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The Vibrio alginolyticus T3SS effectors, Val1686 and Val1680, induce cell rounding, apoptosis and lysis of fish epithelial cells.

ABSTRACT: Vibrio alginolyticus is a Gram-negative bacterium that is an opportunistic pathogen of both marine animals and people. Its pathogenesis likely involves type III secretion system (T3SS) mediated induction of rapid apoptosis, cell rounding and osmotic lysis of infected eukaryotic cells. Herein, we report that effector proteins, Val1686 and Val1680 from V. alginolyticus, were responsible for T3SS-mediated death of fish cells. Val1686 is a Fic-domain containing protein that not only contributed to cell rounding by inhibiting Rho guanosine triphosphatases (GTPases), but was requisite for the induction of apoptosis because the deletion mutant (?val1686) was severely weakened in its ability to induce cell rounding and apoptosis in fish cells. In addition, Val1686 alone was sufficient to induce cell rounding and apoptosis as evidenced by the transfection of Val1686 into fish cells. Importantly, the Fic-domain essential for cell rounding activity was equally important to activation of apoptosis of fish cells, indicating that apoptosis is a downstream event of Val1686-dependent GTPase inhibition. V. alginolyticus infection likely activates JNK and ERK pathways with sequential activation of caspases (caspase-8/-10, -9 and -3) and subsequent apoptosis. Val1680 contributed to T3SS-dependent lysis of fish cells in V. alginolyticus, but did not induce autophagy as has been reported for its homologue (VopQ) in V. parahaemolyticus. Together, Val1686 and Val1680 work together to induce apoptosis, cell rounding and cell lysis of V. alginolyticus-infected fish cells. These findings provide new insights into the mechanism of cell death caused by T3SS of V. alginolyticus.

SUBMITTER: Zhao Z

PROVIDER: S-EPMC5955196 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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The Vibrio alginolyticus T3SS effectors, Val1686 and Val1680, induce cell rounding, apoptosis and lysis of fish epithelial cells.

Zhao Zhe Z Liu Jinxin J Deng Yiqin Y Huang Wen W Ren Chunhua C Call Douglas R DR Hu Chaoqun C

Virulence 20180101 1

Vibrio alginolyticus is a Gram-negative bacterium that is an opportunistic pathogen of both marine animals and people. Its pathogenesis likely involves type III secretion system (T3SS) mediated induction of rapid apoptosis, cell rounding and osmotic lysis of infected eukaryotic cells. Herein, we report that effector proteins, Val1686 and Val1680 from V. alginolyticus, were responsible for T3SS-mediated death of fish cells. Val1686 is a Fic-domain containing protein that not only contributed to c ...[more]

PMID: 29252102

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Project description:BackgroundBiocontrol of bacterial pathogens by bacteriophages (phages) represents a promising strategy. Vibrio alginolyticus, a gram-negative bacterium, is a notorious pathogen responsible for the loss of economically important farmed marine animals. To date, few V. alginolyticus phages have been successfully isolated, and only three complete genome sequences of them have been released. The limited available phage resources and poor genomic data hamper research on V. alginolyticus phages and their applications for the biocontrol of V. alginolyticus.ResultsWe isolated a phage, Vp670, against the V. alginolyticus strain E06333 and obtained its full genomic sequence. It contains 43,121 nucleotides with a GC content of 43.4%, and codes for 49 predicted open reading frames. Observation by electron microscope combined with phylogenetic analysis of DNA polymerase indicates that Vp670 belongs to the subfamily Autographivirinae in the family Podoviridae. orf3 (designated holA) and orf8 (designated cwlQ) are predicted to encode a holin (HolA) and an endolysin (CwlQ), respectively. Expression of holA alone or coexpression of holA and cwlQ from within arrested the growth of Escherichia coli and V. alginolyticus while the expression of cwlQ alone had no effect on the growth of them. Further observation by transmission electron microscopy revealed that the expression of holA vanished the outer membrane and caused the release of cellular contents of V. alginolyticus and the coexpression of holA and cwlQ directly burst the cells and caused a more drastic release of cellular contents. Expression of cwlQ alone in V. alginolyticus did not cause cytomorphological changes.ConclusionsPhage Vp670 is a V. alginolyticus phage belonging to the family of Podoviridae. The genome of Vp670 contains a two-component lysis module, which is comprised of holA and cwlQ. holA is predicted to encode for the holin protein, HolA, and cwlQ is predicted to encode for the endolysin protein, CwlQ. Both holA and cwlQ likely play important roles during the release of phage progeny.

Project description:Vibrio alginolyticus is a major cause of Vibriosis in farmed marine aquatic animals and has caused large economic losses to the Asian aquaculture industry in recent years. Therefore, it is necessary to control V. alginolyticus effectively. The virulence mechanism of V. alginolyticus, the Type III secretion system (T3SS), is closely related to its pathogenicity. In this study, the T3SS gene tyeA was cloned from V. alginolyticus wild-type strain HY9901 and the results showed that the deduced amino acid sequence of V. alginolyticus tyeA shared 75-83% homology with other Vibrio spp. The mutant strain HY9901?tyeA was constructed by Overlap-PCR and homologous recombination techniques. The HY9901?tyeA mutant exhibited an attenuated swarming phenotype and an ~40-fold reduction in virulence to zebrafish. However, the HY9901?tyeA mutant showed no difference in growth, biofilm formation and ECPase activity. Antibiotic susceptibility test was observed that wild and mutant strains were extremely susceptible to Amikacin, Minocycline, Gentamicin, Cefperazone; and resistant to oxacillin, clindamycin, ceftazidime. In contrast wild strains are sensitive to tetracycline, chloramphenicol, kanamycin, doxycycline, while mutant strains are resistant to them. qRT-PCR was employed to analyze the transcription levels of T3SS-related genes, the results showed that compared with HY9901 wild type, ?tyeA had increased expression of vscL, vscK, vscO, vopS, vopN, vscN, and hop. Following vaccination with the mutant strain, zebrafish had significantly higher survival than controls following infection with the wild-type HY9901 (71.2% relative percent survival; RPS). Analysis of immune gene expression by qPCR showed that vaccination with HY9901?tyeA increased the expression of IgM, IL-1?, IL-6, and TNF-? in zebrafish. This study provides evidence of protective efficacy of a live attenuated vaccine targeting the T3SS of V. alginolyticus which may be facilitated by up-regulated pro-inflammatory and immunoglobulin-related genes.

Dataset Information

The Vibrio alginolyticus T3SS effectors, Val1686 and Val1680, induce cell rounding, apoptosis and lysis of fish epithelial cells.

Publications

The Vibrio alginolyticus T3SS effectors, Val1686 and Val1680, induce cell rounding, apoptosis and lysis of fish epithelial cells.

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