1 Department of Medicine, University of Florida College of Medicine, Box 100277 JHMHC, 1600 SW Archer Rd, Gainesville, FL 32610, USA
2 Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Box 100277 JHMHC, 1600 SW Archer Rd, Gainesville, FL 32610, USA
3 the North Florida/South Georgia VA Medical Center, University of Florida College of Medicine, Box 100277 JHMHC, 1600 SW Archer Rd, Gainesville, FL 32610, USA
Correspondence
Cornelius J. Clancy
clancyn{at}medicine.ufl.edu
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ABSTRACT |
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INTRODUCTION |
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We have adapted an antibody-based screening strategy to identify C. albicans genes of diverse functions that are expressed within humans. In earlier studies, we used sera recovered from HIV-infected patients with active OPC to screen a C. albicans genomic DNA expression library, identifying genes that encode immunogenic antigens (Cheng et al., 2003a; Nguyen et al., 2004
). We demonstrated that NOT5, a previously uncharacterized gene, was necessary for normal hyphal formation and complete virulence during disseminated candidiasis (DC) (Cheng et al., 2003a
, b
). In this study, we characterize a previously unreported in vivo expressed gene identified upon further screening.
C. albicans open reading frame IPF8663 is 1917 bp in length and is the sole homologue of two closely related Saccharomyces cerevisiae genes, IRS4 and TAX4. Since IPF8663 is annotated as encoding a protein similar to scIrs4p by the Agabian lab. at UCSF (http://agabian.ucsf.edu/canoDB/anno.php), we have named the corresponding gene C. albicans IRS4. The putative 639 aa protein encoded by C. albicans IRS4 contains a single identifiable domain (an EH domain) of approximately 71 aa at positions 553624. In other eukaryotes, EH domain-containing proteins regulate diverse cellular processes, including cell wall organization (Santolini et al., 1999). Recently, S. cerevisiae IRS4 was demonstrated to contribute to the negative regulation of the cell integrity pathway, which controls cell wall biosynthesis and repair, as well as chitin distribution (Morales-Johansson et al., 2004
). A C. albicans cell integrity pathway has been inferred to contribute to hyphal formation in vitro and virulence during murine DC (Navarro-Garcia et al., 1995
; Diez-Orejas et al., 1997
; Navarro-Garcia et al., 1998
). For these reasons, we hypothesized that disruption of C. albicans IRS4 would result in dysregulation of hyphal formation and cell wall integrity. If so, we further hypothesized that IRS4 would play a role in candidal virulence.
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METHODS |
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In order to obtain a reconstituted strain of one IRS4 allele, we first constructed a modified pMB7 (pMB7-1), in which one copy of hisG had been eliminated by treatment with XbaI followed by religation. Then, nucleotide positions from +820 to +2817 bp relative to ATG of IRS4 were amplified using the primers N-F1F and Reinsert-R (5'-TTGGACGTCGACTCGCTTTCCTCTATGG-3'), introducing a SalI restriction site (underlined). The amplified product was digested with the appropriate restriction enzymes and cloned into pMB7-1 at the PstI/SalI site. The IRS4-URA3-hisG cassette was released by treatment with SphI/SacI and used to transform the Ura irs4 null mutant. Again, results were confirmed by Southern analysis.
Growth rates in vitro.
The in vitro growth rates were determined in YPD and SD media at 30 and 37 °C in microtitre plates as described in our previous publications (Cheng et al., 2003a, b
). We used a BioMate 3 spectrophotometer (Thermo Spectronic) and disposable polystyrene cuvettes (USA Scientific).
Sensitivities to cell wall agents
Each of the following experiments was performed in triplicate.
SDS and calcofluor white.
Cultures from overnight grown organisms were subcultured in YPD liquid medium with 1 % glucose until the exponential phase, and diluted to an OD599 of 0·1. Four microlitres of undiluted and serial 10-fold dilutions of each culture were spotted onto YPD plates containing calcofluor white (40 µg ml1) or SDS (0·02 %). The plates were incubated at 30 °C for 72 h.
