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Pathogenic Helicobacter pylori Strains Translocate DNA and Activate TLR9 via the Cancer-Associated cag Type IV Secretion System

Helicobacter pylori is the strongest identified risk factor for gastric cancer, the third most common cause of cancer-related death worldwide. An H. pylori constituent that augments cancer risk is the strain-specific cag pathogenicity island, which encodes a type IV secretion system (T4SS) that translocates a pro-inflammatory and oncogenic protein, CagA, into epithelial cells. However, the majority of persons colonized with CagA+ H. pylori strains do not develop cancer, suggesting that other microbial effectors also play a role in carcinogenesis. Toll-like receptor 9 (TLR9) is an endosome bound, innate immune receptor that detects and responds to hypo-methylated CpG DNA motifs that are most commonly found in microbial genomes. High expression tlr9 polymorphisms have been linked to the development of premalignant lesions in the stomach. We now demonstrate that levels of H. pylori-mediated TLR9 activation and expression are directly related to gastric cancer risk in human populations. Mechanistically, we show for the first time that the H. pylori cancer-associated cag T4SS is required for TLR9 activation and that H. pylori DNA is actively translocated by the cag T4SS to engage this host receptor. Activation of TLR9 occurs through a contact-dependent mechanism between pathogen and host, and involves transfer of microbial DNA that is both protected as well as exposed during transport. These results indicate that TLR9 activation via the cag island may modify the risk for malignancy within the context of H. pylori infection and provide an important framework for future studies investigating the microbial-epithelial interface in gastric carcinogenesis.


Introduction
Helicobacter pylori is a bacterial carcinogen that incurs the highest known risk for gastric cancer (1). Approximately 80% of the gastric cancer burden and 5.5% of all malignancies worldwide are attributable to H. pylori-induced injury (1,2). However, only a subset of chronically colonized individuals ever develop neoplasia. Enhanced risk for gastric carcinogenesis is related to H. pylori strain differences, inflammatory responses governed by host genetic diversity, and/or specific interactions between host and microbial determinants (3).
One cancer-linked H. pylori locus is the cag pathogenicity island, which encodes a type IV secretion system (T4SS) that forms a syringe-like structure to translocate the effector oncoprotein CagA and peptidoglycan into host cells (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). T4SSs are common among Gram-negative bacteria due to their versatility in terms of the type and destination of secreted substrates. However, despite the ubiquity of T4SS in Gram-negative bacteria, their ability to mediate trans-kingdom DNA transfer is rare. Indeed, only a very few bacterial T4SSs have been shown to facilitate DNA transfer into yeast (20,21) or mammalian cells (22)(23)(24). The T4SS of Agrobacterium tumefaciens is the only known example of a pathogenesis-associated T4SS that facilitates plasmid DNA transfer into a eukaryotic host (25)(26)(27). In this system, A. tumefaciens translocates the tumor-inducing Ti-plasmid into plant cells, which is then incorporated into the genome and ultimately results in malignant transformation. A. tumefaciens T4SS-mediated DNA transfer has also been shown to promote transformation of human cells under non-physiological conditions (28). Of particular interest, the A. tumefaciens archetypal T4SS retains a high level of homology to the cag type IV secretion system, suggesting that H. pylori has the ability to translocate DNA into host cells via the cag island.
In addition to the cag T4SS, host factors have also been implicated in augmenting H. pyloriinduced gastric cancer risk. Toll-like receptors (TLRs) orchestrate host immune responses targeting pathogens via selective recognition of pathogen-associated molecular patterns (PAMPs) (29); however, chronic activation of TLRs in the gastric niche has been implicated in promoting carcinogenesis (30). TLR9 is an intracellular receptor that recognizes hypomethylated CpG motifs (31), which are abundant in DNA of bacterial, viral, or synthetic origin, but are atypical within mammalian genomes (32). TLR9 expression is up-regulated in human gastric cancer specimens, and H. pylori DNA has been shown to directly promote cancer cell invasion (33-38). Moreover, polymorphisms in the tlr9 gene have also been shown to increase the risk for development of both pre-malignant and malignant gastric lesions (30,39). Therefore, we utilized human gastric specimens and in vitro models of microbial-epithelial interactions to define the role of H. pylori DNA translocation and TLR9 activation in gastric carcinogenesis.

