Stefan Moese Matthias Selbach Ursula Zimny-Arndt Peter R. Jungblut Thomas F. Meyer Steffen Backert Max-Planck-Institut für Infektionsbiologie, Abt. Molekulare Biologie, Berlin, Germany Identification of a tyrosine-phosphorylated 35 kDa carboxy-terminal fragment (p35 CagA )ofthe 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 several virulence (Vir) proteins encoded by the cag pathogenicity island (cag 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 sizes of 30 kDa, 38 kDa or 40 kDa, depending on the H. pylori strain. p35 p-Tyr 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 biochemically purified p35 p-Tyr from infected U937 cells. Tryptic peptides of p35 p-Tyr determined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) identified the carboxy (C)-terminal part of the H. pylori CagA protein. Subsequent analysis by two-dimen- sional electrophoresis (2-DE) and immunoblotting using anti-CagA antibodies revealed the presence of three stable CagA protein species in phagocytes: (i) 130–140 kDa full- length CagA (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 pro- cessing leads to the production of p35 CagA and p100 CagA , a process that is enhanced after translocation into host cells. MALDI-MS data and the isoelectric point determined by both 2-DE and sequence analysis suggested that p35 CagA represents the C-terminal part of CagA 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. Keywords: Mass spectrometry / Molecular pathogenesis / Pathogenicity island / Tyrosine phos- phorylation / Two-dimensional gel electrophoresis / Virulence PRO 0061 1 Introduction Helicobacter pylori, a Gram-negative microaerophilic bacterium, is a highly successful pathogen very well adapted to the extreme acidic conditions in the human stomach and can persist for life. H. pylori colonisation of the gastric mucosa induces infiltration of the lamina pro- pria and epithelium with immunocytes and inflammatory cells, a situation referred to as chronic active gastritis. Years after initial infection, chronic gastritis can either remain asymptomatic or may develop into more severe diseases such as peptic ulcer, mucosa-associated lym- phoid tissue (MALT) lymphoma or adenocarcinoma [1–3]. H. pylori-induced gastroduodenal diseases depend on the inflammatory response in the gastric epithelium [4] and on the presence of specific virulence factors from the bacterium [3]. In vivo and in vitro studies showed that H. pylori induces a variety of inflammatory mediators including cytokines (interleukin (IL)-1, IL-6 or TNF-a) and chemokines (IL-8, Gro-a, RANTES, ENA-78, MCP-1, Mip-1a) [5–8]. The inflammatory response and the devel- opment of gastric diseases are more common in patients infected with H. pylori isolates carrying the cag patho- genicity island (cag PAI, a locus of about 40 kb containing up to 31 genes) and the vacuolating cytotoxin VacA (cag + or type I strains) [6, 9]. In contrast, H. pylori strains lacking the cag PAI (cag - or type II strains) do not induce dramatic changes in the host mucosa and resemble commensal bacteria rather than pathogens. Correspondence: Prof. Dr. Thomas F. Meyer, Max-Planck-Insti- tut für Infektionsbiologie, Abt. Molekulare Biologie, Schumann- str. 20/21, D-10117 Berlin, Germany E-mail: meyer@mpiib-berlin.mpg.de Fax: +49-30-28-46-04-01 Abbreviations: AGS, gastric adenocarcinoma cells; cagA, cyto- toxin-associated gene A; Cm R , chloramphenicol resistance; EPEC, enteropathogenic Escherichia coli; MOI, multiplicity of infection; PAI, pathogenicity island; vir, virulence 618 Proteomics 2001, 1, 618–629  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001 1615-9853/01/0404–618 $17.50+.50/0