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1 Department of Medicine, University of Florida College of Medicine, Gainesville, FL, USA
2 Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA
3 North Florida/South Georgia Veterans Health System, Gainesville, FL, USA
4 Department of Pharmaceutics, University of Florida College of Pharmacy, Gainesville, FL, USA
5 Department of Pathology, University of Colorado Health Science Center, Aurora, CO, USA
Correspondence
Cornelius J. Clancy
clancyn{at}dom.pitt.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: University of Pittsburgh School of Medicine, 867 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA.
Two supplementary figures, showing expression of the MEL1 reporter gene and generation of the inp51 null mutant, are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Rather, the pathogenesis of candidiasis requires the coordination of multiple processes in a manner that optimizes proliferation, invasion and tissue damage within a given in vivo environment (Mahan et al., 2000; Staib et al., 2000). Several properties of C. albicans that contribute to virulence have been characterized, including niche-specific regulation of central metabolic pathways, adhesion to host cells, secretion of hydrolytic enzymes, iron sequestration, phenotypic switching and morphogenesis (i.e. reversible transitioning between single-cell blastospores and filamentous pseudohyphae or hyphae) (Barelle et al., 2006; Calderone & Fonzi, 2001; Cheng et al., 2007; Fradin et al., 2003; Lorenz et al., 2004; Rubin-Bejerano et al., 2003). Morphogenesis, in particular, has been extensively studied as a determinant of virulence. The disease-causing capacity of mutant C. albicans strains that are unable to switch between yeast and hyphal morphologies is generally attenuated (Calderone & Fonzi, 2001; Kumamoto & Vinces, 2005). The identification of proteins and protein complexes that might be responsible for coordinating the multiple cellular processes that contribute to efficient morphogenesis has become an active area of research (Brand et al., 2007; Hausauer et al., 2005; Li et al., 2005; Martin & Konopka, 2004; Oberholzer et al., 2006; Walther & Wendland, 2004; Zheng et al., 2003).
In previous reports, we identified C. albicans Irs4p as a protein that is reactive with antibodies in the sera of patients with candidiasis (Badrane et al., 2005; Nguyen et al., 2004). We showed that disruption of IRS4 results in abnormalities of cell wall integrity and chitin distribution, impaired hyphal formation during contact with solid agar and within murine kidneys, and attenuated virulence during disseminated candidiasis. Irs4p consists of 638 amino acids and contains a predicted epidermal growth factor substrate 15 homology (EH) domain. EH domains are highly conserved eukaryotic protein-binding regions that target the amino acid motif Asn-Pro-Phe (NPF) and form the framework of the EH network, an extensive protein interaction network that coordinates pathways regulating cell wall biogenesis and other cellular processes (de Beer et al., 1998; Confalonieri & Di Fiore, 2002; Salcini et al., 1997; Santolini et al., 1999; Tang et al., 2000). C. albicans Irs4p is the sole homologue of the duplicated Saccharomyces cerevisiae proteins Irs4p and Tax4p, which were recently shown to bind and activate Inp51p, a phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] 5-phosphatase (Morales-Johansson et al., 2004). Levels of PI(4,5)P2 are elevated in both S. cerevisiae irs4/tax4 and inp51 null mutants. These strains, however, do not exhibit readily apparent phenotypes unless they are constructed in the presence of mutations to other phosphatases or disturbances in the cell integrity pathway (Böttcher et al., 2006; Morales-Johansson et al., 2004; Singer-Kruger et al., 1998; Stefan et al., 2002, 2005; Stolz et al., 1998a, b).
Like S. cerevisiae INP51, C. albicans INP51 encodes a synaptojanin-like protein with a C-terminal 5-phosphatase domain and an N-terminal SacI-like domain. C. albicans Inp51p also possesses an NPF motif, suggesting that it is likely to be targeted by the EH domain of Irs4p. In the present study, we tested the hypothesis that C. albicans Inp51p and Irs4p interact and regulate levels of PI(4,5)P2. In addition, we investigated the effects of INP51 disruption on cell wall integrity, chitin distribution, hyphal formation and virulence during murine disseminated candidiasis. Finally, we determined whether Inp51p and Irs4p regulate activation of the cell integrity pathway.
