Dr. Luis Ielpi
Instituto Fundación Leloir

Genomic variability and pathogenesis process in Helicobacter pylori

Pathogen DNA as target for host generated oxidative stress: role for repair of bacterial DNA damage in Helicobacter pylori colonization
Helicobacter pylori colonizes the gastric mucosa of more than one half of the human population, resulting in chronic gastritis, ulcers and cancer. One of the notable characteristics of H. pylori is the highest degree of genetic diversity reported for any bacterial species. H. pylori is characterized by the highly unusual combination of high mutation and recombination rate. Mutation is the ultimate source of allelic variants. Bacteria have evolved a variety of DNA damage-repair mechanisms which act together to remove almost any DNA damage. The absence of some ORFs with the capacity to encode for DNA glycosylases from the base excision repair system (BER) in H. pylori has been proposed as a possible cause of genetic diversity. In the present thesis the predicted DNA glycosylases were biochemically characterized and those that were supposed to be absent were identified. By genetic and biochemical approaches it was determined that all the DNA glycosylases from BER were present in H. pylori. We proved in this way that the absence of BER components is not a source of genetic variability in H. pylori. The predicted DNA glycosylases, Ung, MutY and EndoIII, were purified and their enzymatic activities were analyzed. DNA glycosylase-deficient H. pylori were constructed. Their phenotypes were studied in normal and stress conditions. The relevance of the DNA glycosylases from H. pylori in the maintenance of genetic information was confirmed. The impaired ability of mutant strains to remove oxidized pyrimidines from their DNA led to a reduced colonization capacity in a mouse infection model when the bacterial genetic background was mouse-adapted. The results support the hypothesis that the host effectively induces lethal and premutagenic, oxidative DNA adducts on H. pylori genome. The removal of such lesions by the bacterial base excision repair system is required for the efficient colonization of a stable environment. By contrast, when mutants constructed in a non adapted genetic background were compared to the non adapted parental, the mutants were clearly more efficient in colonization. This result suggests that in a non adapted strain, promutagenic lesions could allow a faster adaptation.

DNA transformation machinery components
H. pylori is naturally competent for transformation. Natural genetic transformation is believed to be essential for the genetic plasticity observed in this species. While the relevance of horizontal gene transfer in H. pylori adaptiveness and antibiotic resistance is well documented, the DNA transformation machinery components are barely known. No enzymatic activity associated to the transformation process has been identified. We have isolated from H. pylori, microsequenced, and cloned a major DNA nuclease. The protein, coded by the open reading frame hp0323, was expressed in E. coli. The purified protein, NucT, showed a cation independent thermostable nuclease activity that preferentially cleaves single stranded DNA. NucT is associated to the membrane and its presence is required for efficient transformation. NucT-deficient H. pylori strains are one or more orders of magnitude less efficient than the parental strain for transformation with either chromosomal or self-replicating plasmidic DNA. NucT would be the first enzymatic partner reported for the DNA transfer machinery from H. pylori, a phenomenon of critical relevance in the genetic variability and in consequence in the adaptability and the long term persistence of this pathogen in its host.