J Med Microbiol 57 (2008), 563-569; DOI: 10.1099/jmm.0.47705-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Signal-mediated interactions between Pseudomonas aeruginosa and Candida albicans
Gordon McAlester1,
Fergal O'Gara1,2 and
John P. Morrissey1
1 Department of Microbiology, University College Cork, Ireland
2 BIOMERIT Research Centre, Biosciences Institute, University College Cork, Ireland
Correspondence
John P. Morrissey
j.morrissey{at}ucc.ie
Received 18 October 2007
Accepted 29 January 2008
Pseudomonas aeruginosa causes infections in a wide variety of hosts and is the leading cause of mortality in cystic fibrosis (CF) patients. Although most clinical isolates of P. aeruginosa share common virulence determinants, it is known that strains evolve and change phenotypically during CF lung infections. These changes can include alterations in the levels of N-acyl homoserine lactones (HSLs), which are secreted signal molecules. In the CF lung, fungi, especially Candida albicans and Aspergillus fumigatus, may coexist with P. aeruginosa but the implications for disease are not known. Recent studies have established that signalling can occur between P. aeruginosa and C. albicans, with the bacterial molecule 3-oxo-C12HSL affecting Candida morphology, and the fungal metabolite farnesol reducing levels of the Pseudomonas quinolone signal and pyocyanin in Pseudomonas. Whether these interactions are common and typical in clinical strains of P. aeruginosa was addressed using CF isolates that produced varied levels of HSLs. It was found that, whereas some clinical P. aeruginosa strains affected C. albicans morphology, others did not. This correlated closely with the amounts of 3-oxo-C12HSL produced by the isolates. Furthermore, it was established that signalling is bidirectional and that the C. albicans molecule farnesol inhibits swarming motility in P. aeruginosa CF strains. This work demonstrates that clinical isolates of these opportunistic pathogens can interact in strain-specific ways via secreted signals and illustrates the importance of studying these interactions to fully understand the microbial contribution to disease in polymicrobial infections.
Abbreviations: CF, cystic fibrosis; HSL, N-acyl homoserine lactone; PQS, Pseudomonas quinolone signal; PVC, polyvinyl chloride; QS, quorum sensing.
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INTRODUCTION
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Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that is found in a myriad of environments. As well as being an opportunistic human pathogen, it is also capable of causing disease in eukaryotes such as plants, mammals, insects and nematodes (Diggle et al., 2003; Giamarellou, 2000; Rahme et al., 1995, 2000; Tan & Ausubel, 2000; Whitchurch et al., 2005). It is also the principal cause of mortality in cystic fibrosis (CF) populations (Diggle et al., 2003; Drenkard & Ausubel, 2002; Govan & Deretic, 1996; Smith et al., 1996). P. aeruginosa communicates using chemical signals to sense cell density and alter gene expression and virulence factors in a process known as quorum sensing (QS) (Heurlier et al., 2006; Schuster & Greenberg, 2006; Shiner et al., 2005; Smith & Iglewski, 2003; Williams, 2002). The major cell–cell signalling molecules in P. aeruginosa are the N-acyl homoserine lactones (HSLs) 3-oxo-C12HSL and C4HSL, and the quinolone 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal; PQS). QS regulates many virulence features including the formation of biofilms and production of factors such as hydrogen cyanide, pyocyanin, proteases and rhamnolipids (Calfee et al., 2005; Cugini et al., 2007; Diggle et al., 2003, 2006, 2007; McGrath et al., 2004; Wade et al., 2005).
