Identification of a tyrosine-phosphorylated 35 kDa carboxy-terminal fragment (p35(CagA)) of the Helicobacter pylori CagA protein in phagocytic cells:Processing or breakage?

Helicobacter pylori is a very common bacterial pathogen that causes gastric disease by inducing the infiltration of immune cells as an initial event. Virulent H. pylori strains express a type IV secretion system composed of s everal Virulence (Vir) proteins encoded by the cag pathogenicity island (ca g PAI). During infection of phagocytic cells (U937, Josk-M and J774A.1) we have detected a de novo tyrosine-phosphorylated protein (p35(p-Tyr) with si zes Of 30 kDa, 38 kDa or 40 kDa, depending on the H. pylori strain. p35(p-T yr) occurrence required functional virB4, virB7, virB10, virB11, virD4 and cagA (cytotoxin-associated gene A) genes encoded by the cag PAI suggesting that p35(p-Tyr) is a bacterial protein of variable size. We have biochemica lly purified p35(p-Tyr) from infected U937 cells. Tryptic peptides of p35p- Tyr determined by matrix-assisted laser desorption/ionization-mass spectrom etry (MALDI-MS) identified the carboxy (C)-terminal part of the H. pylori C agA protein. Subsequent analysis by two-dimensional electrophoresis (2-DE) and immunoblotting using anti-CagA antibodies revealed the presence of thre e stable CagA protein species in phagocytes: (i) 130-140 kDa full-length Ca gA (p135(CagA)), (ii) a 100-105 kDa fragment (p100(CagA)) and (iii) a 30-40 kDa fragment (p35(CagA)). Unlike p135(CagA), p35(CagA) and p100(CagA) were also detected in much lower amounts in H. pylori without host cell contact . Therefore, breakage or processing leads to the production of p35(CagA) an d p100(CagA), a process that is enhanced after translocation into host cell s. MALDI-MS data and the isoelectric point determined by both 2-DE and sequ ence analysis suggested that p35(CagA) represents the C-terminal part of Ca gA and p100(CagA) corresponds to the remaining amino (N)-terminal fragment. The possible function of CagA in host signal transduction and development of gastric disease is discussed.