A bacterial small RNA regulates the adaptation of Helicobacter pylori to the host environment

Abstract

Long-term infection of the stomach with Helicobacter pylori can cause gastric cancer. However, the mechanisms by which the bacteria adapt to the stomach environment are poorly understood. Here, we show that a small non-coding RNA of H. pylori (HPnc4160, also known as IsoB or NikS) regulates the pathogen's adaptation to the host environment as well as bacterial oncoprotein production. In a rodent model of H. pylori infection, the genomes of bacteria isolated from the stomach possess an increased number of T-repeats upstream of the HPnc4160-coding region, and this leads to reduced HPnc4160 expression. We use RNA-seq and iTRAQ analyses to identify eight targets of HPnc4160, including genes encoding outer membrane proteins and oncoprotein CagA. Mutant strains with HPnc4160 deficiency display increased colonization ability of the mouse stomach, in comparison with the wild-type strain. Furthermore, HPnc4160 expression is lower in clinical isolates from gastric cancer patients than in isolates derived from non-cancer patients, while the expression of HPnc4160's targets is higher in the isolates from gastric cancer patients. Therefore, the small RNA HPnc4160 regulates H. pylori adaptation to the host environment and, potentially, gastric carcinogenesis.

Fig. 1. H. pylori acquires T-repeat extension upstream of HPnc4160 to decrease its expression during infection in vivo.

a Schematic showing experimental strategy. b RNA expression levels of ORFs or nearby genes of genome regions mutated in >50% of the strains recovered from gerbils, plotted against mutation rates. c DNA sequence around the HPnc4160 and T-repeat sequence of the strains recovered from gerbils. d–g Relative hpnc4160 expression in H. pylori strains recovered from gerbils (T repeat number of 14, n = 12; 16, n = 5; 17, n = 17; 18, n = 6) and genetically modified T-repeat lengths (n = 3) in RT-PCR (d, f) and Northern blot (e, g). Shown are the means ± s.d (d, f). Data were processed by Grubbs’ test for outliers. *P = 0.0379, **P = 0.0088, ****P < 0.0001 by Sidak’s multiple comparison tests (two sided) (d). Data are representative of two independent experiments (d–g). Source data are provided as a Source Data file.

Fig. 2. HPnc4160 downregulates the expression of bacterial pathogenic factors.

a MA plot of ratios [Δhpnc4160-hpnc4170 / wild-type (WT) H. pylori] versus their normalized average mRNA expression determined by RNA-sequencing (RNA-seq). Red dots, P < 0.001 by a Fisher Exact Test (two-sided). b Volcano plot of proteins quantified by isobaric tags for relative and absolute quantification analysis comparing WT and Δhpnc4160-hpnc4170. Each point represents the difference in protein expression (fold-change) between the two groups plotted against the level of statistical significance. Red dots, P < 0.01 by the paired t-test (two-sided). c Venn diagram representing the number of significantly differentially expressed factors between Δhpnc4160-hpnc4170 and WT. Source data are provided as a Source Data file.

Fig. 3. HPnc4160 binds to target mRNA.

a Electrophoretic mobility shift assay (EMSA) analysis of HPnc4160 binding to the 5′UTR region of each candidate mRNA. Data are representative of two independent experiments. b Schematic of CagA motifs, HPnc4160 binding regions, and HPnc4160-non-binding cagA (NB-cagA). c, d Schematic of predicted HPnc4160 binding sites in the corresponding CDS sequence of cagA TYPE 1 (c) and TYPE 2 (d). Upper sequences indicate target cagA mRNA sequences; lower sequences indicate the HPnc4160 sequence with base numbers. Colored sequences correspond to the loop structures indicated in Supplementary Fig. 4a. e EMSA analysis of HPnc4160 WT or HPnc4160 mut (a compensatory form of NB-cagA) bindings to RNA of partial cagA WT or NB-cagA. Data are representative of two independent experiments. f–h Schematic of CDS sequence of the NB-cagA of TYPE 1 (f) and TYPE 2 (g) and HPnc4160 mut (h). Upper sequences indicate target cagA mRNA sequences; lower sequences indicate the HPnc4160 sequence with base numbers. Green-colored sequences correspond to the loop structures of HPnc4160 indicated in Supplementary Fig. 4a. Mutated nucleotides in the cagA mRNA sequence are shown in red. Red-colored sequence correspond to mutated nucleotides in the hpnc4160 sequence (h). i RNase protection assay with HPnc4160, cagA mRNA, and recombinant RNase III. Data are representative of two independent experiments. Source data are provided as a Source Data file.

Fig. 4. HPnc4160 regulates bacterial-host adaptation and pathogenesis.

a, b Three days after infection, quantitative culture assays (a) and RT-PCR (b) were performed on mouse gastric specimens. c Effect of HPnc4160 deletion of H. pylori on bacterial load in mouse stomach at indicated days of post infection (dpi). Statistical significance determined by non-parametric Dunn’s multiple comparison test (two-sided) (a, b) or two-tailed Mann–Whitney test (c). *P = 0.0485 (a); *P = 0.0309 (b); *P = 0.0159 (c); ns, not significant. Source data are provided as a Source Data file.

Fig. 5. Time-dependent changes in bacterial-host adaptation and the T-repeat length.

a, b Time-dependent changes in the bacterial load in the infected mouse stomach (a) and length of the T-repeat region (b) in T1, T4, T16 and T18 strains recovered from mice. Data are presented as the median with interquartile range. Statistical significance determined by uncollected Dunn’s multiple comparison test (two-sided). *P = 0.0174, **P = 0.0066 (a); *P = 0.0294, **P = 0.0014, ****P < 0.0001 (b, T16); **P = 0.0012, ***P = 0.0002 (b, T18); ns, not significant. Source data are provided as a Source Data file.

Fig. 6. T-repeat length and HPnc4160 expression in clinical isolates.

a, b T-repeat length upstream of HPnc4160 (a) and expression of indicated mRNAs (b) in clinical isolates from non-cancer (non-cancer) and patients with cancer (cancer). Data are presented as means with 95% confidence interval (a) or medians with interquartile range (b). Statistical significance determined by two-tailed Mann–Whitney test (a, b). *P = 0.0157 (a); *P = 0.0215 (hpnc4160), ****P < 0.0001 (cagA), ***P = 0.0004 (horB), *P = 0.0025 (hopE), *P = 0.0110 (omp14), *P = 0.0122 (hofC), *P = 0.0205 (HP0410) (b). Source data are provided as a Source Data file.

Fig. 7. Infection-induced silencing of HPnc4160 upregulates target gene expression and promotes bacterial-host adaptation and, potentially, oncogenesis during chronic H. pylori infection.

H. pylori infection in vivo leads to elongation of T-repeats upstream of the HPnc4160 sRNA coding region, which results in decreased expression of HPnc4160 sRNA. Gene silencing of HPnc4160 results in increased target gene expression, coding for OMPs and CagA. As a result, bacterial colonization and CagA translocation into the attached host cells increase, promoting bacterial adaptation to the host and, possibly, oncogenesis.