Survival Characteristics and Transcriptomic Analyses Reveal the Adaptive Response of the Aquatic Pathogen Non-O1/O139 Vibrio cholerae to Starvation Stress

Abstract

Non-O1/O139 Vibrio cholerae is a pathogen of various aquatic organisms but requires major self-regulation to overcome environmental stress in the aquatic environment. However, its survival strategies under environmental stress are not well understood. The objective of this study was to describe the survival characteristics and changes in expression of stress resistance-related genes of non-O1/O139 V. cholerae after 6 months of starvation at room temperature. The results demonstrated that starved cells were still viable, exhibited shortened rods and shrinking surface, and maintained virulence to Macrobrachium rosenbergii. To investigate the changes in gene expression in non-O1/O139 V. cholerae under starvation stress, especially those involved in stress resistance, transcriptome profiles of starved and wild-type cells were determined. The differentially expressed genes (DEGs) in starved cells were identified, including 191 upregulated genes and 180 downregulated genes. Among these DEGs, the well-known stress resistance-related genes were upregulated significantly, including rpoS, rpoD, rpoN, rpoE, uspA, uspC, cspD, hslJ, etc. Gene Ontology (GO) analysis of the DEGs demonstrated that environmental adaptation-related categories, such as response to stimulus and signal transduction, were upregulated significantly in the starved cells, while cell motility was downregulated significantly. These DEGs were also enriched into 54 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, including biofilm formation, two-component system, quorum sensing, flagellar assembly, bacterial chemotaxis stress resistance-related pathways, etc. The potential existence of long-starved non-O1/O139 V. cholerae bacteria in the aquatic environment may raise new concerns about this devastating pathogen in aquaculture. IMPORTANCE Non-O1/O139 V. cholerae is a causal agent of vibriosis that can be subject to nutrient insufficiency and cause high rates of mortality in aquatic animals. However, its molecular mechanisms of survival in response to starvation stress have been investigated only partially. Here, we demonstrate that under starvation stress, non-O1/O139 V. cholerae can survive over the long term and cause disease by dwarfing of the cell structure, upregulation of a series of stress resistance-related genes, and downregulation of flagellum assembly-related genes. This knowledge can help the development of intervention strategies to control non-O1/O139 V. cholerae infection in aquaculture.

Keywords: non-O1/O139 V. cholerae; starvation; stress responses; survival; transcriptome.

FIG 1

Survival curve of non-O1/O139 V. cholerae GXFL1-4 cells under starvation stress.

FIG 2

Phenotype changes of non-O1/O139 V. cholerae cells after starvation. (A) Morphology of wild-type cells revealed by scanning electron microscopy. (B) Morphology of starved cells by scanning electron microscopy. (C) Motility of starved cells after starvation. (D) Migration diameters of starved and wild-type cells. Values marked with different letters (a, b) are significantly different (P < 0.05). Error bars show standard deviations.

FIG 3

Growth curves of starved and wild-type non-O1/O139 V. cholerae. Data are presented as mean values ± SD from three independent biological replicates.

FIG 4

Differentially expressed genes (DEGs) of non-O1/O139 V. cholerae in starved and wild-type cells. (A) Volcano plot of DEGs. Red circles represent upregulated genes, green circles represent downregulated genes, and blue circles indicate no DEGs. (B) Venn diagram showing the overlap of gene expression levels in starved and wild-type cells. (C) DEGs involved in response to starvation based on the transcriptome analyses.

FIG 5

GO and KEGG analyses of genes differentially expressed between the 6-months-starved and wild-type non-O1/O139 V. cholerae strains. (A) Upregulated GO terms. (B) Downregulated GO terms. (C) Upregulated KEGG pathways. (D) Downregulated KEGG pathways.

FIG 6

Pathway maps of flagellar assembly (A) and bacterial chemotaxis (B) pathways in KEGG. Green boxes represent downregulated genes.