Identification of Helicobacter pylori HopQ as a novel T4SS-associated virulence factor

Helicobacter pylori is a micro-aerophilic spiral-shaped motile Gram-negative bacterium colonizing gastrointestinal tracts of about 50% of the human population worldwide and causing severe gastric diseases such as gastritis, peptic and gastric ulcers, gastric cancer and mucosaassociated lymphoid tissue (MALT) lymphoma. Disease outcome is strongly dependent on the expression of a type IV secretion system (T4SS), by which the pathogen translocates virulence factor cytotoxin-associated gene A (CagA) into the host cell. The aim of the present work was to identify new T4SS-associated virulence factors and to study their contribution to T4SS-mediated pathogenesis. In a previous work, the screening of ca. 3000 H. pylori strain G27 transposon mutants revealed that inactivation of outer membrane protein HopQ resulted in strong decrease in the T4SS dependent activation of the proinflammatory transcription factor NF-κB (nuclear factor kappa B). With this hint, a possible contribution of HopQ to the T4SSmediated pathogenesis was studied in details. It has been found that HopQ was important for the induction of T4SS-mediated processes. The most prominent effect was complete failure of CagA translocation and the absence of further CagA-mediated host cell responses such as hummingbird phenotype formation and cell scattering. Furthermore, the deletion of hopQ gene negatively affected the activation of NF-κB, MAPK signaling pathways and IL-8 secretion in the AGS cells infected with H. pylori mutant. In particular, p65 nuclear translocation and IL-8 secretion were ~2.5-fold lower in comparison to wild-type and the peak of NF-κB activation shifted from 45 to 75 min post infection. HopQ deletion mutant also failed to activate JNK1/2 and activation of ERK1/2 and p38 was reduced and delayed. In contrast to that, cell shape, adhesion and motility of mutant were not change compared to the parental strain. Important, the wild-type infectious phenotype was restored by transformation of HopQ mutant with shuttle vector pHel3 expressing the hopQ of the wild-type strain. The functional link between HopQ and T4SS was described in the present work for the first time and therefore represents a scientific novelty. Even though the mechanism of interaction between HopQ and T4SS remains unknown, the discovery of HopQ as a new virulence factor is important for better understanding of H. pylori-driven pathogenic mechanisms.