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pH-responsive transcription factors of the Rim101/PacC family govern virulence in many fungal pathogens. These family members control expression of target genes with diverse functions in growth, morphology, and environmental adaptation, so the mechanistic relationship between Rim101/PacC and infection is unclear. We have focused on Rim101 from Candida albicans, which we find to be required for virulence in an oropharyngeal candidiasis (OPC) model. Rim101 affects the yeast-hyphal morphological transition, a major virulence requirement in disseminated infection models. However, virulence in the OPC model is independent of the yeast-hyphal transition because it is unaffected by an nrg1 mutation, which prevents formation of yeast cells. Here we have identified Rim101 target genes in an nrg1Δ/Δ mutant background and surveyed function using an overexpression-rescue approach. Increased expression of Rim101 target genes ALS3, CHT2, PGA7/RBT6, SKN1, or ZRT1 can partially restore pathogenic interaction of a rim101Δ/Δ mutant with oral epithelial cells. Four of these five genes govern cell wall structure. Our results indicate that Rim101-dependent cell wall alteration contributes to C. albicans pathogenic interactions with oral epithelial cells, independently of cell morphology.
Candida albicans is the major invasive fungal pathogen of humans, with the capacity to cause both disseminated and mucosal infection. Distinct risk factors govern susceptibility to each type of infection (Enoch et al., 2006, Edmond et al., 1999). Hence, it is probable that different attributes of C. albicans may be critical for virulence in each type of infection. Numerous fungal genes and regulatory pathways that contribute to the capacity of C. albicans to cause disseminated infections have been defined through mutant analysis with animal models and, increasingly, with in vitro cell or tissue interaction systems (Ramirez et al., 2007, MacCallum et al., 2006, Schaller et al., 2005, Calera et al., 1999). Much less is known about the fungal determinants required for mucosal infection.
Our focus here is oropharyngeal candidiasis (OPC), a candidal infection of the oral mucosa. We have recently employed in vitro epithelial cell interaction assays to identify steps in host cell-pathogen interaction that may be critical for establishment of OPC. These steps include the binding of C. albicans to oral epithelial cells, endocytosis of C. albicans, and the resulting loss of epithelial cell integrity, or damage (Park et al., 2005). The overall ability to cause epithelial cell damage in vitro has served empirically as a predictor of the capacity of C. albicans strains to cause OPC in an immunosuppressed mouse infection model. Such analysis led to our finding that the C. albicans protein kinases Tpk2 and Cka2 are required for virulence during OPC (Chiang et al., 2007, Park et al., 2005). Interestingly, Tpk2 and Cka2 have minor roles in virulence during hematogenously disseminated candidiasis (Chiang et al., 2007, Park et al., 2005). These findings support the significance of the in vitro epithelial cell interaction assays and illustrate that distinct C. albicans genes govern virulence in the context of OPC.
Among the major regulators of the capacity of C. albicans to cause disease during a disseminated infection are the transcription factors Rim101 and Nrg1 (Davis et al., 2000a, Saville et al., 2003). Rim101 is a member of the Rim101/PacC family of C2H2 zinc finger transcription factors that govern gene expression responses to extracellular pH (Penalva et al., 2004). Rim101/PacC family members have diverse roles in virulence of fungal pathogens. In mice, C. albicans Rim101 and Aspergillus nidulans PacC are required for virulence during disseminated candidiasis and invasive pulmonary aspergillosis, respectively (Davis et al., 2000a, Bignell et al., 2005). In plants, Sclerotinia sclerotiorum Pac1 and Colletotrichum acutatum Klap2, a Rim101 homolog, are required for pathogenicity (Rollins, 2003, You et al., 2007). However, Ustilago maydis Rim101 has no detectable role in plant infection, and Fusarium oxysporum PacC functions as a negative regulator of virulence in plant infection (Arechiga-Carvajal et al., 2005, Caracuel et al., 2003). Thus the precise roles of Rim101/PacC family members in infection vary significantly.
Nrg1 is also a C2H2 zinc finger transcription factor. C. albicans Nrg1 is a negative regulator of hyphal formation under in vitro and in vivo growth conditions (Braun et al., 2001, Murad et al., 2001, Saville et al., 2003). The ability to form hyphae is a critical C. albicans pathogenicity trait in disseminated infection models (Saville et al., 2003, Lo et al., 1997), so one might expect an nrg1Δ/Δ mutant to be hypervirulent. However, the mutant is attenuated in a disseminated infection model (Murad et al., 2001). These findings suggest that the capacity to produce yeast form cells, as well as hyphae, is required for disseminated infection. In fact, a recent study showed that although hyphae are required for the mortality of a C. albicans infection, it is the yeast form cells that are most important for disseminating the infection (Saville et al., 2003).
In one context, Rim101 and Nrg1 have opposite functions: Rim101 promotes hyphal formation in response to neutral or alkaline growth conditions; Nrg1 inhibits hyphal formation under all conditions. In keeping with these observations, of seven hyphal-specific genes that are under positive control by Rim101, four are also under negative control by Nrg1 (Bensen et al., 2004). However, Bensen et al. have argued that Rim101 and Nrg1 act in parallel hyphal regulatory pathways (Bensen et al., 2004). This inference was based on the finding that C. albicans Rim101 does not repress NRG1 expression, in contrast to the situation in S. cerevisiae (Lamb et al., 2003). In addition, a rim101Δ/Δ nrg1Δ/Δ double mutant was slightly less filamentous than an nrg1Δ/Δ single mutant in M199 medium at pH 8. The existence of Rim101 targets that are independent of Nrg1 is substantiated by microarray analysis (Bensen et al., 2004). For example, of ten cell wall genes that are under positive control by Rim101, only one is under negative control by Nrg1. Examination of Rim101 targets that are independent of Nrg1 may yield mechanistic insight into Rim101 functions that are independent of hyphal formation.
