Quorum-Sensing Regulator OpaR Directly Represses Seven Protease Genes in Vibrio parahaemolyticus

Abstract

Proteases play a key role in numerous bacterial physiological events. Microbial proteases are used in the pharmaceutical industry and in biomedical applications. The genus Vibrio comprises protease-producing bacteria. Proteases transform polypeptides into shorter chains for easier utilization. They also function as a virulence factor in pathogens. The mechanism by which protease genes are regulated in Vibrio parahaemolyticus, an emerging world-wide human pathogen, however, still remains unclear. Quorum sensing is the communication system of bacteria. OpaR is the master quorum-sensing regulator in V. parahaemolyticus. In the present study, quantitative reverse transcriptase-polymerase chain reaction and protease gene promoter-fusion reporter assays revealed that OpaR represses seven protease genes-three metalloprotease genes and four serine protease genes-which are involved in environmental survival and bacterial virulence. Furthermore, the electrophoresis mobility shift assay demonstrated that OpaR is bound directly to the promoter region of each of the seven protease genes. DNase I footprinting identified the sequence of these OpaR-binding sites. ChIP-seq analyses revealed 435 and 835 OpaR-binding sites in the late-log and stationary phases, respectively. These OpaR-binding sequences indicated a conserved OpaR-binding motif: TATTGATAAAATTATCAATA. These results advance our understanding of the protease regulation system in V. parahaemolyticus. This study is the first to reveal the OpaR motif within V. parahaemolyticus in vivo, using ChIP-seq, and to provide a database for OpaR direct regulon.

Keywords: ChIP-seq; DNase I footprinting; EMSA; OpaR-binding motif; Vibrio parahaemolyticus; proteases regulation; quorum sensing.

FIGURE 1

ChIP-seq data revealed in vivo OpaR-binding sites in Vibrio parahaemolyticus. (A) Distribution of OpaR-binding sites of ChIP-seq in the late-log and stationary phases. (B) Conserved OpaR-binding motif was identified from 838 OpaR-binding sequence of ChIP-seq data and analyzed using Multiple Expectation Maximization for Motif Elicitation. The height of each base represents its frequency. Conserved OpaR-binding motif aligned using TOMTOM (http://memesuite.org//tools/tomtom) with other Vibrio quorum-sensing regulators: (C) LuxR of V. campbellii and (D) HapR of V. cholera.

FIGURE 2

OpaR-binding peak distribution of the seven protease genes. Yellow spots represent in vivo OpaR-binding positions on the protease promoter or 3′region, as identified using ChIP-seq. White and red arrows denote the protease genes, and blue arrows illustrate the nearby genes. Navy blue peak represents the binding strength of the samples (ChIP-seq enriched by anti-FLAG antibody); light blue peak is the control signal (ChIP-seq without anti-FLAG antibody enrichment). Numbers below the peak diagrams represent the reads per million (RPM) value of ChIP-seq. Central OpaR-binding sites are shown in brown boxes. (A) OpaR bound to five protease genes—lytM, mcp02, m6 protease, serine protease, and degS—both in the late-log and stationary phases. (B) OpaR bound to both protease II and periplasmic protease only in the stationary phase.

FIGURE 3

Protease gene expression levels at different growth phases, as examined using qRT-PCR. Total RNA isolated from VP93 at log (OD₆₀₀ 1.7), late-log (OD₆₀₀ 2.8), and stationary (OD₆₀₀ 4) phases was purified and analyzed using specific primer sets for qRT-PCR: (A) lytM, (B) mcp02, (C) m6 protease, (D) serine protease, (E) degS, (F) protease II, and (G) periplasmic protease. (H) Growth curves of VP93 monitored by measuring OD₆₀₀ at every growth stage. Values represent mean of three independent experiments, and error bars indicate standard deviation. All values were relative to the log phase, which had the value of 1. Asterisks denote statistical significance (*p < 0.01).

FIGURE 4

OpaR regulates the expression of the seven protease genes. Total RNA isolated from the VP93 and ΔopaR strains was purified, and gene expression was detected via qRT-PCR at the (A) late-log (OD₆₀₀ 2.8) and (B) stationary (OD₆₀₀ 4) phases. A luciferase assay was performed using VP93 and ΔopaR strains with the protease promoter-luxAB or 3’region-luxAB fusion plasmid at the (C) late-log (OD₆₀₀ 2.8) and (D) stationary (OD₆₀₀ 4) phases. Values represent mean of three independent experiments, and error bars indicate standard deviation. Asterisks denote statistical significance (*p < 0.05, **p < 0.01).

FIGURE 5

OpaR bound to the promoter region of the protease genes in vitro. An OpaR-binding reaction was performed with the promoter sequences of the protease genes. (A) lytM, (B) m6 protease, (C) protease II, (D) degS, (E) periplasmic protease, (F) mcp02, and (G) serine protease. The more sensitive electrophoretic mobility shift assay (EMSA) probes of each protease gene are labeled with a yellow box. The blue box indicates the other EMSA probe. Digoxigenin-labeled EMSA probes were incubated without (lane 1) or with increasing concentrations of purified OpaR (lanes 2–5) for the yellow and blue boxes. Lane 5 included 100 times more unlabeled probes as competitors.

FIGURE 6

OpaR-binding sequences determined using footprinting assays. The footprinting assay involved capillary electrophoresis analyses on each protease gene promoter sequence containing the ChIP-seq and EMSA-defined OpaR-binding region. Capillary electrophoresis diagrams are shown: (A) lytM, (B) m6 protease, (C) protease II, (D) degS, (E) periplasmic protease, (F) mcp02, and (G) serine protease. Number in yellow circles on the gene promoter and 3’region are consistent with those in the peak diagram. The blue peak shows footprinting results with OpaR. The light blue peak is the control (without OpaR). The arrow (→) represents the predicted transcriptional start site of the protease genes.

FIGURE 7

Schematic of how OpaR directly regulates protease genes. Black arrows represent positive regulation of protease genes by OpaR. Protease genes were categorized depending on activation/repression, periplasmic/extracellular localization, and metalloprotease/serine protease. Proteases that might involve bacterial virulence are depicted on a purple background. The pink region includes the environmental survival genes. Dashed and solid lines indicate the regulation pathway at a low and high cell density, respectively. Signal peptides were predicted by SignalP v5.0 (Armenteros et al., 2019) and are labeled as ∼SP.