1 Center for Medical Mycology, Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH 44106-5028, USA
2 Division of Infectious Diseases, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH 44106-5028, USA
Correspondence
Mahmoud A. Ghannoum
mag3{at}po.cwru.edu
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ABSTRACT |
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Abbreviations: SB, Sabouraud dextrose broth; YPD, yeast extract/peptone/dextrose; YNB, yeast nitrogen base
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INTRODUCTION |
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Several virulence factors potentially representing molecular targets for new anti-Candida drugs have been proposed for Candida albicans infections, which include extracellular phospholipases, secretory aspartic proteinases (Saps), adhesion proteins and proteins involved in germ-tube formation (Cutler, 1991; Fallon et al., 1997
; De Bernardis et al., 1999
; Ghannoum, 2000
; Calderone et al., 2000
; Mukherjee & Ghannoum, 2001
; Asleson et al., 2001
; Bahn & Sundstrom, 2001
). Secretion and activity of candidal Saps have been shown to be influenced by environmental factors including pH, growth media, morphology, nutrients and substrate concentration (Tsuboi et al., 1989
; Hube et al., 1994
; Wu & Samaranayake, 1999
; De Bernardis et al., 1999
; Naglik et al., 1999
).
We have previously shown that C. albicans strains exhibiting increased phospholipase B activity are associated with higher virulence in murine models of disseminated candidiasis (Ibrahim et al., 1995). We have also cloned and disrupted PLB1, the gene encoding C. albicans phospholipase B, and shown that the
plb1 null-mutant has significantly attenuated virulence compared to the isogenic parental strain when tested in a murine model of haematogenously disseminated candidiasis (Leidich et al., 1998
). The reintroduction of a functional PLB1 gene into the
plb1 null-mutant restored virulence to levels similar to those observed for the parental strain, in both haematogenously disseminated and oralintragastric infant mouse models of candidiasis (Mukherjee et al., 2001
). These studies showed that candidal Plb1p is an important candidal virulence factor.
To develop a detailed picture of the multifactorial phenomenon of Candida pathogenesis, in this study we analysed the factors affecting the expression of the C. albicans PLB1 gene and the production of the corresponding protein. Our results showed that PLB1 mRNA was detected in C. albicans grown in rich media only at 30 °C, while the corresponding protein was present at 30 and 37 °C. Growth in chemically defined media failed to induce the expression of PLB1 mRNA at either temperature. However, different environmental conditions induced this gene in C. albicans grown in yeast nitrogen base (YNB). Supplementation of YNB with glucose, serum and a mixture of phospholipids was essential to induce PLB1 expression in C. albicans at 37 °C. Additionally, acidic pH induced higher levels of PLB1 mRNA expression compared to neutral pH, while the morphological form of C. albicans did not have any influence on the expression of this gene. These results indicate that PLB1 is differentially expressed in C. albicans under varying environmental and physiologically relevant conditions.
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METHODS |
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Reagents.
Restriction endonucleases and Taq DNA polymerase (Klenow fragment) were purchased from Roche Molecular Biochemicals. Oligonucleotide DNA primers were custom-ordered from BioSynthesis (Lewisville, TX, USA). Glass beads (25600 µm diameter), used for total chromosomal DNA extraction, and all other chemicals were purchased from Sigma.
Growth conditions.
C. albicans cells were grown in rich or defined media under different nutrient and environmental conditions. The rich media used for cell growth were Sabouraud dextrose broth (SB; 1 % yeast extract, 1 % polypeptone, 4 % glucose) and YPD (1 % yeast extract, 2 % peptone, 2 % dextrose). The chemically defined media used were Lee's medium (Lee et al., 1975), RPMI-1640 (Cellgro MediaTech) and YNB (Difco Laboratories). Media were supplemented as required with glucose, mannose, galactose, serum, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI) or a phospholipid mixture (PE : PC : PI, 1 : 2 : 1). C. albicans cells (6x106 cells ml-1) were grown with shaking (200 r.p.m.) up to mid-exponential phase (7 h), at which time the cells were harvested for RNA extraction. C. albicans cell morphology under each growth condition was monitored microscopically at the end of the incubation period.
PCR amplification of the PLB1-specific region.
