RESEARCH ARTICLE
The Pseudomonas quinolone signal (PQS), and its precursor HHQ, modulate interspecies and interkingdom behaviour F. Jerry Reen1, Marlies J. Mooij1, Lucy J. Holcombe1, Christina M. McSweeney2, Gerard P. McGlacken2, John P. Morrissey3 & Fergal O’Gara1 1
BIOMERIT Research Centre, Department of Microbiology, University College Cork, Ireland; 2Department of Chemistry and Analytical and Biological Research Facility, University College Cork, Ireland; and 3Department of Microbiology, University College Cork, Ireland
Correspondence: Fergal O’Gara, BIOMERIT Research Centre, Department of Microbiology, University College Cork, Ireland. Tel.: 1353 21 490 2646; fax: 1353 21 427 5934; e-mail:
[email protected] Received 11 December 2010; revised 22 March 2011; accepted 22 April 2011. Final version published online 26 May 2011. DOI:10.1111/j.1574-6941.2011.01121.x
MICROBIOLOGY ECOLOGY
Editor: Julian Marchesi Keywords interspecies/interkingdom; Pseudomonas quinolone signal; PQS; HHQ; biofilm; motility.
Abstract The Pseudomonas quinolone signal (PQS), and its precursor 2-heptyl-4-quinolone (HHQ), play a key role in coordinating virulence in the important cystic fibrosis pathogen Pseudomonas aeruginosa. The discovery of HHQ analogues in Burkholderia and other microorganisms led us to investigate the possiblity that these compounds can influence interspecies behaviour. We found that surface-associated phenotypes were repressed in Gram-positive and Gram-negative bacteria as well as in pathogenic yeast in response to PQS and HHQ. Motility was repressed in a broad range of bacteria, while biofilm formation in Bacillus subtilis and Candida albicans was repressed in the presence of HHQ, though initial adhesion was unaffected. Furthermore, HHQ exhibited potent bacteriostatic activity against several Gram-negative bacteria, including pathogenic Vibrio vulnificus. Structure– function analysis using synthetic analogues provided an insght into the molecular properties that underpin the ability of these compounds to influence microbial behaviour, revealing the alkyl chain to be fundamental. Defining the influence of these molecules on microbial–eukaryotic-host interactions will facilitate future therapeutic strategies which seek to combat microorganisms that are recalcitrant to conventional antimicrobial agents.
Introduction Cooperative behaviour has changed our perception of how bacteria interact and cohabit within diverse ecological and clinical environments. The mobilization of diffusible signal molecules among populations of bacteria facilitates coordination of cellular activities towards the benefit of the population as a whole rather than the individual cell. While these signalling molecules are often species-specific, the ability to ‘listen in’ and decipher a competitor’s messages is a valuable asset in mixed microbial communites, such as those that exist during infection of the cystic fibrosis (CF) lung. This phenomenon, often referred as interspecies or interkingdom signalling, is emerging as a key influence on the outcome of infectious diseases, although currently a dearth of knowledge exists regarding the signals involved in many of these interactions. Pseudomonas aeruginosa is a highly adaptable organism, capable of colonizing a wide variety of niches including burn FEMS Microbiol Ecol 77 (2011) 413–428
