Abstract
The clinical outcome of Helicobacter pylori infections is determined by multiple host-pathogen interactions that may develop to chronic gastritis, and sometimes peptic ulcers or gastric cancer. Highly virulent strains encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation at EPIYA-sequence motifs, called A, B and C in Western-type strains, by members of the oncogenic Src and Abl host kinases. Phosphorylated EPIYA-motifs mediate interactions of CagA with host signaling factors – in particular various SH2-domain containing human proteins – thereby hijacking multiple downstream signaling cascades. Observations of tyrosine-phosphorylated CagA are mainly based on the use of commercial phosphotyrosine antibodies, which originally were selected to detect phosphotyrosines in mammalian proteins. Systematic studies of phosphorylated EPIYA-motif detection by the different antibodies would be very useful, but are not yet available. To address this issue, we synthesized phospho- and non-phosphopeptides representing each predominant Western CagA EPIYA-motif, and determined the recognition patterns of seven different phosphotyrosine antibodies in Western blots, and also performed infection studies with diverse representative Western H. pylori strains. Our results show that a total of 9–11 amino acids containing the phosphorylated EPIYA-motifs are necessary and sufficient for specific detection by these antibodies, but revealed great variability in sequence recognition. Three of the antibodies recognized phosphorylated EPIYA-motifs A, B and C similarly well; whereas preferential binding to phosphorylated motif A and motifs A and C was found with two and one antibodies, respectively, and the seventh anti-phosphotyrosine antibody did not recognize any phosphorylated EPIYA-motif. Controls showed that none of the antibodies recognized the corresponding non-phospho CagA peptides, and that all of them recognized phosphotyrosines in mammalian proteins. These data are valuable in judicious application of commercial anti-phosphotyrosine antibodies and in characterization of CagA phosphorylation during infection and disease development.
Highlights
Posttranslational modification of proteins by kinases is important in many cell signaling processes
To systematically analyze the recognition capabilities of phosphorylated CagA EPIYA-motifs by a-phosphotyrosine antibodies we first synthesized a series of peptides derived from the EPIYA-A motif exhibiting the phosphotyrosine residue in the middle +/2 five, four, three or two flanking amino acids, including the STEPIYAKVNK (11-mer), TEPIYAKVN (9-mer), EPIYAKV (7mer) and PIYAK (5-mer) sequences as indicated (Figure 1B, top)
The CagA protein and its EPIYA-motifs are known for long time as important virulence markers of H. pylori [5,6,18,23,24,27,28,29,30,31,32,33,34,67,68]
Summary
Posttranslational modification of proteins by kinases is important in many cell signaling processes. Effector protein phosphotyrosines together with some flanking residues act as recognition motifs for eukaryotic signaling factors They recruit in particular cellular binding partners that contain SH2 (Src homology 2) domains, but not PTB (phosphotyrosine binding) domains, and thereby target and subvert eukaryotic signal transduction pathways in ways that benefit the pathogen [2,4]. This virulence strategy is well-established for six different bacterial pathogens: enteropathogenic Escherichia coli (EPEC), Helicobacter pylori, Chlamydia trachomatis, Bartonella henselae, Anaplasma phagocytophilum and Ehrlichia chaffeensis [7,8,9,10,11,12,13]
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