In this study, we examine the relationship between Rim101 and Nrg1 in OPC infection models. Our results indicate that the role of Rim101 in the pathogenesis of OPC is independent of Nrg1, thus prompting a focus on Rim101 target genes that are independent of Nrg1. Our findings point to the cell surface as a major mediator of the role of Rim101 in pathogenicity during OPC.
Rim101 and Nrg1 are each required for virulence of C. albicans in disseminated infection models (Davis et al., 2000a, Saville et al., 2003, Braun et al., 2001, Murad et al., 2001). To determine whether they may also have roles in virulence during OPC, we investigated the capacity of single and double mutant strains to damage the FaDu oral epithelial cell line (Figure 1A), a trait that is correlated with virulence in a mouse OPC model (Park et al., 2005, Chiang et al., 2007). The rim101Δ/Δ mutant caused little epithelial cell damage compared to either the wild-type or rim101Δ/Δ + pRIM101 complemented strain. In contrast, the nrg1Δ/Δ mutant caused similar levels of epithelial cell damage to the wild-type and complemented strains. To investigate potential interactions of RIM101 and NRG1, we measured the extent of epithelial cell damage induced by a rim101Δ/Δ nrg1Δ/Δ double mutant. This strain caused almost no detectable damage to the epithelial cells (Figure 1B). Complementing this mutant with a wild-type copy of RIM101, but not NRG1, restored its capacity to damage epithelial cells to same level as the wild-type strain. These results indicate that RIM101 is required, independently of NRG1, for C. albicans to damage oral epithelial cells in vitro.
In several in vitro infection models, host cell damage capacity correlates with C. albicans morphology (Phan et al., 2000, Park et al., 2005, Villar et al., 2004). Thus we examined the morphology of each strain after a 3-hour incubation on oral epithelial cells. As expected, the wild-type strain formed true hyphae on these cells (Figure 2A) (Park et al., 2005, Chiang et al., 2007). The rim101Δ/Δ strain formed a mixture of true hyphae and pseudohyphae that were slightly shorter than those of the wild-type strain (Figure 2B). As expected, the nrg1Δ/Δ strain grew as pseudohyphae even under normally non-hyphal inducing conditions (data not shown). On the epithelial cells, the pseudohyphae of this strain were considerably longer and contained more branches than the true hyphae of the wild-type strain (Figure 2C). The rim101Δ/Δ nrg1Δ/Δ double mutant formed pseudohyphae that were highly branched and slightly shorter than those of the nrg1Δ/Δ mutant, but longer than the hyphae of the wild-type strain (Figure 2D). Complementation of each single mutant restored the wild-type phenotype (Figure 2E, 2H). Also, introduction of a wild-type RIM101 allele into the rim101Δ/Δ nrg1Δ/Δ double mutant produced a phenotype similar that of the nrg1Δ/Δ mutant (Figure 2G); introduction of a wild-type NRG1 allele into the rim101Δ/Δ nrg1Δ/Δ double mutant yielded a phenotype similar that of the rim101Δ/Δ mutant (Figure 2F). Thus it is possible that the altered morphology of the rim101Δ/Δ strain may contribute to its host damage defect. However, the finding that pseudohyphae of the nrg1Δ/Δ single mutant damaged epithelial cells, whereas pseudohyphae of the rim101Δ/Δ nrg1Δ/Δ double mutant did not suggests that RIM101 is required for host cell damage independently of its role in cell morphology.
C. albicans invades oral epithelial cells in vitro by inducing its own endocytosis (Drago et al., 2000, Park et al., 2005, Chiang et al., 2007), and cell damage is closely associated with the endocytosis of live organisms (Park et al., 2005). Therefore, we investigated whether RIM101 is required for epithelial cell damage because it is required to induce endocytosis. These experiments were performed using the rim101Δ/Δ nrg1Δ/Δ double mutant and two complemented strains (a rim101Δ/Δ nrg1Δ/Δ + pRIM101 strain and a rim101Δ/Δ nrg1Δ/Δ + pNRG1 strain). Significantly fewer cells of the rim101Δ/Δ nrg1Δ/Δ double mutant were endocytosed by the epithelial cells compared to those of the wild-type strain (Figure 3A). Introduction of a wild-type copy of RIM101, but not NRG1, into the rim101Δ/Δ nrg1Δ/Δ double mutant increased its capacity to induce endocytosis to greater than the wild-type levels. Therefore, RIM101 is necessary for C. albicans to induce maximal endocytosis by oral epithelial cells. Furthermore, NRG1 is a negative regulator of C. albicans invasion of epithelial cells.
We also measured the number of organisms that were associated with the epithelial cells, which was defined as the sum of the adherent and endocytosed organisms (Figure 3B). Over 2-fold more cells of the rim101Δ/Δ nrg1Δ/Δ double mutant were cell-associated compared to the wild-type strain, despite the finding that the double mutant is defective in inducing endocytosis. Introduction of either RIM101 or NRG1 wild-type alleles caused significant reduction in cell association. However, the number of cell-associated organisms of either complemented strain was still greater than that of the wild-type strain (Figure 3B). These observations indicate that both RIM101 and NRG1 are negative regulators of C. albicans adherence, and that and adherence defect is not the cause of the rim101Δ/Δ-associated endocytosis defect.
