A Novel DSF-Like Signal from Burkholderia Cenocepacia Interferes with Candida Albicans Morphological Transition

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A Novel DSF-Like Signal from Burkholderia Cenocepacia Interferes with Candida Albicans Morphological Transition The ISME Journal (2008) 2, 27–36 & 2008 International Society for Microbial Ecology All rights reserved 1751-7362/08 $30.00 www.nature.com/ismej ORIGINAL ARTICLE A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition Calvin Boon1,3, Yinyue Deng1,2,3, Lian-Hui Wang1, Yawen He1, Jin-Ling Xu1, Yang Fan1, Shen Q Pan2 and Lian-Hui Zhang1,2 1Institute of Molecular and Cell Biology, Singapore and 2Department of Biological Sciences, National University of Singapore, Singapore In addition to producing lethal antibiotics, microorganisms may also use a new form of antagonistic mechanism in which signal molecules are exported to influence the gene expression and hence the ecological competence of their competitors. We report here the isolation and characterization of a novel signaling molecule, cis-2-dodecenoic acid (BDSF), from Burkholderia cenocepacia. BDSF is structurally similar to the diffusible signal factor (DSF) that is produced by the RpfF enzyme of Xanthomonas campestris. Deletion analysis demonstrated that Bcam0581, which encodes an RpfF homologue, was essential for BDSF production. The gene is highly conserved and widespread in the Burkholderia cepacia complex. Exogenous addition of BDSF restored the biofilm and extracellular polysaccharide production phenotypes of Xanthomonas campestris pv. campestris DSF-deficient mutants, highlighting its potential role in inter-species signaling. Further analyses showed that Candida albicans germ tube formation was strongly inhibited by either coculture with B. cenocepacia or by exogenous addition of physiological relevant levels of BDSF, whereas deletion of Bcam0581 abrogated the inhibitory ability of the bacterial pathogen. As B. cenocepacia and C. albicans are frequently encountered human pathogens, identification of the BDSF signal and its activity thus provides a new insight into the molecular grounds of their antagonistic interactions whose importance to microbial ecology and pathogenesis is now becoming evident. The ISME Journal (2008) 2, 27–36; doi:10.1038/ISMEJ.2007.76; published online 29 November 2007 Subject Category: microbe-microbe and microbe-host interactions Keywords: BDSF; DSF; quorum sensing; signal interference; antagonism Introduction microorganisms appear to produce, release and respond collectively to species-specific small signal Microbe–microbe interactions are ubiquitous in molecules to coordinate a range of important various natural ecosystems. Not surprisingly, var- activities, such as virulence factor production, ious forms of symbiosis and antagonism have antibiotics biosynthesis and biofilm formation. This surfaced. It has been known for a long time that cell–cell communication mechanism is commonly microorganisms may produce antibiotics to inhibit known as quorum sensing (Whitehead et al., 2001; or stop the growth of their competitors to gain Fuqua and Greenberg, 2002). Logically, microorgan- competitive advantages. Recently, evidence is accu- isms might also boost their competitive strength in mulating for a new form of microbial antagonism, ecosystems by interfering with the quorum-sensing which was tentatively designated as signal inter- signaling of their competitors (Zhang and Dong, ference (Zhang and Dong, 2004). This type of 2004). antagonism acts not by killing, but instead by Candida albicans causes various forms of candia- influencing the signal-mediated gene expression of sis ranging from mucosal infections to serious the competitors and thus tips the balance of systemic infections. Mutants defective in morpho- interaction (Dong et al., 2004; Hogan et al., 2004). logical transitions during infection are avirulent For keeping up their competitive advantages, many (Lo et al., 1997; Braun et al., 2000; Saville et al., 2003; Zheng et al., 2003). Therefore, this ability to switch between yeast and hyphal-form is an im- Correspondence: L-H Zhang, Institute of Molecular and Cell portant aspect of its pathogenesis. Intriguingly, the Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore. chance of infection by C. albicans seems to be E-mail: [email protected] 3These authors have contributed equally to this work. influenced by the presence of certain bacterial Received 2 July 2007; revised 27 August 2007; accepted 27 August pathogens. While the fungal pathogen can be found 2007; published online 29 November 2007 in various groups of patients who have undergone A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans C Boon et al 28 treatments with broad-spectrum antibiotics, it is grown either in GMM medium consisting of 6.7 g of rarely found in individuals suffering from