Abstract
The genomes of the gastric bacterial pathogen Helicobacter pylori harbor multiple type-IV secretion systems (T4SSs). Here we analyzed components of three T4SSs, the cytotoxin-associated genes (cag) T4SS, TFS3 and TFS4. The cag T4SS delivers the effector protein CagA and the LPS-metabolite ADP-heptose into gastric epithelial cells, which plays a pivotal role in chronic infection and development of gastric disease. In addition, the cag T4SS was reported to facilitate conjugative transport of chromosomal bacterial DNA into the host cell cytoplasm, where injected DNA activates intracellular toll-like receptor 9 (TLR9) and triggers anti-inflammatory signaling. Canonical DNA-delivering T4SSs in a variety of bacteria are composed of 11 VirB proteins (VirB1-11) which assemble and engage VirD2 relaxase and VirD4 coupling proteins that mediate DNA processing and guiding of the covalently bound DNA through the T4SS channel. Nevertheless, the role of the latter components in H. pylori is unclear. Here, we utilized isogenic knockout mutants of various virB (virB9 and virB10, corresponding to cagX and cagY), virD2 (rlx1 and rlx2), virD4 (cag5, traG1/2) and xerD recombinase genes in H. pylori laboratory strain P12 and studied their role in TLR9 activation by reporter assays. While inactivation of the structural cag T4SS genes cagX and cagY abolished TLR9 activation, the deletion of rlx1, rlx2, cag5, traG or xerD genes had no effect. The latter mutants activated TLR9 similar to wild-type bacteria, suggesting the presence of a unique non-canonical T4SS-dependent mechanism of TLR9 stimulation by H. pylori that is not mediated by VirD2, VirD4 and XerD proteins. These findings were confirmed by the analysis of TLR9 activation by H. pylori strains of worldwide origin that possess different sets of T4SS genes. The exact mechanism of TLR9 activation should be explored in future studies.
Highlights
Helicobacter pylori represents a Gram-negative bacterium that colonizes the human stomach
The cag T4SS mediates the transport of the CagA effector protein into host cells, which is followed by tyrosine phosphorylation by host cell kinases [8, 12]
Pairwise tBLASTx comparisons and BLASTp searches of the extracted protein sequences showed the presence of genes encoding VirB proteins (VirB2-VirB4; VirB6-VirB11) as well as the VirD4 (TraG) coupling protein in each of the three T4SS-encoding genomic fragments, encoding the cag T4SS, TFS3 and TFS4 (Fig. 1)
Summary
Helicobacter pylori represents a Gram-negative bacterium that colonizes the human stomach. 121 Page 2 of 11 various type-IV secretion systems (T4SSs) were acquired and/or evolved, each of which with specific functions (reviewed by [7, 8]). The T4SS functions include (i) the transfer of conjugative and other mobile DNA elements, (ii) the release of DNA into the environment, (iii) the uptake of extracellular DNA, (iv) the transport of effector proteins into the supernatant, as well as (v) the direct injection of effector proteins into the cytoplasm of the host cell. TFS3 as well as TFS4 supposedly represent conjugational DNA transfer systems, which is supported by the discovery of two VirD2-like relaxase enzymes, Rlx and Rlx2 [18–20], two VirD4-like coupling proteins (TraG1/2) [18, 19], putative oriT (origin of transfer) sequences detected near the TFS3 and TFS4 genes [8, 20], and tyrosine recombinase (XerD) with recognition sites [21]. TFS3 might deliver the effector protein CtkA, a putative cell-translocating kinase, into target cells [22–24]
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