The virulence of the rim101Δ/Δ nrg1Δ/Δ double mutant and its two complemented strains was tested in a mouse model of OPC. Mice infected with the rim101Δ/Δ nrg1Δ/Δ double mutant had significantly lower oral fungal burden than did mice infected with the wild-type strain (Figure 4). Complementing this double mutant with a wild-type copy of RIM101 restored its virulence to that of the wild-type strain. However, complementing the double mutant with a wild-type allele of NRG1 did not increase its virulence. These results indicate that RIM101 is required for virulence in this model.
The relative extent of oropharyngeal disease in mice infected with the various strains was also assessed by histopathologic examination. Mice infected with the wild-type strain had large oral lesions containing extensive fungal elements (Figure 5A). In contrast, mice infected with the rim101Δ/Δ nrg1Δ/Δ double mutant had much smaller oral lesions that contained proportionally fewer organisms (Figure 5B), in agreement with our quantitative fungal burden determinations (Figure 4). Complementation with RIM101 resulted in large oral lesions that were similar to those induced by the wild-type strain (Figure 5C), whereas complementation with NRG1 resulted in lesions that resembled those induced by the rim101Δ/Δ nrg1Δ/Δ double mutant (Figure 5D). These results indicate that RIM101, but not NRG1, is essential for maximal virulence during OPC.
We used microarray analysis to identify Rim101 target genes that may mediate virulence in the OPC model. Specifically, we identified Rim101-dependent genes in an nrg1Δ/Δ mutant background by comparing gene expression profiles of the rim101Δ/Δ nrg1Δ/Δ double mutant and the rim101Δ/Δ nrg1Δ/Δ + pRIM101 complemented strain (Supplemental data, “All genes” worksheet). We found 366 genes whose expression levels varied more than 1.5-fold between these strains (Supplemental data, “Rim101 regulated” worksheet). Rim101-dependent expression of several genes was verified by independent methods (Supplemental Figure 1). Thirty six of these genes were identified as Rim101-responsive in a previous comparison in an NRG1 background (Bensen et al., 2004) (Supplemental data, “Shared Rim101 regulated” worksheet). We identified many additional Rim101-responsive genes (Supplemental data, “New Rim101 regulated” worksheet), including SAP5, SOD5, CHT2, ALS3, CSA2, and SKN1. Many of these new genes extend known Rim101-regulated gene categories (Bensen et al., 2004), with functional connections to the cell wall, transport, and amino acid synthesis (see examples in Table 2).
We considered the hypothesis that Rim101 promotes virulence through activation of specific target genes. To test this hypothesis, we overexpressed individual Rim101-dependent genes by fusion to the strong TDH3 promoter in a rim101Δ/Δ mutant. We chose nine of the most highly Rim101-dependent genes, including CFL2/FRE2, CHT2, CSA1, ECE1, FAA2, PGA7/RBT6, SAP5, SKN1, and ZRT1. We also chose ALS3, because we had observed that Rim101 is required for N-cadherin binding to C. albicans(Phan, Filler, and Mitchell, unpublished data), a known function of Als3 (Phan et al., 2007). Increased expression of each gene was verified by RTPCR or flow cytometry (Supplemental Figures 2 and 3). We tested each target gene overexpressing (TGO) strain for characteristic rim101Δ/Δ mutant phenotypes, including lack of growth on YPD medium containing LiCl, lack of growth on YPD pH 9.0 medium, and lack of filamentation on M199 pH 8.2 medium. All TGO strains behaved similarly to the rim101Δ/Δ mutant in these assays (data not shown). Therefore, no single target gene tested is responsible for these in vitro phenotypes.
We also tested each TGO strain in the damage and endocytosis assays; positive results were replicated with an independent TGO isolate. Overexpression of ECE1, CSA1, SAP5, or CFL2/FRE2 had no effect on the rim101Δ/Δ defects (data not shown). However, overexpression of ALS3, CHT2, or SKN1 in the rim101Δ/Δ background resulted in greater epithelial cell damage (Figure 6A). In addition, overexpression of ALS3, CHT2, PGA7/RBT6, or ZRT1 consistently caused increased endocytosis by epithelial cells (Figure 7). These results argue that ALS3, CHT2, PGA7/RBT6, SKN1, and ZRT1 contribute to the Rim101-dependent pathogenic interactions with epithelial cells.
There is growing evidence that cell wall perturbation may have complex effects on fungal pathogens that result in hypervirulence (Kamran et al., 2004, MacCallum et al., 2006, Wheeler et al., 2006). We considered this explanation for our results because several of the functional Rim101 target genes affect the cell surface. Thus we investigated whether overexpression of these Rim101 target genes in the wild-type strain influenced the extent of epithelial cell damage. Increased expression of CHT2and SKN1 in DAY185 caused a 3% and 5% increase in epithelial cell damage, respectively (Figure 6B). The magnitude of this increase in damage was much less than occurred when these genes were overexpressed in the rim101Δ/Δ mutant. Furthermore, overexpression of ALS3 in the wild-type strain had no effect on epithelial cell damage, even though overexpression of this gene in the rim101Δ/Δ mutant resulted in a significant increase in damage. Collectively, these results support the model that reduced epithelial cell damage caused by the rim101Δ/Δ mutant is due in part to its reduced expression of ALS3, CHT2, and SKN1.
Transcription factors have been viewed with both delight and disdain for their potential in elucidating the mechanistic basis of such complex traits as pathogenicity. In the study of C. albicans, this dichotomy came to the foreground with studies of Efg1 and Cph1. These transcription factors are required for both hyphal formation and pathogenicity, thus linking cell morphogenesis to virulence (Lo et al., 1997, Phan et al., 2000). However, the more skeptical view was that transcription factors are expected to have multiple target genes, and some Efg1/Cph1 targets might be required for pathogenicity independently of any cellular morphogenetic feature. Indeed, subsequent studies have shown that several Efg1/Cph1-dependent genes that are required for virulence are not required for hyphal morphogenesis (Staib et al., 2002, Felk et al., 2002, Korting et al., 2003, Lane et al., 2001). On the other hand, the utility of studying Efg1 and Cph1 has also been illustrated by these subsequent studies, precisely because so many Efg1/Cph1 target genes have roles in virulence.
