The pathogenic bacterium Helicobacter pylori is genetically highly diverse and a major risk factor for the development of peptic ulcer disease and gastric adenocarcinoma in humans. During evolution, H. pylori has acquired multiple type IV secretion systems (T4SSs). These T4SSs represent remarkable molecular transporter machines, often associated with an extracellular pilus structure present in many bacteria, which are commonly composed of multiple structural proteins spanning the inner and outer membranes. By definition, these T4SSs exhibit central functions mediated through the contact-dependent conjugative transfer of mobile DNA elements, the contact-independent release and uptake of DNA into and from the extracellular environment as well as the secretion of effector proteins in mammalian host target cells. In recent years, numerous features on the molecular functionality of these T4SSs were disclosed. H. pylori encodes up to four T4SSs on its chromosome, namely the Cag T4SS present in the cag pathogenicity island (cagPAI), the ComB system, as well as the Tfs3 and Tfs4 T4SSs, some of which exhibit unique T4SS functions. The Cag T4SS facilitates the delivery of the CagA effector protein and pro-inflammatory signal transduction through translocated ADP-heptose and chromosomal DNA, while various structural pilus proteins can target host cell receptors such as integrins or TLR5. The ComB apparatus mediates the import of free DNA from the extracellular milieu, whereas Tfs3 may accomplish the secretion or translocation of effector protein CtkA. Both Tfs3 and Tfs4 are furthermore presumed to act as conjugative DNA transfer machineries due to the presence of tyrosine recombinases with cognate recognition sequences, conjugational relaxases, and potential origins of transfer (oriT) found within the tfs3 and tfs4 genome islands. In addition, some extrachromosomal plasmids, transposons and phages have been discovered in multiple H. pylori isolates. The genetic exchange mediated by DNA mobilization events of chromosomal genes and plasmids combined with recombination events could account for much of the genetic diversity found in H. pylori. In this review, we highlight our current knowledge on the four T4SSs and the involved mechanisms with consequences for H. pylori adaptation to the hostile environment in the human stomach.

Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (cag-T4SS) into host cells is a hallmark of infection with Hp and a major risk factor for severe gastric diseases, including gastric cancer. To mediate the injection of CagA, Hp uses a membrane-embedded syringe-like molecular apparatus extended by an external pilus-like rod structure that binds host cell surface integrin heterodimers. It is still largely unclear how the interaction of the cag-T4SS finally mediates translocation of the CagA protein into the cell cytoplasm. Recently certain carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), acting as receptor for the Hp outer membrane adhesin HopQ, have been identified to be involved in the process of CagA host cell injection. Here, we applied the CRISPR/Cas9-knockout technology to generate defined human gastric AGS and KatoIII integrin knockout cell lines. Although confocal laser scanning microscopy revealed a co-localization of Hp and β1 integrin heterodimers on gastric epithelial cells, Hp infection studies using the quantitative and highly sensitive Hp β-lactamase reporter system clearly show that neither β1 integrin heterodimers (α1β1, α2β1 or α5β1), nor any other αβ integrin heterodimers on the cell surface are essential for CagA translocation. In contrast, deletion of the HopQ adhesin in Hp, or the simultaneous knockout of the receptors CEACAM1, CEACAM5 and CEACAM6 in KatoIII cells abolished CagA injection nearly completely, although bacterial binding was only reduced to 50%. These data provide genetic evidence that the cag-T4SS-mediated interaction of Hp with cell surface integrins on human gastric epithelial cells is not essential for CagA translocation, but interaction of Hp with CEACAM receptors is facilitating CagA translocation by the cag-T4SS of this important microbe. The Cag Type IV secretion system of Helicobacter pylori (Hp) interacts with host cell integrins and injects the bacterial oncoprotein CagA into host cells thereby contributing to inflammation and carcinogenesis during chronic infection. Binding of β1 integrin receptors by the CagA protein and the type IV secretion system is well described by many research groups, but its function for CagA translocation is not well understood. We report here that this interaction is not essential for the function of the secretion system and for CagA injection into the gastric epithelial cells lines AGS and KatoIII. However, the bacterial binding to a set of specific receptors called carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) by the Hp outer membrane protein HopQ is a prerequisite for CagA translocation. Interestingly, other bacterial adhesins and the mediated binding events do not have a similar effect on CagA translocation, suggesting a specific feature associated with HopQ mediated binding.

Attachment to the host gastric mucosa is a key step in Helicobacter pylori infection. Recently, a novel adhesin, HopQ, was shown to bind distinct host CEACAM proteins—an interaction that was found to be essential for the translocation of CagA, a key virulence factor of H. pylori. The HopQ–CEACAM1 co-crystal structure revealed a binding mode dependent on loops in HopQ that are clasped by disulfide bonds. In this study, we investigated the importance of these cysteine residues for CEACAM1 engagement by H. pylori. We observed a loss of CEACAM1 binding and CagA translocation upon disruption of the disulfide bond in loop CL1 (connecting C103 to C132 in HopQ). Deletion of the Dsb-like oxidoreductase HP0231 did not affect cell surface expression of HopQ or alter the interaction of H. pylori with target cells. Although HP0231 deletion was previously described to impede CagA translocation, our results indicate that this occurs through a HopQ-independent mechanism. Together, our results open up new avenues to therapeutically target the HopQ–CEACAM1 interaction and reduce the burden of pathogenic H. pylori.

Type IV secretion of effector proteins is an important principle for interaction of human pathogens with their target cells. The corresponding secretion systems may transport a multitude of effector proteins that have to be deployed in the respective spatiotemporal context, or only a single translocated protein, as in the case of the CagA effector protein produced by the human gastric pathogen Helicobacter pylori. For a more detailed analysis of the kinetics and mode of action of CagA type IV secretion by H. pylori, we describe here, a novel, highly sensitive split luciferase‐based translocation reporter which can be easily adapted to different end‐point or real‐time measurements. Using this reporter, we showed that H. pylori cells are able to rapidly inject a limited amount of their CagA supply into cultured gastric epithelial cells. We have further employed the reporter system to address the question whether CagA has to be unfolded prior to translocation by the type IV secretion system. We showed that protein domains co‐translocated with CagA as protein fusions are more readily tolerated as substrates than in other secretion systems, but also provide evidence that unfolding of effector proteins is a prerequisite for their transport.