Vibrio Campbellii Hmga-Mediated Pyomelanization Impairs Quorum Sensing, Virulence, and Cellular Fitness
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Vibrio campbellii hmgA-mediated pyomelanization impairs quorum sensing, virulence, and cellular fitness The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Wang, Zheng, Baochuan Lin, Anahita Mostaghim, Robert A. Rubin, Evan R. Glaser, Pimonsri Mittraparp-arthorn, Janelle R. Thompson, Varaporn Vuddhakul, and Gary J. Vora. “Vibrio Campbellii hmgA- Mediated Pyomelanization Impairs Quorum Sensing, Virulence, and Cellular Fitness.” Frontiers in Microbiology 4 (2013). As Published http://dx.doi.org/10.3389/fmicb.2013.00379 Publisher Frontiers Research Foundation Version Final published version Citable link http://hdl.handle.net/1721.1/85686 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. ORIGINAL RESEARCH ARTICLE published: 11 December 2013 doi: 10.3389/fmicb.2013.00379 Vibrio campbellii hmgA-mediated pyomelanization impairs quorum sensing, virulence, and cellular fitness Zheng Wang 1*, Baochuan Lin 1, Anahita Mostaghim 1,2, Robert A. Rubin 3,EvanR.Glaser4, Pimonsri Mittraparp-arthorn 5, Janelle R. Thompson 6, Varaporn Vuddhakul 5 and Gary J. Vora 1 1 Center for Bio/Molecular Science & Engineering, Naval Research Laboratory, Washington, DC, USA 2 School of Systems Biology, College of Science, George Mason University, Fairfax, VA, USA 3 Mathematics Department, Whittier College, Whittier, CA, USA 4 Division of Electronics Science and Technology, Naval Research Laboratory, Washington, DC, USA 5 Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Thailand 6 Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA Edited by: Melanization due to the inactivation of the homogentisate-1,2-dioxygenase gene (hmgA) Daniela Ceccarelli, University of has been demonstrated to increase stress resistance, persistence, and virulence in some Maryland, USA bacterial species but such pigmented mutants have not been observed in pathogenic Reviewed by: members of the Vibrio Harveyi clade. In this study, we used Vibrio campbellii ATCC Zongze Shao, State Oceanic Administration, China BAA-1116 as model organism to understand how melanization affected cellular phenotype, Xiu-Lan Chen, Shandong university, metabolism, and virulence. An in-frame deletion of the hmgA gene resulted in the China overproduction of a pigment in cell culture supernatants and cellular membranes that was *Correspondence: identified as pyomelanin. Unlike previous demonstrations in Vibrio cholerae, Burkholderia Zheng Wang, Center for cepacia, and Pseudomonas aeruginosa, the pigmented V. campbellii mutant did not Bio/Molecular Science & ∼ Engineering, Naval Research show increased UV resistance and was found to be 2.7 times less virulent than the Laboratory, Code 6900, 4555 wild type strain in Penaeus monodon shrimp virulence assays. However, the extracted Overlook Avenue, SW, Washington, pyomelanin pigment did confer a higher resistance to oxidative stress when incubated DC 20375, USA with wild type cells. Microarray-based transcriptomic analyses revealed that the hmgA e-mail: [email protected] 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. Keywords: Vibrio, melanin, bioluminescence, quorum sensing, tyrosine catabolism INTRODUCTION appears to contribute to virulence by reducing the susceptibility of As a member of the L-tyrosine catabolism pathway in bacterial pigmented bacteria to host defense mechanisms. Because of these and eukaryotic organisms, the enzyme homogentisate 1,2- particular characteristics, it is not surprising that pyomelanin- dioxygenase (HmgA) catalyzes the intermediate homogentisic producing Pseudomonas aeruginosa and Burkholderia cepacia are acid into 4-maleylacetoacetate which is further catabolized to frequently isolated from cystic fibrosis patients (Zughaier et al., yield fumarate and acetoacetate. In some bacterial species, it has 1999; Rodriguez-Rojas et al., 2009). Furthermore, the production been demonstrated that the inactivation of the hmgA gene results of pyomelanin has also been shown to provide greater protec- in the accumulation of homogentisic acid which when auto- tion from other environmental stresses such as hyperosmotic oxidized leads to the formation of the water-soluble brown pig- shock and elevated temperatures (Kotob et al., 1995)andact ment pyomelanin (Rodriguez-Rojas et al., 