Our current study of Rim101 and Nrg1 illustrates some useful features of a focus on transcription factors. First, because Rim101 is required for pathogenicity in the OPC model and Nrg1 is not, we could concentrate on Rim101 target genes in an nrg1Δ/Δ mutant background, thus avoiding a focus on morphogenesis-related genes. In addition, our approach may have circumvented genetic redundancy. For gene families, deletion of several family members may be necessary to cause an altered phenotype. Many Rim101 target genes are in gene families, including ALS, SAP, FRE/CFL, CHT, and RBT5-related genes. The phenotypic impact of overexpressed genes may have resulted from the fact that Rim101 is required for expression of several members of each gene family. In terms of pathogenicity, analysis of these transcription factor mutants has led to new connections between target genes and phenotype.
Nrg1 is required for virulence in a disseminated infection model, a fact established with two independent nrg1Δ/Δ strains in two different labs (Murad et al., 2001, Braun et al., 2001). The constitutive pseudohyphal formation is likely a major factor in the reduced virulence of nrg1Δ/Δ strains when they are inoculated into mice via the tail vein. The nrg1Δ/Δ pseudohyphae probably lodge in the pulmonary capillaries where they are killed by the resident macrophages before they can be carried by the blood to the kidney, the usual target organ in mice with hematogenously disseminated candidiasis. Aberrant trafficking of the nrg1Δ/Δ strain did not occur in our model of OPC because all strains were applied directly to the oral mucosa. We assayed virulence through both fungal burden and histopathology, which gave consistent results. This finding correlated well with our discovery that an nrg1Δ/Δ strain had no defect in damaging an oral epithelial cell line. Thus far, only one other gene, IRS4, is required for disseminated candidiasis but not OPC (Badrane et al., 2005).
Two genes, CKA2 and TPK2, are required for virulence during OPC but not disseminated candidiasis, the converse of NRG1 (Chiang et al., 2007, Park et al., 2005). There are numerous differences between the infection sites in these two diseases, so it seems reasonable to expect genes of these two classes. In the disseminated candidiasis model, mutants locked in either the yeast or filamentous growth forms are attenuated (Navarro-Garcia et al., 2001). This finding indicates that conversion between the forms is required for virulence in the disseminated infection model. In the OPC model, the nonfilamentous efg1Δ/Δ mutant is attenuated (Park et al., 2005), thus arguing that filamentous growth forms are required for OPC. However, our findings with the hyperfilamentous nrg1Δ/Δ mutant suggest that the yeast growth form is not required for virulence in the OPC model.
Many of the most highly regulated Rim101-dependent genes in our data set are hyphal-specific genes, including SOD5, CSA2, PGA7/RBT6, ECE1, CSA1, SAP5, HYR1, and RBT5. This result is consistent with the previous finding that Rim101 is required for hyphal-specific gene expression (Bensen et al., 2004). Our microarray results also demonstrate that Rim101 governs expression of at least some of these genes independently of Nrg1, in keeping with prior studies (Liu, 2001, Bensen et al., 2004).
Our data identified 175 new Rim101-dependent genes. Ninety-one new genes may be direct Rim101 targets because their 5′ regions contain between one and six Rim101 binding site core sequences (CCAAG (Bensen et al., 2004, Ramon et al., 2003)). Several additional sequences are overrepresented among all Rim101-dependent gene 5′ regions (1.5 Kb) in our dataset, as determined by RSAT analysis (van Helden et al., 1998, van Helden et al., 2000), including GGTTAA, CAAGAA, and TCGTCA (see examples in Table 2). In many cases, the CAAGAA is part of a Rim101 consensus site (CCAAGAA (Bensen et al., 2004, Ramon et al., 2003)), but the other sequences may represent binding sites for Rim101-dependent transcription factors that mediate indirect regulation.
We also found 155 new Rim101-repressed genes, out of a total of 164 Rim101-repressed genes in our dataset. Ninety-one of these genes have one or more 5′ region Rim101 binding site core sequences and thus may be direct targets. The sequences AATTGC and TGAAAA are overrepresented in the repressed gene 5′ regions (see examples in Table 2), and may be binding sites for mediators of Rim101 regulation.
Overexpression of any of five genes, ALS3, CHT2, PGA7/RBT6, SKN1, and ZRT1, partially rescued the epithelial cell interaction defects of the rim101Δ/Δ mutant. The extent of epithelial cell damage caused by C. albicans is closely related to the number of fungal cells that are endocytosed by the epithelial cells (Chiang et al., 2007, Park et al., 2005). However, our current data suggest that damage and endocytosis can be dissociated from one another because overexpression of SKN1 restored epithelial cell damage, but not endocytosis. Conversely, overexpression of PGA7/RBT6 and ZRT1 partially restored endocytosis, but not cell damage. Further evidence of the dissociation between endocytosis and damage was seen with the rim101Δ/Δ nrg1Δ/Δ+pRIM101 strain. Although this strain was endocytosed by epithelial cells more avidly than the wild-type strain, it caused less epithelial cell damage. A possible explanation for these results is that the different strains have different surface characteristics and therefore bind to different epithelial cell receptors. These disparate receptors mediate endocytosis into different intracellular compartments within the epithelial cell, which influences the extent of epithelial cell damage.
