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Candida albicans IRS4 encodes a protein that regulates phosphatidylinositol-(4,5)-bisphosphate, which was shown to contribute to hematogenously disseminated candidiasis (DC) after several days in the standard mouse model. Our objective was to more accurately define the temporal contributions of IRS4 to pathogenesis. During competition assays in vitro, an irs4-null (Δirs4) mutant exhibited wild-type fitness. In DC experiments, mice were infected intravenously with the Δirs4 mutant, strain CAI-12 (1 × 105 CFU), or a mixture of the strains (0.5 × 105 CFU each). In single-strain infections, quantitative PCR revealed reduced Δirs4 mutant burdens within kidneys at days 1, 4, and 7 but not 6 h. In competitive infections, the Δirs4 mutant was outcompeted by CAI-12 in each mouse at ≥6 h (competitive indices, P ≤ 0.0001). At 4 and 7 days, the Δirs4 mutant burdens during competitive infections were significantly lower than those during single-strain infections (P = 0.01 and P < 0.001, respectively), suggesting increased susceptibility to inflammatory responses. Phagocytic infiltration of kidneys in response to CAI-12 or competitive infections was significantly greater than that in response to Δirs4 mutant infection at days 1 and 4 (P < 0.001), and the Δirs4 mutant was more susceptible to phagocytosis and killing by human polymorphonuclear cells (P = 0.01 and P = 0.006, respectively) and mouse macrophages in vitro (P = 0.04 and P = 0.01, respectively). Therefore, IRS4 contributes to tissue invasion at early stages of DC and mediates resistance to phagocytosis as DC progresses. Microarray analysis revealed remarkably similar gene expression by the Δirs4 mutant and reference strain CAI-12 within blood, suggesting that IRS4 is not significantly involved in the hematogenous stage of disease. A competitive DC model detects attenuated virulence that is not evident with the standard model.
Candida albicans is the major fungal pathogen among hospitalized patients in the developed world, causing a wide range of superficial, mucosal, and invasive infections (1). Candidemia is the fourth most common bloodstream infection in the United States and is associated with mortality rates approaching or exceeding 40%, despite antifungal therapy (2, 3). The development of new treatment, prevention, and diagnostic strategies against candidemia and disseminated candidiasis depends upon better understanding of the pathogenesis of C. albicans infections.
In previous studies, we demonstrated that C. albicans IRS4 was essential for full virulence during hematogenously disseminated candidiasis in mice. IRS4 encodes an Eps15 homology (EH) domain protein that physically interacts with the 5′-phosphatase Inp51 to regulate the levels and plasma membrane distribution of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] (4–6). Disruption of IRS4 resulted in cell wall derangements and impaired hyphal formation within solid agar and mouse kidneys. The tissue burdens of the irs4-null (Δirs4) mutant within mouse kidneys were similar to those of wild-type C. albicans after 20 h. However, the Δirs4 mutant was unable to form dense mats of hyphae, and tissue burdens at 4 days were significantly attenuated (4, 5). The data suggested that PI(4,5)P2 regulation is required for the progression but not initiation of kidney infection. We hypothesized that the attenuated virulence of the Δirs4 mutant results from a failure to maintain cell wall integrity in the face of ongoing cell wall stress during tissue invasion. At the same time, we recognized that IRS4 may make contributions to pathogenesis within the kidneys that were not apparent in the mouse model. Indeed, mutants were significantly impaired in adherence to various epithelial cell lines in vitro, suggesting that IRS4 is relevant at the early stages of tissue invasion. Furthermore, if the impaired cell wall integrity of the Δirs4 mutant was contributing to attenuated virulence at later time points, it was not clear why such defects would not also be important at early time points.
