Sigma E Regulators Control Hemolytic Activity and Virulence in a Shrimp Pathogenic Vibrio harveyi

Members of the genus Vibrio are important marine and aquaculture pathogens. Hemolytic activity has been identified as a virulence factor in many pathogenic vibrios including V. cholerae, V. parahaemolyticus, V. alginolyticus, V. harveyi and V. vulnificus. We have used transposon mutagenesis to identify genes involved in the hemolytic activity of shrimp-pathogenic V. harveyi strain PSU3316. Out of 1,764 mutants screened, five mutants showed reduced hemolytic activity on sheep blood agar and exhibited virulence attenuation in shrimp (Litopenaeus vannamei). Mutants were identified by comparing transposon junction sequences to a draft of assembly of the PSU3316 genome. Surprisingly none of the disrupted open reading frames or gene neighborhoods contained genes annotated as hemolysins. The gene encoding RseB, a negative regulator of the sigma factor (σE), was interrupted in 2 out of 5 transposon mutants, in addition, the transcription factor CytR, a threonine synthetase, and an efflux-associated cytoplasmic protein were also identified. Knockout mutations introduced into the rpoE operon at the rseB gene exhibited low hemolytic activity in sheep blood agar, and were 3-to 7-fold attenuated for colonization in shrimp. Comparison of whole cell extracted proteins in the rseB mutant (PSU4030) to the wild-type by 2-D gel electrophoresis revealed 6 differentially expressed proteins, including two down-regulated porins (OmpC-like and OmpN) and an upregulated protease (DegQ) which have been associated with σE in other organisms. Our study is the first report linking hemolytic activity to the σE regulators in pathogenic Vibrio species and suggests expression of this virulence-linked phenotype is governed by multiple regulatory pathways within the V. harveyi

Transposon mutants with reduced hemolytic activity on sheep blood agar.

a<p>Based on Rapid Annotation using Subsystem Technology (RAST) of de novo assembled genome fragments from PSU3316.</p>b<p><i>E</i> values determined using NCBI BLASTN using the full open reading frame of PSU3316.</p>c<p>Control challenges with wild-type PSU3316 or transposant mutants with wild-type hemolysis activity exhibited mortality of 100% at the challenge dose.</p>d<p>Hemolysis attenuated mutants exhibited variable mortality that was statistically different from wild-type as evaluated by Fisher's exact test (<i>p</i><0.05).</p

Growth of <i>V. harveyi</i> in LB broth with 1.5% NaCl, pH 7.5 at 30°C.

<p><b>▪</b> Wild type (PSU3545), <b>⧫</b> RseBC⁻ (PSU4030), <b>▴</b> RseBC⁻ (PSU4031). No significant differences were observed in the growth of tested strains.</p

Contigs from the PSU3316 genome draft assembly bearing genes for differentially expressed proteins (A–E) in the <i>rseBC</i> mutant PSU4030 (spots 2 to 6, respectively).

<p>Peptide sequences from differentially expressed proteins were identified by mass spectrometry.</p

Nucleotide sequences of PCR primers for gene disruption mutagenesis.

<p>Nucleotide sequences of PCR primers for gene disruption mutagenesis.</p

Hemolytic activity of wild type and mutants strains of <i>V. harveyi</i> on sheep blood agar.

<p>Images illustrate clear zone around the colony.</p

Percent hemolytic activity of <i>V. harveyi</i> wild type PSU3545 and <i>rseBC</i> mutants relative to controls.

<p>An asterisk indicates that a value is significantly different from the wild-type strain (PSU3545) (<i>p</i><0.05). The error bars indicate standard errors of the means.</p

Summary of MS/MS data for protein spots showing altered expression levels on 2-D gels for wild-type and <i>rseBC</i> mutant cell extracts.

a<p>Spot no. corresponds to region of stained gel in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032523#pone-0032523-g006" target="_blank">Figure 6</a>. Spots 1–3 were under-expressed and spots 4–6 were over expressed in the <i>rseBC</i> mutant relative to WT.</p>b<p>Based on Rapid Annotation using Subsystem Technology (RAST) of de novo assembled genome fragments, or by homology to reference genome BAA-1116.</p>c<p><i>E</i> values determined using NCBI BLASTN using full open reading frame in PSU3316.</p>d<p>Individual ion scores of greater than 53 indicates significant identities (<i>p</i><0.05).</p>e<p>Not available.</p

<i>V. harveyi</i> PSU3316 genome regions containing sites interrupted and the flanking open reading frames.

<p>Each region (A–D) was interrupted by transposon or targeted mutagenesis (orange stars). The corresponding full contigs from the draft genome assembly have been deposited at NCBI.</p