These findings argue that cell surface defects are a key component of the rim101Δ/Δ virulence defect, because four of the five Rim101 target genes that affect epithelial cell interactions govern cell wall features. Two of the genes that increased cell damage, CHT2 and SKN1, have roles in cell wall polysaccharide structure: Cht2 is a chitinase, and Skn1 is a putative glucosidase that is implicated by homology in β-1,6-glucan synthesis (McCreath et al., 1996, Selvaggini et al., 2004, Mio et al., 1997). Two of the genes that enhanced endocytosis, ALS3 and PGA7/RBT6, specify known or predicted GPI-linked cell surface proteins (De Groot et al., 2003, Phan et al., 2007, Hoyer et al., 1998). Als3 is known to function as an adhesin/invasin in several contexts (Zhao et al., 2004, Nobile et al., 2006, Phan et al., 2007), in keeping with the conclusion from our overexpression analysis here. Pga7/Rbt6 has not been recognized previously as a prospective invasin. It has significant similarity to several other Rim101-regulated cell wall proteins, including Rbt5, Pga10, Csa2, and Csa1 (Bensen et al., 2004), all of which share the cysteine-rich CFEM domain (Kulkarni et al., 2003). Most of these proteins are small compared to known adhesins, but Rbt5, Pga10, and Csa1 have all been implicated in biofilm adherence (Nobile et al., 2006, Perez et al., 2006). Our hypothesis is that Rim101 governs multiple cell surface features that contribute to host cell interaction both directly, as adhesins (Als3, Pga7/Rbt6), and indirectly, through cell wall modifications that may improve secretion of cell damage factors or survival after endocytosis (Cht2, Skn1).
Zrt1 is a putative low-affinity zinc transporter that is predicted to be a transmembrane protein (Braun et al., 2005). The effect of Zrt1 on C. albicans endocytosis may be indirect as zinc is required for the function of numerous enzymes and transcription factors. However, Zrt1 biological function is also connected to the cell wall, as several secretory pathway activities (protein folding, GPI anchor addition, proteolytic processing) are zinc-dependent (Eide, 2006). Thus Zrt1 overexpression may ultimately affect the same aspect of host interaction that is affected in the other TGO strains.
It was notable that SAP5 overexpression did not cause increased epithelial cell damage in this study. Prior studies have shown that SAP5 overexpression rescues a rim101Δ/Δ defect in another surrogate virulence model, an assay for invasion of reconstituted human epithelium (Villar et al., 2007). Villar et al. showed that Sap5 is critical for E-cadherin degradation, which in turn disrupts epithelial tissue integrity. Degradation required at least four hours of incubation with C. albicans in that system, whereas our epithelial cell damage assays are completed within three hours. Thus it is reasonable that different contributions to virulence would be most readily detectable in these two systems.
Virulence of C. albicans is thought to be multifactorial (Cutler, 1991), and it is not surprising that a transcriptional regulator like Rim101 governs several virulence mechanisms. Adhesins and secreted proteases are well recognized participants in host cell interaction (Navarro-Garcia et al., 2001). Our studies also point to new aspects of this interaction: the roles of small CFEM proteins and of cell wall polysaccharide. Three functionally significant target genes, CHT2, SKN1, and PGA7/RBT6, have 5′ Rim101 binding sites (Bensen et al., 2004, Ramon et al., 2003). We suggest that the effects of Rim101 on adherence and damage reflect direct roles of the Rim101 pathway in virulence.
All C. albicans strains used in this study were derived from BWP17 (Wilson et al., 1999) and are listed in Table 1. Primer sequences from 5′ to 3′ are listed in Supplemental data, “Primer sequences” worksheet. Construction of DAY25 (rim101Δ/Δ) (Davis et al., 2000a), DAY5 (Davis et al., 2000b), DAY44 (rim101Δ/Δ +pRIM101) (Davis et al., 2000b), and DAY185 (reference strain) (Davis et al., 2000b) was described previously. Construction of CJN649 (nrg1Δ/Δ) was made by PCR-product-directed gene deletion (Wilson et al., 1999) with 100-mer oligonucleotides NRG1null-5DR and NRG1null-3DR via consecutive rounds of transformation into BWP17. For gene complementation, PCR was used to generate a fragment for NRG1 from 1000 bp upstream of the start codon to 500 bp downstream of the stop codon. This fragment was inserted into the pGEMT-Easy vector (Promega), digested with NgoMIV and AlwNI, and subsequently inserted by in vivo recombination in S. cerevisiae into NotI- and EcoRI-digested HIS1 vector pDDB78 (Spreghini et al., 2003), yielding plasmid pCJN104. The complemented strain CJN706 was made by transforming CJN649 with Nru1-digested plasmid pCJN104, directing integration to the HIS1 locus. The nrg1Δ/Δ mutant strain was made His+ by transforming CJN649 with Nru1-digested pDDB78 to yield strain CJN721. Strain CJN759 (rim101Δ/Δ nrg1Δ/Δ double mutant) was generated via consecutive rounds of transformation into VIC18 (rim101Δ;::dpl200/rim101Δ;::dpl200) (Davis et al., 2002) using NRG1null-5DR and NRG1null-3DR oligonucleotides. CJN793, the NRG1 complemented strain in the rim101Δ/Δ nrg1Δ/Δ double mutant, was made by transforming CJN759 with Nru1-digested plasmid pCJN104. CJN783, the RIM101 complemented strain in the rim101Δ/Δ nrg1Δ/Δ double mutant, was made by transforming CJN759 with Nru1-digested plasmid pDDB61 (Davis et al., 2000b). CJN775, the His+ rim101Δ/Δ nrg1Δ/Δ double mutant was made by transforming CJN759 with Nru1-digested pDDB78. C. albicans transformants were selected for on synthetic medium (2% dextrose, 6.7% yeast nitrogen base with ammonium sulfate, and auxotrophic supplements). Genotypes were verified by colony PCR.