Results and Discussion
In many areas of the world, geographic differences in gastric cancer rates are present despite similarly high H. pylori prevalence rates (40). This has been well described in Colombia, where residents of the Andean Mountain region have an extraordinarily high incidence of gastric cancer (150/100 000), compared to inhabitants of the Pacific coast region (6/100 000), despite the fact that greater than 90% of the collective population is infected with H. pylori (41,42). This disparity in gastric cancer risk but not H. pylori prevalence (15,(43)(44)(45) provides a unique opportunity to define microbial and host determinants that play a role in gastric carcinogenesis. Therefore, we first analyzed H. pylori strains isolated from the high and low gastric cancer risk regions for their ability to activate TLR9 using a specific HEK293-hTLR9 reporter assay system. In this reporter assay, HEK293 cells, which are inherently devoid of most innate immune receptors, are stably transfected with either a TLR9 expression plasmid and an NFκB/AP-1-linked SEAP reporter (TLR9+), or a control NFκB/AP-1 SEAP reporter alone (Parental). Cells were challenged with H. pylori strains isolated from patients residing in either the high-risk or low-risk region. H. pylori strains isolated from the high-risk region induced significantly higher levels of TLR9 activation compared to strains harvested from patients in the low-risk region (Figure 1a). To determine whether the ability of high-risk H. pylori isolates to activate TLR9 in vitro translated into the cognate gastric niche, levels of epithelial TLR9 expression in gastric biopsies obtained from infected patients were quantified. Gastric epithelial TLR9 expression levels were significantly increased in patients residing in the high-risk region compared to the low-risk region (Figure 1b,c), findings that mirror the in vitro data. Patients residing in the high cancer-risk region also displayed significantly increased histological scores compared to patients in the low cancer-risk region (mean ± SEM; 3.89±0.26 vs. 2.05±0.11, respectively; p≤0.0001). Additionally we observed that TLR9 expression was more extensive and involved more regions of the gastric glands in patients residing in the high-risk region, frequently extending from the base to the gastric pit. Collectively, these results indicate that H. pylori strains linked to an increased risk for gastric cancer induce more intense TLR9 activation in vitro and enhanced TLR9 expression in vivo.
Despite the evolutionary homology of the cag T4SS to the archetypal A. tumefaciens T4SS (46), DNA translocation by the H. pylori cag system has never been demonstrated. Therefore, to define mechanisms through which H. pylori activates TLR9, we subsequently conducted experiments using the well-characterized wild-type and mutant H. pylori cag + strains J166, 26695, and 7.13 (12,47,48). Previous reports have shown that H. pylori DNA induces TLR9 activation in immune cells (49)(50)(51); thus, we first confirmed that purified H. pylori genomic DNA (gDNA) could induce TLR9 activation in our HEK293 reporter assay ( Figure 2a). We also demonstrated that viable wild-type cag + H. pylori could activate TLR9 to significantly increased levels compared to controls (Figure 2b, 2c, S1). The specificity of the TLR9-dependent response was then validated in HEK293-hTLR9 reporter cells that were pre-treated with the endosomal inhibitor chloroquine, which blocks TLR9 activation.
H. pylori-infected reporter cells pre-challenged with chloroquine displayed a significant decrease in TLR9 activation compared to control cells ( Figure S2).
H. pylori possesses up to four potential T4SSs with the putative ability to translocate DNA to and from the bacterial cell: the comB DNA uptake (competence) system, the cag system, as well as the tfs3 and tfs4 secretion systems (52)(53)(54). Analysis of an H. pylori comB DNA uptake mutant revealed no differences in levels of TLR9 activation compared to the H. pylori wild-type strain J166 (Figure 2b). Therefore, we next investigated the role of the cag T4SS in mediating TLR9 activation. TLR9 reporter cells were challenged with either an isogenic cagA − mutant (which lacks the effector protein CagA), cagE − or cagY − mutants (which encode essential proteins for T4SS assembly), or a pgdA − mutant (which reduces peptidoglycan-mediated NOD1 activation (55)). Loss of cagA or pgdA had no effect on TLR9 activation; however, cagE − or cagY − mutants were incapable of activating TLR9, suggesting that DNA translocation to the host cell and subsequent TLR9 activation may occur via the cag T4SS ( Figure 2b). The requirement for a functional cag T4SS to induce TLR9-dependent responses was also demonstrated in two independent H. pylori cag + strains, 26695 and 7.13, as mutations in cag T4SS structural components abrogated TLR9 activation (Figure 2c, S1). Importantly, H. pylori strain 26695, which lacks intact tfs3 and tfs4 gene clusters (53), still induced robust TLR9 activation ( Figure 2c). We also observed TLR9 activation following infection with H. pylori strains devoid of plasmid DNA (strain J166, 26695, Figure 2b, 2c (12, 47)), suggesting that other forms of DNA can be translocated by the cag T4SS. This observation is of particular importance because it highlights fundamental differences between H. pylori DNA translocation and DNA translocation that occurs in other bacterial species, which only translocate plasmid DNA into host cells. To extend these findings, we also infected AGS gastric epithelial cells with wildtype H. pylori cag + strains (J166 or 26695), their respective cagE − mutants, or their purified genomic DNA and assessed the levels of IL-8 secretion as a potential downstream target of TLR9 mediated NF-κB activation (Figure 2d). We found that the cagE − mutants induced significantly decreased levels of IL-8 compared to wild-type H. pylori strains. Additionally, purified gDNA from the wild-type strains induced a significantly increased level of IL-8 production compared to vehicle control, suggesting that H. pylori gDNA can not only activate TLR9, but can also induce production of a known downstream target of this receptor. Collectively, these data indicate that a functional H. pylori cancer-linked T4SS is required for TLR9 activation, but the known effector molecules translocated by this system (CagA and peptidoglycan) are dispensable for this phenotype.