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METHODS |
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(Badrane et al., 2005; Fonzi & Irwin, 1993; Gillum et al., 1984). The inp51 null mutant strains were created using the SAT1 flipper method (details below), and C. albicans SC5314 served as reference wild-type strain. The irs4 null mutant had been created previously using the ura-blaster method (Badrane et al., 2005), and C. albicans CAI-12 was used as reference wild-type strain. All strains were routinely grown in yeast peptone dextrose (YPD) liquid medium (1 % yeast extract, 1 % bactopeptone, 2 % 
[D]-glucose), or on YPD agar or Sabouraud dextrose agar (SDA) at 30 °C. To induce hyphal formation in liquid media, C. albicans strains grown overnight on YPD were subcultured into liquid YPD supplemented with 5 or 10 % fetal calf serum (FCS) or liquid RPMI-1640 (Sigma-Aldrich) at 37 °C. To induce hyphal formation on solid media, overnight-grown C. albicans were spotted on Spider medium, Medium 199 (M-199) (Gibco–BRL, adjusted to pH 7.5), modified Lee's, and YPD medium supplemented with 5 % FCS and grown at 37 °C. To evaluate growth under embedded conditions, 
100 C. albicans cells subcultured at early exponential phase were mixed into 20 ml molten YPD, YPD+5 % FCS, YPD-reverse [BASF pluronic polyol F-127, kindly provided by the Fungal genetics Stock Center (FGSC); www.fgsc.net], Spider or M-199 agar, and incubated for 3 days at 30 or 37 °C. Reverse agar is a lock polymer of polyoxypropylene and polyoxyethylene that can be used as a replacement for conventional agar in solid media. For targeted homologous recombination in C. albicans, we used either transformation by electroporation (Reuss et al., 2004) or the lithium/caesium acetate protocol provided with the Alkali–Cation Yeast kit (QBiogene).
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Gene disruption.
The SAT1 flipper tool was used for INP51 gene disruption (Reuss et al., 2004). Briefly, fragments at the 5' (F1) and 3' (F2) ends of the targeted gene were amplified by PCR using primers that introduced appropriate restriction sites. These fragments were subcloned into pSFS-2A between KpnI and XhoI, and SacII and SacI sites, respectively. In parallel, the full gene with 1 kb of downstream non-coding sequence was amplified and used as F1 for gene reinsertion. The resulting plasmid was extracted and linearized with KpnI and SacI, and introduced into competent C. albicans cells by electroporation. Transformants were grown on selective medium plates containing 200 µg streptothricin ml–1 (Alexis Biochemicals) for 2–3 days, and then screened by PCR. Positive transformants were grown overnight in maltose-based medium to induce excision of the plasmid cassette, plated on selective medium plates for 3–4 days, and screened for positive excisions to enable the strain for another round of targeted recombination. All positive events of homologous recombination and excision were confirmed by Southern blotting.
Myo-[2-3H]inositol labelling, extraction of inositol lipids, and HPLC of deacylated lipids.