In assessing the contribution of IRS4 to virulence, we used the standard mouse model of hematogenously disseminated candidiasis (7). In this model, C. albicans strains are inoculated via the lateral tail vein, and the kidneys are the primary target organ. Groups of mice are typically infected with a wild-type, isogenic gene disruption, or gene reinsertion strain, and endpoints like mortality and tissue burdens are compared between groups at serial time points (7). The model has advantages of simplicity and reproducibility, but it is limited by relatively insensitive endpoints. Moreover, large numbers of mice are typically required to demonstrate that virulence is attenuated in mutant strains, although expenditure of lives may be reduced by the technical skill of investigators and in cases of large differences between strains. Competitive infection models, in which individual hosts are infected with a mixture of microbial strains, are powerful tools for identifying small differences in fitness (i.e., relative virulence) between strains (8–16). By eliminating interhost variability, they achieve high levels of sensitivity and spare animals. In recent years, investigators have begun to use competitive models of oral, gastrointestinal, and disseminated candidiasis in lieu of conventional mouse models to compare the virulence of wild-type and mutant C. albicans strains (8, 10, 11, 13, 15, 16). These studies have conclusively identified C. albicans genes that encode virulence determinants, but they have not tapped the full potential of competitive models to characterize the pathogen-host interaction. In particular, the studies have not compared C. albicans strain behavior and host responses during both competitive and single-strain infections, which is a particularly useful strategy for defining the contribution of a specific gene to the pathogenic process over the time course of disease.
The primary objective of the present study was to define more accurately the temporal contribution of C. albicans IRS4 to the pathogenesis of disseminated candidiasis. Toward this end, we developed a competitive infection model of hematogenously disseminated candidiasis in mice and characterized gene expression by the Δirs4 mutant within blood. We hypothesized that these methods would demonstrate that the virulence of the Δirs4 mutant is attenuated at the early stages of kidney invasion, rather than after several days, as suggested in our prior studies.
C. albicans Δirs4 and an IRS4 reinsertion strain were created and characterized as described in our previous publications (4, 5) (Table 1). C. albicans CAI-12 was the IRS4-intact, isogenic strain used for comparisons throughout the study. Growth rates in vitro were determined in yeast peptone dextrose (YPD) and Sabouraud dextrose (SD) media at 30 and 37°C in microtiter plates, as described previously (19). Extended growth curves in vitro were assessed through four dilution and regrowth cycles of 24 h each. To induce hyphal formation in liquid media, C. albicans strains grown overnight on YPD agar were subcultured into liquid YPD supplemented with 5% fetal calf serum (FCS) and liquid RPMI 1640 at 37°C (4, 5). In preparation for intravenous challenge of mice, all strains were grown to stationary phase overnight in YPD medium at 35°C. C. albicans cells were washed in sterile saline, and inocula were prepared at the desired concentration in sterile saline.
For the standard curves relating changes in threshold cycle (ΔCT) values to C. albicans cell numbers, DNA samples were prepared from sterile saline or naive kidneys spiked with cells from a mid-logarithmic-phase culture of C. albicans CAI-12 grown at 30°C in YPD medium. The cell density of the undiluted culture was determined by direct counting with a hemocytometer and verified by plating. The culture was serially diluted in YPD medium. In both spiking and disseminated candidiasis experiments, kidneys were homogenized and subjected to lyticase treatment (30 min at 37°C). Genomic DNA was isolated using a DNeasy blood and tissue kit (Qiagen). Our analyses included at least three biological replicates for all samples, each from independent collections. The quantitative real-time PCR was performed using PerfeCTa SYBR green Fast Mix (Quanta Biosciences Inc.) with an ABI 7500 sequence detection system (Applied Biosystems). For each 20-μl PCR mixture, 2 μl genomic DNA was used with 6 μl nuclease-free water, 10 μl PerfeCTa SYBR green Fast Mix, and 1 μl of each primer. Gene-specific primers