Research in my laboratory focuses on the understanding of the mechanisms of bacterial pathogenesis. Our current focus is on the gram-negative bacterium, Helicobacter pylori, that chronically infects a large percentage of the worlds population (50 to 90%) and is the causative agent for gastritis, ulcer disease and some gastric cancers. To date, the mechanism of H. pylori pathogenesis is not completely understood. With animal model, we know that H. pylori can infect and survive in the stomachs of mice. In human, the infection can last a life-time suggesting that it can successfully evade the immune responses. Although, previously thought to be an extracellular organism, several recent in vitro studies suggest that H. pylori may be a facultative intracellular bacterium. This observation may partially explain its immune and therapeutic evasion.? ? Identification of the genes underlying cell growth, stress responses and evasion of host immune responses is fundamental to understanding the mechanisms of pathogenesis? We are using C57BL/6 mice and the murine macrophage cell line, RAW 264.7 to screen for additional H. pylori virulence factors. Initial experiments involving H. pylori infection of macrophage and subsequent gentamicin treatment demonstrated that H. pylori can infect macrophage cells and can survive intracellularly for more than 24 hours. Fluorescence and confocal microscopic studies confirmed that H. pylori were intracellular.? ? In collaboration with Drs. Ding Jin and Yan Zhou, NCI, we are testing whether the spoT gene of H. pylori is important for environmental stresses and intracellular survival. The spoT gene of H. pylori encodes a bifunctional enzyme with both (p)ppGpp synthetase and hydrolase activities. The response to stress by bacteria is mediated by increased intracellular levels of hyperphosphorylated guanine nucleotides, specifically the 3-pyrophosphate derivative of GDP (ppGpp) and the 3-pyrophosphate derivative of GTP (pppGpp). We constructed spoT null mutation in the Sydney strain 1 (SS1) and G27 H. pylori strains. We made the following observations: 1) SpoT mutants failed to produce (p)ppGpp when shifted from rich to minimal media lacking carbon sources and phosphates. 2) ATP levels increased with growth for 48 hours in all strains but the peak value for ATP in the spoT mutant was reproducibly lower than wt. 3) The spoT mutant converts from spiral form to coccoid form prematurely. 4) SpoT mutants have a longer lag time when grown microaeophilically with low CO2 (2.5% vs. 10%). Finally, the macrophage intracellular survival of spoT mutants decreased 4 to 10-fold relative to isogenic wt H. pylori strains after 24 hour. In contrast, both spoT mutant and isogenic wt strains invaded at the same rate with equal numbers of invading bacteria when observed after 1 hour. These results suggest that the spoT mutation reduced the intracellular survival in macrophage only. ? ? Finally, we are employing a genetic method known as in vivo expression technology (IVET) to probe microbial genes important in pathogenicity. IVET is a practical strategy for identifying a subset of genes induced preferentially during infection of an animal host. We use an antibiotic-based variant of IVET involving random DNA fragments of H. pylori fused to a tandem-reporter system of chloramphenicol acetyltransferase (cat) and beta-galactosidase (lacZ). We constructed unique H. pylori promoter-screening vectors (pIVET11 and pIVET12). A H. pylori genomic bank constructed in pIVET11 or pIVET12 was transformed in H. pylori strain HP1061 to generate a library of co-integrated strains in which plasmid recombinants integrated by homologous recombination into the HP1061 genome at different loci. Strains containing fusions of the cat gene that are transcriptionally active in mouse stomachs or within macrophage were selected in the presence of chloramphenicol. A subset of the in vivo induced genes (ivi genes) may play a role in H. pylori infection, survival and pathogenesis. The IVET screening in mice and macrophage cell line RAW264.7 identified two novel putative ivi genes (flhF and virA) and the previously reported virulence vacA. We are now focused on this novel ivi gene flhF. Recently in a H. pylori genome analysis studies it was reported that flhF is a master regulator of H. pylori flagellar biosynthesis. The flagellar system of Helicobacter pylori comprises more than 40 mostly unclustered genes. H. pylori depends on a functional flagellar motility system in its natural habitat, i.e, the mouse or human stomach mucus. The in vivo induced gene, flhF, may be essential for colonization in the stomach mucus. RT-PCR and mutagenesis experiments are ongoing to confirm flhF in vivo induction and its role in stomach colonization.
