H. pylori has a relatively small genome with approximately 1500 genes. It had been long observed that H. pylori clinical isolates are heterogeneous at the sequence level and using a H. pylori microarray we showed that this variability extends to the presence and absence of whole genes. We further showed that even in the context of a single human stomach there exist multiple clones with unique gene complements. We are currently investigating how this diversity is generated and the consequences of this diversity on patient outcome. This includes new efforts to detect genetic variation non-invasively from stool samples and tracking of genetic changes during transmission of infection.
We have developed a number of mutant libraries including random transposon mutant libraries and a sequenced defined mutant library encompassing most non-essential genes. We are using these libraries in a variety of in vitro and in vivo systems to probe H. pylori phenotypes important for pathogenesis. We use gastric epithelial tissue culture cells to monitor wild-type and mutant bacteria binding to host cells and stimulation of host cell signaling pathways including those activating innate immunity and host cell shape changes. To understand bacterial-host interactions in the complex environment of the stomach, which includes many cell types, we employ a mouse model of infection with wild-type and genetically modified mice that perturb immune pathways or stomach differentiation. This allows us to look at the relative fitness of different mutants, their location and their ability to induce host inflammation and pathology associated with gastric cancer.
In follow up to our genetic and population based screens, we are exploring the mechanistic details of how H. pylori genes contribute to persistent colonization:
Our shape mutants (straight or slightly curved rods instead of helical rods) have colonization defects in our mouse infection model. Based on sequence motifs and structural modeling, we hypothesized that some of these proteins affect cell shape by modifying the peptidoglycan cell wall. We have subsequently shown a subset of shape mutants have altered peptidoglycan profiles. We currently are testing motility in viscous solutions, susceptibility to various stresses and peptidoglycan-mediated innate immune signaling to tease out how these proteins and cell morphology contribute to survival in the host. We are also probing the biophysical and biochemical mechanisms by which cell shape proteins drive helical shape.
A surprising outcome of our colonization screen was that genes involved in DNA metabolism contribute to stomach colonization. Natural transformation and restriction modification are thought to contribute to and limit, respectively, the remarkable genetic diversity of H. pylori isolates found around the world. How these processes contribute to survival in short-term infection experiments is not clear. We currently are testing several models including genetic interactions between DNA competence and recombination-based DNA repair. To this end we have identified previously unrecognized components of the recombination-based repair machinery and shown that they: i) confer protection from DNA damaging agents; ii) have enhanced survival during stomach colonization, and iii) stimulate at least one gene conversion event in H. pylori.