Analysis of core protein clusters identifies candidate variable sites conferring metronidazole resistance in Helicobacter pylori

Abstract

Background: Metronidazole is one of the first-line drugs of choice in the standard triple therapy used to eradicate Helicobacter pylori infection. Hence, the global emergence of metronidazole resistance in Hp poses a major challenge to health professionals. Inactivation of RdxA is known to be a major mechanism of conferring metronidazole resistance in H. pylori. However, metronidazole resistance can also arise in H. pylori strains expressing functional RdxA protein, suggesting that there are other mechanisms that may confer resistance to this drug.

Methods: We performed whole-genome sequencing on 121 H. pylori clinical strains, among which 73 were metronidazole-resistant. Sequence-alignment analysis of core protein clusters derived from clinical strains containing full-length RdxA was performed. Variable sites in each alignment were statistically compared between the resistant and susceptible groups to determine candidate genes along with their respective amino-acid changes that may account for the development of metronidazole resistance in H. pylori.

Results: Resistance due to RdxA truncation was identified in 34% of metronidazole-resistant strains. Analysis of core protein clusters derived from the remaining 48 metronidazole-resistant strains and 48 metronidazole-susceptible identified four variable sites significantly associated with metronidazole resistance. These sites included R16H/C in RdxA, D85N in the inner-membrane protein RclC (HP0565), V265I in a biotin carboxylase protein (HP0370) and A51V/T in a putative threonylcarbamoyl-AMP synthase (HP0918).

Conclusions: Our approach identified new potential mechanisms for metronidazole resistance in H. pylori that merit further investigation.

Keywords: Helicobacter pylori; antibiotic resistance; core protein clusters; metronidazole; sequence alignment; whole-genome sequencing.

Figure 1.

Structural alignment comparing the model of HP0918 computed by Phyre2 based on 1HRU and 1HRU itself. Highlighted with red stars are the conserved amino acids of the TsaC protein family, identified by Teplova et al. [26], which are identical in HP0918. Highlighted in blue are amino acids that are found in the YrdC subgroup, which are also found in HP0918 or for which there are conservative substitutions.

Figure 2.

Proposed mechanism of superoxide anion radical neutralization mediated by HP0370, superoxide dismutase and catalase enzymes in H. pylori. In this diagram, we propose that the hydrogen ions generated by biotin carboxylation can be utilized by the superoxide dismutase enzyme to convert superoxide radicals into hydrogen peroxide. This can be further inactivated by AhpC alkyl hydrogen peroxidase into non-deleterious water molecules.