Simultaneous Inhibition of Rhamnolipid and Polyhydroxyalkanoic Acid Synthesis and Biofilm Formation in Pseudomonas aeruginosa by 2-Bromoalkanoic Acids: Effect of Inhibitor Alkyl-Chain-Length

et al. (2013) Simultaneous Inhibition of Rhamnolipid and Polyhydroxyalkanoic Acid Synthesis and Biofilm Formation in Pseudomonas aeruginosa by 2-Bromoalkanoic Acids: Effect of Inhibitor Alkyl-Chain-Length. PLoS ONE 8(9): e73986. doi:10.1371/journal.pone.0073986 Simultaneous Inhibition of Rhamnolipid and Polyhydroxyalkanoic Acid Synthesis and Biofilm Formation in Pseudomonas aeruginosa by 2- Bromoalkanoic Acids: Effect of Inhibitor Alkyl-Chain- Length Merced Gutierrez 0 Mun Hwan Choi 0 Baoxia Tian 0 Ju Xu 0 Jong Kook Rho 0 Myeong Ok Kim 0 You-Hee Cho 0 Sung Chul Yoon 0 Gunnar F. Kaufmann, The Scripps Research Institute and Sorrento Therapeutics, Inc., United States of America 0 1 Nano-Biomaterials Science Laboratory, Division of Applied Life Sciences, Graduate School, Gyeongsang National University , Jinju , Republic of Korea, 2 National Research Foundation Funded Pioneer Research Center for Alzheimer Disease Control, Gyeongsang National University , Jinju , Republic of Korea, 3 Neurobiology Laboratory, Division of Applied Life Sciences, Graduate School, Gyeongsang National University , Jinju , Republic of Korea, 4 Laboratory of Antiinfective Agents and Phage Therapy, College of Pharmacy, CHA University , Gyeonggi-do , Republic of Korea Pseudomonas aeruginosa, an opportunistic human pathogen is known to synthesize rhamnolipid and polyhydroxyalkanoic acid (PHA) of which the acyl-group precursors (e.g., (R)-3-hydroxydecanoic acid) are provided through RhlA and PhaG enzyme, respectively, which have 57% gene sequence homology. The inhibitory effect of three 2-bromo-fatty acids of 2bromohexanoic acid (2-BrHA), 2-bromooctanoic acid (2-BrOA) and 2-bromodecanoic acid (2-BrDA) was compared to get an insight into the biochemical nature of their probable dual inhibition against the two enzymes. The 2-bromo-compounds were found to inhibit rhamnolipid and PHA synthesis simultaneously in alkyl-chain-length dependent manner at several millimolar concentrations. The separate and dual inhibition of the RhlA and PhaG pathway by the 2-bromo-compounds in the wild-type cells was verified by investigating their inhibitory effects on the rhamnolipid and PHA synthesis in P. aeruginosa DphaG and DrhlA mutants. Unexpectedly, the order of inhibition strength was found 2-BrHA ($90% at 2 mM) . 2-BrOA . 2-BrDA, equally for all of the rhamnolipids and PHA synthesis, swarming motility and biofilm formation. We suggest that the novel strongest inhibitor 2-BrHA could be potentially exploited to control the rhamnolipid-associated group behaviors of this pathogen as well as for its utilization as a lead compound in screening for antimicrobial agents based on new antimicrobial targets. - Funding: This study was supported by grants (NRF grant #: 2009-0070747 and 2012-0009522) from the Ministry of Science, ICT & Future Planning/NRF. M.G., B.T., and J.X. were supported by a graduate scholarship through the BK21 program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Pseudomonas aeruginosa is a typical opportunistic human pathogen which colonizes the lungs of cystic fibrosis patients and causes serious infections in immuno-compromised hosts [1]. It can simultaneously produce two biotechnologically important compounds, namely polyhydroxyalkanoic acids (PHAs) and rhamnolipids [2]. PHAs, which are promising materials for biodegradable plastics, have been studied extensively as replacements for conventional petrochemical-based plastics [3]. The Rhamnolipids, which represent one of the most important classes of microbial surfactants, are of increasing industrial interest because of their broad range of potential applications including use as surface coatings and also additives for environmental remediation [4,5]. They serve as extracellular virulence factors that play multiple roles [46]. For example, they enhance uptake of hydrophobic substrates in an energy-dependent manner [7], display antibiotic activities, and contribute to pathogenesis [810]. Along with its precursor, b-hydroxyalkanoyl-b-hydroxyalkanoic acid (HAA) in which b-hydroxydecanoic acid (C10) is the major component, rhamnolipids have been demonstrated to play a central role in swarming motility [1114].They are also implicated in various steps of biofilm development [1519]. Two types of rhamnolipids are known: the monorhamnolipids (Rha-C10-C10), which contain one unit of rhamnose linked to HAA, and the dirhamnolipids (Rha-Rha-C10-C10), which contain two units of rhamnose (Figure 1) [9]. When P. aeruginosa is grown on glycerol and saccharides, (R)-b-hydroxyalkanoyl-acyl carrier protein ((R)-b-hydroxyalkanoyl-ACP) is utilized by RhlA (HAA synthase) to produce HAAs from two molecules of (R)-bhydroxyalkanoyl-ACP [5,12,20]. In medium-chain-length (MCL, 614 carbon atoms)-polyhydroxyalkanoic acid (PHA) producing bacteria, such as Pseudomonas spp. belonging to rRNA group I, MCL-type (R)-b-hydroxyalkanoyl monomers are derived as the form of (R)-b-hydroxyalkanoyl-coenzyme A (CoA) which is the substrate of MCL-PHA synthase. The coenzyme A monomer is derived from ACP intermediates of the fatty acid de novo synthesis pathway via the enzyme (R)-b-hydroxyalkanoyl-ACP:CoA transacylase (PhaG) [21]. Thus, PhaG and RhlA may compete for (R)-bhydroxyalkanoyl-ACP, especially (R)-b-hydroxydecanoyl-ACP which is the major acyl component of rhamnolipid [20]. However, it has been suggested that RhlA can produce CoA-linked fatty acid dimers using ACP-linked fatty acids [22,23] and could also contribute to PHA synthesis by the RhlA activity which is analogous to that of PhaG. This suggestion is based on the fact that PHA synthesis in P. aeruginosa phaG mutants is not completely abrogated and phaG mutants of other Pseudomonas spp. completely lack PHA production when grown with a sugar as the carbon source. The gene rhlB, which encodes a rhamnosyltransferase, is known to be responsible for the synthesis of rhamnolipids by transferring a rhamnosyl group to HAA [5]. The gene rhlC encodes the rhamnosyltransferase II responsible for the addition of the second rhamnosyl group to the monorhamnolipid [5]. The close metabolic relationship between PHA and rhamnolipid synthesis was experimentally confirmed on the basis of comparative 13C NMR analysis of them in wild-type and mutants [24]. Higher PHA accumulation was found in the rhamnolipid-negative mutants than in the wild-type strains, suggesting that 3-hydroxy fatty acid precursors become more available for PHA synthesis when rhamnolipid synthesis is lacking. However, compared to the wild-type strains, rhamnolipid production was not enhanced in the four pha mutants of P. aeruginosa PA14 and PAO1 which indicates that rhamnolipid production in P. aeruginosa could be tightly regulated. This may be ascribable to transcriptio (...truncated)