for IRS4 detection in CAI-12 (primers IRS-qPCR-For [5′-ACC AGC AAT CTT CCA CTG AGA TCA ACA-3′] and IRS-qPCR-Rev [5′-CTT CCA TGG CTT CAA CTC ATT AAA CCT TGA-3′], yielding a 138-bp product) and primers specific to the Δirs4 mutant (primers Δirs-qPCR For [5′-CTG AGG TGG AAG TTG GAG AAA CAA CC-3′] and Δirs-qPCR-Rev [5′-CCC TGA TTG ACT GGA ACA GGA TCC TC-3′], yielding a 122-bp product) were designed. The thermocycler program was 95°C for 10 min and 40 cycles of 95°C for 30 s and 58°C for 45 s. Quantitative PCR (qPCR) assays were run according to the manufacturer's directions, and results were analyzed with sequence detection system software (v2.0.1; Applied Biosystems). Each sample was assigned a CT value, which identifies the cycle number during PCR when fluorescence exceeds a threshold value determined by the software. Differences in DNA recovery between samples were normalized by determining the total DNA concentration of each infected kidney sample ([DNA]sample), comparing that concentration to the DNA concentration values from uninfected kidney ([DNA]uninfected), and adjusting each sample CT value according to the formula CT adjusted = CT + x, where x is log2([DNA]sample/[DNA]uninfected) (20). ΔCT values were used to calculate genomic equivalents (GE) from a standard curve generated from DNA samples prepared from uninfected kidneys spiked individually with CAI-12, the Δirs4 mutant, and a reinsertion strain yeast. All qPCR results for samples from infected tissues are expressed as GE per gram equivalent of tissue. Since a full copy of IRS4 was reintroduced into a disrupted native locus in the reinsertion strain, both wild-type (IRS4)-specific and Δirs4 mutant-specific primers amplified signals. Therefore, in the mixed infection (infection with CAI-12 plus the reinsertion strain), the IRS4 primer set amplified both wild-type alleles from CAI-12 and one wild-type allele from the reinsertion strain (wild-typetotal GE), and the Δirs4 mutant primer set amplified one allele from the reinsertion strain (reinsertion strain GE). The actual number of wild-type GE in the mixed infection was calculated using the formula wild-typetotal GE − reinsertion strain GE. No signal was detected when the following templates were tested as negative controls: genomic DNA prepared from kidneys of uninfected mice, mutant primers for kidneys spiked or infected with CAI-12, and CAI-12 primers for kidneys spiked or infected with the Δirs4 mutant.
Mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh. Groups of 7-week-old male ICR mice (8 to 10 mice per group; Harlan-Sprague) were individually inoculated by single intravenous injections of the lateral tail vein with 200 μl of sterile saline containing (i) 1 × 105 CFU of CAI-12, the Δirs4 mutant, or the IRS4 reinsertion strain; (ii) 0.5 × 105 CFU each of CAI12 and the Δirs4 mutant, or (iii) 0.5 × 105 CFU each of CAI-12 and the reinsertion strain. Mice were randomly selected to be included in a given group. Strain concentrations were confirmed by serial dilution of inocula and enumeration of CFU. Mice in the CAI-12, the Δirs4 mutant, and the CAI-12–Δirs4 mutant groups were sacrificed at 6 h, 1 day, 4 days, and 7 days after intravenous inoculation, and their kidneys were aseptically removed. Reinsertion strain-infected groups were sacrificed on days 1 and 4 after challenge. C. albicans CAI-12 does not kill mice at this inoculum over 7 days, which ensured that interpretation of tissue burdens would not be obscured by death. Kidneys were sectioned such that one-half of the left kidney and one-half of the right kidney were combined and used for CFU determination, while the other halves were combined and used for qPCR analysis. The organs were weighed and homogenized in 2 ml sterile phosphate-buffered saline (PBS). For CFU enumeration, serial dilutions were plated onto SD plates containing piperacillin (60 mg ml−1) and amikacin (60 mg ml−1). The plates were incubated at 30°C for 48 h. Values were expressed as the log number of CFU per gram kidney. Differences in tissue burdens (numbers of GE or CFU) between strains were compared using analysis of variance (ANOVA). Pairwise comparisons were calculated using a Bonferroni adjustment. Within individual mice during competitive infections, results were expressed as the competitive index (CI). CI was defined as [(log10 GE/g kidney for mutant strain/log10 GE/g kidney for CAI-12)/(log10 GE/ml inoculum for mutant strain/log10 GE/ml inoculum for CAI-12)] (9). Results were compared using Student's t test.
