Transcriptomic and Functional Approaches Unveil the Role of tmRNA in Zinc Acetate Mediated Levofloxacin Sensitivity in Helicobacter pylori

Abstract

Rapid increase in resistance of Helicobacter pylori (H. pylori) has hindered antibiotics-based eradication efforts worldwide and raises the need for additional approaches. Here, we investigate the role of zinc-based compounds in inhibiting H. pylori growth and modulating antibiotic sensitivities, interrogate their downstream transcriptomic changes, and highlight the potential mechanism driving the observed effects. We showed that zinc acetate inhibited H. pylori growth and increased H. pylori sensitivity to levofloxacin. Transcriptomic profiling showed distinct gene expression patterns between zinc acetate treated groups versus controls. In particular, we independently replicated the association between zinc acetate treatment and increased ssrA expression. Knockdown of ssrA restored levofloxacin resistance to levels of the control group. In this study, we first demonstrated the role of zinc acetate in H. pylori growth and antibiotic sensitivities. Additionally, we explored the transcriptomic perturbations of zinc acetate followed by functional knockdown follow-up of differentially expressed ssrA, highlighting the role of tmRNA and trans-translation in H. pylori levofloxacin resistance. Our results provide alternative and complementary strategies for H. pylori treatment and shed light on the underlying mechanisms driving these effects. IMPORTANCE Helicobacter pylori (H. pylori) eradication plays an important role in gastric cancer prevention, but the antimicrobial resistance of H. pylori is fast becoming a growing concern. In this study, we investigated the role of zinc acetate in inhibiting H. pylori growth and modulating antibiotic sensitivities in vitro. Additionally, we explored the transcriptomic perturbations of zinc acetate followed by functional knockdown follow-up of differentially expressed ssrA, highlighting the role of tmRNA and trans-translation in H. pylori levofloxacin resistance. Our results open up a new horizon for the treatment of antibiotic-resistant H. pylori.

Keywords: Helicobacter pylori; levofloxacin resistance; ssrA; transcriptomics; zinc acetate.

FIG 1

Zinc compounds inhibit the growth of H. pylori (NCTC 11637).

FIG 2

Differentially expressed genes (DEGs) identified by transcriptomic (RNA) sequencing. (A) Principal-component analysis (PCA) analysis of tested samples. (Con, control group; ZA, zinc acetate treated group). (B) Hierarchical clustered heatmap of differentially expressed genes (n = 227) between control and treated group. (C) Volcano plot exhibition of DEGs. The vertical axis represents the -log₁₀ (P-value), the horizontal axis displays the log₂ fold change. Red points signify the upregulated transcripts (n = 121), green points stand for downregulated transcripts (n = 106). Vertical dashed lines indicate absolute log₂ fold change = 0 and horizontal dashed lines indicate P-value = 0.05.

FIG 3

Validation of selected candidate genes using qRT-PCR. (A) Expression of 6 selected genes measured by RNA sequencing and assessed by Fragments Per Kilobase of exon model per Million mapped fragments (FPKM). Error bars indicate standard deviation. (B) Expression of 6 selected genes measured by qRT-PCR. 2^-ΔΔCt method was applied to evaluate gene expression differences. Error bars indicate standard deviation. Con, control group; ZA, zinc acetate treated group. (* P < 0.05).

FIG 4

(A) The levels of ssrA expression after knockdown evaluated by qRT-PCR. 2^-ΔΔCt was used to evaluate the difference of gene expression (P-value by independent samples t test). (B) The impact of ssrA knockdown on levofloxacin sensitivity. Left: H. pylori electroporation at 2500 V for 5 ms as control; the MIC of levofloxacin was 4.0 μg/mL. Right: H. pylori electroporation at 2500 V for 5 ms and transformed by siRNA to ssrA for 1 h; the MIC of levofloxacin was 6.0–8.0 μg/mL.