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

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付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21794号 / 工博第4611号 / 新制||工||1718(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 梅田 眞郷, 教授 跡見 晴幸, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Animais de companhia têm sido apontados como reservatórios de bactérias gram-negativas resistentes a antibióticos utilizados em medicina humana e veterinária. O objetivo deste estudo foi investigar a prevalência da resistência mediada por plasmídeos em bactérias Gram-negativas isoladas de animais de companhia no Brasil e na França, elucidando o papel potencial desses animais como portadores assintomáticos. Amostras de DNA extraídas de quatro coleções de bactérias Gram-negativas produtoras de ESBL foram analisadas por tipagem e sub-tipagem baseados em PCR, análise do polimorfismo de comprimento de fragmentos de restrição (RFLP), dimensionamento baseado em PFGE de nuclease S1 e hibridação Southern blot. Adicionalmente, isolados de Escherichia coli, Klebsiella pneumoniae e Enterobacter cloacae foram caracterizados por PFGE (Pulsed-field Gel Electrophoresis) e Multilocus Sequence Typing, agrupamento filogenético e tipagem de O25b. A presença de plasmídeos IncH12 (~600-kb) e IncFIB (~210-290-kp) carregando genes blaCTX-M-15 e blaCTX-M-9, foi confirmada entre cepas de E. coli isoladas de animais brasileiros, enquanto uma predominância de plasmídeos IncI1 (~200 kb) pertencentes ao complexo clonal (CC) CC12 contendo o gene blaCMY-2 foi observado entre linhagens de E. coli, em filogrupos de baixa virulência A e B1. A presença de plasmídeos do tipo IncHI2 (~ 600kb) carregando o gene blaCTX-M-15 foi confirmada em cepas de E. cloacae ST927 isoladas de fezes e saliva de cães assintomáticos no Brasil. Entre os animais franceses com infecções, os isolados de E. coli pertencentes ao filogrupo A, B1 e B2 apresentaram tamanho de plasmídeo IncF de ~210-290 kb, carregando principalmente genes blaCTX-M-15, além da presença de plasmídeo IncI1 carregando em sua maioria genes blaCTX-M-1, blaCTX-M-9 e blaCMY-2. Em animais franceses saudáveis, além das associações blaCTX-M-15/IncI1, blaCTX-M-1/IncFIB, blaCTX-M-14/IncF e da presença de blaCMY-2 e blaTEM-52b (não tipáveis), foi identificada uma cepa de E. coli carregando um plasmídeo IncL (~60kb) contendo o gene blaOXA-48, sendo esta a primeira descrição desse gene em animais na França. Além disso, os genes blaCTX-M-15, blaCTX-M-2 e blaCTX-M-9 foram localizados no cromossomo em cepas brasileiras e francesas, como observado por Southern blot e Sequenciamento de Nova Geração (NGS). Em resumo, em ambos os países, a prevalência de cepas positivas para a resistência tipo ESBL é grande. Os animais de companhia podem ter um papel importante na disseminação dos genes AmpC e ESBL mediados por plasmídeos. / Companion animals can be reservoirs of Gram-negative bacteria resistant to antibiotics used in human and veterinary medicine. The aim of this study was to investigate the genetic context of plasmid-mediated resistance in Gram-negative bacteria isolated from companion animals in Brazil and France, elucidating the potential role of these animals as asymptomatic carriers. DNA samples, extracted from a collection of ESBL-producing Gram-negative bacteria, were analyzed by PCR-based typing and sub-typing schemes, restriction fragment length polymorphism analysis, S1 nuclease PFGE-based sizing and Southern blot hybridization. Additionally, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae isolates were characterized by PFGE (Pulsed-field Gel Electrophoresis), Multilocus Sequence Typing, phylogenetic grouping and O25b typing. Presence of IncH12 (~600-kb) and IncFIB (~210-290-kp) plasmids carrying blaCTX-M-15 and blaCTX-M-9, respectively, was confirmed among E. coli strains isolated from Brazilian pets, whereas predominance of IncI1 plasmids (~200 kb) belonging to the clonal complex (CC) CC12 and carrying blaCMY-2 gene was observed among E. coli strains of low-virulence phylogrups A and B1. The presence of IncHI2-type (~ 600-kb) plasmids carrying blaCTX-M-15 gene was confirmed in E. cloacae strains ST927 isolated from feces and saliva from asymptomatic dogs in Brazil. Among French diseased companion animals, E. coli isolates belonging to phylogroup A, B1 and B2 were found carrying IncF-type plasmid with size of ~210-290-kb, which harbored mainly blaCTX-M-15 genes. In addition, presence of IncI1 plasmids carrying blaCTX-M-1, blaCTX-M-9 and blaCMY-2 genes was identified. In healthy French animals, besides associations blaCTX-M-15/IncI1, blaCTX-M-1/IncFIB, blaCTX-M-14/IncF, and the presence of blaCMY-2 and blaTEM-52b (non-typable), it was observed an E. coli strain carrying an IncL plasmid (~ 60kbp) containing the blaOXA-48 gene, representing the first description of this gene in a French dog. In addition, blaCTX-M-15, blaCTX-M-2 and blaCTX-M-9 genes were located on the chromosome in Brazilian and French strains, as observed by Southern Blot and New Generation Sequencing (NGS) analysis. In summary, in both countries, the prevalence of positive strains for ESBL-type resistance in cats and dogs is high. Companion animals may play an important role in the dissemination of the plasmid mediated AmpC and ESBL genes.