FBPAII and rpoBC, the Two Novel Secreted Proteins Identified by the Proteomic Approach from a Comparative Study between Antibiotic-Sensitive and Antibiotic-Resistant Helicobacter pylori-Associated Gastritis Strains

Abstract

Helicobacter pylori infection is the leading cause of chronic gastritis, which can develop into gastric cancer. Eliminating H. pylori infection with antibiotics achieves the prevention of gastric cancer. Currently, the prevalence of H. pylori resistance to clarithromycin and metronidazole, and the dual resistance to metronidazole and clarithromycin (C_R, M_R, and C/M_R, respectively), remains at a high level worldwide. As a means of exploring new candidate proteins for the management of H. pylori infection, secreted proteins from antibiotic-susceptible and antibiotic-resistant H. pylori-associated gastritis strains were obtained by in-solution tryptic digestion coupled with nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS). A total of 583, 582, 590, and 578 differential expressed proteins were identified from C_R, M_R, C/M_R, and antibiotic-sensitive strain (S_S) samples, respectively. Of these, 23 overlapping proteins were found by Venn diagram analysis. Based on heat map analyses, the most and least differing protein expressions were observed from C/M_R strains and S_S strains, respectively. Of the proteins secreted by the S_S strain, only nine were found. After predicting the protein interaction with metronidazole and clarithromycin via the STITCH database, the two most interesting proteins were found to be rpoBC and FBPAII. After quantitative real-time reverse transcription PCR (qRT-PCR) analysis, a downregulation of rpoB from M_R strains was observed, suggesting a relationship of rpoB to metronidazole sensitivity. Inversely, an upregulation of fba from C_R, M_R, and C/M_R strains was noticed, suggesting the paradoxical expression of FBPAII and the fba gene. This report is the first to demonstrate the association of these two novel secreted proteins, namely, rpoBC and FBPAII, with antibiotic-sensitive H. pylori-associated gastritis strains.

Keywords: Helicobacter pylori-associated gastritis; antibiotic resistance; antibiotic-sensitive; clarithromycin; dual resistance to metronidazole and clarithromycin; in-solution digestion proteomic study; metronidazole.

FIG 1

Heat map analysis (a) and biological function of 592 differentially expressed proteins (b), 23 overlapping proteins from the four strains (c), and 9 identified proteins from the S_S strains (d). (a) A total of 583, 582, 590, and 578 different proteins are displayed as a fold change in heat map analyses. Biological function is based on the PANTER database classifications of (b) 592 secreted proteins, (c) 23 overlapping proteins and (d) 9 identified proteins from the S_S strains. These classifications demonstrate the first two functions—the cellular process (46%, 51%, and 61%, respectively) and the metabolic process (40%, 46%, and 38%, respectively).

FIG 2

Venn diagram of 592 differentially expressed proteins from (a) nano-LC-MS/MS analysis and (b) heat map of 23 overlapping proteins among C_R, M_R, C/M_R, and S_S strains. (a) A total of 583, 582, 590, and 578 unique proteins were identified from the C_R, M_R, C/M_R, and S_S samples, respectively. (b) A higher than average abundance of a protein (indicated by log₂) is displayed in shades of red, whereas reduced abundance is displayed in shades of green. The heat map analysis revealed that FBPAII is the only secreted protein found exclusively in the S_S strain.

FIG 3

The interaction of the chemical and proteins network of 9 proteins secreted by antibiotic-susceptible strains (numbers 1 to 9 in Table 1) and other known proteins, including clarithromycin and metronidazole, by STITCH 4.0. Protein-protein interactions are shown in gray, chemical-protein interactions are shown in in green, and interactions between chemicals are shown in in red. (a) Interaction between four secreted proteins from the S_S strains but not the M_R strains (numbers 1 to 4 in Table 1). (b) Interaction between three secreted proteins from the S_S strains but not the C_R strains (numbers 5 to 7 in Table 1). (c) Interaction between 1 secreted protein from S_S strains but not from the M_R and C_R strains (number 8 in Table 1). (d) Interaction between one unique protein found only in the S_S strain (number 9 in Table 1).

FIG 4

mRNA expression pattern of fba and rpoB. (a and b) The fold change values in mRNA expression of (a) fba and (b) rpoB were analyzed by qRT-PCR from the four strains used in this study; the C_R, M_R, C/M_R, and S_S samples (*, P < 0.05, versus S_S as the control). Error bars indicate the standard deviation values from three independent experiments. The fold changes represented in this figure were calculated by using the expression level at the S_S strains as a reference.

FIG 5

(a and b) Schematic diagram of bacterial strain cultures with C_R clinical strain as an example (a) and overview of the experimental workflow in this study (b). The number of experimental repeats in this study was 9 for each of the H. pylori samples (numbers C_R 1–3, M_R 4-6, C/M_R 7-9, and S_S 10-12). The oval enclosures represent H. pylori clinical strains (C_R, M_R, C/M_R, and S_S); the diamond enclosures represent the patient number (e.g., C_R 1 = T2629, C_R 2 = T3086, C_R 2 = T3383); the rounded rectangular and rectangular enclosures represent the triplicate in each experimental workflow.