Zymolyase.
Exponentially grown C. albicans cells at OD599 0·8 were incubated with 100 µg zymolyase 20T (Sigma) ml1 in 10 ml Tris/HCl, pH 7·5. An aliquot was removed at timed intervals and the OD599 was measured. We used a BioMate 3 spectrophotometer (Thermo Spectronic) and disposable polystyrene cuvettes (USA Scientific). The OD599 was plotted against time of incubation.
Caspofungin.
Sensitivity to caspofungin (Merck) was measured in a 48-well microtitre plate. Organisms grown overnight in YPD at 30 °C were diluted to OD600 of 0·1 in YPD. Caspofungin was added at concentrations ranging from 0·075 to 20 µg ml1 and transferred to the microtitre plate (800 µl per well). The plate was incubated at 30 °C with shaking at 250 r.p.m. and OD620 was measured every hour. We used a Beckman Coulter AD340 spectrometer and 48-well tissue culture cluster plates.
Chitin staining and microscopy.
Cells were grown at 30 or 37 °C, either embedded in YPD-reverse agar for 3 days or in liquid YPD to exponential phase. For chitin staining, cells were resuspended in a 0·1 mg ml1 calcofluor white solution and incubated at room temperature for 25 min, then washed three times in cold PBS. Assays were performed on three separate occasions.
Adherence assay.
We evaluated the adherence ability of C. albicans strains using the colonic adenocarcinoma cell line HT-29, cervical carcinoma cell line HeLa and pharyngeal squamous cell carcinoma cell line FaDu (purchased from ATCC). HT-29, HeLa and FaDu cells were cultivated in ATCC complete growth medium (minimal essential medium; Eagle) with 2 mM L-glutamine and Earle's balanced salt solution adjusted to contain 1·5 g sodium bicarbonate l1, 0·1 mM non-essential amino acids and 1·0 mM sodium pyruvate supplemented with 10 % fetal bovine serum. Cells (5x105) were seeded in each well of six-well plastic dishes. The cells were grown to confluencey at 37 °C and 5 % CO2. Monolayers were washed twice with 2 ml Dulbecco's PBS (DPBS), incubated with 150 C. albicans cells in 1 ml DPBS for 45 min at 37 °C and 5 % CO2. Monolayers were washed three times with warm DPBS to remove non-adhering cells, then covered with 2 ml YPD agar. Yeast colonies appearing after 48 h of growth at 30 °C were counted. Each experiment was performed in triplicate, on three separate occasions. Adherence for each strain was determined as the percentage of fungal cells attached to monolayers. Results for each strain were expressed as the percentage of the adherence noted for control strain CAI-12.
Murine OPC.
As described by Kamai et al. (2001), 7-week-old male ICR mice (Harlan-Sprague) were immunosuppressed with 4 mg cortisone acetate (Sigma Aldrich) in saline with 0·05 % Tween 80 administered subcutaneously on the day before, 1 and 3 days after inoculation. Mice received tetracycline hydrochloride (Fisher) in their drinking water (0·5 mg ml1) starting the day before inoculation. For inoculation, mice were anaesthetized by intraperitoneal injection with 3 mg pentobarbital sodium solution (Abbott Laboratories), and cotton wool balls (3 mm diameter) containing 108 c.f.u. of C. albicans were placed sublingually in the oral cavity for 2 h. To determine tissue fungal burden, the mice were sacrificed at day 7 post-infection and the mandibular soft tissue, including the tongue, was dissected free of the teeth and bone. The excised tissue was homogenized in saline, after which serial dilutions were plated onto SDA plates containing 60 µg amikacin ml1 for colony counting. For the histopathological study, tissues were fixed with formalin and embedded in paraffin, after which thin sections were prepared and stained with gomori methenamine silver (GMS) stain.
Murine DC.
