NCTC9343, 638R and YCH46 displayed evidence of extensive HGT, demonstrated by the presence of 28 divergent capsular polysaccharide-associated biosynthesis loci. The genomes of a further four B. fragilis strains, LS66, GNAB92, RD48 and BE1 were sequenced and analysed. Genomic comparisons of BE1 and GNAB92 with NCTC9343, 638R and YCH46 identified ten new divergent polysaccharide biosynthesis loci. There is consequently, the potential to express 38 different polysaccharides amongst these five strains. Such a high level of variation in capsular polysaccharides, in so few strains has not been previously observed. HGT has occurred in B. fragilis despite the presence of diverse Restriction-Modification systems. The genome sequences of NCTC9343 and 638R contained a gene, ubb, the product of which, BfUbb, has 63% identity to human ubiquitin. The closest DNA sequence homology is to a migratory grasshopper entomopox virus, suggesting acquisition of this gene was via inter-kingdom HGT. The ubb gene was also identified in the newly sequenced genomes of B. fragilis strains LS66 and RD48. BfUbb had a predicted signal sequence; both full-length and processed forms were detected in whole-cell extracts by Western blot analysis. The inability to detect BfUbb in periplasmic extracts isolated from a B. fragilis strain containing an ubb signal sequence deletion construct, supported the periplasmic location of the processed form of the protein and the requirement for the signal peptide for transport from the cytoplasm. BfUbb was also detected in concentrated supernatants containing outer membrane vesicles, suggesting a mechanism by which the protein may be delivered to the host. This is the first example of ubiquitin being produced by a prokaryote. Transduction by bacteriophages is one mechanism by which horizontal gene transfer can occur and can also be a useful tool for genetic manipulation. Fifteen potentially new B. fragilis-specific bacteriophages were isolated from filtered sewage and characterised by phage titres and restriction endonuclease cleavage profiles. Of the fifteen, seven phages appeared to be different to the previously identified phage ФED01. None of the bacteriophages were capable of transduction. B. fragilis is a predominant member of the gastrointestinal microbiota. To survive within this specific niche, bacteria must successfully compete with other organisms for nutrients and space, and withstand attacks from bacteriophages. HGT may aid in the survival of B. fragilis as a commensal.

The commensal intestinal microbiota contributes substantially to intestinal development in the early neonatal period by mechanisms that are not yet elucidated but could contribute to novel strategies to improve intestinal health. A series of gnotobiotic experiments using isolator-reared caesarian section-derived piglets inoculated at 1 d of age with selected bacteria and euthanized at 14 or 15 days of age were performed to investigate intestinal morphology, inflammation and digestive function. In Experiment 1, piglets were maintained germfree (GF), mono-associated with Escherichia coli (EC), mono-associated with Lactobacillus fermentum (LF) or conventionalized with sow feces (CV). Increased (P<0.05) gene expression of Fas ligand (FasL) and tumor necrosis factor (TNF?) in EC and CV as compared to LF and GF pigs coincided with increased apoptotic and proliferative activity. Toll-like receptors (TLR) 2, 4 and 9 were differentially regulated (P<0.05) by colonizing species. In Experiment 2 using the same animals as Exp. 1, increased turnover of brush border enzymes was indicated by reduced (P<0.05) specific activity of aminopeptidase N (APN) and lactase (LPH) and increased expression of APN in CV and EC as compared to GF and LF pigs. Reduced enzyme activity to gene expression ratio corresponded with an in vitro assay of microbial inactivation of APN. In Experiment 3, probiotic Lactobacillus sp., L3777, and Bifidobacteria sp., B5445, did not induce expression of inflammatory cytokines in mono-association but di-association with E. coli increased (P<0.05) inflammatory and anti-inflammatory mediators and resulted in a high rate of sepsis (50%) relative to E. coli mono-association. Induced expression of inflammatory cytokines by commensal bacteria through TLR and other means, appear to play a substantial role in microbially-induced enterocyte turnover. Enterocyte immaturity did not account for reduced enzyme activity associated with inflammation as increased expression of APN in response to microbial colonization was observed, suggesting a host response pathway enabling effective competition with the intestinal microbiota for available peptide nutrients. Probiotic bacteria were relatively benign in mono-association but may have facilitated increased translocation of <i>E. coli</i> in di-association. Gnotobiotic animal models are essential to demonstrate outcomes of host response characterized by communication among numerous cell types, although are of significant technical difficulty.