cystic Bacto yeast nitrogen base (Difco, Sparks, MD, USA) fibrosis that are chronically infected with Pseudo- and 0.2% glucose or in minimum medium (pH 7.2). monas aeruginosa and Burkholderia cepacia (Kerr, The latter consists of K2PO4,60mM;KH2PO4,30mM; 1994). This putative antagonism seems to be at least citrate, 20 mM; (NH4)2SO4,15mM; MgSO4 Á 7H2O, partially explained by the recent finding that the 0.8 mM; CaCl2,90mM;FeSO4,30mM;MnCl2,15mM; long-chain quorum-sensing signal 3-oxo-C12HSL and Casamino acids, 0.5%. DSF was added to the produced by P. aeruginosa at physiological relevant medium in a final concentration of 5 mM unless level was sufficient to inhibit C. albicans yeast-to- otherwise indicated. hyphae transition (Hogan et al., 2004). However, C8- HSL, the major quorum-sensing signal produced by B. cepacia (Lewenza et al., 1999; Riedel et al., 2001), Bioassay of BDSF was not able to suppress the fungal filamentation The assay was performed as described previously (Hogan et al., 2004). using the biosensor strain FE58 (Wang et al., 2004). Burkholderia cepacia complex (Bcc) has emerged Briefly, 4-mm diameter wells were introduced on as a major opportunist pathogen for immunocom- prepared bioassay plates and 20 ml concentrated promised individuals in particular the patients with culture was added to each well. Alternatively, single cystic fibrosis and chronic granulomatous disease colonies were spotted on bioassay plates. The plates (Isles et al., 1984; Goldmann and Klinger, 1986; were incubated at 30 1C overnight. DSF activity is Mahenthiralingam et al., 2005). The Bcc consist of at indicated by the presence of a blue halo around the least nine species that are phenotypically similar well or colony. but genetically distinct, and all the species are capable of causing infections (Coenye et al., 2001; Mahenthiralingam et al., 2005). Among them, Burkholderia cenocepacia constitutes the majority BDSF purification of the transmissible and epidemic strains and is B. cenocepacia J2315 was grown in LB overnight 1 highly virulent (Mahenthiralingam et al., 2001, with agitation at 37 C. Seventy liters of culture 2002). In this study, we report the identification supernatant was collected by centrifugation and and characterization of a novel signaling molecule extracted with equal volume of ethyl acetate. The designated as BDSF from B. cenocepacia. BDSF is a crude extract (organic phase) was dried using a structural homologue of diffusible signal factor rotary evaporator and dissolved with methanol. The (DSF), which is the quorum-sensing signal produced mixture was subjected to flash column chromato- by the plant bacterial pathogen Xanthomonas graphy using a silica gel column (12 Â 150 mm, campestris pv. campestris (Xcc). In addition, we Biotage Flash 12 M cartridge) and eluted with ethyl demonstrate that the ORF Bcam0581 is essential for acetate–hexane (20:80 v/v). The active fractions BDSF production and can genetically substitute for were detected using the DSF bioassay and pooled. the DSF synthase gene rpfF in Xcc. Furthermore, we These were then concentrated, subjected to flash show that the dimorphic transition of C. albicans column chromatography again and eluted with ethyl was inhibited by either exogenous addition of BDSF acetate–hexane (10:90 v/v). The purity of the col- or coculturing with B. cenocepacia. lected active components was analyzed by high- performance liquid chromatography using a C18 reverse-phase column (4.8 Â 250 mm, Waters, Milford, MA, USA), eluted with methanol–water Materials and methods (80:20 v/v) at a flow rate of 1 ml minÀ1. Bacteria strains and growth conditions B. cenocepacia J2315, a cystic fibrosis clinical isolate, was obtained from the American Type Structure analysis and synthesis of BDSF Culture Collection, Manassas, VA, USA. It is the 1H, 13C, 1H-1H COSY, distortionless enhancement by representative strain of the highly transmissible polarization transfer, heteronuclear multiple bond ET12 clone (Govan et al., 1993). X. campestris pv. coherence and heteronuclear multiple quantum campestris strain 8004 and its rpfF deletion mutant coherence (HMQC) nuclear magnetic resonance 8004dF were described previously (Wang et al., (NMR) spectra in CDCl3 solution were obtained 2004; He et al., 2006b). Xcc strains were maintained using a Bruker DRX400 spectrometer operating at at 30 1C in YEB medium (Zhang et al., 2002), 400 MHz for 1H or 100.5 MHz for 13C. High-resolu- while Escherichia coli and B. cenocepacia strains tion electrospray ionization mass spectrometry was were grown in Luria-Bertani (LB) broth at 37 1C. performed on a Finnigan/MAT MAT 95XL-T mass The following antibiotics were supplemented spectrometer. Conditions used were as stated before when necessary: rifampicin, 50 mgmlÀ1; tetra- (Wang et al., 2004). BDSF was synthesized by cycline, 10
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