Together with P. aeruginosa, the dimorphic yeast Candida albicans is one of the most commonly isolated micro-organisms from CF patient sputum (Bakare et al., 2003; Bauernfeind et al., 1987; Hughes & Kim, 1973). Although C. albicans is a common non-pathogenic commensal of the skin and mucosal flora of healthy individuals, in compromised individuals it is able to initiate invasive growth that may result in serious disease and death (Hube, 2006; Naglik et al., 2004; Pfaller & Diekema, 2007). C. albicans can exist as yeast or filamentous forms and, in response to certain cues, including temperature and host factors, it switches from yeast to hyphal (filamentous) growth. Interestingly, both morphological forms are important for virulence and thus the ability to morphologically transform is an important virulence trait (Calderone & Fonzi, 2001; Gow, 1997; Gow et al., 2002; Liu, 2002; Mitchell, 1998; Whiteway & Oberholzer, 2004). Like P. aeruginosa, C. albicans employs secreted signals to regulate gene expression and virulence. Actively growing yeast cells secrete farnesol, a 12-carbon sesquiterpene, which acts as a virulence factor and a repressor of the switch from yeast to hyphal growth in C. albicans (Enjalbert & Whiteway, 2005; Hogan, 2006a, b; Hornby et al., 2001; Navarathna et al., 2007a, b; Nickerson et al., 2006).
P. aeruginosa and C. albicans are frequently co-isolated from CF patient sputum and a variety of in vitro and in vivo studies have established that these two species can interact. In vitro and in vivo, there is evidence to suggest that P. aeruginosa may inhibit C. albicans growth in the host (Bauernfeind et al., 1987; Burns et al., 1999; Gupta et al., 2005; Kaleli et al., 2007; Kerr, 1994a, b). Furthermore, several in vitro studies using well-characterized laboratory strains have identified more complex interactions between these human pathogens. Certain strains of P. aeruginosa are cytotoxic to the filamentous form of C. albicans but are unable to attach to or kill C. albicans yeast cells (Hogan & Kolter, 2002). In addition, it has been reported that 3-oxo-C12HSL and structurally related molecules can inhibit and even reverse the switch from yeast to hyphal growth in C. albicans (Hogan et al., 2004). That study also showed that a mutant defective in producing 3-oxo-C12HSL lost the ability to influence C. albicans morphology (Hogan et al., 2004). Interestingly, recent research has shown that these signalling interactions between P. aeruginosa and C. albicans may in fact be bidirectional. It was found that addition of farnesol leads to decreased PQS production in P. aeruginosa and that this decrease leads to a reduction in the PQS-regulated virulence factor pyocyanin (Cugini et al., 2007). Those studies open up an intriguing scenario whereby secreted factors from P. aeruginosa and C. albicans have mutually antagonistic effects on important virulence traits.
It is known, however, that strains of P. aeruginosa isolated from CF patients show significant phenotypic and genotypic variation (Finnan et al., 2004; Martin et al., 1995; Oliver et al., 2000; Wolfgang et al., 2003). These variations in P. aeruginosa CF strains are likely to alter how P. aeruginosa interacts with the host and other organisms including C. albicans. In this study, we took advantage of a set of seven characterized P. aeruginosa CF clinical isolates to investigate possible interaction between bacterial isolates and the yeast C. albicans. We addressed specifically the question of whether secreted factors from clinical isolates would affect C. albicans growth or morphology. Using cell-free supernatants we were able to focus on the role of HSL molecules in mediating these effects. In addition, we examined the effect of the yeast signal, farnesol, on these bacterial isolates.
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METHODS
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Strains, culture conditions and compounds.
All strains used in this study (apart from the HSL– mutant) were clinical isolates and are listed in Table 1
. All strains were characterized genetically previously and were shown to be genotypically distinct (Adams et al., 1998; Finnan et al., 2004). P. aeruginosa PAO1 and CF strains were routinely cultured at 37 °C in Luria–Bertani (LB) broth or on LB plates. Antibiotics for selection of the P. aeruginosa mutant strain were used at the following concentrations: 100 µg gentamicin ml–1 and 200 µg tetracycline ml–1. C. albicans was grown in non-filament-inducing medium (YNB) and filament-inducing medium (YNBNP) as described by Hogan et al. (2004). Farnesol (trans, trans-farnesol) was purchased from Fluka and diluted in methanol as required. Purified C12HSL was purchased from Sigma and diluted in acetonitrile as required.
Swarming motility assay.