The commensal intestinal microbiota contributes substantially to intestinal development in the early neonatal period by mechanisms that are not yet elucidated but could contribute to novel strategies to improve intestinal health. A series of gnotobiotic experiments using isolator-reared caesarian section-derived piglets inoculated at 1 d of age with selected bacteria and euthanized at 14 or 15 days of age were performed to investigate intestinal morphology, inflammation and digestive function. In Experiment 1, piglets were maintained germfree (GF), mono-associated with Escherichia coli (EC), mono-associated with Lactobacillus fermentum (LF) or conventionalized with sow feces (CV). Increased (P<0.05) gene expression of Fas ligand (FasL) and tumor necrosis factor (TNF?) in EC and CV as compared to LF and GF pigs coincided with increased apoptotic and proliferative activity. Toll-like receptors (TLR) 2, 4 and 9 were differentially regulated (P<0.05) by colonizing species. In Experiment 2 using the same animals as Exp. 1, increased turnover of brush border enzymes was indicated by reduced (P<0.05) specific activity of aminopeptidase N (APN) and lactase (LPH) and increased expression of APN in CV and EC as compared to GF and LF pigs. Reduced enzyme activity to gene expression ratio corresponded with an in vitro assay of microbial inactivation of APN. In Experiment 3, probiotic Lactobacillus sp., L3777, and Bifidobacteria sp., B5445, did not induce expression of inflammatory cytokines in mono-association but di-association with E. coli increased (P<0.05) inflammatory and anti-inflammatory mediators and resulted in a high rate of sepsis (50%) relative to E. coli mono-association. Induced expression of inflammatory cytokines by commensal bacteria through TLR and other means, appear to play a substantial role in microbially-induced enterocyte turnover. Enterocyte immaturity did not account for reduced enzyme activity associated with inflammation as increased expression of APN in response to microbial colonization was observed, suggesting a host response pathway enabling effective competition with the intestinal microbiota for available peptide nutrients. Probiotic bacteria were relatively benign in mono-association but may have facilitated increased translocation of <i>E. coli</i> in di-association. Gnotobiotic animal models are essential to demonstrate outcomes of host response characterized by communication among numerous cell types, although are of significant technical difficulty.

<p>The humoral immune system plays a critical role in the clearance of numerous pathogens. In the setting of HIV-1 infection, the virus infects, integrates its genome into the host's cells, replicates, and establishes a reservoir of virus-infected cells. The initial antibody response to HIV-1 infection is targeted to non-neutralizing epitopes on HIV-1 Env gp41, and when a neutralizing response does develop months after transmission, it is specific for the autologous founder virus and the virus escapes rapidly. After continuous waves of antibody mediated neutralization and viral escape, a small subset of infected individuals eventually develop broad and potent heterologous neutralizing antibodies years after infection. In this dissertation, I have studied the ontogeny of mucosal and systemic antibody responses to HIV-1 infection by means of three distinct aims: 1. Determine the origin of the initial antibody response to HIV-1 infection. 2. Characterize the role of restricted VH and VL gene segment usage in shaping the antibody response to HIV-1 infection. 3. Determine the role of persistence of B cell clonal lineages in shaping the mutation frequencies of HIV-1 reactive antibodies. </p><p>After the introduction (Chapter 1) and methods (Chapter 2), Chapter 3 of this dissertation describes a study of the antibody response of terminal ileum B cells to HIV-1 envelope (Env) in early and chronic HIV-1 infection and provides evidence for the role of environmental antigens in shaping the repertoire of B cells that respond to HIV-1 infection. Previous work by Liao et al. demonstrated that the initial plasma cell response in the blood to acute HIV-1 infection is to gp41 and is derived from a polyreactive memory B cell pool. Many of these antibodies cross-reacted with commensal bacteria, Therefore, in Chapter 3, the relationship of intestinal B cell reactivity with commensal bacteria to HIV-1 infection-induced antibody response was probed using single B cell