wounds and immunocompromised patients and it is the main pathogen associated with morbidity and mortality in CF patients (Govan & Deretic, 1996). Pseudomonas aeruginosa produces 4 50 alkylquinolones that differ structurally on the basis of substitution at the C3 position, N-oxide substitution of the quinolone nitrogen and modification of the alkyl side chain (Pesci et al., 1999; Lepine et al., 2004). Many of these alkylquinolones have been characterized with respect to their antibiotic activities (Wratten et al., 1977; Leisinger and Margraff, 1979; Lepine et al., 2004), while a role as signal molecules in cell–cell communication has been revealed for 2-heptyl-3-hydroxy-4-quinolone [Pseudomonas quinolone signal (PQS)] and its immediate precursor 2heptyl-4-quinolone (HHQ) (Pesci et al., 1999; McKnight et al., 2000; Diggle et al., 2003; Deziel et al., 2004). PQS signalling is pleiotropic, regulating biofilm formation, secondary metabolite production, pigment and virulence factor production, motility and membrane vesicle formation (Diggle et al., 2003, 2007b; Dubern & Diggle, 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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2008; Mashburn-Warren et al., 2009). Many of these phenotypes are mediated through the LysR-type transcriptional regulator PqsR, for which both HHQ and PQS act as coinducers (Wade et al., 2005; Xiao et al., 2006). Several important phenotypes such as iron chelation and prooxidant activities have since been attributed to PQS (Diggle et al., 2003; Bredenbruch et al., 2006; Haussler and Becker, 2008). While PQS has poor solubility in aqueous solution and is packaged into self-promoted vesicles to facilitate trafficking (Mashburn-Warren et al., 2009), HHQ is known to passively diffuse out of the cell into the extracellular milieu (Deziel et al., 2004). Recent studies have identified HHQ biosynthetic systems in non-Pseudomonas species (Diggle et al., 2006; Vial et al., 2008). While the function of HHQ analogues in these species remains to be elucidated, strong structural similarities with P. aeruginosa HHQ suggest the existence of a conserved interspecies signalling system. However, although the spectrum of influence of alkylquinolones has been extensively reviewed in recent years (most recently by Heeb et al., 2010 and Huse & Whiteley, 2010), the question remains as to whether HHQ and PQS signal molecules have an interspecies/interkingdom communication role. In this study we reveal a role for both PQS and HHQ as modulators of key phenotypes in Gram-positive and Gramnegative bacteria, as well as towards the eukaryotic yeast Candida albicans. In addition, we provide evidence for the structural requirements that define the interkingdom role of these molecules.
F.J. Reen et al.
of peptone, 15 g of Bacto agar, 33.3 g of InstantOcean (Aquarium Systems, Mentor, OH) and 1 L of distilled water. Candida albicans was grown in non-filament-inducing media; YPD [2% (w/v) Bacto peptone, 1% (w/v) yeast extract and 2% (w/v) glucose] or YNB [1 YNB salts with ammonium sulphate (Difco 291940), 0.2% (w/v) glucose and 0.1% (w/v) maltose] and filament-inducing media; YNBNP [YNB supplemented with 25 mM phosphate buffer (pH 7) and 2.5 mM N-acetylglucosamine (Sigma A-8625)], or Spider media (10 g of nutrient broth, 10 g of mannitol and 2 g of K2HPO4 in 1 L distilled water pH 7.2) as described previously (Liu et al., 1994; McAlester et al., 2008).
Chemical synthesis of alkylquinolone derivatives
Materials and methods
HHQ was prepared using a procedure we have recently reported (McGlacken et al., 2010) starting with Meldrum’s acid in a four-step procedure. Conversion to the aldehyde proceeded using a Duff formylation reaction using a modified method to that described by Pesci et al. (1999). Transformation to PQS occurred as described previously (Pesci et al., 1999). Synthesis of the methyl derivative of PQS was achieved through reaction of the appropriate anthranilic acid with chloroacetone under basic conditions followed by cyclization of the corresponding anthranilate in refluxing N-methylpyrrolidone. This procedure described by Hradil et al. (1999) gave the quinolone in good yield ( 4 50% over two steps). Synthesis of the methyl derivative of HHQ was achieved by reaction of ethyl acetoacetate and aniline followed by cyclization in refluxing diphenylether. All alkylquinolone compounds (Supporting Information, Table S1) were solubilized in methanol and stored at 20 1C.