The TDH3 promoter was chosen for overexpression analysis because it was found to be highly and constitutively expressed during epithelial cell damage in a previous array experiment (Filler, unpublished data). The NAT1-TDH3 promoter plasmid pCJN542 (for gene overexpression) was generated as follows. PCR was done using primers TDH3-Fpro-SpeI and TDH3-Rpro-NdeI to generate an 800 bp product containing the C. albicans TDH3 promoter (abbreviated TDH3p) with flanking NdeI and SpeI restriction sites around the promoter. This PCR fragment was digested with NdeI and SpeI and ligated into NdeI- and SpeI-digested plasmid pCJN498 (Nobile et al., 2006) to create pCJN542 containing the Ashbya gossypii TEF1 promoter (abbreviated TEF1p) next to the C. albicans NAT1 ORF, followed by the A. gossypii TEF1 terminator, followed by the C. albicans TDH3p in the correct orientation. The TDH3-ZRT1 overexpression strains, CJN1364 and CJN1365, were constructed by transforming CJN793, the NRG1 complemented strain in the rim101Δ/Δ nrg1Δ/Δ double mutant, using PCR products from template plasmid pCJN542 and primers ZRT1-F-OE-Ag-NAT-Ag-TEF1p-CJN and ZRT1-R-OE-Ag-NAT-Ag-TDH3p-CJN. These primers amplify the entire A. gossypii TEF1p, the C. albicans NAT1 ORF, the A. gossypii TEF1 terminator, and the C. albicans TDH3p with 100 bp of hanging homology to 500 bp upstream into the promoter of ZRT1 for the forward primer and 100 bp of hanging homology from exactly the start codon of ZRT1. The homology in these primers allows for homologous recombination of the entire cassette directly upstream of the natural locus of ZRT1 so that its expression is driven by the TDH3p instead of its natural promoter. By the same method, primers ECE1-F-OE-Ag-NAT-Ag-TEF1p and ECE1-R-OE-Ag-NAT-Ag-TDH3p-CJN were used for overexpression of ECE1 to produce strains CJN1371 and CJN1372; SAP5-F-OE-Ag-NAT-Ag-TEF1p and SAP5-R-OE-Ag-NAT-Ag-TDH3p-CJN for overexpression of SAP5 (strains CJN1379 and CJN1380); CHT2-F-OE-Ag-NAT-Ag-TEF1p and CHT2-R-OE-Ag-NAT-Ag-TDH3p-CJN for overexpression of CHT2 (strains CJN1387 and CJN1388); SKN1-F-OE-Ag-NAT-Ag-TEF1p-CJN and SKN1-R-OE-Ag-NAT-Ag-TDH3p-CJN for overexpression of SKN1 (strains CJN1395 and CJN1396); CSA1-F-OE-Ag-NAT-Ag-TEF1p-CJN and CSA1-R-OE-Ag-NAT-Ag-TDH3p-CJN for overexpression of CSA1 (strains CJN1403 and CJN1404); PGA7-F-OE-Ag-NAT-Ag-TEF1p-CJN and PGA7-R-OE-Ag-NAT-Ag-TDH3p-CJN for overexpression of PGA7/RBT6 (strains CJN1411 and CJN1412); CFL2-F-OE-Ag-NAT-Ag-TEF1p-CJN and CFL2-R-OE-Ag-NAT-Ag-TDH3p-CJN for overexpression of CFL2 (strains CJN1419 and CJN1420); FAA2-F-OE-Ag-NAT-Ag-TEF1p-CJN and FAA2-R-OE-Ag-NAT-Ag-TDH3p-CJN for overexpression of FAA2 (strains CJN1427 and CJN1428); and ALS3-F-OE-Ag-NAT-Ag-TEF1p and ALS3-R-OE-Ag-NAT-Ag-TDH3p for overexpression of ALS3 (strains CJN1321 and CJN1322). Each overexpression strain was produced by an independent transformation. The transformation into C. albicans strains and selection on YPD+clonNAT (2% Bacto Peptone, 2% dextrose,1% yeast extract, and 400 μg/mL clonNAT (Werner BioAgents) plates was done as previously described (Nobile et al., 2006). Correct integration of the constructs was verified by colony PCR using a forward detection primer annealing to a sequence within the promoter of each gene of interest for each gene (Supplemental data, “Primer sequences” worksheet) in combination with the reverse primer Nat-OE-R-det2-CJN annealing to a sequence found in the NAT gene. Function of this overexpression strategy was verified for ZRT1, ECE1, SAP5, CHT2, SKN1, CSA1, PGA7/RBT6, CFL2/FRE2, and FAA2 by real-time RT-PCR (Supplemental Figure 2) and for ALS3 by flow cytometry (Supplemental Figure 3).
Prior to use in the experiments, all strains were grown overnight in liquid YPD medium in a shaking incubator at 30°C. The organisms were harvested by centrifugation, washed twice in phosphate buffered saline (PBS), resuspended in RPMI 1640 medium (Irvine Scientific). They were sonicated briefly to produce singlet cells and then counted using a hemacytometer.