Swarming was analysed on agar plates containing 0.5 % Eiken agar and 8 g l–1 Eiken nutrient broth (Eiken Chemical Co.) supplemented with 5 g glucose l–1. Bacteria from an overnight culture were inoculated onto the centre of plates using a sterile pipette tip and the plates were incubated overnight at 37 °C. A positive result was recorded as growth over the agar surface away from the inoculation point. To determine the effect of farnesol, swarming media were supplemented with 100 µM farnesol.
Growth and adhesion assays.
P. aeruginosa growth curves were performed in LB medium at 37 °C using a Bioscreen C microtiterplate reader. To determine the effect of farnesol, LB media were supplemented with 30, 100 or 300 µM farnesol. Adhesion on polyvinyl chloride (PVC) was measured after 12 h static incubation using a crystal violet assay essentially as described previously (Burrowes et al., 2006). In experiments to determine the effects of farnesol, 300 µM farnesol was added at each starting inoculation.
Detection of HSLs by TLC.
HSL extraction with dichloromethane and detection by TLC was carried out as described previously (Baysse et al., 2005) and by Shaw et al. (1997) using the reporter strains Chromobacterium violaceum CV026, capable of sensing short-chain HSL molecules (C4HSL and C6HSL) through the production of a violet pigment (Ravn et al., 2001), and Agrobacterium tumefaciens NTI, which carries a lacZ transcriptional fusion under the control of an HSL-inducible promoter for long-chain HSLs (3-oxo-C12HSL) (Shaw et al., 1997).
Supernatant preparation and morphology studies.
P. aeruginosa strains were grown overnight for 16 h in LB liquid media until an approximate OD600 1.2 was reached. Cells were harvested by centrifugation and the supernatant was passed through a Nalgene vacuum filter unit with a 0.2 µM polyethersulfone membrane (Sigma). The sterile supernatant was immediately lyophilized and resuspended at a concentration of x20 in sterile purified water. The sterile, concentrated supernatant was used immediately or stored for short periods of time at –80 °C before use. Analysis of C. albicans morphology in liquid medium was carried out as described by Hogan et al. (2004) with minor adjustments. Briefly, C. albicans was grown as yeast cultures overnight at 30 °C in YNB medium. Yeast cells were diluted in YNBNP filament-inducing medium to OD600 0.05 and incubated at 37 °C. Upon inoculation from YNB to YNBNP, sterile concentrated supernatant from the P. aeruginosa strains was added at a final concentration of x2 from a x20 stock. After 6 h growth, cells were examined microscopically at x400 magnification and the images were digitally captured.
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RESULTS AND DISCUSSION
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Production of HSLs is strain dependent
In a previous study, we surveyed phenotypic characteristics including motility and production of specific virulence factors in a small collection of P. aeruginosa CF isolates (Finnan et al., 2004). No defining virulence traits were identified but, as HSLs have since been shown to exert effects on C. albicans (Hogan et al., 2004), we tested each of the strains for their ability to produce both long- and short-chain HSLs (Fig. 1). HSL production is known to be growth-phase dependent; therefore, we tested each strain at three stages of growth in liquid medium using a well-established bioassay. Although not strictly quantitative, this bioassay facilitates comparisons of relative HSL levels among strains. All strains produced the long-chain molecule 3-oxo-C12HSL (Fig. 1, top panel), with clear variation between strains observed. Only three strains, PAO1, CF144 and CF242, produced significant amounts of the short-chain molecules C4HSL and C6HSL, although trace levels could be detected in all strains. Another minor spot, perhaps C5HSL or 3-oxo-C4HSL, was also visible in these strains (Fig. 1, bottom panel, circled spot). HSL production was growth-phase dependent in all cases, with the highest levels being typically associated with stationary phase cultures. It is clear from this analysis that significant variation in HSL production exists within this small group of clinical isolates, with two strains, CF144 and CF242, resembling the laboratory strain PAO1 and categorized as ‘high producers’ and the five other clinical strains as ‘low producers’ of long-chain HSLs, short-chain HSLs, or both. These data are consistent with how P. aeruginosa is believed to behave in the CF lung. In this condition, the same lineage of P. aeruginosa can persist in the lung for years, even decades, undergoing many evolutionary changes due to selective and adaptive pressures within the host (Nguyen & Singh, 2006; Smith et al., 2006). As an infection progresses from the acute to chronic phase, virulence factors, including production of HSLs, appear to be counter-selected by the host (Nguyen & Singh, 2006; Smith et al., 2006). Interestingly, our set of CF isolates showed a large degree of variation in their production of both long- and short-chain HSLs (Fig. 1) and this is consistent with adaptation of these strains to the host.
