In vivo RNA sequencing reveals a crucial role of Fus3-Kss1 MAPK pathway in Candida glabrata pathogenicity

ABSTRACT

Candida glabrata is an important and increasingly common pathogen of humans, particularly in immunocompromised hosts. Despite this, little is known about how this fungus causes disease. Here, we applied RNA sequencing and an in vivo invasive infection model to identify the attributes that allow this organism to infect hosts. Fungal transcriptomes show a dramatic increase in the expression of Fus3 and Kss1, two mitogen-activated protein kinases (MAPKs), during invasive infection. We further demonstrate that they are both highly induced under a combination of serum and high CO₂ conditions. Deletion of both FUS3 and KSS1, but neither gene alone, results in a reduced fungal burden in organs, as well as in the gastrointestinal tract in the DSS (Dextran Sulfate Sodium)-induced colitis model. Similarly, the defect in persistence in macrophages and attenuated adhesion to epithelial cells are observed when FUS3 and KSS1 are both disrupted. The fus3 kss1 double mutant also displays defects in the induction of virulence attributes such as genes required for iron acquisition and adhesion and in the anti-fungal drug tolerance. The putative downstream transcription factors Ste12 (1), Ste12 (2), Tec1, and Tec2 are found to be involved in the regulation of these virulence attributes. Collectively, our study indicates that an evolutionary conserved MAPK pathway, which regulates mating and filamentous growth in Saccharomyces cerevisiae, is critical for C. glabrata pathogenicity.

IMPORTANCE

The MAPK signaling pathway, mediated by closely related kinases Fus3 and Kss1, is crucial for controlling mating and filamentous growth in Saccharomyces cerevisiae, but this pathway does not significantly impact hyphal development and pathogenicity in Candida albicans, a commensal-pathogenic fungus of humans. Furthermore, deletion of Cpk1, the ortholog of Fus3 in pathogenic fungus Cryptococcus neoformans, has no effect on virulence. Here, we demonstrate that the MAPK pathway is crucial for the pathogenicity of Candida glabrata, a fungus that causes approximately one-third of cases of hematogenously disseminated candidiasis in the United States. This pathway regulates multiple virulence attributes including the induction of iron acquisition genes and adhesins, as well as persistence in macrophages and organs. Our work provides insights into C. glabrata pathogenesis and highlights an example in which regulatory rewiring of a conserved pathway confers a virulent phenotype in a pathogen.

INTRODUCTION

Candida glabrata (Nakaseomyces glabratus), commonly residing in the oral and gastrointestinal regions of healthy humans, serves as a prevalent commensal organism (1). Nonetheless, it possesses the potential to transform into a pathogenic yeast, causing a range of infections from superficial to life-threatening systemic ones, with high morbidity and mortality rates. Factors such as weakened immune systems, cancer, the use of antibiotics, and the presence of intravenous catheters can increase the risk of contracting infections caused by C. glabrata (2). Among the emerging fungal pathogens, the prevalence of C. glabrata infections is on the rise, positioning it as the second most common source of candidiasis, ranking only behind Candida albicans (3). The challenge of treating infections caused by C. glabrata is increased by its natural resistance to antifungal treatments, a situation further complicated by its ability to adapt to azole-based fungistatic medications (4, 5).

