Transposable elements (TEs) contribute importantly to genome organization and evolution, regulation, developmental programming, and human health and disease -- most famously, as bacterial drug resistance transposons, oncogenic retroviruses, and V(D)J immune system recombination. TEs are diverse in mechanisms and regulation of movement, and their analyses provide valuable insights into protein-nucleic acid interactions and cellular regulatory mechanisms. TEs in pathogens often contain auxiliary genes that affect phenotypes such as virulence. This proposal stems from our discovery of novel """"""""plasticity zone"""""""" transposons (TnPZs) in the gastric pathogen Helicobacter pylori. These TEs encode a novel member of the XerC/XerD branch of the tyrosine recombinase family (""""""""XerT""""""""), a type IV secretion system (""""""""tfs3""""""""), and a large protein (OrfQ) (2800 - 4000 residues, depending on strain) that contains prominent DNA methylase and RNA helicase motifs. OrfQ-like proteins are evident in genome sequences of other unrelated bacterial pathogens. We hypothesize that (i) TnPZs are conjugative transposons, passed between bacterial cells via their Tfs3 protein complex, and inserted into new sites using XerT protein;and (ii) that the putative DNA methylase OrfQ may cause epigenetic (DNA modification) changes in target tissues that could impact on gastric pathology and disease.
In Specific Aim 1 we will study the mechanism and control of TnPZ excision and transposition in H. pylori and E. coli. Since prototype (E. coli) XerC/XerD proteins are specific for one unique chromosomal sequence whereas TnPZs insert into many sites, studies of XerT action should enhance understanding of protein-nucleic acid specificity and its evolution.
In Specific Aim 2 we will test for OrfQ-mediated DNA methylation in infected mammalian cells and in bacterial cells. OrfQ's domain structure suggests that studies of its action could give new insights into the dynamics of bacterial-host interactions during chronic infection. In conclusion, results of these R21 studies should enhance understanding of transposition-related phenomena, and of infection and virulence mechanisms, and also provide data needed to support an anticipated larger RO1-type application in coming years.
The fundamental insights to be gained from transposon TnPZ studies will enrich understanding of microbial pathogen evolution and human disease. Studies of OrfQ, in particular, could reveal a new dimension of bacterial-host interactions and the origin of human epigenetic changes important in disease pathology, and result in improved diagnosis and therapy for infections and associated pathologies, including gastric cancer.
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