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

Jang, Kyung Ku; Lee, Zee Won; Kim, Bityeoul; Jung, Young Hyun; Han, Ho Jae; Kim, Myung Hee; Kim, Byoung Sik; Choi, Sang Ho

doi:10.1074/jbc.m117.791657

Cited by 49 publications

(74 citation statements)

References 67 publications

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“…VvPlpA is significantly induced in a host environment. It can disrupt the phospholipid membrane and thus lead to host cell lysis, contributing to systemic infection (6). Therefore, VvPlpA is an essential virulence factor of V. vulnificus and may serve as a promising antivirulence drug target.…”

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confidence: 99%

Structural analysis of a Vibrio phospholipase reveals an unusual Ser–His–chloride catalytic triad

Wan

Liu

2019

Journal of Biological Chemistry

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Phospholipases can disrupt host membranes and are important virulence factors in many pathogens. VvPlpA is a phospholipase A 2 secreted by Vibrio vulnificus and essential for virulence. Its homologs, termed thermolabile hemolysins (TLHs), are widely distributed in Vibrio bacteria, but no structural information for this virulence factor class is available. Herein, we report the crystal structure of VvPlpA to 1.4-Å resolution, revealing that VvPlpA contains an N-terminal domain of unknown function and a C-terminal phospholipase domain and that these two domains are packed closely together. The phospholipase domain adopts a typical SGNH hydrolase fold, containing the four conserved catalytic residues Ser, Gly, Asn, and His. Interestingly, the structure also disclosed that the phospholipase domain accommodates a chloride ion near the catalytic His residue. The chloride is five-coordinated in a distorted bipyramid geometry, accepting hydrogen bonds from a water molecule and the amino groups of surrounding residues. This chloride substitutes for the most common Asp/Glu residue and forms an unusual Ser-His-chloride catalytic triad in VvPlpA. The chloride may orient the catalytic His and stabilize the charge on its imidazole ring during catalysis. Indeed, VvPlpA activity depended on chloride concentration, confirming the important role of chloride in catalysis. The VvPlpA structure also revealed a large hydrophobic substrate-binding pocket that is capable of accommodating a long-chain acyl group. Our results provide the first structure of the TLH family and uncover an unusual Ser-His-chloride catalytic triad, expanding our knowledge on the biological role of chloride.

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confidence: 99%

Structural analysis of a Vibrio phospholipase reveals an unusual Ser–His–chloride catalytic triad

Wan

Liu

2019

Journal of Biological Chemistry

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“…As expected, FTH inhibited the transcription of rtxA1 and vvhA genes in a dose-dependent manner (Figures 3A,B ). HlyU regulates another virulence determinant phospholipase A 2 ( plpA 2 ) in V. vulnificus which has been recently reported (Jang et al, 2017 ). The phospholipase A 2 is essential for host cell lysis and necrotic epithelial cell death.…”

Section: Resultsmentioning

confidence: 87%

“…The strains and plasmids used in this study are listed in Table S1 . Vibrio vulnificus MO6-24/O wild type (Wright et al, 1990 ) (hereafter termed as WT V. vulnificus ), deletion mutants Δ hlyU (ZW141) and Δ rtxA1 (MW064) of V. vulnificus MO6-24/O were used in this study (Lee et al, 2007 ; Jang et al, 2017 ). V. vulnificus was revived on a Vibrio sp .…”

Section: Methodsmentioning

confidence: 99%

Identification and Validation of an Antivirulence Agent Targeting HlyU-Regulated Virulence in Vibrio vulnificus

Imdad

Chaurasia

Kim

2018

Front. Cell. Infect. Microbiol.