For phosphoinositide analysis, we followed a procedure reported elsewhere (Dove et al., 1997). For labelling of inositols, C. albicans mid-exponential cultures were diluted to 5x104 cells ml–1 in inositol-free SD medium supplemented with 10 µCi (370 kBq) myo-[2-3H]inositol ml–1 (American Radiolabelled Chemicals) and grown to 2–3x106 cells ml–1. Cells were harvested after addition of methanol to 50 %. Lipids were extracted by vortexing in the presence of glass beads and 100 % methanol, followed by twice adding a mixture of chloroform, methanol and HCl-tetrabutylammonium hydrogen sulfate to recover the lower phases, and drying in a speed vacuum. Dried lipids were deacylated by vigorous vortexing with methylamine reagent, incubated at 53 °C for 30 min then dried again. Samples were resuspended in H2O, extracted twice with butanol/petroleum-ether/ethyl formate, and dried and suspended in 0.1 mM EDTA by vigorous vortexing. Samples were processed by HPLC, either immediately or after a few days storage at –70 °C. The sample analysis was performed using an HPLC system consisting of a quaternary pump (Series 200, Perkin Elmer) and a manual injector with 5 ml stainless steel loop (Rheodyne). Chromatographic separation was achieved on a Partisphere SAX column (5 µm, 4.6 mmx125 mm, Whatman) using a gradient of distilled water (solvent A) and 1.25 M NaH2PO4 (solvent B) at a flow rate of 1 ml min–1. The buffer was filtered through a 0.22 µm filter (Millipore) and de-gassed in a sonicator (Fisher Scientific) for 15 min before use. The dried samples were reconstituted in 500 µl buffer, vortex-mixed for 1 min, and 1 ml distilled water was added to make up the volume to 1.5 ml. The sample was injected onto the HPLC. The gradient was run and fractions were collected at 0.5 min intervals up to 86 min in scintillation vials (Fisher Scientific). Each fraction was added to 2 ml Packard Ultima Flo AP (Perkin Elmer) liquid scintillation cocktail and mixed gently. The radioactivity in samples was determined on a scintillation counter (Beckman LS 6500, Beckman Coulter). Each strain was tested twice and phosphoinositide levels were normalized to the total signal.
Sensitivity to cell wall agents
Each of the following experiments was performed in triplicate.
SDS and calcofluor white.
Cultures from overnight grown cells were subcultured in YPD liquid medium with 1 % glucose until the exponential phase and diluted to OD599 0.1. Samples (4 µl) of undiluted and serial 10-fold dilutions of each culture were spotted onto YPD plates containing calcofluor white (40 µg ml–1) 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 ml–1 (Sigma-Aldrich) in 10 ml Tris-HCl, pH 7.5. An aliquot was removed at timed intervals and the OD599 was measured. The OD599 was plotted against time of incubation.
Caspofungin.
Sensitivity to caspofungin (Merck Research Laboratories) was measured in a 48-well microtitre plate. Organisms grown overnight in YPD at 30 °C were diluted to OD600 =0.1 in YPD. Caspofungin was added at concentrations ranging from 0.075 to 20 µg ml–1 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.
Staining of chitin.
C. albicans cells were grown either as a suspension in YPD liquid culture to exponential phase or embedded in solid medium for 3 days. Cells were washed three times for staining and imaging. For chitin staining, cells were incubated for 5 min at room temperature in 0.1 mg calcofluor white ml–1, and then washed three times in cold PBS (1x) (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.3). Slides were viewed in a Zeiss AxioKop2 microscope equipped for epifluorescence, using the appropriate filter for each fluorescent compound, and images were digitized with a camera connected to Epilab software, at x1000 magnification.
Murine disseminated candidiasis and histopathology.
Groups of 10–12 seven-week-old male ICR mice (Harlan-Sprague) were inoculated by intravenous injection of the lateral tail vein with 1x106 c.f.u. C. albicans. For mortality studies, mice were followed until they were moribund, at which point they were sacrificed, or for 42 days. Survival curves were calculated according to the Kaplan–Meier method using the PRISM program (GraphPad Software) and compared using Newman Keuls analysis; a P value of <0.05 was considered significant. For tissue burden, mice were infected as above with 5x105 c.f.u. Mice (6–8 per group per time point) were sacrificed 24 h and 4 days post-inoculation, and their kidneys were aseptically removed. The kidneys were weighed, homogenized in 2 ml sterile PBS (1x) (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.3), and serial dilutions were plated on SDA plates containing pipericillin (60 µg ml–1) and amikacin (60 µg ml–1). The plates were incubated at 30 °C for 48 h, after which the number of c.f.u. was determined. Values were expressed as log(c.f.u. per gram kidney). The differences in kidney burden between strains were determined by the Wilcoxon rank sum test; a P value <0.05 was determined to be statistically significant.
