Temperature regulation of virulence factors in the pathogen Vibrio coralliilyticus

Abstract

Sea surface temperatures (SST) are rising because of global climate change. As a result, pathogenic Vibrio species that infect humans and marine organisms during warmer summer months are of growing concern. Coral reefs, in particular, are already experiencing unprecedented degradation worldwide due in part to infectious disease outbreaks and bleaching episodes that are exacerbated by increasing SST. For example, Vibrio coralliilyticus, a globally distributed bacterium associated with multiple coral diseases, infects corals at temperatures above 27 °C. The mechanisms underlying this temperature-dependent pathogenicity, however, are unknown. In this study, we identify potential virulence mechanisms using whole genome sequencing of V. coralliilyticus ATCC (American Type Culture Collection) BAA-450. Furthermore, we demonstrate direct temperature regulation of numerous virulence factors using proteomic analysis and bioassays. Virulence factors involved in motility, host degradation, secretion, antimicrobial resistance and transcriptional regulation are upregulated at the higher virulent temperature of 27 °C, concurrent with phenotypic changes in motility, antibiotic resistance, hemolysis, cytotoxicity and bioluminescence. These results provide evidence that temperature regulates multiple virulence mechanisms in V. coralliilyticus, independent of abundance. The ecological and biological significance of this temperature-dependent virulence response is reinforced by climate change models that predict tropical SST to consistently exceed 27 °C during the spring, summer and fall seasons. We propose V. coralliilyticus as a model Gram-negative bacterium to study temperature-dependent pathogenicity in Vibrio-related diseases.

Figure 1

Global distribution of V. coralliilyticus strains. The V. coralliilyticus strains represented here are (a) type strains (Ben-Haim et al., 2003a), as well as strains identified using (b) DnaJ PCR (Vezzulli et al., 2010), (c, d) 16S rRNA sequencing (Sussman et al., 2008; Kesarcodi-Watson et al., 2009), (e) multi-locus sequencing (Alves et al., 2010) and (f) multiple molecular analyzes, that is, 16S rRNA sequencing, recA PCR and repetitive extragenic palindromic - polymerase chain reaction (REP-PCR) (Vizcaino et al., 2010).

Figure 2

The growth rate and total protein production of Vc450 is similar whether grown at 24 °C or 27 °C. Vc450 was grown in GASW media at 24 °C and 27 °C. The growth curves were prepared using optical density at time points 0, 2, 4, 6, 8, 10, 12 and 24 h for both 24 °C ( ) and 27 °C (- - - ). Protein production was measured using the Bradford assay for both 24 °C ( ) and 27 °C (—) from time points 2–24 h. Error bars represent the standard deviation of three replicate samples.

Figure 3

Transmission electron photomicrograph of Vc450 T6SS tubular structure. Vc450 cell stained with 0.5% sodium phosphotungstic acid, pH 6.8. (a) A VipA/VipB-like T6SS tubular structure, similar to that described for V. cholerae, is evident in the cytoplasm (black arrowheads). (b) A Vc450 VipA/VipB-like T6SS tubular structure found outside of a cell.

Figure 4

Schematic representation of Vc450 CPI-1 and comparison with VcP1 using Artemis Comparison Tool (ACT, Wellcome Trust Sanger Institute, Hinxton, UK). Coding sequences of CPI-1 from Vc450 are shown on the top, with G+C content directly below. Two large regions are present in Vc450 and not in VcP1, one with mostly hypothetical proteins (fuschia) and the other containing an RTX homolog, transporter and associated genes (green). Additionally, there were three regions of gene-level divergence (light gray) at VIC_4016, VIC_4037 and within VIC_4065. CPI-1 of VcP1 is composed of nine contigs: AEQS01000075, −105, −183, −138, −135, −025, −161, −224 and −071, with contig gaps (asterisks) indicated on the bottom ACT scale.

Figure 5

Proposed Vc450 QS systems. This figure illustrates the potential QS mechanisms utilized by V. coralliilyticus at high density. (a) The QseBC system is a two-component system, in which the QseC histidine kinase receptor becomes phosphorylated when bound to cognate ligands and subsequently activates QseB through phosphorelay. The activated QseB molecule binds DNA, acting as a direct transcriptional regulator. (b) The three two-component histidine kinase receptor systems previously described in Vibrio species, each produce and detect a specified class of AI. Ligand binding, at levels above the density threshold, blocks the kinase activity of the membrane-bound receptors, reversing the phosphorelay. This results in the dephosphorylation of the response regulator, LuxO, via LuxU. In its unactivated state, LuxO is unable to transcriptionally activate the sRNAs that degrade the messenger RNA (mRNA) of LuxR-type genes. Thus, LuxR-type mRNA is stabilized and proteins are produced. The LuxR-type proteins in turn act as transcriptional regulators of virulence-associated genes. All of the proteins shown are present in the Vc450 genome. The shaded (blue and red) proteins are present in the Vc450 proteome, with (red) proteins representing those significantly affected by temperature.