H. pylori is predominantly an extracellular pathogen and TLR9 is an endosomal receptor, implying that DNA is either directly injected into the host cell, or is taken up via a different mechanism that is enhanced by a functional T4SS. To determine whether TLR9 was activated by H. pylori DNA entering host cells via a host-induced pathway, TLR9 reporter cells were incubated with bacterial cell-free, H. pylori conditioned media at concentrations up to 30%; however, no changes in TLR9 activation were observed compared to control cells ( Figure 2e). These data indicate that activating DNA is not secreted, in contrast to strategies employed by Neisseria in which DNA is translocated into the extracellular milieu (56). Although primarily extracellular, H. pylori does retain the capacity to invade host epithelial cells in a limited fashion (55). Therefore, we next quantified intracellular H. pylori viability within HEK293-hTLR9 reporter cells. Wild-type H. pylori survived equally well compared to the corresponding cag mutants (Figure 2f), despite marked differences in TLR9 activation induced by these strains (Figure 2b). These results indicate that microbial endocytosis does not affect H. pylori-induced TLR9 activation since only wild-type H. pylori were capable of activating this endosome-bound receptor.
To determine whether the cag T4SS is required for direct DNA delivery into host cells, H. pylori DNA was labeled with BrdU and bacteria were subsequently co-cultured with AGS gastric epithelial cells. Using this technique, bacterial DNA can be easily distinguished from host DNA via incorporation of BrdU. Structured illumination microscopy (SIM) demonstrated that wild-type H. pylori translocated BrdU-labeled DNA into host cells ( Figure 3a, Figure S3, Figure S4 movie a,b). However, intracellular BrdU-labeled DNA was not observed in host cells infected with the H. pylori cagE − mutant (Figure 3a, Fig S5   movie). To confirm and quantify these results using an independent methodology, host intracellular levels of BrdU-labeled H. pylori DNA were assessed via flow cytometry. Levels of intracellular DNA were significantly increased in H. pylori wild type-infected compared to uninfected AGS cells. A significant reduction was observed in cells infected with the cagE − mutant compared to wild-type infected AGS cells (Figure 3b), albeit not to levels observed in uninfected cells, which may be due to adherent and/or invasive H. pylori that could not be completely removed prior to analysis. Collectively, these data demonstrate that H. pylori utilizes the cag T4SS to translocate microbial DNA into eukaryotic cells, thereby activating TLR9.
Elaboration of the cancer-associated cag T4SS develops in response to direct host cell contact, and effector translocation is dependent on assembly of the T4SS apparatus. We therefore performed orthologous mechanistic studies to investigate whether TLR9 activation required de novo synthesis of Cag proteins for apparatus biogenesis, or whether cag T4SSdependent DNA transfer could be achieved using pre-assembled protein complexes. Treatment of H. pylori-HEK293 co-cultures with bacteriostatic, sub-lethal concentrations of chloramphenicol, an inhibitor of bacterial protein synthesis, revealed a significant reduction in levels of TLR9 activation (Figure 4a). Chloramphenicol-mediated inhibition of H. pyloridriven TLR9 activation occurred in a dose-dependent manner but did not affect H. pylori viability (Figure 4b). These data indicate that cag T4SS-mediated DNA translocation requires new protein synthesis.
DNA transfer by A. tumefaciens occurs via substrate delivery both within and on the external surface of the T4SS conduit (25). To further investigate the mechanisms of cag T4SS-dependent DNA transfer into host cells in greater depth, we assessed whether exogenous DNase I treatment would compromise the integrity of transported DNA and impair TLR9 activation. Compared to untreated controls, levels of TLR9 activation decreased by approximately 50% when DNase I was added during H. pylori-HEK293 cell co-culture (Figure 4c). In parallel, we analyzed the ability of monoclonal anti-DNA antibodies to functionally block H. pylori-dependent TLR9 activation. Compared to untreated controls, H. pylori-mediated TLR9 activation was significantly reduced in the presence of pan anti-DNA antibodies, an effect that was titratible (Figure 4d). Taken together, these data indicate that cag T4SS-dependent DNA translocation occurs through a contact-dependent mechanism that involves transfer of bacterial DNA that is both protected as well as exposed during transport.
In conclusion, we have demonstrated that patients residing within a high gastric cancer risk region of Colombia express significantly higher levels of TLR9 within gastric epithelial cells compared to patients residing in the low risk region, and that the H. pylori strains isolated from these patients concomitantly induce higher levels of TLR9 activation. We further demonstrate that H. pylori utilizes the cag island, a locus known to augment cancer risk, to translocate bacterial DNA into host cells. Collectively these results define a mechanism that may explain previous epidemiologic data linking TLR9 to the development of gastric cancer.