RESUMEN
BACKGROUND: Amoxicillin-resistant H. pylori strains are increasing worldwide. To explore the potential resistance mechanisms involved, the 3D structure modeling and access tunnel prediction for penicillin-binding proteins (PBP1A) was performed, based on the Streptococcus pneumoniae, PBP 3D structure. Molecular covalent docking was used to determine the interactions between amoxicillin (AMX) and PBP1A. RESULTS: The AMX-Ser368 covalent complex interacts with the binding site residues (Gly367, Ala369, ILE370, Lys371, Tyr416, Ser433, Thr541, Thr556, Gly557, Thr558, and Asn560) of PBP1A, non-covalently. Six tunnel-like structures, accessing the PBP1A binding site, were characterized, using the CAVER algorithm. Tunnel-1 was the ultimate access route, leading to the drug catalytic binding residue (Ser368). This tunnel comprises of eighteen amino acid residues, 8 of which are shared with the drug binding site. Subsequently, to screen the presence of PBP1A mutations, in the binding site and tunnel residues, in our clinical strains, in vitro assays were performed. H. pylori strains, isolated under gastroscopy, underwent AMX susceptibility testing by E-test. Of the 100 clinical strains tested, 4 were AMX-resistant. The transpeptidase domain of the pbp1a gene of these resistant, plus 10 randomly selected AMX-susceptible strains, were amplified and sequenced. Of the amino acids lining the tunnel-1 and binding site residues, three (Ser414Arg, Val469Met and Thr556Ser) substitutions, were detected in 2 of the 4 resistant and none of the sequenced susceptible strains, respectively. CONCLUSIONS: We hypothesize that mutations in amino acid residues lining the binding site and/or tunnel-1, resulting in conformational/spatial changes, may block drug binding to PBP1A and cause AMX resistance.
RESUMEN
OBJECTIVES: Disruption of protein synthesis, by drug-mediated restriction of the ribosomal nascent peptide exit tunnel (NPET), may inhibit bacterial growth. Here, we have studied the secondary and tertiary structures of domain V of the 23S rRNA in the wild-type and mutant (resistant) H. pylori strains and their mechanisms of interaction with clarithromycin (CLA). METHODS: H pylori strains, isolated from cultured gastric biopsies, underwent CLA susceptibility testing by E test, followed by PCR amplification and sequencing of domain V of 23S rRNA. The homology model of this domain in H pylori, in complex with L4 and L22 accessory proteins, was determined based on the E. coli ribosome 3D structure. The interactions between CLA and 23S rRNA complex were determined by molecular docking studies. RESULTS: Of the 70 H pylori strains, isolated from 200 dyspeptic patients, 11 (16%) were CLA-resistant. DNA sequencing identified categories with no (A), A2142G (B), and A2143G (C) mutations. Docking studies of our homology model of 23S rRNA complex with CLA showed deviated positions for categories B and C, in reference to category A, with 12.19 Å and 7.92 Å RMSD values, respectively. In both mutant categories, CLA lost its interactions at positions 2142 and 2587 and gained two new bonds with the L4 accessory protein. CONCLUSION: Our data suggest that, in mutant H pylori strains, once the nucleotides at positions 2142 and 2587 are detached from the drug, CLA interacts with and is peeled back by the L4 accessory protein, removing the drug-imposed spatial restriction of the NPET.