Histopathology was performed by a pathologist blinded to the experimental design. Kidneys were fixed with formalin and embedded in paraffin, after which thin sections were prepared and stained with hematoxylin-eosin (H&E) or periodic acid-Schiff (PAS) (21). For each strain, kidneys from three mice were chosen for image analysis. TIFF images of all the tissue on each of the slides were captured. The images were analyzed on a Windows XP personal computer using the public domain National Institutes of Health (NIH) image program ImageJ (http://rsb.info.nih.gov/nih-image/). 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 (5). At least 20 images for each kidney were analyzed. The percentage of the total area with inflammation was calculated and expressed as the mean ± standard deviation.
Phagocytosis and killing assays were performed as previously described (19, 22), with slight modifications. Polymorphonuclear cells (PMNs) were isolated from heparinized blood by dextran sedimentation, followed by centrifugation through Ficoll-Hypaque. After removing contaminating erythrocytes by hypotonic lysis, the PMNs were resuspended in RPMI 1640. Prior to phagocytosis and killing assays, C. albicans strains were opsonized with 50% normal human serum at 30°C for 30 min. For phagocytosis assay, 0.5 ml of opsonized C. albicans cells was incubated with 0.5 ml of PMNs at a PMN/C. albicans ratio of 1:1 in 1 ml RPMI 1640 at 37°C for 15 min on a shaker. Three drops of the sample were then cytospun and Gram stained. Percent phagocytosis was calculated as the proportion of PMNs containing one or more yeast cells after counting of 100 PMNs. For the killing assay, opsonized C. albicans strains were incubated with 106 PMNs at a PMN/C. albicans ratio of 50:1 in 1 ml of RPMI 1640 containing 5% human serum at 37°C for 2 h with gentle shaking. After complete lysis of PMNs with sterile water, serial 10-fold dilutions were made and colony counts were enumerated. For mouse macrophage (J774A.1) studies, cells procured from ATCC were generated in a 96-well plate (5 × 105 cells/well) by incubation for 30 min at 37°C in 5% CO2. Prior to phagocytosis and killing assays, C. albicans strains suspended in modified Eagle's medium (MEM) were opsonized with 5% normal human serum at room temperature for 30 min. For the phagocytosis assay, 200 μl opsonized C. albicans cells was added to wells containing the monolayer (effector/target cell ratio, ~10:1) and the cells were incubated for 15 min at 37°C in 5% CO2. After incubation, the supernatant was aspirated and washed with prewarmed MEM. Serial 10-fold dilutions were made, and colony counts were enumerated. For the killing assay, 200 μl MEM-Sabouraud broth (1:1) was added to wells containing monolayers with phagocytosed C. albicans cells, and the plates were incubated for 3 h at 37°C in 5% CO2. The monolayers were scraped, and after complete lysis of macrophages with sterile water, serial 10-fold dilutions were made and colony counts were enumerated. The phagocytosis and killing experiments were performed in triplicate and repeated at least twice. The percentage of phagocytized C. albicans cells was defined as [1 − (number of uningested CFU/number of CFU at the start of incubation)] × 100. The percentage of C. albicans cells killed by the phagocytes was defined as [1 − (number of CFU after incubation in the presence of phagocytes/number of CFU phagocytosed)] × 100. The fungicidal activity was calculated as the percent survival of C. albicans after 2 h of incubation with PMNs.