sorting, reverse transcription and nested polymerase chain reaction (RT- PCR) methods, and recombinant antibody technology. The dominant B cell response in the terminal ileum was to HIV-1 envelope (Env) gp41, and 82% of gp41- reactive antibodies cross-reacted with commensal bacteria whole cell lysates. Pyrosequencing of blood B cells revealed HIV-1 antibody clonal lineages shared between ileum and blood. Mutated IgG antibodies cross-reactive with both Env gp41 and commensal bacteria could also be isolated from the terminal ileum of HIV-1 uninfected individuals. Thus, the antibody response to HIV-1 can be shaped by intestinal B cells stimulated by commensal bacteria prior to HIV-1 infection to develop a pre-infection pool of memory B cells cross-reactive with HIV-1 gp41.</p><p>Chapter 4 details the study of restricted VH and VL gene segment usage for gp41 and gp120 antibody induction following acute HIV-1 infection; mutations in gp41 lead to virus enhanced neutralization sensitivity. The B cell repertoire of antibodies induced in a HIV-1 infected African individual, CAP206, who developed broadly neutralizing antibodies (bnAbs) directed to the HIV-1 envelope gp41 membrane proximal external region (MPER), is characterized. Understanding the selection of virus mutants by neutralizing antibodies is critical to understanding the role of antibodies in control of HIV-1 replication and prevention from HIV-1 infection. Previously, an MPER neutralizing antibody, CAP206-CH12, with the binding footprint identical to that of MPER broadly neutralizing antibody 4E10, that like 4E10 utilized the VH1-69 and VK3-20 variable gene segments was isolated from this individual (Morris et al., 2011). Using single B cell sorting, RT- PCR methods, and recombinant antibody technology, Chapter 4 describes the isolation of a VH1-69, Vk3-20 glycan-dependent clonal lineage from CAP206, targeted to gp120, that has the property of neutralizing a neutralization sensitive CAP206 transmitted/founder (T/F) and heterologous viruses with mutations at amino acids 680 or 681 in the MPER 4E10/CH12 binding site. These data demonstrate sites within the MPER bnAb epitope (aa 680-681) in which mutations can be selected that lead to viruses with enhanced sensitivity to autologous and heterologous neutralizing antibodies. </p><p>In Chapter 5, I have completed a comparison of evolution of B cell clonal lineages in two HIV-1 infected individuals who have a predominant VH1-69 response to HIV-1 infection--one who produces broadly neutralizing MPER-reactive mAbs and one who does not. Autologous neutralization in the plasma takes ~12 weeks to develop (Gray et al., 2007; Tomaras et al., 2008b). Only a small subset of HIV-1 infected individuals develops high plasma levels of broad and potent heterologous neutralization, and when it does occur, it typically takes 3-4 years to develop (Euler et al., 2010; Gray et al., 2007; 2011; Tomaras et al., 2011). The HIV-1 bnAbs that have been isolated to date have a number of unusual characteristics including, autoreactivity and high levels of somatic hypermutations, which are typically tightly regulated by immune control mechanisms (Haynes et al., 2005; 2012b; Kwong and Mascola, 2012; Scheid et al., 2009a). The VH mutation frequencies of bnAbs average ~15% but have been shown to be as high as 32% (reviewed in Mascola and Haynes, 2013; Kwong and Mascola, 2012). The high frequency of somatic hypermutations suggests that the B cell clonal lineages that eventually produce bnAbs undergo high-levels of affinity maturation, implying prolonged germinal center (GC) reactions and high levels of T cell help. To study the duration of HIV-1- reactive B cell clonal persistence, HIV-1 reactive and non HIV-1- reactive B cell clonal lineages were isolated from an HIV-1 infected individual that produces bnAbs, CAP206, and an HIV-1 infected individual who does not produce bnAbs, 004-0. Single B cell sorting, RT-PCR and recombinant antibody technology was used to isolate and produce monoclonal antibodies from multiple time points from each individual. B cell sequences clonally related to mAbs isolated by single cell PCR were identified within pyrosequences of longitudinal samples of these two individuals. Both individuals produced long-lived B cell clones that persisted from 0-232 weeks in CAP206, and 0-238 weeks in 004-0. The average length of persistence of clones containing members isolated from two separate time points was 91.5 weeks both individuals. Examples