Despite the rising incidence of C. glabrata infections, the molecular mechanisms underlying its virulence remain poorly understood. Unlike C. albicans, C. glabrata does not rely on a reversible morphogenetic switch between yeast and hyphal or pseudohyphal forms for pathogenicity. Although C. glabrata can switch to a pseudohyphal form under nitrogen-starvation conditions in vitro (6, 7), this filamentation process has not been observed under the in vivo conditions (8). Instead, the ability of C. glabrata to adhere strongly to various substrates has been found to be a key virulence attribute (9), which is mediated by a large number of adhesins. These adhesins are glycosylphosphatidylinositol (GPI)-modified proteins covalently incorporated into the cell wall (10) and play a critical role in the initial stages of infection, serving as the primary point of contact with the host. They are also crucial for biofilm formation on abiotic substrates, particularly on medical devices like catheters (11). The most well-studied adhesin family in C. glabrata is the Epithelial Adhesin (EPA) family, comprising approximately 17–23 genes depending on the isolate, which facilitates attachment to epithelial cells and macrophages (12–15). Among these EPA family adhesins, Epa1 is predominantly responsible for in vitro adherence to epithelial cells, as evidenced by a 95% reduction in adherence in epa1 mutant cells (12). The YPS gene cluster, encoding extracellular GPI-linked aspartyl proteinases known as yapsins, is implicated in the removal and release of Epa1 (16). However, deletion of EPA1 alone results in only a minor, non-significant reduction in organ colonization in a murine urinary tract infection model, likely due to the involvement of other adhesins in C. glabrata infection (17–19). Indeed, a triple mutant strain lacking EPA1, EPA6, and EPA7 exhibits significantly reduced bladder colonization in this model (17).

In the model yeast Saccharomyces cerevisiae, adhesion and biofilm formation are mediated by the GPI-anchored cell wall protein FLO11, whose expression is regulated by the MAPK pathway and the cAMP/PKA pathway (20). The regulation via the MAPK pathway requires the transcription factors Ste12 and Tec1, whereas cAMP-mediated activation requires a distinct factor, Flo8 (21). One of the STE12 homologs in C. glabrata, STE12 (1) has been found to be necessary for filamentation under the nitrogen-starvation condition and maintaining wild-type virulence in a murine model of candidiasis (22). A recent study reported a stronger biofilm-impaired phenotype, resulting from the loss of both STE12-homologous genes in the double mutant, compared with the single mutant with the deletion of STE12(1) (23). However, C. albicans mutant strain with the deletion of CPH1, the ortholog of S. cerevisiae STE12, is still able to cause lethal infections in mice (24). Instead, Flo8 functions downstream of the cAMP/PKA pathway, and together with a basic helix-loop-helix protein Efg1, are essential for the virulence of C. albicans (25). The mechanism of how the interconnections are established between downstream transcription factors of MAPK pathway and the C. glabrata pathogenicity needs to be further investigated.

MAP Kinases Fus3 and Kss1 play an essential role in maintaining the specificity of MAPK signaling in S. cerevisiae (26). These closely related kinases are activated by the common upstream MAPK kinase Ste7 yet generate distinct output responses, mating, and filamentous growth in S. cerevisiae, respectively (27). For the important human pathogenic fungus Cryptococcus neoformans, deletion of the S. cerevisiae Fus3 ortholog Cpk1 results in defects in mating pheromone production, cell fusion, and filamentous growth but does not affect virulence (28). Although it has been reported that deleting the S. cerevisiae Fus3 ortholog Cek1 in C. albicans led to a virulence defect in a mouse model of systemic infection (29), this finding may be confounded by the effect of the selectable marker gene (30). In the C. glabrata genome, homologs of S. cerevisiae FUS3 and KSS1 are present, but so far, they are not characterized.

Here, we found that the expression of MAPKs Fus3 and Kss1 was activated over the course of invasive infection of C. glabrata. Importantly, deletion of both FUS3 and KSS1 led to a reduced colonization in murine models of systemic candidiasis and DSS-induced colitis. Moreover, we demonstrate the critical role of C. glabrata MAPKs Fus3 and Kss1 in the resistance to intracellular killing by macrophages and adhesion to the plastic surface and human epithelial cells. The downstream transcription factors of MAPK signaling pathway have also been identified to be involved in the regulation of C. glabrata virulence. Our findings suggest that the activation of MAPK signaling pathway during invasive candidiasis facilitates C. glabrata infection.