Seven-week-old male ICR mice (Harlan-Sprague) were inoculated by intravenous injection of the lateral tail vein with 1x106 c.f.u. of C. albicans. Mice were monitored until they were moribund, at which point they were sacrificed, or for 30 days. Survival curves were calculated according to the KaplanMeier method using the PRISM program (GraphPad Software) and compared using the Newman Keuls analysis; a P-value of <0·05 was considered significant. To determine C. albicans tissue burdens, mice were infected in the preceding manner. Mice in each group were sacrificed 20 h or 4 days after intravenous inoculation and their kidneys were aseptically removed. The kidneys were weighed, homogenized in 2 ml sterile PBS, and serial dilutions were plated onto SDA plates containing pipericillin (60 µg ml1) and amikacin (60 µg ml1). The plates were incubated at 30 °C for 48 h, after which the number of c.f.u. was determined. Values were expressed as log10 c.f.u. g1 kidney. The difference in kidney burden between strains was determined by Wilcoxon rank sum test; a P-value <0·05 was determined statistically significant. In a separate experiment, murine kidneys collected 20 h and 4 days after intravenous inoculation were fixed with formalin and embedded in paraffin, after which thin sections were prepared and stained with GMS stain (Churukian & Schenk, 1977; Carson, 1996
).
RT-PCR
OPC.
Pseudomembranes were recovered from the oral cavities of HIV-infected patients, total RNA extracted and treated with RNase-free DNase, and RT-PCR performed as previously described (Cheng et al., 2003a). We used the following PCR protocol: 1 min at 94 °C, 1 min at 50 °C and 1 min at 72 °C, preceded by denaturation for 5 min at 94 °C and followed by a final extension cycle for 10 min at 72 °C. Reactions of 35 and 40 amplification cycles were performed using primers IRS-1300F (5'-CTACCAGCAATCTTCCACTGAGAT-3') and IRS-CRev (5'-CAGTGACATTAACACCAATTCCACCTAAACT-3'), which should result in an amplification product of 633 nt. We included the constitutively expressed housekeeping gene EFB1 (elongation factor 1
) as an internal mRNA control using primers EFB1-For (5'-AATTCTTGGCTGACAAATCATACAT-3') and EFB1-Rev (5'-CTAAAGCATCTTCAACAACCAAGTT-3') (Schaller et al., 1998
; Naglik et al., 1999
; Ripeau et al., 2002
). In the event of genomic DNA contamination, an 891 bp fragment containing an intron would be amplified. PCR products were migrated and visualized in a 1·5 % agarose gel.
Murine kidneys.
Mice were infected intravenously with strain CAI-12 (106 c.f.u.) as described for the DC model, and sacrificed at 20 h (three mice) or 4 days (two mice) post-infection. Kidneys were harvested aseptically and homogenized, and total RNA extracted using RiboPure-Yeast kit (Ambion), which includes RNase-free DNase. First-strand cDNA synthesis using the primers listed in the preceding paragraph was performed with SuperScript III reverse transcriptase (Invitrogen) following the manual. PCR amplification of the targeted genes was performed for 30 cycles using Taq DNA polymerase (Eppendorf) and the manufacturer's instructions, using the amplification protocol described above. As in the OPC RT-PCR experiments, EFB1 was included as an internal mRNA control, and PCR products were migrated and visualized in a 1·5 % agarose gel.
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RESULTS |
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In YPD supplemented with 5 % FCS and RPMI-1640 liquid media, we noted no significant differences in the formation of true hyphae by CAI-12 and the heterozygous mutant, null mutant and reinsertion strains after 4 h of incubation at 37 °C. Furthermore, all strains reverted to yeast morphology after 1012 h of incubation. Clear phenotypic differences, however, were noted when strains were grown in contact with solid media, both on the surfaces of agar and under microaerophilic conditions of embedded growth. During surface growth, CAI-12 exhibited extensive hyphae after 24 days on YPD+5 % FCS, Spider, modified Lee's and M-199 agar at 37 °C. The null mutant strain, on the other hand, either did not form hyphae or exhibited markedly truncated hyphae (Fig. 2). The heterozygous mutant and reinsertion strains exhibited intermediate phenotypes under these conditions. Similar findings for the strains were noted after 3 days of growth under embedded conditions within YPD, YPD+5 % FCS, YPD-reverse, Spider and M-199 agar at 30 and 37 °C (Fig. 2
). Of note, differences between strains in hyphal formation on the surface of and within solid agar continued to be maintained until plates were disposed of after 7 days, indicating that the earlier observations reflected differences in morphogenesis rather than growth rates.