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Antimicrobial resistance (AMR) in pathogens is the result of indiscriminate use of antibiotics and consequent metabolic/genetic modulation to evolve survival strategies and clonal-selection in AMR strains. As an alternative to antibiotic treatment, antivirulence strategies are being developed, not only to combat bacterial pathogenesis, but also to avoid emerging antibiotic resistance. Vibrio vulnificus is a foodborne pathogen that causes gastroenteritis, necrotizing wound infections, and sepsis with a high rate of mortality. Here, we developed an inhibitor-screening reporter platform to target HlyU, a master transcriptional regulator of virulence factors in V. vulnificus by assessing rtxA1 transcription under its control. The inhibitor-screening platform includes wild type and ΔhlyU mutant strains of V. vulnificus harboring the reporter construct PrtxA1::luxCDABE for desired luminescence signal detection and control background luminescence, respectively. Using the inhibitor-screening platform, we identified a small molecule, fursultiamine hydrochloride (FTH), that inhibits the transcription of the highly invasive repeat-in-toxin (rtxA1) and hemolysin (vvhA) along with other HlyU regulated virulence genes. FTH has no cytotoxic effects on either host cells or pathogen at the tested concentrations. FTH rescues host cells from the necrotic cell-death induced by RtxA1 and decreases the hemolytic activity under in vitro conditions. The most important point is that FTH treatment does not induce the antivirulence resistance. Current study validated the antivirulence strategy targeting the HlyU virulence transcription factor and toxin-network of V. vulnificus and demonstrated that FTH, exhibits a potential to inhibit the pathogenesis of deadly, opportunistic human pathogen, V. vulnificus without inducing AMR.

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“…V. vulnificus MO6-24/O [ 26 ] (hereafter termed as V. vulnificus ) and ∆ hlyU deletion mutant [ 27 ] strains were routinely cultured in Luria–Bertani (LB) medium supplemented with 2% sodium chloride (LBS) or on LBS solid agar plates (1.5% agar) at 37 °C. The growth of V. vulnificus was assessed by measuring turbidity at 600 nm (OD 600 ) and counting the colony forming units (CFU).…”

Section: Methodsmentioning

confidence: 99%

Identification of 2′,4′-Dihydroxychalcone as an Antivirulence Agent Targeting HlyU, a Master Virulence Regulator in Vibrio vulnificus

et al. 2018

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The emergence of antimicrobial resistance and rapid acclimation allows Vibrio vulnificus to rapidly propagate in the host. This problematic pathological scenario can be circumvented by employing an antivirulence strategy, treating Vibrio infections without hindering the bacterial growth. We developed a genome-integrated orthogonal inhibitor screening platform in E. coli to identify antivirulence agents targeting a master virulence regulator of V. vulnificus. We identified 2′,4′-dihydroxychalcone (DHC) from the natural compound library and verified that it decreases the expression of the major toxin network which is equivalent to the ∆hlyU deletion mutant. 2′,4′-DHC also reduced the hemolytic activity of V. vulnificus which was tested as an example of virulence phenotype. The electrophoretic mobility shift assay confirmed that 2′,4′-DHC specifically targeted HlyU and inhibited its binding to PrtxA1 promoter. Under in vivo conditions, a single dose of 2′,4′-DHC protected ~50% wax-worm larvae from V. vulnificus infection at a non-toxic concentration to both V. vulnificus and wax-worm larvae. In the current study, we demonstrated that an orthogonal reporter system is suitable for the identification of antivirulence compounds with accuracy, and identified 2′,4′-DHC as a potent antivirulence agent that specifically targets the HlyU virulence transcriptional regulator and significantly reduces the virulence and infection potential of V. vulnificus.

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

Cited by 49 publications

References 67 publications

Structural analysis of a Vibrio phospholipase reveals an unusual Ser–His–chloride catalytic triad

Structural analysis of a Vibrio phospholipase reveals an unusual Ser–His–chloride catalytic triad

Identification and Validation of an Antivirulence Agent Targeting HlyU-Regulated Virulence in Vibrio vulnificus

Identification of 2′,4′-Dihydroxychalcone as an Antivirulence Agent Targeting HlyU, a Master Virulence Regulator in Vibrio vulnificus

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