RESULT

The increased expression of MAPKs Fus3 and Kss1 during C. glabrata infection in the host

To uncover the signaling pathway contributing to the virulence of C. glabrata, we performed in vivo RNA sequencing. Because the kidney is frequently the most heavily infected organ with hematogenously disseminated candidiasis, the RNA-seq analysis was applied to this organ at 24 h and 48 h post-infection (pi) (Fig. 1A, n = 3 mice). For each sample, a minimum of 65 million mRNA-Seq reads were mapped to the C. glabrata genome, which covered roughly 96% of the predicted genome. Cells grown under in vitro growth conditions (YPD 30°C) were used as a control to identify differentially expressed genes during in vivo infection. Two hundred and forty-nine genes upregulated in C. glabrata cells by 2-fold or more at both 24 h and 48 h pi (Table S1). These activated genes throughout the prompt response of C. glabrata upon infection (24 h and 48 h) were then subjected to KEGG analysis. As shown in Fig. 1A, they were significantly enriched in genes for glycolysis/gluconeogenesis (P = 2.49 × 10⁻⁴), cell cycle (P = 7.67 × 10⁻⁴), MAPK signaling pathway (P = 0.0199), and arginine biosynthesis (P = 0.0266). Among them, we focused on CAGL0J04290g and CAGL0K04169g (Fig. 1B). These two genes encode proteins sharing a high sequence homology with Fus3 and Kss1, two MAPKs in S. cerevisiae (Fig. 1A and B). We therefore designated CAGL0J04290g as FUS3 and CAGL0K04169g as KSS1 in C. glabrata.

Fig 1.

The expressions of MAPKs FUS3 and KSS1 are activated upon invasive infection of C. glabrata. (A) KEGG analysis of genes upregulated at both 24 h and 48 h during C. glabrata infection was performed using KOBAS (http://bioinfo.org/kobas/). (B) Heatmap showing the expression levels of genes for MAPK signaling pathway in (A), in z-score normalized to fragments per kilobase per million mapped reads (FPKM). UI, uninfected. (C) qRT-PCR analysis for the expression of FUS3 and KSS1 during invasive infection of C. glabrata. WT C. glabrata cells were grown in liquid YPD medium at 30°C for an in vitro control and then subjected to 19–21 g male BABL/c mice by tail vein injection. At 24 h post-infection, total RNA of the infected kidneys and livers was extracted for the in vivo analysis. (D) qRT-PCR analysis of FUS3 and KSS1 mRNA in WT C. glabrata cells incubated in DMEM medium with or without 10% serum in the absence or presence of 5% CO₂ at 37°C. (C and D) The signals obtained from ACT1 mRNA were used for normalization. Error bars represent standard deviations from the means of three experiments. Significance was measured with an unpaired t-test in GraphPad Prism. ns, no significance; *, P  <  0.05; **, P  <  0.01; ***, P  <  0.001.

The increased expression of FUS3 and KSS1 in murine kidneys during the invasive infection relative to in vitro growth condition (YPD medium) was confirmed by qRT-PCR (Fig. 1C). No significant difference was observed in the expression of FUS3 and KSS1 in RPMI medium compared with that in YPD medium (Fig. S2). In addition to kidneys, the enhanced expression of FUS3 and KSS1 was also observed in the infected livers, suggesting that host-associated signals might account for the in vitro–in vivo difference observed in the FUS3 and KSS1 transcripts. Indeed, FUS3 and KSS1 showed increased expression in response to host-associated signals, such as a combination of serum and high CO₂ (Fig. 1D). Interestingly, serum or high CO₂ alone was not sufficient to induce FUS3 expression, indicating that FUS3 expression is synergistically regulated by multiple signals within the host. Taken together, our transcription profiling data suggest that the MAPK signaling pathway is activated during C. glabrata infection.

The MAPKs Fus3 and Kss1 are critical for the pathogenicity of C. glabrata

To elucidate the impact of MAPK pathway on C. glabrata virulence, fus3 and kss1 single mutants were constructed. Since Fus3 and Kss1 are two paralogous MAPKs that have overlapping functions in pheromone response in S. cerevisiae (31), we also constructed fus3 kss1 double mutant strain to further investigate their roles. It is found that single mutants fus3 and kss1, and double mutant fus3 kss1 exhibited no obvious growth defect in yeast extract-peptone glucose (YPD) liquid medium and YNB glucose plates at 30°C (Fig. S3; Fig. 2A). Considering the complex carbon sources in host niches, we next examined the growth of these mutants in fermentable carbon source maltose and nonfermentable carbon sources glycerol and ethanol, as well as the short-chain organic acid such as lactate. As shown in Fig. 2A, these mutants grew well on media containing indicated carbon sources.