C. albicans Genome Oligo Set v1.1 (Qiagen, Valencia, CA) was resuspended at 30 μM in ArrayIt Micro Spotting Solution Plus (Telechem, San Jose, CA) and printed on UltraGAPS slides (Corning, Corning, NY) using a Genemachine Omnigrid arrayer (Genomic Instrumentation Services, San Carlos, CA). Each slide had two replicated arrays with 6,936 features, including 70-mer oligonucleotides representing 5,948 predicted open reading frames (ORFs), 318 cloned genes, and random 70-mer controls. Transcriptional profiling experiments in human blood were performed as previously described (23). In brief, three biological replicates of C. albicans CAI-12 and the Δirs4 mutant were grown to mid-exponential phase in YPD medium at 30°C, washed once in 0.1 M PBS, pH 7.0, and resuspended in this buffer at a density of 37.5 × 108 cells/ml. After incubation for 30 min at 37°C, 100 μl of the suspension was inoculated into 7.5 ml of fresh whole human blood from one of the investigators (C.J.C.), which was collected in a heparinized tube. After incubation of the tube with gentle shaking for 2 h at 37°C, cells were harvested and mixed with 1 g of 425- to 600-μm-diameter glass beads (Sigma Chemical Co., St. Louis, MO). Total RNA was extracted using an RNeasy minikit (Qiagen) and a Mini-BeadBeater (Biospec Products, Bartlesville, OK), and on-column DNase digestion with an RNase-free DNase set (Qiagen) was systematically performed. Twenty to 25 μg of total RNA was used to generate labeled cDNA with a SuperScript indirect cDNA labeling system (Invitrogen, Carlsbad, CA). Two cDNAs from different samples labeled with different dyes (Cy3 and Cy5) were mixed, denatured in 3.4× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.3% SDS, 0.5 μg/μl salmon sperm DNA, and hybridized overnight (14 to 16 h) to microarray slides. A dye-flip strategy was employed. After washing and drying, the slides were scanned in a GenePix 4000B scanner (Axon Instruments, Union City, CA). GenePix Pro v3.0 software (Axon) was used to extract the spot intensities for both fluorescent dyes, and data were archived in Microsoft Access software. For individual slides, the threshold intensity for each fluorescent channel was established as the average intensity of spots corresponding to negative-control 70-mers plus 1.5 times the standard deviation. Only intensities higher than the threshold were considered. Replicated spots in each slide had to have a coefficient of variation in intensities of <65%. The intensities of the replicated spots in each slide that passed the criterion were averaged and used for normalization. We performed a global normalization among all slides so that the total slide intensity for each channel was identical among slides and channels. The ratio of the intensity of the Δirs4 mutant to that of strain CAI-12 was used for further analysis.
We applied significance analysis of microarrays (SAM) to detect differentially expressed ORFs (24). SAM assigns a score to each gene on the basis of the change in gene expression relative to the standard deviation of repeated measurements. For genes with scores greater than an adjustable threshold, SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance, the false discovery rate (FDR). SAM has been shown to be superior to conventional microarray analysis based on pairwise fold change methods. Cluster software and Treeview software were used for cluster analysis and display of genome-wide expression data (25). Differentially expressed ORFs were assigned gene names, descriptions, and pathways on the basis of assignments in the Candida Genome Database (http://www.candidagenome.org/).
Gene expression by the strains was confirmed for eight genes by reverse transcription-PTR (GCA2, ERG16, RNR22, CWH8, SKN1, CHT2, RHO1, and ERK1), using primers designed and synthesized by Invitrogen (see Table S1 in the supplemental material). cDNA was synthesized using 1 μg total RNA, 2.5 μM reverse primers, 500 μM deoxynucleoside triphosphate (dNTP) mix, 5 mM dithiothreitol, 40 U RNaseOUT, 1× first-strand buffer, and 100 U SuperScript III reverse transcriptase (Invitrogen) at 46°C for 2 h. Amplification was performed for 30 cycles, which gave band intensities linearly proportional to the amount of cDNA. The reaction mixtures included 1 μl a 1/10 dilution of cDNA in 0.2 mM dNTP mix, 1.5 mM MgCl2, 0.2 μM each primer, 1× PCR buffer, and 1.5 U Platinum Taq DNA polymerase (Invitrogen). Ten microliters of each PCR product was run in a 1.5% agarose gel, and the gel was stained with ethidium bromide. Images were digitalized using a transilluminator connected to a camera and Quantity One software (Bio-Rad). The software estimated the band intensity for each PCR product, which was used to calculate the ratio of Δirs4 mutant expression/CAI-12 expression for each ORF.