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Disruption of C. albicans IRS4 attenuates hyphal formation and virulence during murine DC but not OPC
To study the contribution of IRS4 to virulence in vivo, we first used a murine model of OPC. We applied cotton balls saturated with 108 c.f.u. to the oropharynges of 7-week-old ICR mice immunocompromised with subcutaneous cortisol acetate (strain CAI-12, 12 mice; irs4 null mutant, 13 mice) (Kamai et al., 2001). After 7 days, the mice were sacrificed, and the oropharynges and oesophagi removed for c.f.u. enumeration. We demonstrated that there were no significant differences in the candidal tissue burdens for mice infected with CAI-12 or the null mutant strain (log10 c.f.u. g1 tissue mean±SD, n=6: 5·81±0·60 vs 5·91±0·64 %, respectively; P=not significant). Furthermore, histopathological studies of resected tongues and oesophagi revealed no differences in candidal morphologies, with clusters of yeast and hyphae present for both strains (Fig. 5
). Similarly, we found no differences in the degree of tissue invasion, numbers of infecting organisms or host tissue damage. We obtained similar results for both tissue burdens and histopathology upon repeating the murine experiments.
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Finally, we recovered kidneys from non-moribund mice infected with either strain after 20 h and 4 days for histopathological evaluation (three mice per group). Our results were consistent with the tissue burden data. At 20 h, all kidneys exhibited small clusters of C. albicans growing as yeasts, germ tubes and short hyphae, which were widely separated (Fig. 7). Normal tissue histopathology was maintained. Furthermore, kidneys from mice infected with CAI-12 and the null mutant could not be distinguished based on the number of infected clusters, number of cells within clusters or candidal morphology. By 4 days, however, differences were striking (Fig. 7
). Kidneys infected with CAI-12 showed multiple clusters of C. albicans, consisting of dense mats of hyphae interspersed with yeasts. These were surrounded by a brisk inflammatory response that disrupted normal tissue architecture. Kidneys infected with the null mutant, on the other hand, showed widely dispersed pockets composed of yeast cells that stained poorly with GMS. Germ tubes and truncated hyphae were extremely rare, isolated and very poorly stained. The inflammatory response and damage to host cells within the kidneys were minimal. Of note, the length of hyphal elements and the number of germ tubes and hyphae for the null mutant strain were markedly less at 4 days than at 20 h.
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DISCUSSION |
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Our findings support a paradigm in which the pathogenesis of diverse candidal diseases depends upon both generalized virulence factors and those that function in specific environments dictated by tissue site, immune function and other host factors. The former group, for example, would include the transglutaminase substrate encoded by HWP1 and the adhesin protein encoded by ALS1, which contribute to virulence at mucosal and deep organ sites (Staab et al., 1999; Sundstrom et al., 2002
; Kamai et al., 2002
; Fu et al., 2002
). It is notable that IRS4 appears to differ from a number of other genes in the latter group, for which temporalspatial contributions to virulence are determined by expression patterns. For example, PHR1 and PHR2 encode cell surface proteins and are expressed at neutralalkaline and acidic pH, respectively. Consistent with this expression, phr1 null mutant strains are avirulent during murine DC and phr2 null mutants are avirulent during vaginal candidiasis in rats (Ghannoum et al., 1995
; Muhlschlegel & Fonzi, 1997
; De Bernardis et al., 1998
). Individual members of the SAP family of secreted aspartyl proteases and genes encoding phospholipases are also regulated at the transcriptional level in vivo, with expression patterns varying depending on tissue sites and stages of infection (Staib et al., 2000a
; Fradin et al., 2003
; Naglik et al., 2003
). Presumably, Irs4p is present and performs functions in the oral cavity and in the kidneys in the first day of DC, but these do not contribute to hyphal formation, tissue invasion or damage. Within the unique in vivo milieu of the kidneys after 4 days of DC, however, the protein appears to play a role in these processes. In this regard, our data indicate that the host environment is likely to be the principle determinant of whether Irs4p contributes to virulence.
