Architectural asymmetry enables DNA transport through the Helicobacter pylori cag type IV secretion system.

Structural asymmetry within secretion system architecture is fundamentally important for apparatus diversification and biological function. However, the mechanism by which symmetry mismatch contributes to nanomachine assembly and interkingdom effector translocation are undefined. Here, we show that architectural asymmetry orchestrates dynamic substrate selection and enables trans-kingdom DNA conjugation through the Helicobacter pylori cag type IV secretion system (cag T4SS). Structural analyses of asymmetric units within the cag T4SS periplasmic ring complex (PRC) revealed intermolecular π-π stacking interactions that coordinate DNA binding and license trans-kingdom conjugation without disrupting the translocation of protein and peptidoglycan effector molecules. Additionally, we identified a novel proximal translocation channel gating mechanism that regulates cargo loading and governs substrate transport across the outer membrane. We thus propose a model whereby the organization and geometry of architectural symmetry mismatch exposes π-π interfaces within the PRC to facilitate DNA transit through the cag T4SS translocation channel.

DNA Transport through the Dynamic Type IV Secretion System.

The versatile type IV secretion system (T4SS) nanomachine plays a pivotal role in bacterial pathogenesis and the propagation of antibiotic resistance determinants throughout microbial populations. In addition to paradigmatic DNA conjugation machineries, diverse T4SSs enable the delivery of multifarious effector proteins to target prokaryotic and eukaryotic cells, mediate DNA export and uptake from the extracellular milieu, and in rare examples, facilitate transkingdom DNA translocation. Recent advances have identified new mechanisms underlying unilateral nucleic acid transport through the T4SS apparatus, highlighting both functional plasticity and evolutionary adaptations that enable novel capabilities. In this review, we describe the molecular mechanisms underscoring DNA translocation through diverse T4SS machineries, emphasizing the architectural features that implement DNA exchange across the bacterial membrane and license transverse DNA release across kingdom boundaries. We further detail how recent studies have addressed outstanding questions surrounding the mechanisms by which nanomachine architectures and substrate recruitment strategies contribute to T4SS functional diversity.

Immunostimulatory membrane proteins potentiate H. pylori-induced carcinogenesis by enabling CagA translocation.

Infection with Helicobacter pylori is the single greatest risk factor for developing gastric adenocarcinoma. In prospective, population-based studies, seropositivity to the uncharacterized H. pylori proteins Hp0305 and Hp1564 was significantly associated with cancer risk in East Asia. However, the mechanism underlying this observation has not been elucidated. Here, we show that Hp0305 and Hp1564 act in concert with previously ascribed H. pylori virulence mechanisms to orchestrate cellular alterations that promote gastric carcinogenesis. In samples from 546 patients exhibiting premalignant gastric lesions, seropositivity to Hp0305 and Hp1564 was significantly associated with increased gastric atrophy across all stomach conditions. In vitro, depletion of Hp0305 and Hp1564 significantly reduced levels of gastric cell-associated bacteria and markedly impaired the ability of H. pylori to stimulate pro-inflammatory cytokine production. Remarkably, our studies revealed that Hp1564 is required for translocation of the oncoprotein CagA into gastric epithelial cells. Our data provide experimental insight into the molecular mechanisms governing novel H. pylori pathogenicity factors that are strongly associated with gastric disease and highlight the potential of Hp0305 and Hp1564 as robust molecular tools that can improve identification of individuals that are highly susceptible to gastric cancer. We demonstrate that Hp0305 and Hp1564 augment H. pylori-mediated inflammation and gastric cancer risk by promoting key bacteria-gastric cell interactions that facilitate delivery of oncogenic microbial cargo to target cells. Thus, therapeutically targeting microbial interactions driven by Hp0305/Hp1564 may enable focused H. pylori eradication strategies to prevent development of gastric malignancies in high-risk populations.