Helicobacter pylori Uptake and Efflux: Basis for Intrinsic Susceptibility to Antibiotics In Vitro

Abstract

We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63: 1567-1572, 1995) that Helicobacter pylori has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels. H. pylori is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that H. pylori is able to take up higher basal levels of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN) than Pseudomonas aeruginosa or Escherichia coli, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake in H. pylori (6 to 8 g/ml) than in P. aeruginosa or E. coli (0.3 to 0.5 g/ml), suggesting that H. pylori has a less efficient selfpromoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic ␤-lactamase (which H. pylori lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in H. pylori 11637 the presence of portions of three genes with homology to potential restriction-nodulationdivision (RND) efflux systems. It was confirmed that H. pylori contained only these three putative RND efflux systems, named here hefABC, hefDEF, and hefGHI, and that the hefGHI system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in H. pylori. Independent mutagenesis of the three putative RND efflux operons in the chromosome of H. pylori had no effect on the in vitro susceptibility of H. pylori to 19 antibiotics. These results, in contrast to what is observed in E. coli, P. aeruginosa, and other clinically important gram-negative bacteria, suggest that active efflux does not play a role in the intrinsic resistance of H. pylori to antibiotics.

Identification of Surface Proteins of Helicobacter pylori by Selective Biotinylation, Affinity Purification, and Two-dimensional Gel Electrophoresis

“…The large overlap between surface-exposed, firmly bound proteins (this study) and previously characterized secreted proteins is consistent with the concept of re-adsorption of released proteins at the H. pylori surface in vitro (25,26). HefA is homologous to TolC, an outer membrane protein from E. coli (37,38), while HP1564 is homologous to an outer membrane protein of Pasteurella hemolytica (39). In contrast, the ABC transporter of iron, CeuE, is likely to be localized in the periplasm based on data from E. coli (40).…”

Comparative Genomics ofHelicobacter pylori: Analysis of the Outer Membrane Protein Families

“…Each system contains an outer membrane component with some homology to the E. coli TolC protein, and these proteins (HefA, -D, and -G) share Ͼ92% amino acid identity between the J99 and 26695 strains (Table 1). These proposed efflux systems have been shown to be highly conserved in sequence and organization between multiple H. pylori strains (9).…”

“…Both sequenced H. pylori strains contain three clusters (hefA-C, hefD-F, and hefG-I) which encode homologs of resistance-nodulation-division efflux pump systems (9). Each system contains an outer membrane component with some homology to the E. coli TolC protein, and these proteins (HefA, -D, and -G) share Ͼ92% amino acid identity between the J99 and 26695 strains (Table 1).…”

Vibrio cholerae tolC Is Required for Bile Resistance and Colonization

“…For example, the P. aeruginosa mexAB-oprM (16), mexCD-oprJ (25), and mexEF-oprN operons (14) each encode their own cognate outer membrane pore proteins (oprM, oprJ, and oprN, respectively) that are TolC homologues. Similar systems can be found in Helicobacter pylori where each RND system contains a gene encoding a distinct outer membrane protein (2). In these bacterial species, the functions attributed to TolC in E. coli are not linked to a single tolC allele but are distributed among the various tolC homologues and their cognate transport system.…”