Total chromosomal DNA isolated from C. albicans cells was subjected to PCR amplification with oligonucleotide primers targeting a 751 bp region representing the 5' half of the PLB1 gene, as described previously (Mukherjee et al., 2001). The oligonucleotide primers used were 5'-ATGATTTTGCATCATTTG-3' (forward) and 5'-AGTATCTGGAGCTCTACC-3' (reverse). PCR amplification reaction mixtures consisted of 100 µl volumes containing 10 mM Tris/HCl (pH 8·4), 50 mM KCl, 1·5 mM MgCl2, dNTPs (1·0 mM each dNTP), oligonucleotide primers (1 µM each), template DNA (10100 ng) and 2 U Taq DNA polymerase. The PCR conditions used were an initial denaturation (95 °C, 2 min) followed by 33 cycles of denaturation (94 °C, 1 min), annealing (50 °C, 1 min) and extension (72 °C, 3 min), with a final extension (72 °C, 10 min). Reaction mixtures were analysed for the presence of the 751 bp DNA fragment on 1·2 % agarose gels stained with ethidium bromide.
Northern blot analyses.
Total RNA was extracted from C. albicans cells as described by Collart & Oliviero (1993). Briefly, 2 mg harvested cells were resuspended in 1·0 ml buffer and lysed by vortexing with 0·5 mm diameter glass beads; the lysate was recovered by centrifugation. Total RNA was isolated from the lysate using the RNeasy Kit (Qiagen) according to manufacturer's instructions. Ten micrograms of the isolated total RNA were electrophoresed through 1 % agarose/formaldehyde gels with circulating buffer and then transferred to nylon membranes (Boehringer Manheim). The transferred RNA was cross-linked to the membrane using a UV Stratalinker 228 (Stratagene), pre-hybridized for 1 h and then hybridized overnight with the PLB1-specific PCR-amplified 751 bp probe at 42 °C. After stringency washes with 0·2xSSC, the hybridized RNA signals were detected by autoradiography.
Western blot analyses.
Immunodetection of the Plb1p protein secreted by C. albicans cells into the media was performed by Western blot analyses as described previously (Leidich et al., 1998; Mukherjee et al., 2001
). Briefly, extracellular proteins secreted by the C. albicans cells were concentrated using a Millipore Ultrafree centrifugation column (molecular mass cut-off, 10 000 Da). Ten micrograms of total protein were separated on a 412 % polyacrylamide gel by SDS-PAGE and transferred to a nitrocellulose membrane using a SemiDry TransBlot apparatus (Bio-Rad) according to manufacturer's instructions. The transferred blots were incubated with 5 % non-fat dry milk and probed with an anti-Plb1p antibody. Plb1p on the nitrocellulose membrane was detected using goat-anti-rabbit IgG as the secondary antibody (1 : 1000 dilution) and a chemiluminescence assay (ECL Amersham) according to the manufacturer's instructions.
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RESULTS |
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Plb1p is secreted at 37 °C in rich media
Expression of mRNA is not always correlated at the protein level in yeasts (Gygi et al., 1999). Since our results indicated that PLB1 mRNA was not detected at 37 °C in C. albicans cells grown in rich medium under different conditions, we decided to investigate whether this observation was supported at the protein level also. Western blot analyses of proteins secreted by C. albicans under different conditions revealed that Plb1 was produced at 37 °C in C. albicans grown in rich media (SB and YPD) supplemented with 0·5, 2 or 4 % glucose (Fig. 3
). Thus, although PLB1 mRNA was not detected at 37 °C, the corresponding protein was present at this temperature, suggesting the possibility that the mRNA may be unstable or expressed at an earlier stage of growth.
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DISCUSSION |
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Expression of yeast genes at the mRNA level is not always correlated with the amount of the translated functional protein. A study to determine the relationship between mRNA and protein levels for selected genes expressed in the yeast Saccharomyces cerevisiae reported that expression levels of proteins coded by mRNA with comparable abundance varied by up to 30-fold, while mRNA levels coding for proteins with comparable expression levels varied by as much as 20-fold (Gygi et al., 1999). Thus, the inability to detect PLB1 mRNA at 37 °C does not necessarily indicate the absence of the gene product. Indeed, Western blot analysis confirmed this contention since the Plb1p protein was present at 37 °C in rich medium conditions in which the corresponding mRNA was not detected. Previously, using Western blot analyses, we reported the extracellular secretion of Plb1p by C. albicans cells grown at 30 °C (Leidich et al., 1998
; Mukherjee et al., 2001
). Therefore, PLB1 is expressed at 30 and 37 °C, although its mRNA may not be clearly detectable at the latter temperature. Recently, it was reported that phospholipase activities in another opportunistic fungal pathogen, Cryptococcus neoformans, vary with strain type and growth temperature (Wright et al., 2002
). Thus, the temperature-responsive expression of virulence factors may be a common mode of regulation in yeasts.
