Helicobacter pylori persistently colonizes the human stomach, and is associated with inflammation-induced gastric cancer. Bacterial crosstalk with the host immune system produces various inflammatory mediators and subsequent reactions in the host, but not bacterial clearance. Interleukin-1β (IL-1β) is implicated in gastric cancer development and certain gene polymorphisms play a role in this scenario. Mature IL-1β production depends on inflammasome activation, and the NLRP3 inflammasome is a major driver in H. pylori-infected mice, while recent studies demonstrated the down-regulation of NLRP3 expression in human immune cells, indicating a differential NLRP3 regulation in human vs. mice. In addition to the formation of mature IL-1β or IL-18, inflammasome activation induces pyroptotic death in cells. We demonstrate that H. pylori infection indeed upregulated the expression of pro-IL-1β in human immune cells, but secreted only very low amounts of mature IL-1β. However, application of exogenous control activators such as Nigericin or ATP to infected cells readily induced NLRP3 inflammasome formation and secretion of high amounts of mature IL-1β. This suggests that chronic H. pylori infection in humans manipulates inflammasome activation and pyroptosis for bacterial persistence. This inflammasome deregulation during H. pylori infection, however, is prone to external stimulation by microbial, environmental or host molecules of inflammasome activators for the production of high amounts of mature IL-1β and signaling-mediated gastric tumorigenesis in humans.

The gastric pathogen and carcinogen Helicobacter pylori (H. pylori) encodes a type IV secretion system for translocation of the effector protein CagA into host cells. Injected CagA becomes tyrosine-phosphorylated at the five amino acid residue Glutamate-Proline- Isoleucine-Tyrosine-Alanine (EPIYA)-sequence motifs. These phosphorylated EPIYA-sites represent recognition motifs for binding of multiple host factors, which then manipulate signaling pathways to trigger gastric disease. Thus, efficient detection of single phosphorylated EPIYA-motifs in CagA is required. Detection of phospho-CagA is primarily performed using commercial pan-phosphotyrosine antibodies. However, those antibodies were originally generated to recognize many phosphotyrosines in various mammalian proteins and are not optimized for use in bacteria. To address this important limitation, we synthesized 11-mer phospho- and non-phospho-peptides from EPIYA-motifs A, B, and C, and produced three phospho-specific and three non-phospho-specific rabbit polyclonal CagA antibodies. These antibodies specifically recognized the corresponding phosphorylated and non-phosphorylated EPIYA-motifs, while the EPIYA-C antibodies also recognized the related East-Asian EPIYA-D motif. Otherwise, no cross-reactivity of the antibodies among EPIYAs was observed. Western blotting demonstrated that each EPIYA-motif can be predominantly phosphorylated during H. pylori infection. This represents the first complete set of phospho-specific antibodies for an effector protein in bacteria, providing useful tools to gather information for the categorization of CagA phosphorylation, cancer signaling, and gastric disease progression.