Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis

Abstract

The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A₂ secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase-dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA Molecular biological analyses revealed that plpA expression is controlled via the promoter, P _plpA , and that HlyU and CRP directly bind to P _plpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A₂ regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.

Keywords: CRP; HlyU; Phospholipase A; PlpA; Vibrio vulnificus; gene regulation; microbial pathogenesis; transcription factor; virulence factor.

Figure 1.

Analysis of amino acid sequence, secretion, and enzymatic activity of VvPlpA. A, the amino acid sequences retrieved from the NCBI protein database (http://www.ncbi.nlm.nih.gov) (accession numbers ADV88279 for VvPlpA and AAY26144 for VaPlpA) were aligned using the Clustal Omega and Genedoc program. Identical residues (gray boxes) and missing sequence (dash) are indicated. The putative signal peptides and consensus blocks for SGNH hydrolases are boxed by a black line. The GDSL motif is indicated by asterisks. The four residues conserved in the SGNH hydrolases are indicated by black-filled inverse triangles. B, VvPlpA in the supernatants of the V. vulnificus strains grown to an A₆₀₀ of 0.9 was detected by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. C and D, PLA₂ activity was examined by measuring fluorescence intensity emitted from the fluorogenic phosphatidylcholine substrate (red/green BODIPY PC-A2) after incubation with various amounts of the rPlpA (C) or supernatants of the V. vulnificus strains (D) for 0.5 h. Error bars, S.D. Statistical significance was determined by Student's t test. *, p < 0.05 relative to the wild type. WT pJH0311 and WT pRK415, wild type; pilD pJH0311, pilD mutant; plpA pJH0311 and plpA pRK415, plpA mutant; pilD pMS0908 and plpA pKK1320, complemented strains.

Figure 2.

VvPlpA is essential for pathogenesis of V. vulnificus. A, the wild type grown to an A₆₀₀ of 0.5 was exposed to either LBS or blood of uninfected mice (n = 3) for 1 or 2 h and then used to isolate total RNAs. The plpA mRNA was determined by real-time qPCR analyses, and the plpA mRNA level in the bacteria exposed to LBS was set as 1. B, 7-week-old female ICR mice (n = 15 per group), injected intraperitoneally with iron-dextran, were intragastrically infected with the wild type and plpA mutant at doses of 10⁸ cfu. C–F, the mice, without an iron-dextran administration, were intraperitoneally infected with the wild type (n = 11), plpA mutant (n = 11), or PBS (n = 5, control) at doses of 10⁶ cfu and then sacrificed to obtain blood at 4 h postinfection. C, the number of either the wild type or plpA mutant in the blood of infected mice (n = 6 per group) was enumerated as cfu/100 μl of blood. Each open symbol represents an individual mouse. Black-filled inverted triangles indicate median values. D–F, the levels of MPXI (D), ALB (E), and AST/ALT (F) in the wild type– or plpA mutant–infected or PBS-treated mice (n = 5 per group) were determined by hematological analyses. Error bars, S.D. Statistical significance was determined by Student's t test for A and C–F, and by log-rank test for B. *, p < 0.05; **, p < 0.005; ns, not significant. WT, wild type; plpA, plpA mutant; PBS, control.

Figure 3.

VvPlpA is essential for necrotic cell death in tissue culture. A and B, INT-407 cells were infected with the V. vulnificus strains at various MOIs for 2 h (A) or at an MOI of 10 for various incubation times (B). The cytotoxicity was expressed using the total LDH activity of the cells completely lysed by 1% Triton X-100 as 100%. Error bars, S.D. Statistical significance was determined by Student's t test. **, p < 0.005 relative to groups infected with the wild type at each MOI or incubation time. C, INT-407 cells were infected with the V. vulnificus strains at an MOI of 10 for 1.5 h as indicated, stained using Texas Red®-X–conjugated WGA (for membrane, red) and Hoechst 33342 (for nucleus, blue), and then photographed using a fluorescence microscope. D, INT-407 cells were infected with the V. vulnificus strains at an MOI of 10 for 2 h, and actin and CypA released in the culture supernatants were analyzed by Western blot analysis. Molecular size markers (GenDEPOT, Barker, TX) are shown in kDa. WT (pRK415), wild type; plpA (pRK415), plpA mutant; plpA (pKK1320), complemented strain; PBS, control (uninfected).

Figure 4.