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Fig. 1. Production of HSLs is strain dependent. Strains were grown to mid-exponential phase (A600 0.6), late-exponential phase (A600 1.0) and stationary phase (A600 1.6) and HSLs were extracted twice and detected using TLC as described in Methods. For each image, three lanes represent three growth phases (A600 0.6, 1.0, 1.6) in that order, as shown for strain CF139. The top panel shows long-chain HSLs and the bottom panel short-chain HSLs. The arrow and circle indicate an extra uncharacterized HSL.
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Cell-free supernatant from P. aeruginosa can influence C. albicans morphology in an HSL-dependent manner
Pure HSLs and structurally similar molecules can prevent the yeast to hyphal switch in C. albicans but it was not known whether this effect could also occur with complex bacterial secretions from clinical strains. The variation in levels of HSLs is also likely to impact on how different strains of P. aeruginosa interact with both the host and with other micro-organisms such as C. albicans that may be present in the lung. In support of this, we found that supernatants from some strains, when added to actively growing cultures of C. albicans, inhibited the yeast to hyphal switch in an HSL-dependent manner (Fig. 2). Supernatants from the high HSL producer, CF144, and the low HSL producer, CF177, were added to Candida cultures as they were induced to switch from the yeast to the hyphal form. We also included supernatant from the characterized strain P. aeruginosa PAO1, which produces high levels of HSLs (Fig. 1
). We found that strains that produced high amounts of HSLs (PAO1 and CF144) inhibited the yeast to hyphal switch (Fig. 2c, d), whereas the low/non-producing strain (CF177) was unable to influence the transition (Fig. 2a, b). These data suggest that this is an HSL-dependent phenomenon. To rule out the possibility that other molecules in the supernatant may prevent CF177 from mediating the effect, we performed an add-back experiment in which pure C12HSL was added to CF177 supernatant. This supplemented supernatant now inhibited the yeast to hyphal transition (data not shown). Further support for the role for long-chain HSLs came from experiments that showed that a HSL– mutant of PAO1, PAO1
lasRI::GmrhlRI::Tc, also failed to inhibit the morphological switch (data not shown). Similar effects were reported previously with pure HSLs but the mechanism by which this effect is mediated is not known (Hogan & Kolter, 2002; Hogan et al., 2004). The morphological switch in C. albicans involves the activity of two distinct signal transduction systems, a RAS-cAMP-protein kinase A pathway and a mitogen-activated protein kinase pathway, and it is believed that farnesol modulates one or both of these pathways in some way (Biswas et al., 2007; Enjalbert & Whiteway, 2005; Hornby et al., 2001; Liu, 2001; Sato et al., 2004; Whiteway, 2000). As farnesol is also a 12-carbon molecule, it is tempting to speculate that 3-oxo-C12HSL recognizes the same target as the C. albicans auto-signal, but this remains to be determined.
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Fig. 2. Cell-free supernatant from P. aeruginosa influences C. albicans morphology. Supernatants from P. aeruginosa strains were sterilized by filtration and concentrated x20 by lyophilization and resuspension in purified water. Sterile, concentrated supernatants, at a final concentration of x2, were then added to C. albicans yeast cells that were induced to grow in filament form. Supernatants from strains PAO1 and CF144 inhibited the yeast to hyphal switch (c, d), whereas supernatant from strain CF177 (b) did not. (a) C. albicans hyphal control, (b) C. albicans+CF177 supernatant, (c) C. albicans+PAO1 supernatant and (d) C. albicans+CF144 supernatant. Magnification x400.