For staining of C. albicans cells recovered from murine kidneys, aliquots from homogenized kidneys were fixed in formalin, washed in cold water and stained with calcofluor white as described above. Samples were visualized under a fluorescence microscope to search for C. albicans hyphae. Histopathology was performed by a pathologist blinded to the experimental design. Kidneys were collected 4 days post-inoculation, and fixed with formalin and embedded in paraffin, after which thin sections were prepared and stained with periodic acid–Schiff (Churukian et al., 1986; Churukian & Schenk, 1977). For each strain, kidneys from three mice were chosen for image analysis. TIFF images were captured of all the tissue on each of the slides. The images were analysed on a Windows XP PC using the public domain National Institutes of Health (NIH) image program Image J (http://rsb.info.nih.gov/nih-image/) developed at the NIH. For each image, outline splines were traced around the total area(s) of tissues, and another series of outline splines was traced around the area(s) involved in acute inflammation. At least 20 images for each kidney were analysed. The percentage of the total area with the inflammation was calculated and expressed as mean±SD. Values were compared by t test.
Protein extraction and Western blotting.
C. albicans cells were grown to mid-exponential phase in YPD medium, then harvested and centrifuged at 4 °C, and processed as previously described (Navarro-Garcia et al., 1998). Protein concentration was determined using the Bio-Rad Protein Assay and equal amounts of proteins (usually 150–200 µg per lane) were loaded. After SDS-PAGE migration and transfer to a nitrocellulose membrane, Mkc1p was detected with either an anti-Mkc1p (kindly provided by Jesús Pla, Universidad Complutense de Madrid) or the anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody (Cell Signaling Technology).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-galactosidase) reporters in S. cerevisiae strain AH109. Such experiments are complicated by C. albicans non-canonical codon usage, in which CTG codons encode serine instead of leucine. To account for this, we substituted five CTG codons in C. albicans IRS4 with TCT to match universal codon usage. C. albicans INP51 and the modified IRS4 were subcloned into pGBKT7-Rec (binding domain vector, expressing TRP1 as a selection marker) and pGADT7 (activation domain vector, expressing LEU2), respectively. Co-transformation of S. cerevisiae AH109 (auxotroph for Trp and Leu) with both plasmids yielded viable transformants on SDA (Trp–, Leu–, Ade–, His–) medium, as did the positive control plasmids carrying SV40 large T antigen-HA and murine p53-c-Myc. In this system, the adenine and histidine nutritional selection markers add a level of stringency that minimizes false positives, as S. cerevisiae AH109 transformants containing both pGBKT7-Rec and pGADT7 vectors will only grow on SDA (Trp–, Leu–, Ade–, His–) medium if the tested proteins interact physically. Moreover, the transformants were positive for -galactosidase activity, as indicated by the presence of blue colonies in the presence of X--gal. As negative control, co-transformation of AH109 with pGADT7 carrying the modified IRS4 and pGBKT7-Rec carrying INP51 in the reverse orientation did not yield transformants on SDA (Trp–, Leu–, Ade–, His–) medium, since the ADE2 and HIS3 reporters were not activated. As anticipated, the negative control was able to grow on SDA (Trp–, Leu–) medium. Upon the addition of X--gal to this medium, the large T antigen/p53 positive control and Inp51/Irs4 co-transformants clearly grew as blue colonies, whereas the negative control co-transformants did not (see Supplementary Fig. S1).
To verify the Inp51p–Irs4p interaction that was suggested by the yeast two-hybrid results, we performed co-immunoprecipitation experiments following co-transcription/translation. We initially amplified complete ORF sequences of IRS4 and INP51 from their plasmids so that the resulting fragments encompassed optimal signal sequences for in vitro transcription and translation, as well as c-Myc and HA tags, respectively. Expression of the full-length INP51, however, resulted in low yields. For this reason, the C-terminal fragment encompassing the last 475 residues of Inp51p (Inp51p-C) was then amplified, and sufficient yield was obtained after in vitro expression (Fig. 1a). The in vitro expression products were mixed, and both Irs4p-c-Myc and Inp51p-C-HA were immunoprecipitated using either anti-c-Myc or anti-HA antibodies (Fig. 1b). As positive controls, SV40 large T antigen-HA and murine p53-c-Myc were co-immunoprecipitated using either of the two antibodies, consistent with the anticipated interaction between these proteins. As negative controls, Irs4p-c-Myc was mixed with SV40 large T antigen-HA and Inp51p-C-HA with p53 c-Myc. Upon immunoprecipitating the first mixture with anti-HA, only large T antigen was pulled down (Fig. 1b). Likewise, only p53 was pulled down when the second mixture was immunoprecipitated with anti-c-Myc.
