Boampong, Kwadwo
(2019)
Identification and characterisation of toxin-antitoxin systems in Helicobacter pylori tfs ICEs.
PhD thesis, University of Nottingham.
Abstract
The tfs gene clusters that largely comprise the main plasticity regions of H. pylori encode Type IV secretion systems (T4SS); Tfs3 T4SS and Tfs4 T4SS. Both tfs3 and tfs4 have been described as integrative and conjugative elements (ICEs) that can excise from a host chromosome and transfer to a recipient chromosome, thereby contributing to genetic variation of H. pylori population. Although many tfs-encoded genes are uncharacterised, the tfs3-encoded gene ctkA has been associated with induction of pro-inflammatory cytokines such as IL-8 and TNF-α, thereby increasing disease risk. The tfs4-encoded gene dupA is also a disease marker for duodenal ulcer disease.
The tfs ICEs like other mobile genetic elements are susceptible to loss when extrachromosomal. Many such genetic elements harbour toxin-antitoxin (TA) systems which function in their maintenance and stability. Considering that the tfs ICEs might similarly employ TA systems, this study aimed to identify and characterise tfs-encoded TA systems in H. pylori that could be responsible for ICE maintenance.
Initial bioinformatics studies revealed that both H. pylori tfs ICEs harbour genes encoding for putative proteins with homology to known TA toxins. The tfs4-encoded TA toxin, termed TfiT, was shown to inhibit the growth of E. coli cells while the presence of the cognate antitoxin TfiA abrogated toxicity. TfiT toxin activity was shown to be reversible and therefore bacteriostatic since inhibitory effects could be recovered by TfiA antitoxin expression.
Consistent with activity of other Type II toxins, TfiT was shown to have RNase activity. Notably, however, TfiT was also demonstrated to have a broader nuclease activity as it also nicked supercoiled, but not linear plasmid DNA. The C-terminal region of TfiT was demonstrated to be important for activity, with mutation of an active site His87 residue inhibiting toxicity as well as nuclease activity.
The tfs3 ICE lacked a Type II TA system but was however found to encode a putative Type IV TA system, with the toxin component, termed TtiT, homologous to the Type IV TA toxin AbiEii. TtiT was also shown to inhibit E. coli cell growth while co-expression of its cognate antitoxin, TtiA, was sufficient to inhibit toxicity. As shown for tfs4 TfiT, the growth inhibiting effect of TtiT was bacteriostatic and could be recovered by the presence of TtiA.
Investigating a possible target of TtiT activity, TtiT was shown to have weak interaction with the cytoskeletal protein FtsZ, although this interaction did not affect E. coli cell morphology. TtiT also had no nuclease activity towards mRNA or DNA.
Intriguingly, partial TA domains could also be identified in other tfs3-encoded proteins CtkA, Fic and PZ39. These proteins all share a similar carboxyl terminus that has been demonstrated in CtkA to be essential for CtkA-mediated pro-inflammatory signalling by gastric epithelial cells as well as T4SS-mediated secretion. Over-expression of CtkA, Fic and PZ39 did not inhibit the growth of E. coli cells suggesting that the TA domains of these proteins do not function in the context of a TA system.
Transfected PZ39 was however shown to localize inside the nucleus of gastric epithelial AGS cells, promote the formation of giant multinucleated cells and reduce IFN-γ-induced STAT1 phosphorylation. These collective observations suggest that PZ39 might also be a secreted effector encoded by the tfs3 ICE which contributes to disease outcomes of H. pylori infection.
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