of the continued evolution of clonal lineages were observed in both the bnAb and non-bnAb individual. These data indicated that the ability to generate persistent and evolving B cell clonal lineages occurs in both bnAb and non-bnAb individuals, suggesting that some alternative host or viral factor is critical for the generation of highly mutated broadly neutralizing antibodies. </p><p> Together the studies described in Chapter 3-5 show that multiple factors influence the antibody response to HIV-1 infection. The initial antibody response to HIV-1 Env gp41 can be shaped by a B cell response to intestinal commensal bacteria prior to HIV-1 infection. VH and VL gene segment restriction can impact the B cell response to multiple HIV-1 antigens, and virus escape mutations in the MPER can confer enhanced neutralization sensitivity to autologous and heterologous antibodies. Finally, the ability to generate long-lived HIV-1 clonal lineages in and of itself does not confer on the host the ability to produce bnAbs.</p> / Dissertation

An investigation was conducted to determine the contribution of the gastrointestinal microbiota to variation in bioefficacy of methionine sources and the interrelationship between intestinal microbiota and cereal grain type with respect to gastrointestinal physiology. Apparent gastrointestinal absorption of DL-methionine (MET) and 2-hydroxy-4-methylthiobutanoic acid (MHA-FA), post-weaning intestinal morphology, digestive physiology, mucin dynamics and digesta flow were studied in a series of experiments using conventional and gnotobiotic pigs. At 14 d of age, sow - reared conventional (CON) pigs and isolator - reared monoassociated gnotobiotic pigs (EF) were weaned to corn or wheat/barley based diets supplemented with MET or MHA-FA. At 24 d of age, after an overnight fast, pigs were fed experimental diet supplemented with 107 Bq of either 3H-L-MET or 3H-L-MHA-FA per kg of feed and chromic oxide (0.5% wt/wt). Pigs were killed 3 h after consuming the meal to collect digesta and tissue samples from the stomach and along the small intestinal (SI) length. Conventional pigs fed a wheat/barley-based diet had increased (P < 0.05) total aerobes, whereas supplementation with MHA-FA increased (P < 0.05) total aerobes and lactobacilli populations in proximal SI. Among the gnotobiotic pigs, 8 pigs (2 isolators) were monoassociated with a bacteria closely related to <i>Providencia</i> spp. and 16 pigs (4 isolators) were monoassociated with <i>Enterococcus faecium</i> (EF). Species of bacterial contaminant and diet composition did not affect residual MET or MHA-FA in digesta. Decreased (P < 0.05) apparent residual MET in digesta compared with MHA-FA in CON but not monoasscoiated pigs, along with significantly higher (P<0.05) MET associated radioactivity at 5% SI tissue suggested that microbial metabolism of MHA-FA increases its retention in small intestinal digesta and contributes in part to the lower bioefficacy of MHA-FA compared to MET. A comparison of CON and EF pigs showed that wheat/barley diets increased digesta viscosity (<i>P</i> < 0.01) and proliferating cell nuclear antigen (PCNA) expression (<i>P</i> < 0.001) and tended to decrease (<i>P</i> < 0.07) aminopeptidase N (APN) activity. Monoassociation decreased (<i>P</i> < 0.01) body weight, relative spleen weight, crypt depth, PCNA expression, caspase-3 activity, sucrase expression, total goblet cells in crypts and mucin gene expression and increased (<i>P</i> < 0.01) relative SI length, digesta viscosity, villus height, APN and sucrase activity. Interactive effects between cereal grain type and microbial status were observed only as trends (<i>P</i> < 0.1) for PCNA, Muc2, APN and sucrase suggesting these effects were mediated indirectly through microbial changes. Decreased % retained chromic oxide in digesta at all SI locations and no chromic oxide at 95% SI length in monoassociated pigs indicated slower small intestinal transit of digesta in monoassociated pigs. We successfully developed the chromic oxide microassay for estimating chromic oxide in 1/20th of original sample size (2.0 g). Results of this study indicate that microbial metabolism of MHA-FA contributes in part to the lower bioefficacy of MHA-FA compared to MET. Monoassociation had major effects on intestinal physiology whereas limited indirectly mediated effects of cereal type were observed suggesting no major influences of cereal grain type during the short early post-weaning phase.