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NPF-HA, which has a deletion of the last 22 amino acids including the NPF motif. This truncated version of Inp51p-C no longer showed the interaction with Irs4p-c-Myc (Fig. 1b
). Sufficient expression of Inp51p-C-NPF-HA was confirmed by immunoprecipitation with anti-HA antibody (Fig. 1b).
Irs4p and Inp51p negatively regulate levels of PI(4,5)P2
To determine whether C. albicans Inp51p and Irs4p regulate PI(4,5)P2, we measured intracellular phosphoinositide levels for isogenic null mutant and complemented strains using HPLC (Dove et al., 1997). In our previous study, using the ura-blaster method, we created an irs4 null mutant strain, which was then complemented with the full IRS4 ORF (Badrane et al., 2005). In this study, we used the SAT1 flipper technique for targeted homologous recombination to successively knock out both alleles of INP51, creating the null mutant strain inp51_2KO. The full gene with 1 kb downstream non-coding DNA was then reinserted in the null mutant background (creating strain 2KO_Rei-INP51), and all recombination events were verified using Southern blotting (see Supplementary Fig. S2). For both genes, the null mutant and complemented strains showed growth rates that were indistinguishable from those of wild-type strains when cultured in YPD media.
We were able to detect four different phosphoinositides in all strains: phosphatidylinositol 3-phosphate (PI3P), phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], and PI(4,5)P2. Under routine growth conditions, PI4P was the major peak (7.1 % of total signal), followed by PI(4,5)P2 (4.9 %), PI3P (2.7 %) and PI(3,5)P2 (<0.1 %). The number and relative distribution of phosphoinositides was similar to that of S. cerevisiae, with significantly lower levels of PI(3,5)P2 than the others (Bonangelino et al., 2002; Morales-Johansson et al., 2004). In both C. albicans inp51 and irs4 null mutant strains, we observed a significant increase in PI(4,5)P2 levels to approximately twice the levels of wild-type strains (Fig. 2a). Levels of PI(4,5)P2 were re-established to wild-type levels in the irs4 null mutant complemented with one copy of IRS4. The complementation was partial upon reintroduction of one copy of INP51 to the inp51 null mutant, indicating a possible dosage effect. The null mutants did not show any significant changes in the three other detected phosphoinositides (Fig. 2b).
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). Like the irs4 null mutant, the inp51 mutant was indistinguishable from strain SC5314 when grown under hyphal-inducing conditions in liquid media (5 and 10 % FCS, data not shown). Finally, calcofluor white staining of inp51 mutant cells grown under embedded conditions revealed abnormal distribution of chitin along the hyphal walls (Fig. 4). No defects in chitin distribution were detected in yeast, hyphal or pseudohyphal cells among our strains when grown in YPD liquid media at 30 or 37 °C (data not shown). For each of the phenotypes tested, the effects of disrupting INP51 were identical to those previously observed with IRS4 disruption (Badrane et al., 2005). Moreover, similar results were obtained using independently created inp51 null mutant and complemented strains.
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). In contrast, null mutant strains (inp51_2KO) killed only one-third of mice by the end of the study (P<0.001). The complemented strain killed all mice within 8 days (P<0.001 versus null mutant).
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).
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). The complemented strain yielded intermediate results. Extended filamentous morphologies of both wild-type and complemented strains were clearly evident within areas of acute inflammation (Fig. 6). The null mutant, on the other hand, was difficult to locate within areas of inflammation, and filamentous forms were rarely identified. The null mutant also demonstrated abnormal chitin distribution along the hyphal walls upon calcofluor white staining of cells recovered from infected kidneys, consistent with the findings during in vitro embedded growth (Fig. 7).
