Enteric Disease Pathogenesis and Vaccine Development

许多肠道细菌病原体具有穿透肠道上皮细胞的能力，这是疾病发生的早期和必要步骤。我的实验室在20世纪80年代早期的工作确定了志贺氏菌触发入侵的遗传机制是在一个大的毒力相关质粒上编码的。最近的研究表明，其他肠道细菌的入侵能力是由染色体编码的。

Many bacterial pathogens of the intestinal tract have as an early and necessary step in initiation of disease the ability to penetrate gut epithelial cells. My lab's work in the early 1980's established that Shigella's genetic machinery to trigger invasion is encoded on a large virulence-associated plasmid. More recent studies have shown that the invasion ability of other enteric bacteria is chromosomally encoded. This program is aimed at understanding the prokaryotic and eukaryotic requirements for bacterial internalization, with the ultimate aim being a thorough molecular definition of the events involved in bacterial invasion of eukaryotic tissues. Current experimental approaches involve the use of assays measuring bacterial entry into cultured lines of human epithelial cells of various tissue origins. Biochemical inhibitors of prokaryotic structure/function or of eukaryotic cell processes are employed in these tissue culture invasion assays to examine the requirements for bacterial uptake. Direct visualization of bacterial entry is measured via transmission electron microscopy, video microscopy, fluorescent microscopy, and confocal microscopy. In addition to the approaches described above, genetic techniques are employed to clone the responsible bacterial genes. Eukaryotic receptors for bacterial ligands and specific eukaryotic cell responses to bacterial invasion are measured via inhibitor competition assays, ligand binding assays, and mRNA analyses of infected eukaryotic cells. The information gained from each of these approaches is integrated to provide a mechanistic understanding of bacterial entry for each pathway studied. Recent progress has revealed that Campylobacter jejuni invasion is dependent upon host microtubules but not microfilaments. Apparent signalling by the approaching bacterium triggers the host cell to extend a microtubule-based, fingerlike projection. C. jejuni interacts with a host receptor located in membrane caveolae at the tip of this membrane extension. This bacterial-host "ligand -receptor" interaction activates a signal transduction cascade that releases Ca++ from intracellular stores and activates PI-3 kinase, calmodulin, and protein kinase C, which are required for C. jejuni internalization. The entering bacterium within an endosome moves via dynein and microtubules to the perinuclear region over 4 hrs. Bacterial invasion of of eukaryotic tissues. Many bacterial pathogens of the intestinal tract have as an early and necessary step in initiation of disease the ability to penetrate gut epithelial cells. My lab's work in the early 1980's established that Shigella's genetic machinery to trigger invasion is encoded on a large virulence-associated plasmid. More recent studies have shown that the invasion ability of other enteric bacteria is chromosomally encoded. This project is aimed at understanding the prokaryotic and eukaryotic requirements for bacterial internalization, with the ultimate aim being a thorough molecular definition of the events involved in bacterial invasion of eukaryotic tissues. Current experimental approaches involve the use of assays measuring bacterial entry into cultured lines of human epithelial cells of various tissue origins. Biochemical inhibitors of prokaryotic structure/function or of eukaryotic cell processes are employed in these tissue culture invasion assays to examine the requirements for bacterial uptake. Direct visualization of bacterial entry is measured via transmission electron microscopy, video microscopy, fluorescent microscopy, and confocal microscopy. In addition to the approaches described above, genetic techniques are employed to clone the responsible bacterial genes. Eukaryotic receptors for bacterial ligands and specific eukaryotic cell responses to bacterial invasion are measured via inhibitor competition assays, ligand binding assays, and mRNA analyses of infected eukaryotic cells. The information gained from each of these approaches is integrated to provide a mechanistic understanding of bacterial entry for each pathway studied. Recent progress has revealed that Campylobacter jejuni invasion is dependent upon host microtubules but not microfilaments. Apparent signalling by the approaching bacterium triggers the host cell to extend a microtubule-based, fingerlike projection. C. jejuni interacts with a host receptor located in membrane caveolae at the tip of this membrane extension. This bacterial-host "ligand -receptor" interaction activates a signal transduction cascade that releases Ca++ from intracellular stores and activates PI-3 kinase, calmodulin, and protein kinase C, which are required for C. jejuni internalization. The entering bacterium within an endosome moves via dynein and microtubules to the perinuclear region over 4 hrs. Characterization of chronic, asymptomatic intestinal shedding of Shigella; application to vaccine safety. These studies are directed at utilizing specific virulence gene regions of Shigella as DNA probes or their sequence information in polymerase chain reaction assays to detect Shigella in stools. Our early studies showed that DNA probes were very sensitive, quick, and effective in detecting Shigella in the stool of diarrheal disease patients in Peru. In more recent studies utilizing polymerase chain reaction analysis of stools of healthy (i.e. asymptomatic) nonhuman primates and their healthy human caretakers, we have found a high rate of chronic, low level( i.e. below bacteriologic detection) asymptomatic carriage of Shigella. Our specific objectives are to determine (1) if these Shigella have full virulence potential,(2) the exact state in which Shigella are residing in human or primate carriers( e.g. inside mucosal epithelial cells in the colon?), (3) the immune status of the carrier to Shigella, and (4) if this chronic low level carrier state is responsible for maintaining Shigella endemicity in the U.S. where there are now over 480,000 cases per year estimated by the CDC. Genetic and biochemical analyses of adaptive mutations in Shigella and other enteric bacteria. Genetic and biochemical studies of Escherichia coli over the past 5 years have revealed novel mutations, termed adaptive mutational events, that occur at high frequency(i.e. generally 3 to 5 logs higher than mutations occurring during logarithmic growth), only under special adaptive conditions, and in the stationary phase of bacterial growth. Our recent studies of Shigella indicate that this organism also undergoes adaptive mutations in a large variety of oligosaccharide utilization pathways and in other metabolic and virulence gene regions. This project is aimed at characterizing genetically and biochemically adaptive mutational events in Shigella at various metabolic and virulence gene loci for frequency and chemical nature. Involved loci will be DNA sequenced to examine the precise nature of the mutational event, with an emphasis on understanding the mechanisms involved in stimulating these high frequency (e.g. 10-3) mutational events in stationary phase cells. Similar studies of other related enteric bacteria will be conducted to determine if adaptive mutations are a cause of misidentification of pathogens, via altered biochemical pathways, or a method by which bacteria modulate their virulence. Development of vaccines against anthrax. This project is aimed at introducing genes encoding key protective antigens of Bacillus anthracis into a safe, attenuated live Salmonella typhi vaccine vector. The anthrax protective antigen gene(s) have been inserted into several plasmid vectors behind assorted promoters that allow for a low or high level of constituitive expression. Promoters that "turn on" the anthrax gene(s) only upon infection or only inside host cells are being employed. These plasmid constructs have initially been examined for level of antigen production and genetic stability in S. typhi vaccine vectors. Several selected candidate live, oral Salmonella-based vaccines against anthrax are currently being assessed for immunogenicity in mice and for protective efficacy in small animal models. This project incorporates FY2002 projects 1Z01BJ005007-09, 1Z01BJ005008-09, 1Z01BJ005009-09, and 1Z01BJ005013-02.