Strains and growth conditions
Antibacterial activity assays
Strains, media composition and growth conditions are described in Table 1. One of the Bacillus subtilis strains used in this study, NCTC 10073, has been renamed Bacillus atrophaeus, with both species being indistinguishable using standard characterization methods (Fritze & Pukall 2001). However, as this strain is still listed as B. subtilis in the NCTC collection, we have used that designation throughout the paper. Trypticase soy agar (TSA) (Merck, Germany) was routinely used to culture Bacillus, Escherichia and Staphylococcus species. Listeria monocytogenes was cultured on brain–heart infusion (BHI) agar. Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus were cultured on Luria–Bertani (LB) [tryptone 1% (w/v), yeast extract 0.5% (w/v), agar 1.5% (w/v)] supplemented with 0.5%, 2% and 3% (w/v) NaCl, respectively. Vibrio fischeri was cultured on LBS [Tryptone 1% (w/v), yeast extract 0.5% (w/v), NaCl 2% (w/v), agar 1.5% (w/v) and 20 mM Tris-HCl (pH 7.5)]. Marine agar was used to culture sea-sponge isolates and was constituted as 10 g of soluble starch, 4 g of yeast extract, 2 g
Antibacterial activity of all alkylquinolone derivatives was initially investigated using agar plate assays. Bacteria were streaked onto growth agar (Table 1) and incubated overnight at an appropriate temperature (Table 1). Where growth was altered on plates, kinetics were measured in liquid culture in microtitre plates using a BioScreen C analyser (Oy Growth Curves Ab Ltd, Helsinki). To initiate time kill-curve assays, overnight cultures were standardized to OD600 nm 0.2, to which HHQ was added at 1, 10 or 50 mM. Equal volumes of methanol were added to control cultures. Samples were taken at 1 h intervals and viable cell counts were enumerated on LBS (V. fischeri) or LB (Escherichia coli and V. cholerae) agar.
2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Semi-solid motility assays Motility of B. subtilis and Bacillus cereus was analysed on LB and TSA 0.3% (w/v) agar, Staphylococcus aureus colony spreading was assessed on LB and TSA 0.24% (w/v) agar, FEMS Microbiol Ecol 77 (2011) 413–428
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Quinolone signal molecules modulate interkingdom behaviour
Table 1. List of strains and routine growth conditions used in this study Strain Gram negative Pseudomonas aeruginosa PA14 P. aeruginosa PA14 pqsH Escherichia coli NCIMB11943 Vibrio fischeri ES114 V. fischeri MJ11 V. cholerae 0395 V. vulnificus YJ016 V. parahaemolyticus RIMD2210633 Salmonella Typhimurium LT2 Serratia marcescens Serratia sp. 39006 Proteus vulgaris NCIMB12426 Gram positive Bacillus subtilis NCTC10073 B. subtilis NCDO1789 B. cereus NCIMB9373 Staphylococcus aureus NCDO949 S. epidermidis DMSZ3095 S. gallinarum DMSZ4616 Micrococcus luteus NCIMB9278 Listeria monocytogenes LO28 Marine sponge isolates Vibrio sp. Algoriphagus sp. Bacillus sp. Pseudoalteromonas sp. Spongibacter sp. Shewanella sp. Micrococcus sp. Pseudovibrio sp. Yeast Candida albicans SC5314 C. albicans BCa2-10 C. glabrata ATCC2001 Kluyveromyces marxianus CBS608 Saccharomyces cerevisiae BY4741
Description
Temperature ( 1C)
Media
Source/reference
37 37 37 25 25 30 30 30 37 30 30 37
LB LB LB LBS LBS LB LB 2% NaCl LB 3% NaCl LB LB LB LB
Liberati et al. (2006) Liberati et al. (2006) NCIMB E. Ruby lab, UW E. Ruby lab, UW UCC Collection UCC Collection UCC Collection UCC Collection J. Clair, Mercy Hospital Poulter et al. (2010) NCIMB
30
TSB
NCTC
Serotype 1/2c
30 30 37 37 37 37 37
TSB TSB TSB TSB TSB TSB BHI
NCDO NCIMB NCDO DMSZ DMSZ NCIMB C. Hill lab, UCC
P. boletiformis isolate P. boletiformis isolate P. boletiformis isolate P. boletiformis isolate P. boletiformis isolate P. boletiformis isolate P. boletiformis isolate A. dissimilis isolate
23 23 23 23 23 23 23 23
MA MA MA MA MA MA MA MA
UCC Collection UCC Collection UCC Collection UCC Collection UCC Collection UCC Collection UCC Collection UCC Collection
37 37 37 37 37
YPD YPD YPD YPD YPD
Gillum et al. (1984) Braun & Johnson (1997) ATCC CBS Brachmann et al. (1998)
pqsH