For comparisons across groups (see Fig. 1, ,4,4, and and5),5), ANOVA was used. Pairwise comparisons were then made using a Bonferroni adjustment. Otherwise, comparisons were made using t test. All analyses were performed using STATA v11 software (College Station, TX). P values of <0.05 were considered significant.
Microarray data were deposited in the NCBI GEO database under accession number GSM967204 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM967204).
To distinguish between C. albicans strains in competition assays, we designed qPCR primers that targeted specific segments of IRS4 and the hisG cassette used for gene disruption. In titration experiments, serial dilutions of yeasts of strain CAI-12, the Δirs4 mutant, or the IRS4 reinsertion strain were suspended in sterile saline and spiked into explanted, uninfected mouse kidneys. Genomic DNA extracted from suspensions and spiked kidneys was used as a template for qPCR assays that established standard curves (see Fig. S1 in the supplemental material). The signals from these reactions were linear over at least 7 orders of magnitude for each strain (all R2 values were ≥0.96). The limits of detection in suspension and spiked kidneys were at or near 1 and 10 yeast cells, respectively.
As we showed previously, growth rates of CAI-12 and the Δirs4 mutant were indistinguishable after the strains were independently inoculated into YPD or SD medium at 30°C (see Fig. S2 in the supplemental material). The growth curves for a mixed culture of CAI-12 and the Δirs4 mutant were similar to those of the individual strains. At all time points, qPCR confirmed that the concentrations of the strains were the same whether they were grown alone or in mixed cultures. Similar results were obtained in SD medium at 30°C and at 37°C (not shown). In addition, there were no significant differences in the growth curves of the strains through four dilution and regrowth cycles of 24 h each. The formation of hyphae by the strains in liquid medium was comparable, consistent with our previous reports (10, 11).
To establish our competitive infection model, we infected groups of mice intravenously with CAI-12 alone (1 × 105 CFU/mouse), the Δirs4 mutant alone (1 × 105 CFU/mouse), or a 1:1 mixture of CAI-12 and the Δirs4 mutant (0.5 × 105 CFU/strain/mouse). Using conventional methods for enumerating the CFU within the kidneys, we corroborated our earlier findings for single-strain infections: tissue burdens of the the Δirs4 mutant were diminished compared to those of CAI-12 at 4 and 7 days but comparable to those of CAI-12 at 6 h or 1 day (Fig. 1A). The qPCR method of quantitating genome equivalents (GE) detected absolute candidal burdens during single-strain infections that were approximately 1 log unit higher than those suggested by the numbers of CFU (Fig. 1B). By qPCR, the Δirs4 mutant inoculated alone caused significantly lower tissue burdens than CAI-12 at 1 day, as well as 4 and 7 days. At 6 h, there were still no differences in tissue burdens between mice infected with the Δirs4 mutant and mice infected with CAI-12.
In the competitive model, the tissue burdens of the Δirs4 mutant and strain CAI-12 at 6 h and 1 day, as determined by qPCR, were identical to those observed when the strains were given alone (Fig. 1B). At days 4 and 7, however, the tissue burdens of the Δirs4 mutant in the competitive model were significantly attenuated compared to those from the Δirs4 mutant single-strain infection. The burdens of CAI-12 at each time point were identical whether the strain was given alone or with the mutant. Of note, the tissue burdens of an IRS4 reinsertion strain were identical to those of CAI-12 in both single-strain and competitive infections, indicating that the attenuated virulence of the Δirs4 mutant was a result of gene disruption (see Fig. S3 in the supplemental material).
Of course, the real power of competitive infection models comes from comparing strains within individual animals, rather than across groups. In these intrahost analyses, results are expressed as the competitive index, which is the ratio of the mutant to a reference strain. Using the competitive index, the Δirs4 mutant was significantly attenuated at each time point, including 6 h, compared to CAI-12 (Fig. 2). Again, there were no differences between the reinsertion strain and CAI-12.
