Comparative Transcriptome Analysis of Vibrio splendidus JZ6 Reveals the Mechanism of Its Pathogenicity at Low Temperatures

Abstract

Yesso scallop-pathogenic Vibrio splendidus strain JZ6 was found to have the highest virulence at 10°C, while its pathogenicity was significantly reduced with increased temperature and completely incapacitated at 28°C. In the present study, comparative transcriptome analyses of JZ6 and another nonpathogenic V. splendidus strain, TZ19, were conducted at two crucial culture temperatures (10°C and 28°C) in order to determine the possible mechanism of temperature regulation of virulence. Comparisons among four libraries, constructed from JZ6 and TZ19 cultured at 10°C and 28°C (designated JZ6_10, JZ6_28, TZ19_10, and TZ19_28), revealed that 241 genes were possibly related to the increased virulence of JZ6 at 10°C. There were 10 genes, including 2 encoding Flp pilus assembly proteins (FlhG and VS_2437), 6 encoding proteins of the "Vibrio cholerae pathogenic cycle" (ToxS, CqsA, CqsS, RpoS, HapR, and Vsm), and 2 encoding proteins in the Sec-dependent pathway (SecE and FtsY), that were significantly upregulated in JZ6_10 (P < 0.05) compared to those in JZ6_28, TZ19_10, and TZ19_28, which were supposed to be responsible for adhesion, quorum sensing, virulence, and protein secretion of V. splendidus. When cultured at 10°C, JZ6 cells were larger and tended to aggregate more than those cultured at 28°C. The virulence factor (extracellular metalloprotease) was also found to be highly expressed in the extracellular product (ECP) of JZ6 at 10°C, and this ECP exhibited obvious cytotoxicity to oyster primary hemocytes, A549 cells, and L929 cells. These results indicated that low temperatures (10°C) could enhance adhesion, activate the quorum sensing systems, upregulate virulence factor synthesis and secretion, and, lastly, increase the pathogenicity of JZ6.

FIG 1

Grouping of differentially expressed genes among the three comparison groups, JZ6_10/JZ6_28, TZ19_10/TZ19_28, and JZ6_10/TZ19_10.

FIG 2

Distribution analysis of differentially expressed genes in JZ6_10/JZ6_28, TZ19_10/TZ19_28, and JZ6_10/TZ19_10 comparison groups with histogram presentation of gene ontology classifications.

FIG 3

Partial list of key genes involved in the heat shock response and motility of JZ6 upregulated at 28°C.

FIG 4

Expression pattern of virulence-related genes involved in the Vibrio cholerae pathogenic cycle. (A) KEGG pathway diagram of virulence-related genes in the Vibrio cholerae pathogenic cycle (generated using the KEGG PATHWAY database [Kanehisa Laboratories]). Genes highlighted in gray are upregulated in JZ6 at 10°C. cAMP, cyclic AMP; MSHA, mannose-sensitive hemagglutinin; TCP, toxin-coregulated pilin. (B) Detection of relative expression levels of six upregulated genes of the Vibrio cholerae pathogenic cycle by qRT-PCR.

FIG 5

Scanning electron microscope photographs of V. splendidus strains JZ6 and TZ19 cultured at 10°C and 28°C. (A) JZ6 at 10°C. (B) JZ6 at 28°C. (C) TZ19 at 10°C. (D) TZ19 at 28°C. (Magnification, ×10,000.)

FIG 6

FACS flow cytometry analysis of the size and complexity of JZ6 cells at 10°C and 28°C. (A) Scatter diagram for FSC and SSC analyses. (B) Peak diagram for single-FSC analysis. (C) Peak diagram for single-SSC analysis.

FIG 7

ECPs of JZ6 and TZ19 cultured at 10°C and 28°C analyzed by SDS-PAGE and Western blotting. Lane M, protein molecular mass standards (in kilodaltons). P_{JZ6_10}, ECP of JZ6 cultured at 10°C; P_{JZ6_28}, ECP of JZ6 cultured at 28°C; P_{TZ19_10}, ECP of TZ19 cultured at 10°C; P_{TZ19_28}, ECP of TZ19 cultured at 28°C. Western blotting detected the extracellular metalloprotease (Vsm) of V. splendidus with polyclonal antiserum (rat anti-Vsm) diluted 1:1,000.

FIG 8

Cytotoxicity of ECPs from different bacterial samples to oyster primary hemolymph cells, human lung cancer cells (A549), and mouse fibroblast cells (L929).

FIG 9

Viability analysis of cultured cells treated with ECP samples P_{JZ6_10}, P_{JZ6_28}, P_{TZ19_10}, and P_{TZ19_28} by using Cell Counting kit 8. (A) Viability of the A549 cell line. (B) Viability of the L929 cell line.