Vibrio campbellii hmgA-mediated pyomelanization impairs quorum sensing, virulence, and cellular fitness

Zheng Wang,Evan R Glaser,Gary J Vora,Robert A Rubin,Janelle R Thompson,Varaporn Vuddhakul,Anahita Mostaghim,Baochuan Lin,Pimonsri Mittraparp-Arthorn

doi:10.3389/fmicb.2013.00379

Abstract

Melanization due to the inactivation of the homogentisate-1,2-dioxygenase gene (hmgA) has been demonstrated to increase stress resistance, persistence, and virulence in some bacterial species but such pigmented mutants have not been observed in pathogenic members of the Vibrio Harveyi clade. In this study, we used Vibrio campbellii ATCC BAA-1116 as model organism to understand how melanization affected cellular phenotype, metabolism, and virulence. An in-frame deletion of the hmgA gene resulted in the overproduction of a pigment in cell culture supernatants and cellular membranes that was identified as pyomelanin. Unlike previous demonstrations in Vibrio cholerae, Burkholderia cepacia, and Pseudomonas aeruginosa, the pigmented V. campbellii mutant did not show increased UV resistance and was found to be ~2.7 times less virulent than the wild type strain in Penaeus monodon shrimp virulence assays. However, the extracted pyomelanin pigment did confer a higher resistance to oxidative stress when incubated with wild type cells. Microarray-based transcriptomic analyses revealed that the hmgA gene deletion and subsequent pyomelanin production negatively effected the expression of 129 genes primarily involved in energy production, amino acid, and lipid metabolism, and protein translation and turnover. This transcriptional response was mediated in part by an impairment of the quorum sensing regulon as transcripts of the quorum sensing high cell density master regulator LuxR and other operonic members of this regulon were significantly less abundant in the hmgA mutant. Taken together, the results suggest that the pyomelanization of V. campbellii sufficiently impairs the metabolic activities of this organism and renders it less fit and virulent than its isogenic wild type strain.

Highlights

As a member of the L-tyrosine catabolism pathway in bacterial and eukaryotic organisms, the enzyme homogentisate 1,2dioxygenase (HmgA) catalyzes the intermediate homogentisic acid into 4-maleylacetoacetate which is further catabolized to yield fumarate and acetoacetate
CHARACTERIZATION OF hmgA PIGMENT AND PHENOTYPES An examination of the V. campbellii ATCC BAA-1116 genome revealed that 4 genes in the catabolic pathway of tyrosine metabolism, hmgA (M892_02450), hppd (4hydroxyphenylpyruvate dioxygenase, M892_02455), fahA, and maiA, appeared to form an operon and shared the same genetic synteny as other sequenced Vibrio species
While the material properties of V. campbellii pyomelanin were similar to previous descriptions, there did not appear to be a generality of pyomelanin-mediated phenotypes as pyomelanization had either a neutral or deleterious effect on cell survival

Summary

Introduction

As a member of the L-tyrosine catabolism pathway in bacterial and eukaryotic organisms, the enzyme homogentisate 1,2dioxygenase (HmgA) catalyzes the intermediate homogentisic acid into 4-maleylacetoacetate which is further catabolized to yield fumarate and acetoacetate. It has been demonstrated that the inactivation of the hmgA gene results in the accumulation of homogentisic acid which when autooxidized leads to the formation of the water-soluble brown pigment pyomelanin (Rodriguez-Rojas et al, 2009; Schmaler-Ripcke et al, 2009; Turick et al, 2009; Valeru et al, 2009; Wang et al, 2011) This phenotype has been observed in naturally pigmented environmental and clinical strains of Vibrio cholerae and has been shown to be due to mutations in the hmgA gene (Wang et al, 2011). The production of pyomelanin has been shown to provide greater protection from other environmental stresses such as hyperosmotic shock and elevated temperatures (Kotob et al, 1995) and act as a sole terminal electron acceptor and soluble electron shuttle to iron which may provide an additional fitness advantage to pyomelanin-producing mutants in anaerobic environments (Turick et al, 2002)

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Results

Conclusion