PMNs and mononuclear cells are the first line of innate immune defenses against invasive candidiasis. To determine the impact of IRS4 on PMN and mononuclear cell responses, we first assessed the histopathology of infected kidneys at days 1, 4, and 7 (Fig. 3 and and4).4). Indeed, PMN and mononuclear cell infiltration into the kidneys over the first 4 days was significantly greater for mice infected with CAI-12 alone or the CAI-12–Δirs4 mutant mixture than mice infected with the Δirs4 mutant alone (Fig. 4). Of note, histopathology confirmed our earlier findings that the Δirs4 mutant failed to form extended mats of hyphal elements during the course of kidney invasion (4). Next, we measured the susceptibility of CAI-12 and the Δirs4 mutant to phagocytosis and killing by human PMNs and J774A.1 mouse macrophages in vitro. Indeed, the Δirs4 mutant was significantly more sensitive to phagocytosis and killing by both PMNs and macrophages (Fig. 5). Results with the IRS4 reinsertion strain were similar to those with CAI-12 in histopathology and phagocytosis studies.
Having defined the temporal contribution of C. albicans IRS4 to the pathogenesis of disseminated candidiasis within kidneys, we studied the hematogenous stage of disease by assessing gene expression by the Δirs4 mutant and strain CAI-12 within blood. We incubated the strains separately in blood from a healthy human for 2 h, prior to extracting RNA for whole-genome microarray experiments. Overall, the global gene expression patterns for the Δirs4 mutant and CAI-12 were remarkably similar, as determined using significance analysis of microarrays (SAMs) (see Fig. S4 in the supplemental material). Indeed, only 14 genes were identified to be significantly downregulated in the null mutant (~0.2% of unique ORFs on the array). Gene names, descriptions, and pathways for differentially expressed ORFs are presented in Table 2.
In this study, we used a competitive infection model of hematogenously disseminated candidiasis in mice to redefine the role of C. albicans IRS4 in pathogenesis. Most notably, we demonstrated that IRS4 contributes to pathogenesis at the early stages of kidney invasion, rather than after several days, as suggested in our previous study (4, 5). Significant reductions in the tissue burdens of the Δirs4 mutant compared to CAI-12 (an isogenic, IRS4-expressing strain) were evident within the kidneys at as early as 6 h after intravenous inoculation. Taken with our previous finding that the Δirs4 mutant adheres poorly to a variety of cell lines (4, 5), the data suggest that IRS4 is engaged in the pathogenic process from the initial contact of C. albicans with the kidney. The attenuated virulence of the Δirs4 mutant did not reflect a generalized loss of fitness, as the mutant was not outcompeted by CAI-12 during routine growth in vitro. Moreover, IRS4 does not appear to play a role during the hematogenous stage of disseminated disease, as gene expression by the mutant was not significantly altered within blood.
We also showed that IRS4 makes an additional, previously unrecognized contribution to later stages of kidney invasion. By day 4 of disseminated candidiasis, the burdens of the Δirs4 mutant within the kidneys were significantly lower during mixed infections with CAI-12 than mutant-only infections. This finding was in contrast to that at day 1, at which point the burdens of the Δirs4 mutant were similar in the presence or absence of CAI-12. Histopathology of infected kidneys over the first 4 days revealed significantly greater infiltration of PMNs and mononuclear cells, the front-line effectors of the innate immune system, in response to mixed infections than to mutant-only infections. Along these lines, the Δirs4 mutant was significantly more susceptible than CAI-12 to phagocytosis and killing by human PMNs and mouse macrophages in vitro. Therefore, data from the competitive infection model suggest that IRS4 makes distinct early and later contributions to virulence. The early contributions likely reflect roles in processes like adherence, tissue penetration, and progressive hyphal formation. The later contributions are likely to also include effects in mediating resistance to phagocytosis as disseminated candidiasis progresses.