Hyphal induction assays were done in liquid M199 medium (Invitrogen), pH 8.0 at 37°C. To produce germ tubes, yeast form cells were incubated in RPMI 1640 medium at 37°C in a shaking incubator for 90 min for the epithelial cell damage assays or for 75 min for the flow cytometry assays. For phenotypic characterization, strains were grown overnight in YPD at 30°C, and OD600 values were determined. Solid media was prepared with the addition of 2% Bacto-agar. To analyze the response of the strains to lithium and alkaline pH, 5 μL of the overnight culture was spotted onto plates of YPD, YPD + 150 mM LiCl, YPD + 150 mM HEPES buffered at pH 9.0, and M199 medium + 150 mM HEPES buffered at pH 8.2; and streaked for singles on the plate. Plates were grown at 37°C for 2 days for all assays, except M199 medium, which was grown at 37°C for 5 days.
The FaDu oral epithelial cell line, which was originally derived from a pharyngeal carcinoma, was obtained from the ATCC (ATCC Number HTB-43). The cells were grown in MEM Earl’s salts (Irvine Scientific) containing 10% fetal bovine serum, 1 mM pyruvic acid, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 100 IU/ml penicillin, and 100 IU/ml streptomycin. All experiments were performed using cells at 90% confluency and incubated in 5% CO2 at 37°C.
The extent of damage to epithelial cells caused by the various C. albicans strains was determined using a chromium release assay as described previously (Park et al., 2005, Martinez-Lopez et al., 2006, Chiang et al., 2007). The epithelial were grown in 96-well tissue culture plates, loaded with 51Cr and infected with 105 organisms per well. When the overexpression strains were tested in this assay, they were added to the host cells as germ tubes because we found that there was stronger expression of the Rim101 target genes in germ tubes (data not shown). After 3 hr of incubation, the medium and cells were collected, and their respective 51Cr content was measured by γ-counting. Wells containing uninfected epithelial cells were processed in parallel to determine the spontaneous release of 51Cr. After correcting for well-to-well differences in the incorporation of 51Cr, the percent specific release of 51Cr was calculated using the following formula: (experimental release - spontaneous release)/(total incorporation - spontaneous release). Experimental release was the amount of 51Cr released into the medium by epithelial cells infected with C. albicans. Spontaneous release was the amount of 51Cr released into the medium by uninfected epithelial cells. Total incorporation was the sum of the amount of 51Cr released into the medium and remaining in the epithelial cells. Each assay was performed in triplicate at least three times, and differences in epithelial cell damage caused by the various strains were evaluated by analysis of variance. P values of ≤ 0.05 were considered to be significant.
The various strains were imaged using differential interference contrast to determine their morphology while growing on FaDu oral epithelial cells. The epithelial cells were grown on 12 mm diameter glass coverslips that had been coated with fibronectin and placed in a 24-well tissue culture plate. Next, 105 cells of each C. albicans strain suspended in RPMI 1640 medium were added to different wells. After 90 min of incubation, the medium above the cells was aspirated and the cells were fixed in 3% paraformaldehyde. The coverslips were mounted inverted onto slides, after which the organisms were viewed by differential interference contrast.
The capacity of each strain of C. albicans to adhere to and invade epithelial cells was measured using our previously described differential fluorescence assay (Park et al., 2005, Martinez-Lopez et al., 2006, Chiang et al., 2007). Briefly, 105 cells of each strain were added to epithelial cells that were grown on fibronectin coated glass coverslips as in the microscopy experiments. When the rim101Δ/Δ nrg1Δ/Δ strains were tested, the organisms were added to the epithelial cells in the state in which they grew in the YPD liquid culture, and the incubation period was 90 min. When the overexpression strains were tested, the organisms were added to the epithelial cells as germ tubes, and the incubation period was 45 min. At the end of the incubation period, the cells were rinsed twice with Hank’s balanced salt solution (HBSS; Irvine Scientific) in a standardized manner and then fixed with 3% paraformaldehyde. The adherent, but non-endocytosed organisms were labeled with rabbit polyclonal anti-C. albicans antibodies (Biodesign International) that had been conjugated with the red fluorescent dye, Alexa 568 (Invitrogen). Next, the cells were permeablized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS, and the cell-associated organisms (the endocytosed plus non-endocytosed organisms) were labeled with anti-C. albicans antibodies conjugated with the green fluorescent dye, Alexa 488 (Invitrogen). The coverslips were viewed by epifluorescence and the number endocytosed organisms was determined by subtracting the number of non-endocytosed organisms (which fluoresced red) from the number of cell-associated organisms (which fluoresced green). At least 100 organisms were examined on each coverslip and the results were expressed as number of endocytosed or cell-associated organisms per high-powered field. An organism was considered to be endocytosed when all or part of it was internalized by an epithelial cell. Each assay was performed in triplicate at least three times, and differences among the various strains were evaluated by analysis of variance. P values of ≤ 0.05 were considered to be significant.
To assess the virulence of the various strains of C. albicans, the mouse model of OPC was used (Park et al., 2005, Chiang et al., 2007). This study was approved by the Animal Use Committee of the Los Angeles Biomedical Institute in compliance with NIH guidelines for the ethical treatment of animals. Male Balb/c mice (National Cancer Institute) weighing approximately 20 gm were immunosuppressed with cortisone acetate (Sigma-Aldrich) at a dose of 225 mg/kg administered subcutaneously on days - 1, +1 and +3 relative to the day of infection. To induce OPC, the mice were anesthetized with xylazine and ketamine (both from Phoenix pharmaceuticals) administered intraperitoneally. Next, calcium alginate urethral swabs (Type 4 Calgiswab; Puritan Medical Products Company LLC) were saturated with C. albicans by placing them in HBSS containing 106 organisms per ml. The saturated swabs were placed sublingually in the anesthetized mice for 75 min. Each strain of C. albicans was inoculated into 7 to 9 mice. After the mice recovered from anesthesia, they were given food and water ad libitum. The mice were sacrificed after 5 days of infection, after which their tongues and adjacent sublingual tissue were excised and then divided in half. One half was weighed, homogenized, and quantitatively cultured on Sabauroud dextrose agar containing chloramphenicol. The other half was used for histopathologic examination. The tissue was fixed in zinc-buffered formalin and embedded in paraffin for thin sectioning. The sections were stained with periodic acid-Schiff. Differences in oral fungal burden among mice infected with different strains were analyzed using the Wilcoxon rank sum test. P values of ≤ 0.05 were considered to be significant.