An investigation was conducted to determine the contribution of the gastrointestinal microbiota to variation in bioefficacy of methionine sources and the interrelationship between intestinal microbiota and cereal grain type with respect to gastrointestinal physiology. Apparent gastrointestinal absorption of DL-methionine (MET) and 2-hydroxy-4-methylthiobutanoic acid (MHA-FA), post-weaning intestinal morphology, digestive physiology, mucin dynamics and digesta flow were studied in a series of experiments using conventional and gnotobiotic pigs. At 14 d of age, sow - reared conventional (CON) pigs and isolator - reared monoassociated gnotobiotic pigs (EF) were weaned to corn or wheat/barley based diets supplemented with MET or MHA-FA. At 24 d of age, after an overnight fast, pigs were fed experimental diet supplemented with 107 Bq of either 3H-L-MET or 3H-L-MHA-FA per kg of feed and chromic oxide (0.5% wt/wt). Pigs were killed 3 h after consuming the meal to collect digesta and tissue samples from the stomach and along the small intestinal (SI) length. Conventional pigs fed a wheat/barley-based diet had increased (P < 0.05) total aerobes, whereas supplementation with MHA-FA increased (P < 0.05) total aerobes and lactobacilli populations in proximal SI. Among the gnotobiotic pigs, 8 pigs (2 isolators) were monoassociated with a bacteria closely related to <i>Providencia</i> spp. and 16 pigs (4 isolators) were monoassociated with <i>Enterococcus faecium</i> (EF). Species of bacterial contaminant and diet composition did not affect residual MET or MHA-FA in digesta. Decreased (P < 0.05) apparent residual MET in digesta compared with MHA-FA in CON but not monoasscoiated pigs, along with significantly higher (P<0.05) MET associated radioactivity at 5% SI tissue suggested that microbial metabolism of MHA-FA increases its retention in small intestinal digesta and contributes in part to the lower bioefficacy of MHA-FA compared to MET. A comparison of CON and EF pigs showed that wheat/barley diets increased digesta viscosity (<i>P</i> < 0.01) and proliferating cell nuclear antigen (PCNA) expression (<i>P</i> < 0.001) and tended to decrease (<i>P</i> < 0.07) aminopeptidase N (APN) activity. Monoassociation decreased (<i>P</i> < 0.01) body weight, relative spleen weight, crypt depth, PCNA expression, caspase-3 activity, sucrase expression, total goblet cells in crypts and mucin gene expression and increased (<i>P</i> < 0.01) relative SI length, digesta viscosity, villus height, APN and sucrase activity. Interactive effects between cereal grain type and microbial status were observed only as trends (<i>P</i> < 0.1) for PCNA, Muc2, APN and sucrase suggesting these effects were mediated indirectly through microbial changes. Decreased % retained chromic oxide in digesta at all SI locations and no chromic oxide at 95% SI length in monoassociated pigs indicated slower small intestinal transit of digesta in monoassociated pigs. We successfully developed the chromic oxide microassay for estimating chromic oxide in 1/20th of original sample size (2.0 g). Results of this study indicate that microbial metabolism of MHA-FA contributes in part to the lower bioefficacy of MHA-FA compared to MET. Monoassociation had major effects on intestinal physiology whereas limited indirectly mediated effects of cereal type were observed suggesting no major influences of cereal grain type during the short early post-weaning phase.

No description available.