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Farnesol reduces swarming motility while having no effect on adhesion or growth in P. aeruginosa
We were also interested in the effects of the Candida secreted signal, the 12-carbon molecule farnesol, on the clinical strains of P. aeruginosa. We therefore examined the effects that addition of farnesol had on three P. aeruginosa traits that have been reported as being important for virulence: swarming motility, growth and biofilm formation. As biofilm formation is influenced by HSLs and PQS, and the addition of farnesol reduces PQS levels, we investigated the ability of each strain to adhere to polyvinyl chloride (PVC) plates in the presence and absence of farnesol. Despite the phenotypic differences in the strains (growth rates, HSL production and colony morphology), no significant difference in the ability of the bacteria to adhere to PVC was detected among the strains and addition of 300 µM farnesol had no effect on this adherence (data not shown). We also observed no effect on the growth of P. aeruginosa in the presence of up to 300 µM farnesol (data not shown). Differences between strains and effects of farnesol were observed, however, when swarming motility was assessed (Fig. 3). The high HSL-producing strains (PAO1, CF144 and CF242) were strong swarmers, the low producer CF95 a moderate swarmer, and the low producers CF139, CF198, CF93 and CF177 were unable to swarm (Fig. 3a, arrows mark the edge of the swarming colony). The ability of strains PAO1, CF144, CF242 and CF95 to swarm was almost completely abolished by the addition of 100 µM farnesol. The effect of farnesol may be mediated via rhamnolipids as rhamnolipids are known to be required for swarming and are part-regulated by PQS, which is known to be downregulated by farnesol. The reduction in PQS, pyocyanin production and swarming motility may also have implications for the interaction between P. aeruginosa and the host.
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Fig. 3. Farnesol inhibits swarming in P. aeruginosa. (a) Swarming motility of P. aeruginosa on 0.5 % Eiken agar plates. (b) Media were supplemented with 100 µM farnesol and swarming was almost abolished. Swarm edges are indicated by arrows.
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Importance of interspecies signalling in disease
From the data above and in the literature, it is clear that the in vivo interaction between P. aeruginosa and C. albicans is likely to be complex and that it is possible to predict some consequences of these interactions. During the acute phase of infection when a strain of P. aeruginosa is secreting high levels of HSLs, any Candida cells in that niche will remain in the yeast form. In contrast, during a chronic infection when HSL concentrations are likely to drop, C. albicans may be able to trigger a switch to filamentous growth, possibly leading to formation of a fungal biofilm with the potential for invasive growth. Relatively little is known about the importance of fungal biofilms in disease, but this is an emerging topic of research. The possible cytotoxic effects of some strains of P. aeruginosa are an additional consideration as it has been shown that some strains of P. aeruginosa destroy Candida hyphae (but not yeast cells) (Hogan & Kolter, 2002). In contrast, fungal signals can affect Pseudomonas traits important for gene regulation and motility, and are likely to impact on the ultrastructure of the bacterial biofilm.
In conclusion, it is emerging that the structure of the microbial community in the lung may have significant consequences for the nature of disease in CF patients. The key challenges now are to determine mechanistically precise details of how Pseudomonas and Candida interact, and to devise methods to study this interaction in vivo. The ultimate aim will be to use this knowledge to manipulate, modify, or disrupt microbial ecology in the CF lung to the benefit of the patient. These data also have implications for interactions between bacteria and fungi that may occur in other clinical settings, for example the burn wound.
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ACKNOWLEDGEMENTS
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We thank J. S. Mattick for generous provision of the HSL– mutant, Pat Higgins for excellent technical support, and Lucy Holcombe for critical reading of the manuscript. We also thank F. Boyd and colleagues in UCC for helpful discussions over the course of this research. Work in F. O'Gara's and J. Morrissey's laboratories is supported by grants awarded by the European Union (05/EDIV/FP107; MTKD-CT-2006-042062; O36314), the Higher Education Authority of Ireland (PRTI3), Science Foundation of Ireland (SFI 02/IN.1/B1261; 04/BR/B0597), the Irish Environmental Protection Agency (EPA 2006-PhD-S-21), Enterprise Ireland (SC/02/517) and the Department of Agriculture (06RDC459; 06RDC506; RSF06321; RSF06377). G. McAlester thanks the Society for General Microbiology for funding support.
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