RPMI 1640 medium was inoculated with organisms from a YPD 30°C overnight culture to obtain a starting OD600 of 0.05 and was incubated at 37°C. Cells were harvested by vacuum filtration when the OD600 was 1. RNA was isolated using a hot-phenol method as previously described (Spellman et al., 1998) for Northern analysis, and using the RiboPure-Yeast RNA extraction kit (Ambion) as per the manufacturers instructions for microarray, RT-PCR, and real time RT-PCR analyses. Northern analysis was performed as described previously to verify the expression levels of CHT2, ECE1, and RBT5 (Nobile et al., 2005). RT-PCR analysis was performed as described previously (Nobile et al., 2005) to verify the expression levels of SAP5, SAP6, and ZRT1 using the primers SAP5F195 and SAP5R1075 for SAP5; SAP6F614 and SAP6R1197 for SAP6; and ZRT1FATG and ZRT1RUTR for ZRT1 listed in Supplemental data, “Primer sequences” worksheet. Eleven 2-fold dilutions of cDNA were used for this analysis, as well as a no RT control. Flow cytometry was used to verify the surface expression levels of ALS3, as previously described (Phan et al., 2007). For quantitative real time RT-PCR analysis, 10 μg total RNA was DNase treated at 37°C for 1 hr using the DNA-free kit (Ambion), cDNA was synthesized using the AffinityScript multiple temperature cDNA synthesis kit (Stratagene), and quantitative real time RT-PCR was done using the iQ SYBR Green Supermix (Bio-Rad) as previously described (Norice et al., 2007) using the primers ZRT1FATG and ZRT1RUTR for ZRT1; ECE1-F-qRT-CJN and ECE1-R-qRT-CJN for ECE1; SAP5F195 and SAP5R1075 for SAP5; CHT2 FWD PR and CHT2 REV PR for CHT2; SKN1-F-qRT-CJN and SKN1-R-qRT-CJN for SKN1; CSA1-F-qRT-CJN and CSA1-R-qRT-CJN for CSA1; PGA7F-RT-CJN and PGA7R-RT-CJN for PGA7/RBT6, CFL2-F-qRT-CJN and CFL2-R-qRT-CJN for CFL2/FRE2; and FAA2-F-qRT-CJN and FAA2-R-qRT-CJN for FAA2 listed in Supplemental data, “Primer sequences” worksheet. In order to control for DNA contamination, reverse transcriptase was omitted from a control set of samples. Samples were processed in triplicate, and real time RT-PCR was performed using the iCycler iQ detection system (Bio-Rad) with the following program: initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 45 sec, 58°C for 30 sec, and 72°C for 30 sec. Amplification specificity was determined by melting curve analysis. Bio-Rad iQ5 software was used to calculate normalized gene expression values using the ΔΔCt method, using TDH3 as a reference gene. For ease of interpretation, the reference strain expression level values were set to 1.0 for each target gene set, and the normalized expression of each gene relative to TDH3 expression is shown in Supplemental Figure 2.
Transcriptional profiling on long oligonucleotide microarrays was performed as previously described (Nantel et al., 2006). We conducted four individual hybridization experiments from four pairs of independently-produced RNA samples of CJN775 versus CJN793. LOWESS normalization and statistical analysis of the data was conducted in GeneSpring GX version 7.3 (Agilent Technologies). A volcano-plot algorithm was used to identify genes that exhibited statistical significance (P values < 0.05) with a change in transcript abundance of at least 1.5-fold. The results of this analysis with adjusted P values < 0.05 are listed in the Supplemental data, “Rim101 regulated” worksheet.
Flow cytometry was used to quantify the amount of Als3 expressed on the surface of the various strains. Germ tubes were produced by incubating yeast form cells of each strain in RPMI 1640 medium at 37°C for 75 min. Next, the germ tubes were fixed in 3% paraformaldehyde, blocked with 1% goat serum, and then incubated with a rabbit anti-Als3 antiserum that had been adsorbed on hyphae of an als3Δ/Δ mutant strain of C. albicans (Phan et al., 2007). After being rinsed extensively with PBS, the germ tubes were incubated with an Alexa 488-labeled goat anti-rabbit IgG secondary antibody. The cells were analyzed using a Becton Dickenson FACS calibur flow cytometer with gating on germ tubes with the same forward and side scatter characteristics. Ten thousand germ tubes of each strain were analyzed.
We thank our anonymous reviewers for useful critics and comments on this manuscript. We thank all members of the Mitchell and Filler labs for comments and advice. We are indebted to Aaron Hernday for his help in analysis of the quantitative real time RT-PCR data, to Jill Blankenship, Jason Rauceo, and Carmelle Norice for providing primers and guidance for the quantitative real time RT-PCR analysis; and to Frank Smith and Jessica Hamaker for technical assistance. We also gratefully acknowledge the assistance of Hyunsook Park and Quynh T. Phan for animal experiments. We are grateful for the availability of the Candida Genome Database, without which this work would not have been possible. This is NRC publication number 49559. This study was supported by grants 1R01DE017088 and 9R01AI070272 from the National Institutes of Health.