Expression of plpA in V. vulnificus under different growth phases (A) or with different genetic backgrounds (B and C). A, total RNAs from the V. vulnificus wild type under different growth phases were isolated, and the plpA mRNA levels were determined by real-time qPCR analyses. The plpA mRNA level in the cells grown to an A₆₀₀ of 0.3 was set as 1. B and C, V. vulnificus strains were grown to an A₆₀₀ of 0.9 and used to determine the plpA mRNA and VvPlpA levels. The plpA mRNA levels in the total RNAs isolated from the cultures were determined by real-time qPCR analyses, and the plpA mRNA level in the wild type was set as 1. The VvPlpA levels in the supernatants harvested form the cultures were determined by Western blot analyses. Molecular size markers (Bio-Rad) are shown in kDa. Error bars, S.D. Statistical significance was determined by Student's t test. **, p < 0.005 relative to the cells grown to an A₆₀₀ of 0.3 (for A) or to the wild type (for B and C); ns, not significant. WT pJH0311, wild type; hlyU pJH0311, hlyU mutant; crp pJH0311, crp mutant; hlyU pZW1510 and crp pKK1502, complemented strains.

Figure 5.

HlyU and CRP mediate additive activation and host-dependent induction of plpA. A and B, V. vulnificus strains were grown to an A₆₀₀ of 0.9 and used to determine the plpA mRNA levels and VvPlpA, HlyU, CRP, and DnaK protein levels. A, the plpA mRNA levels were determined by real-time qPCR analyses, and the plpA mRNA level in the wild type was set as 1. B, the secreted VvPlpA and cellular HlyU, CRP, and DnaK (as an internal control) levels were determined by Western blot analyses. Molecular size markers (Bio-Rad) are shown in kDa. C, V. vulnificus strains were exposed to MEM (control) or INT-407 cells at an MOI of 10 for 1 h and then used to isolate total RNAs. The plpA mRNA levels were determined by real-time qPCR, and the plpA mRNA level in the wild type exposed to MEM was set as 1. Error bars, S.D. Statistical significance was determined by Student's t test. *, p < 0.05; **, p < 0.005; ns, not significant. WT and WT pJH0311, wild type; hlyU and hlyU pJH0311, hlyU mutant; crp and crp pJH0311, crp mutant; hlyU crp pJH0311, hlyU crp double mutant; hlyU crp pZW1510 or hlyU crp pKK1502, hlyU crp double mutant overexpressing HlyU or CRP, respectively.

Figure 6.

Transcription start site and sequences of the plpA-regulatory region. A, the transcription start site of plpA was determined by primer extension of the RNA isolated from the wild type grown to an A₆₀₀ of 0.9. Lanes C, T, A, and G, nucleotide sequencing ladders of pKK1505. The asterisk indicates the transcription start site of plpA. B, the transcription start site of plpA is indicated by a bent arrow, and the positions of the putative −10 and −35 regions are underlined. The sequences for binding of HlyU (HLYUB1, HLYUB2, and HLYUB3; gray boxes) and CRP (CRPB; white box) were determined later in this study (Fig. 8, C and D). The consensus sequences for binding of CRP (32) are indicated above the V. vulnificus DNA sequence. x, any nucleotide.

Figure 7.

Deletion analysis of the P_plpA regulatory region. A, construction of plpA-lux fusion pKK reporters. PCR fragments carrying the plpA-regulatory region with 5′-end deletions were subcloned into pBBR-lux (64) to create each pKK reporter. Solid lines, the upstream region of plpA; black blocks, the plpA coding region; white blocks, luxCDABE. The wild-type plpA-regulatory region is shown on top with the proposed −10 and −35 regions, and the binding sites for HlyU (HLYUB1, HLYUB2, and HLYUB3; gray boxes) and CRP (CRPB; white box) were determined later in this study (Fig. 8, C and D). B, cellular luminescence was determined from the wild type (black bars), hlyU mutant (gray bars), and crp mutant (white bars) containing each pKK reporter as indicated. Cultures grown to an A₆₀₀ of 0.9 were used to measure the cellular luminescence. Error bars, S.D. Statistical significance was determined by Student's t test. *, p < 0.05; **, p < 0.005; ns, not significant. WT, wild type; hlyU, hlyU mutant; crp, crp mutant.

Figure 8.

Specific bindings of HlyU and CRP to P_plpA. A 461-bp DNA fragment of the plpA-regulatory region was radioactively labeled and then used as a probe DNA. A and B, the radiolabeled probe DNA (5 nm) was incubated with increasing amounts of HlyU (A) and CRP (B) as indicated. For competition analysis, the same but unlabeled 461-bp DNA fragment was used as a self-competitor DNA. Various amounts of the self-competitor DNA were added to a reaction mixture containing the 5 nm labeled DNA before the addition of 40 nm HlyU (A) and 80 nm CRP (B) as indicated. B, bound DNA; F, free DNA. C and D, the same radiolabeled probe DNA (25 nm) was reacted with increasing amounts of HlyU (C) and CRP (D) as indicated. The regions protected from DNase I cleavage by HlyU and CRP are indicated by gray boxes (HLYUB1, HLYUB2, and HLYUB3) and a white box (CRPB), respectively. Lanes C, T, A, and G represent the nucleotide sequencing ladders of pKK1505. Nucleotide numbers shown are relative to the transcription start site of plpA.