Proteins released by Helicobacter pylori in vitro.
Secretion of proteins by Helicobacter pylori may contribute to gastric inflammation and epithelial damage. An in vitro analysis was designed to identify proteins released by mechanisms other than nonspecific lysis. The radioactivity of proteins in the supernatant was compared with that of the intact organism by two-dimensional gel phosphorimaging following a 4-h pulse-chase. The ratio of the amount of UreB, a known cytoplasmic protein, in the supernatant to that in the pellet was found to be 0.25, and this was taken as an index of lysis during the experiments (n = 6). Ratios greater than that of UreB were used to distinguish proteins that were selectively released into the medium. Thus, proteins enriched more than 10-fold in the supernatant compared to UreB were identified by mass spectrometry. Sixteen such proteins were present in the supernatant: VacA; a conserved secreted protein (HP1286); putative peptidyl cis-trans isomerase (HP0175); six proteins encoded by HP0305, HP0231, HP0973, HP0721, HP0129, and HP0902; thioredoxin (HP1458); single-stranded-DNA-binding 12RNP2 precursor (HP0827); histone-like DNA-binding protein HU (HP0835); ribosomal protein L11 (HP1202); a putative outer membrane protein (HP1564); and outer membrane proteins Omp21 (HP0913) and Omp20 (HP0912). All except HP0902, thioredoxin, HP0827, HP0835, and HP1202 had a signal peptide. When nalidixic acid, a DNA synthesis inhibitor, was added to inhibit cell division but not protein synthesis, to decrease possible contamination due to outer membrane shedding, two outer membrane proteins (Omp21 and Omp20) disappeared from the supernatant, and the amount of VacA also decreased. Thus, 13 proteins were still enriched greater than 10-fold in the medium after nalidixic acid treatment, suggesting these were released specifically, possibly by secretion. These proteins may be implicated in H. pylori-induced effects on the gastric epithelium.
Kim
Nayoung
N
Department of Physiology and Medicine, UCLA Digestive Research Center, University of California, Los Angeles, and VA Greater Los Angeles Health Care System, 90073, USA.
Weeks
David L
DL
Shin
Jai Moo
JM
Scott
David R
DR
Young
Mary K
MK
Sachs
George
G
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001431:Bacteriological Techniques; D017077:Culture Media, Conditioned / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D016480:Helicobacter pylori / Q000254:growth & development* / Q000378:metabolism*; D013058:Mass Spectrometry; D008715:Methionine / Q000378:metabolism; D009268:Nalidixic Acid / Q000494:pharmacology; D013462:Sulfur Radioisotopes / Q000378:metabolism
D001426:Bacterial Proteins; D017077:Culture Media, Conditioned; D013462:Sulfur Radioisotopes; D009268:Nalidixic Acid; D008715:Methionine