The role of cellular morphology in the regulation of virulence varies with each determinant. A recent study has shown that phase-specific expression of the transcription regulator Tup1p plays an important role in the whiteopaque' phenotypic switching that affects colony morphology, cellular phenotype and expression of a number of phase-specific genes and virulence traits (Zhao et al., 2002). White & Agabian (1995)
have shown that specific Sap isoenzymes are produced by different cell types and that the level of SAP mRNA detected during hypha formation is low and transient, suggesting that both the yeasthyphal transition and phenotypic switching influence Sap production. In another study, Hoover et al. (1998)
reported that the level of PLB1 mRNA is higher in blastospores and pseudohyphae compared to germ-tube-forming cells of C. albicans grown to early-exponential phase (3 h) in YPD, suggesting that the expression of this gene is morphology-dependent. Our studies revealed that this morphology-dependent expression of PLB1 was not observed after the mid-exponential growth stage (7 h) of C. albicans, since varying morphologies of the fungal cells grown to 7 h did not affect PLB1 expression. The growth-phase-dependent regulation of PLB1 expression by morphogenic transition is notable since Plb1p is believed to aid in host tissue invasion by damaging host cellular membranes (Ghannoum, 2000
; Mukherjee et al., 2001
), and early expression of this virulence gene under physiological conditions is likely to be an advantage for C. albicans during infection.
In this study, we have shown that PLB1 expression is dependent on the pH of the growth medium. The role of pH in regulating Candida virulence factors such as germination and morphology-switching has been well documented (Brown et al., 1999; Ernst, 2000
). The effect of pH on the regulation of C. albicans virulence genes has been shown for PHR1, expressed at pH 5·5 or higher, and PHR2, which is expressed at pH values less than 5·5 (Ghannoum et al., 1995
; Muhlschlegel & Fonzi, 1997
; De Bernardis et al., 1998
). De Bernardis et al. (1998)
showed that a
phr1 null-mutant was avirulent in a mouse model of systemic candidiasis but uncompromised in its ability to cause vaginal infection in rats, while a
phr2 null-mutant was avirulent in a vaginal-infection model but virulent in a systemic-infection model. Since the systemic pH is near neutrality and the vaginal pH is around 4·5, the studies by De Bernardis et al. (1998)
suggested that the pH of the infection site regulates the expression of genes essential to survival within that niche.
Fungal cells grown in chemically defined medium expressed PLB1 mRNA at 30 °C but not at 37 °C. However, we have shown previously that Plb1p is present in the sera of mice infected with C. albicans (Mukherjee et al., 2001) and in sera from patients with systemic candidiasis (M. Hossain, unpublished data). Moreover, Ripeau et al. (2002)
reported that PLB1 was expressed constitutively in a murine model of oropharyngeal candidiasis. Therefore, we reasoned that the presence of in vivo physiological factors such as serum and host phospholipids may be essential to induce PLB1 expression in C. albicans at 37 °C (Nelson, 1967
; Engen & Clark, 1990
; Kurumi et al., 1991
). Indeed, supplementing YNB (+0·5 % glucose) with serum and a mixture of phospholipids commonly found in mammalian cells (Nelson, 1967
; Engen & Clark, 1990
) induced PLB1 expression at 37 °C. Interestingly, supplementing the medium with either serum or the phospholipid mixture separately failed to induce PLB1 expression at the physiologically relevant temperature of 37 °C. Thus, in vitro expression of PLB1 mRNA at 37 °C can only be achieved under physiological conditions. This situation differs from that of SAP regulation, where it was reported that pH and hypha production alone are sufficient for SAP expression and that components of serum are not necessary (White & Agabian, 1995
).
In conclusion, our results suggest that the expression of PLB1 by C. albicans is a complex and closely coordinated phenomenon involving multiple environmental and physiological parameters. Furthermore, expression of this gene at the physiological temperature (37 °C) necessitates the presence of additional host-associated factors including serum and phospholipids. Understanding the mechanism by which environmental stimuli exert their effect on the regulation of virulence factors is likely to aid in the development of novel antifungal agents with increased efficacy in the management of candidiasis.
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ACKNOWLEDGEMENTS |
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Received 24 June 2002;
revised 11 September 2002;
accepted 20 September 2002.
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