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). The regulation of PI(4,5)P2 levels by S. cerevisiae Irs4p–Inp51p potentially plays a role in repressing the cell integrity pathway, but only in the presence of mutations to other negative regulators of the pathway (Bickle et al., 1998; Morales-Johansson et al., 2004). To determine if the cell integrity pathway was affected in our irs4 and inp51 mutant strains, we assayed the phosphorylation of Mkc1p, a MAP kinase that is phosphorylated upon activation of the pathway. Under conditions in which the pathway is known to be inactive in wild-type cells (growth as yeast in liquid YPD medium at 30 °C) (Navarro-Garcia et al., 1998), it was also inactive in the mutant strains (Fig. 8a). As anticipated, the pathway was activated in wild-type strains during embedded growth and in the presence of 10 mM H2O2 (Kumamoto, 2005; Navarro-Garcia et al., 1998). We also demonstrated activation of the pathway in the wild-type strain grown under hyphae-inducing conditions in liquid media (YPD+10 % FCS, 37 °C). Under each of the activating conditions, excess phosphorylated Mkc1p accumulated in both mutant strains, consistent with pathway overactivation (Fig. 8a, b). This overactivation was greater in the irs4 mutant than in the inp51 mutant. It is of note that the expression of Mkc1p did not differ between wild-type and mutant strains for any of the conditions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). At the same time, C. albicans irs4 and inp51 null mutant strains exhibited phenotypes that have not been reported in S. cerevisiae, including impaired contact-induced hyphal formation, increased sensitivity to cell wall-active drugs and attenuated virulence. Moreover, the cell integrity pathway was overactivated in the C. albicans irs4 and inp51 mutants in response to stress and hyphal-inducing stimuli, whereas the S. cerevisiae mutants only demonstrate such overactivation in the presence of mutations to other negative regulators of the pathway (Morales-Johansson et al., 2004). The abnormal phenotypes of the C. albicans null mutants were reversed upon reintroduction of the respective genes and the accompanying reductions in PI(4,5)P2 levels. Taken together, our findings suggest that the interacting C. albicans proteins Irs4p and Inp51p are important in coordinating cellular processes that contribute to invasive growth and virulence under conditions of cell wall tension, such as those encountered within solid agar or during tissue invasion.
It is likely that Irs4p and Inp51p mediate their cellular effects, at least in part, through negative regulation of PI(4,5)P2 levels. PI(4,5)P2 is one of several phosphoinositides that play structural roles in membrane biogenesis and have emerged in the past decade as membrane sensors and second messengers (Daum et al., 1998). PI(4,5)P2 and other phosphoinositides are synthesized in different cell compartments by phosphatidyl-inositol kinases and phosphatases, and they have the ability to interact with specific domains of target proteins (Sprong et al., 2001). Because phosphoinositides are rapidly modified by headgroup phosphorylation–dephosphorylation, they can transiently and locally activate or deactivate signalling pathways. In so doing, they coordinate diverse processes, including cell wall organization, cell signalling, cytoskeleton regulation, and endocytic and exocytic trafficking (Martin, 1998; Lemmon, 2003). It is of note that efficient hyphal formation depends upon the careful coordination of these cellular processes. As such, dysregulation of PI(4,5)P2 is consistent with cell wall abnormalities, impaired hyphal formation and attenuated virulence, as seen in our mutants. In interpreting our results, it is important to recognize that global cellular levels of phosphoinositides, as measured in this study, might be less relevant to the observed phenotypes than local subcellular derangements.
Our data support a model in which Irs4p binds the NPF motif of Inp51p. We used a yeast two-hybrid system that we adapted to account for C. albicans non-canonical codon usage to demonstrate a likely interaction between full-length Irs4p and Inp51p. We then confirmed our findings by co-immunoprecipitating Irs4p (Irs4p-c-Myc) and the C terminus of Inp51p (Inp51p-C-HA). Deletion of the NPF motif from Inp51p eliminated the interaction, as Inp51p-C-NPF-HA failed to co-immunoprecipitate with Irs4p-c-Myc. It is of note that Irs4p contains an EH domain between residues 532 and 627, which is a highly conserved eukaryotic signalling module that binds NPF motifs (de Beer et al., 1998; Confalonieri & Di Fiore, 2002; Salcini et al., 1997; Santolini et al., 1999; Tang et al., 2000). In future studies, we will use site-directed mutagenesis to conclusively demonstrate that the Irs4p–Inp51p interaction is mediated by the EH domain and NPF motif, respectively, and that the interaction is directly responsible for the regulation of PI(4,5)P2 levels.
