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Virulence
2019 Dec 01;101:839-848. doi: 10.1080/21505594.2019.1682761.
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Dihydrolipoamide dehydrogenase of Vibrio splendidus is involved in adhesion to Apostichopus japonicus.
Dai F
,
Zhang W
,
Zhuang Q
,
Shao Y
,
Zhao X
,
Lv Z
,
Li C
.
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Vibrio splendidus is one of the most opportunistic marine pathogens and infects many important marine animals, including the sea cucumber Apostichopus japonicus. In this study, two genes named DLD1 and DLD2, encoding dihydrolipoamide dehydrogenase (DLD) homologues in pathogenic V. splendidus, were cloned, and conditionally expressed in Escherichia coli BL21 (DE3). The enzymatic activities of DLD1 and DLD2 showed that they both belonged to the NADH oxidase family. Both DLD1 and DLD2 were located on the outer membrane of V. splendidus as detected by whole-cell ELISA. To study the adhesion function of DLD1 and DLD2, polyclonal antibodies were prepared, and antibody block assay was performed to detect the normal function of the two proteins. DLD1 and DLD2 were determined to play important roles in adhesion to different matrices and the adhesive ability of V. splendidus reduced more than 50% when DLD1 or DLD2 was defective.
Figure 1. (a): Multiple sequence alignment of DLDs from Vibrio spp. The GenBank accession numbers are as follows: DLDs of Vibrio fischeri WP_069594165.1, Vibrio splendidus (DLD1) WP_010435969.1, Vibrio splendidus (DLD2) WP_004729673.1, Vibrio campbellii WP_012128871.1, Vibrio coralliilyticus WP_006958123.1, Vibrio parahaemolyticus WP_021823119.1, Vibrio alginolyticus AGK62253.1, Vibrio mimicus WP_000031532.1, Vibrio cholerae WP_000031535.1, Vibrio anguillarum WP_013857673.1, Vibrio vulnificus RAH35251.1, Vibrio fluvialis WP_020430460.1. (b and c) showed the domains of DLD1 and DLD2.
Figure 2. Unrooted phylogenetic tree of DLDs from different Vibrio spp. constructed using MEGA 5.0 software. The scale bar represents a distance of 0.1 substitutions per site. The tree was obtained by bootstrap analysis with a neighbor-joining method, and numbers on branches represent bootstrap values for 1000 replications.
Figure 3. (a): the SDS-PAGE analysis of the recombinant proteins of DLD1 (lane 1) and DLD2 (lane 2), and M is the Marker (kDa). (b): the absorbance at OD340 after the reaction was catalyzed by DLD1 or DLD2. The reaction without DLD1 or DLD2 was used as a control (NC). Data are the means of three independent experiments, and are presented as means ± SD.
Figure 4. Determination of DLDs location using whole cell ELISA. The same amount of cells was used as antigen to react with DLD1 or DLD antibody 2. The developed color was measured at 450 nm. (a): a, the cells blocked by DLD1 antibody; b, the negative group; c, the cells blocked by DLD2 antibody; d, the negative group; e, the control group. (b): The absorbance at OD450 of the control group, negative group, V. splendidus reacted with DLD1 antibody and V. splendidus reacted with DLD2 antibody. Data are the means of three independent experiments, and are presented as means ± SD. *P< 0.05, **P< 0.01.
Figure 5. The adhesive ability of V. splendidus to polystyrene demonstrated by fluorescence (a-c) and colony counting (d). The green fluorescence was generated by the labeled Vs-DTAF. a, the cells blocked by DLD1 antibody; b, the cells blocked by DLD2 antibody; c, the control group; d, quantity of the cells that adhered to polystyrene. From the left to right: V. splendidus treated with DLD1 antibody, V. splendidus treated with DLD2 antibody, and V. splendidus.
Figure 7. The adhesive ability of V. splendidus to different tissues of A. japonicus. The tissues of body wall, tentacle, muscle, respiratory tree, and intestine of A. japonicus specimens were collected and homogenized after 24 h immersion infection. The homogenate was 1.0 × 103-fold diluted and 50 μL was spread on 2216E plate. (a): the cells blocked by DLD1 antibody; (b): the cells blocked by DLD2 antibody; Data are means of three independent experiments, and are presented as means ± SD. *P < 0.05, **P< 0.01.
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