The mechanisms by which IRS4 contributes to virulence in the appropriate settings are not clear from this study. It is possible that the irs4 null mutant strain's attenuated virulence correlates, at least in part, with its impaired hyphal formation. The precise relationship between yeast-hyphal morphogenesis and virulence remains controversial (Gow et al., 2002; Sudbery et al., 2004
). As with other genes that regulate hyphal growth, it is uncertain whether the attenuated virulence of the irs4 null mutant strain in the kidneys after 4 days stems from defects in morphogenesis per se, repression of hyphal-associated gene expression, or pleiotropic effects of gene disruption. Nevertheless, it is interesting that the impaired hyphal formation by the null mutant within the kidneys resembles the phenotype observed during embedded growth within agar rather than in liquid media. Indeed, it has been hypothesized that embedded conditions in vitro resemble those encountered by C. albicans in vivo (Brown et al., 1999
; Ernst, 2000
; Doedt et al., 2004
), where the organism grows under reduced oxygen tension, in contact with surrounding extracellular matrix. Our data suggest that the phenotypes exhibited by the null mutant during embedded growth might be more relevant to the pathogenesis of invasive candidiasis than those seen in liquid media. We will investigate this possibility in future studies of gene expression patterns under embedded conditions.
The difficulty in establishing a definite link between IRS4's roles in morphogenesis and virulence is highlighted by our demonstration that the gene is also required for normal cell wall structure and integrity. In fact, cell wall remodelling is integral to hyphal formation, so it is not surprising that IRS4 might contribute to both processes. At the same time, cell wall integrity is likely to play an important role in growth and survival of C. albicans in vivo, independent of cell morphology. For this reason, it might reasonably be hypothesized that IRS4's effects on the cell wall are as important to its virulence function as are the effects on morphogenesis. For example, alterations in the cell wall might contribute to the diminished adherence of the irs4 null mutant to oesophageal, vaginal and colonic adenocarcinoma cells in vitro. Alternatively, mutant cells might form hyphae whose altered cell walls lack sufficient strength to penetrate or cause damage deep within the tissue. Indeed, this could also explain the differences in virulence during OPC and DC, since OPC lesions are normally superficial, as well as the discrepancy between hyphal formation in liquid media and under embedded conditions. Interestingly, our observations in many ways resemble those of investigators studying C. albicans septin mutants, which are only mildly defective for hyphal growth in liquid media but significantly impaired in invasive growth into agar and murine kidneys (Warenda et al., 2003). These investigators have suggested that the ability to undergo invasive growth entails the coordination of hyphal formation and multiple other processes, and is likely to be more important to pathogenesis than hyphal growth alone.
The contribution of IRS4 to virulence in conducive settings is likely to be multifactorial and indirectly mediated. S. cerevisiae IRS4 has recently been shown to encode a component of a complex that negatively regulates the cell integrity mitogen-activated protein (MAP) kinase pathway (Morales-Johansson et al., 2004). This negative regulation is effected through activation of a phosphatase that downregulates levels of the second messenger phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]. PI(4,5)P2, in turn, is an important mediator of signal transduction that is involved in diverse cellular processes. As a follow-up to the present study, we will investigate whether C. albicans IRS4 similarly regulates phosphoinositide levels.
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ACKNOWLEDGEMENTS |
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Received 1 March 2005;
revised 3 June 2005;
accepted 6 June 2005.
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