The attenuation of virulence seen with the disruption of INP51 was evident from multiple end points during intravenously disseminated candidiasis in mice, including mortality, tissue burdens and inflammation. Interestingly, the wild-type C. albicans strain caused significantly higher burdens of infection and larger areas of inflammation than the null mutant within the kidneys after 4 days of disseminated candidiasis, whereas the complemented strain showed trends toward intermediate results. These observations were consistent with the relative levels of PI(4,5)P2 for the strains, suggesting a possible dose effect on proliferation and invasion within the kidneys. It is worth noting that the impaired hyphal formation and abnormal hyphal wall chitin distribution exhibited by irs4 and inp51 mutants within the kidneys after 4 days resembled the phenotypes observed during embedded growth within agar over a similar time period. Indeed, it has been hypothesized that embedded conditions in vitro resemble those encountered by C. albicans in vivo, where the organism grows in contact with a solid matrix under reduced oxygen concentrations (Ernst, 2000).
In addition to confirming that inp51 null mutants resembled irs4 mutants in their increased susceptibility to cell wall-active agents, impaired hyphal formation, abnormal chitin distribution and attenuated virulence, we demonstrated that disruption of either gene resulted in overactivation of the cell integrity pathway. The fungal cell integrity pathway is responsible for initiating and controlling responses to cell wall stresses (Delley & Hall, 1999; Jung & Levin, 1999). Our findings indicate that the Irs4p–Inp51p interaction exerts some degree of control over the pathway, but this effect is not dominant since it was only observed under conditions in which the pathway was already activated. It has been proposed that effects of S. cerevisiae Irs4p/Tax4p and Inp51p on the cell integrity pathway are exerted through the binding of PI(4,5)P2 to the plekstrin homology domain of the GDP/GTP exchange protein Rom2p (Morales-Johansson et al., 2004). In the cell integrity pathway, Rom2p is an upstream activator of the PKC–MAP kinase cascade (Ozaki et al., 1996), which culminates in Mkc1p. Since the pathway components are conserved in C. albicans, a similar hypothesis is plausible.
The C. albicans cell integrity pathway has been linked to the regulation of morphogenesis. C. albicans Mkc1p has been shown to be required for morphogenesis (Navarro-Garcia et al., 1998), and the pathway is activated in candidal cells grown in contact with agar (Kumamoto, 2005). A connection between the S. cerevisiae cell integrity/PKC and RAS/cAMP pathways has been recently demonstrated through the shared protein Rom2p (Kuranda et al., 2006; Park et al., 2005; Verna et al., 1997). In C. albicans, the RAS/cAMP pathway is a major regulator of hyphal formation (Ernst, 2000; Zhao et al., 2002). As such, dysregulation of the C. albicans cell integrity pathway might be associated with dysregulation of hyphal-inducing pathways. Nevertheless, dysregulation of the cell integrity pathway cannot fully account for the phenotypes we observed, as the irs4 and inp51 mutant strains overactivated Mkc1p in liquid media but did not display impaired hyphal growth or abnormal chitin deposition.
In conclusion, we propose a model in which the interaction between Irs4p and the phosphatase Inp51p negatively regulates PI(4,5)P2 levels in C. albicans. The specific increases in PI(4,5)P2 levels in irs4 and inp51 null mutants are associated with abnormal chitin deposition and impaired hyphal formation during contact-induced growth in vitro and within murine kidneys, and attenuated virulence during disseminated candidiasis of mice. Lack of Irs4p and Inp51p also results in overactivation of the cell integrity pathway under normal activating conditions. Future studies of irs4 and inp51 null mutant strains that assess processes such as cell wall biogenesis, secretion, endocytosis, exocytosis and, in particular, the temporal–spatial regulation of phosphoinositides under diverse biological conditions will provide unique insights into the pathogenesis of candidiasis.
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ACKNOWLEDGEMENTS |
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Edited by: J. F. Ernst
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Received 26 February 2008;
revised 10 July 2008;
accepted 10 July 2008.
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