Resumo

The most widely therapeutic regimen for Helicobacter pylori eradication is based on a proton-pump-inhibitor and two antibiotics (clarithromycin plus amoxicillin or metronidazole). The efficacy of the therapy depends on antibiotic susceptibility pattern, and patient compliance. Noncompliance is a frequent problem in outpatient setting. Subinhibitory concentrations of antibiotics might alter host-pathogen interaction. In this study we evaluated how ½ minimal inhibitory concentration (MIC) of amoxicillin affects H. pylori-protein expression profile. H. pylori ATCC 43504 (metronidazole resistant and amoxicillin susceptible) was cultured in presence of 0.125 μg/mL of amoxicillin and absence. Bacterial cells were lyses and protein concentration was quantified by Bradford method. Preliminary evaluation was performed using SDS–PAGE, protein bands stained by colloidal Coomassie Blue, and identified by mass spectrometry. After analysis by ImageMaster 2D Platinum software (GE Healthcare), nine spots showed significant identity (score greater than the limit of significance) with H. pylori and homology with other bacteria when tested against the database of eubacteria protein. The spots differentially expressed were subjected to mass spectrometry analysis. Proteomics analyses revealed that amoxicillin treatment affected the expression (p < 0.05) of heat-shock proteins (HSP), ATP synthase alpha subunit, enolase, argininosuccinate synthase, RNA polymerase alpha subunit, superoxide dismutase, neutrophil activating protein (NAP), and 50S ribosomal protein. Two of the most over-expressed proteins were the molecular chaperones HSP70 and HSP60, both with adhesion function. Enolase is one of the best-characterized human plasminogen receptors that promotes host-pathogen interactions. NAP does not only recruit leukocytes from the vascular lumen but also stimulates them to produce messengers that contribute to the inflammation. Argininosuccinate synthase, an intermediate of urea cycle, is a virulence determinant necessary for colonization. An important finding from our study is that the superoxide dismutase, an enzyme that plays a central part in H. pylori defense mechanisms against reactive oxygen species, was also over expressed. The results show that ½ MIC of amoxicillin produces specific changes on the cell wall proteins of susceptible H. pylori that contribute to the pathogenesis of the bacterium.