2009; Schmaler-Ripcke as a sole terminal electron acceptor and soluble electron shut- et al., 2009; Turick et al., 2009; Valeru et al., 2009; Wang et al., tle to iron which may provide an additional fitness advantage 2011). This phenotype has been observed in naturally pigmented to pyomelanin-producing mutants in anaerobic environments environmental and clinical strains of Vibrio cholerae and has been (Turick et al., 2002). shown to be due to mutations in the hmgA gene (Wang et al., Despite these seemingly advantageous phenotypes, such pig- 2011). Interestingly, pyomelanin pigmented V. cholerae demon- mented mutants have not been reported from pathogenic mem- strate greater UV and oxidative stress resistance, virulence factor bers of the Vibrio Harveyi clade. Two of the most economically expression and infant mouse intestine colonization rates than important Harveyi clade species, V. campbellii and V. har ve y i, their non-pigmented counterparts (Valeru et al., 2009). The abil- are common inhabitants of tropical marine environments and ity of pyomelanin to confer increased resistance to oxidative stress are among the most important bacterial pathogens of many www.frontiersin.org December 2013 | Volume 4 | Article 379 | 1 Wang et al. Vibrio campbellii pyomelanization commercially farmed marine invertebrate and vertebrate species GROWTH CURVE ANALYSES (Thompson et al., 2004; Austin and Zhang, 2006). As certain Bacterial replication was measured using a Bioscreen C analyzer pathogenic members of both species are capable of producing (Growth Curves USA, Piscataway, NJ, USA). Briefly, overnight quorum sensing induced bioluminescence, the disease caused by cultures were diluted 1:5000 (∼105 cells/mL) in pre-warmed LM them is often referred to as luminescent vibriosis (Defoirdt et al., and five 200 μL aliquots of the wild type (WT) and hmgA 2008) and is a disease manifestation that is frequently implicated strains were transferred into a 100-well honeycomb plate. The in outbreaks within penaeid shrimp larval culture facilities world- plate was incubated at 30◦Cfor48hwithcontinuousshaking wide (Austin and Zhang, 2006). Given the importance of shrimp andwidebandOD450−580 nm measurements taken every 30 min. hemocyte-mediated oxidative defense mechanisms in combatting Three independent experiments were performed in this manner. Vibrio infections (Ji et al., 2011), it is not unreasonable to posit that pyomelanization may benefit the survival and perhaps exac- MEASUREMENT OF PIGMENTATION, BIOLUMINESCENCE AND erbate the virulence of vibrios in this host environment. However, CELLULAR SUSCEPTIBILITY TO H2O2 the production of pyomelanin comes at the cost of impairing Matched diluted overnight WT and hmgA cultures were used the tyrosine catabolism pathway and the effect of the inactiva- to inoculate 50 mL LM media in 250 mL flasks and incubated at ◦ tion of hmgA and/or pyomelanin production on global cellular 30 C and 200 rpm. Every 24 h, three 100 μL aliquots of culture metabolism is not known. In this study, we used V. campbellii were collected and bacterial cells were pelleted via centrifuga- ATCC BAA-1116, a bioluminescent marine bacterium that is best tion at 10,000× g for 5 min. Supernatant pigments were mea- known as a model organism for quorum sensing studies (Bassler, sured using a NanoDrop ND-2000c spectrophotometer (Thermo 1999), to begin to determine the generality of pyomelanin- Scientific, Pittsburg, PA, USA) at OD400. Another three 100 μL mediated phenotypes and how the deletion of the hmgA gene and aliquots of culture were placed in a black U96 Nunc MicroWell™ resulting pyomelanin production may affect cellular phenotypes, plate (Thermo Scientific) and measured for bioluminescence virulence, and transcription. using a Luminoskan Ascent Microplate Luminometer (Thermo Scientific). Three independent experiments were performed in MATERIALS AND METHODS this manner. BACTERIAL STRAINS AND GROWTH CONDITIONS At the 48 h time point, WT, and hmgA cells were harvested, V. campbellii (ATCC strain BAA-1116; previously known as V. washed and resuspended in fresh LM media. They were then incu- harveyi BAA-1116 or BB120; Lin et al., 2010)andthehmgA bated with 2 mM H2O2 at room temperature for 15 min. The mutant were grown in Luria Marine (LM) medium (20 g NaCl, percentage survival was calculated by counting colony forming 10 tryptone, 5 g yeast extract per L, pH 7.8)