UC Santa Cruz

The complement system has long been appreciated for its role in bloodborne infection, but its activities in other places, including the gastrointestinal tract, are unknown. Here, we report that complement restricts gastric infection by the pathogen Helicobacter pylori. This bacterium colonized complement-deficient mice to higher levels than wild-type counterparts, particularly in the gastric corpus region. H. pylori used uptake of the host molecule L-lactate to create a complement-resistant state that relied on blocking the deposition of the active complement C4b component on H. pylori’s surface. H. pylori mutants unable to achieve this complement-resistant state had a significant mouse colonization defect that was largely corrected by mutational removal of complement. This work highlights a previously unknown role for complement in the stomach, and has revealed a new mechanism for microbial-derived complement resistance.We further found that to mediate complement resistance, L-lactate played a role as signaling molecule that mediated the upregulation of a small regulon, including the phospholipase A (PldA) coding gene. PldA was critical to prevent killing by complement activation. PldA significantly enhanced H. pylori complement tolerance by efficiently dissociating C4b on the H. pylori surface. Without PldA, H. pylori survival was impaired under complement exposure, and mutants were unable to colonize mouse stomachs. This work highlights a previously unknown function of phospholipases in complement resistance, and a new type of bacterial anti-complement mechanism. Our results suggest PldA may plays essential role in facilitating H. pylori gastric colonization through facilitating complement resistance. Last, to conduct the investigations, we applied genetic manipulation to generate isogenic mutants from WT H. pylori background. Genetic manipulation is a frequently applied approach to study numerous bacterial processes, including H. pylori. However, H. pylori is difficult to manipulate, partially due to robust Restriction-Modification (RM) systems that destroy exogenous incoming DNA. To overcome RM barrier, we developed an easily appliable approach to improve H. pylori transformation efficiency. For this approach, we computationally predicted under-represented short Kmer sequences in the H. pylori genome, with the idea that these sequences reflect restriction enzyme targets. We then used this information to modify an antibiotic resistance cassette by generating synonymous mutations at the predicted restriction sites, and use this modified cassette for transformation. Indeed, antibiotic cassettes with modified under-represented Kmer sites resulted in up to 105-fold higher transformation efficiency compared to a non-modified cassette. Because our approach relies on computational Kmer site prediction, it is readily applicable to any microbe with a sequenced genome. We thus expect this approach will enable genetic manipulation to be more achievable in a wide range of bacterial species.