It is certainly plausible that the cellular processes to which IRS4 contributes are important for tissue invasion and resistance to clearance by the host (4–6). We have shown that disruption of IRS4 results in increased levels of PI(4,5)P2 (but not other phosphoinositides) and focal accumulations of excess PI(4,5)P2, septins, and cell wall protein within plasma membrane and cell wall invaginations (4–6). These abnormalities are associated with profound derangements in cell wall integrity, as evidenced by hypersusceptibility to caspofungin and other cell wall stressors and impaired invasive growth (4–6, 26). Although we found that only 0.2% of genes were differentially expressed by the Δirs4 mutant in blood, it is notable that several downregulated genes are involved in cell wall-related processes such as UDP-N-acetylglucosamine biosynthesis, septin regulation, and invasive growth. Independent of its essential role in maintaining cellular viability, the cell wall is central to the pathogenesis of invasive candidiasis (27–32). It is the interface of the pathogen-host interaction and makes complex contributions to adherence, morphogenesis, and resistance to host defenses. Therefore, it is reasonable that a cell wall-defective mutant would be less able to penetrate or proliferate within target organs, which present ongoing physical and environmental stresses. Along these lines, our data indicate that C. albicans is perceived to be less threatening by the host in the face of IRS4 disruption, as normally protective phagocytic infiltration and inflammatory responses within the kidneys are dampened. At the same time, the hypersusceptibility of the Δirs4 mutant to phagocytic killing is in keeping with impaired cell wall integrity.
The study highlights the major advantages of a competitive infection model over conventional models. First, competitive infections are more sensitive at detecting small differences in virulence between strains. In fact, the absolute differences in tissue burdens between the Δirs4 mutant and strain CAI-12 at 6 h were extremely small (median, ~2-fold). Nevertheless, the competitive model achieved robust statistical significance because the mutant was outcompeted by CAI-12 in each mouse. The sensitivity was heightened by the use of qPCR to quantitate GE rather than reliance upon colony-counting methods. The numbers of GE were approximately 1 log unit higher than the numbers of CFU, which likely reflects the ability of qPCR to more accurately detect higher fungal burdens associated with filamentous morphologies (18). Even in single-strain infections, qPCR revealed statistically significant differences between the Δirs4 mutant and CAI-12 at 24 h that were not evident by CFU enumeration. A second advantage of competitive models is that they can be employed in conjunction with single-strain infections to study how specific factors may impact virulence by altering the interaction with the host immune system. For example, it was apparent from our single-strain infections that IRS4 induced rapid inflammatory responses. Nevertheless, the competitive model demonstrated that the attenuated virulence of the Δirs4 mutant above and beyond that seen in the single-strain infection was correlated with phagocyte infiltration and susceptibility to phagocytosis.
We conducted the competitive and single-strain infections using 8 to 10 mice at each time point, which was consistent with the design of our previous studies. It is important to realize, however, that competitive infections have the potential to spare large numbers of animals. To highlight the power of competitive infections in sparing animals while detecting relatively small differences in virulence between strains, consider an example in which the goal is to detect a 0.6-log-unit (~4-fold) difference in mean tissue burdens with 80% power, assuming a standard deviation of 0.55. In this scenario, 15 animals and one strain per time point would be needed in single-strain infections to detect significant differences in virulence. Therefore, 120 animals would be needed to compare two strains at four time points. In a competitive model with the same assumptions and a correlation coefficient of 0.8, only 5 animals and two strains per time point (or 20 animals to compare two strains at four time points) would be needed. In other words, similar conclusions could be reached using 17% (20/120) of the number of animals. Even if additional animals were used for histopathology or other studies of host responses, the net reduction in lives expended would be substantial.
In conclusion, our data suggest that competitive infection models should be routinely employed in the study of C. albicans virulence. In addition to their superior performance, they are technically straightforward and generate reproducible data. As we demonstrate, competitive infections are particularly useful for studying genes whose contributions to virulence may be subtle, temporally regulated, or influenced by interaction with the host immune system.
Published ahead of print 19 February 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00743-12.