Fig 2.

The fus3 kss1 double mutant strain is defective in the persistence in infected organs of mice. (A) Cells of wild-type, single mutants fus3 and kss1, and double mutant fus3 kss1 were serially diluted 10-fold and spotted onto YNB solid medium containing 2% of the indicated carbon sources and incubated at 30°C. (B) Groups of immunosuppressed male BALB/c mice were infected with 5 × 10⁷ CFU of wild-type, fus3 single mutant, kss1 single mutant, or fus3 kss1 double mutant by tail vein injection, followed by euthanasia of five animals per group after 4 days. BALB/c mice were rendered neutropenic by intraperitoneal administration of cyclophosphamide (150 mg/kg of body weight per day) 3 days before challenge with C. glabrata and on the day of infection. CFUs were determined by plating kidney, liver, or spleen homogenates onto agar plates (supplemented with streptomycin and ampicillin) and counting after incubation at 30°C. (C) The numbers of live C. glabrata recovered from the stomach and colon of mice during the induction of colitis by low doses of DSS. Eight- to 10-week-old female c57 mice were infected by oral gavage with 1 × 10⁸ cells of wild-type or fus3 kss1 double mutant and treated with 2% DSS on the day of infection. The DSS administration occurred over a period of 2 weeks. Schematic overview of the protocol for the induction of colitis by DSS is shown on the left.The values represent means ± SD for n = 11 mice in each group. (B and C) Significance was measured with an unpaired t-test in GraphPad Prism. ns, no significance; *, P  <  0.05; **, P  <  0.01; ***, P  <  0.001; ****, P  <  0.0001.

We further determined the C. glabrata survival in the host during the invasive infection. Tissue fungal burdens were measured 4 days after infection in the immunosuppressed mice using tail-vein injections according to the previously established protocol (32). Mice infected with the fus3 kss1 double mutant, but not fus3 or kss1 single mutant, had significantly lower fungal loads in the kidneys (~75%), livers (~70%), and spleens (~65%) than those infected with the WT strain (Fig. 2B). We next investigated the impact of Fus3 and Kss1 on the colonization of C. glabrata in the intestinal tract. Since all reported C. glabrata strains are eliminated within 2 days after challenge from mice without colitis in terms of fungal colonization, the DSS (Dextran Sulfate Sodium)-induced colitis model was used to experimentally mimic chronic inflammatory bowel diseases as reported previously with minor modification (18, 33). BALB/c mice were treated with 2% DSS on the day of infection with C. glabrata by oral gavage (Fig. 2C). Significantly higher numbers (>8-fold) of viable C. glabrata were detected in the stomach and colon of mice colonized with the WT strain than in mice colonized with the fus3 kss1 double mutant strain after treatment with DSS for 14 d. Together, our data revealed a crucial role of Fus3 and Kss1 in C. glabrata survival in the host.

The role of MAPKs Fus3 and Kss1 in C. glabrata survival in macrophages

To understand the mechanism by which MAPKs regulate C. glabrata virulence, we first investigated the fungistatic activity of macrophages toward wild-type, single mutants fus3 and kss1, and double mutant fus3 kss1. Interestingly, a significant reduction (~35%) in the fungal burden was observed in kss1 single mutant compared with the wild-type after exposure to macrophages for 8 h (Fig. 3A), although this mutant exhibited no phenotype for the survival in the infected organs during invasive infection (Fig. 2B), suggesting that multiple virulence factors may work together to determine the C. glabrata virulence in the host. Furthermore, the fus3 kss1 double mutant displayed a more severe deficiency in resistance to intracellular killing by phagocytic cells than the kss1 single mutant (Fig. 3A). However, all these mutants grew normally upon stress challenges under the in vitro condition, including osmotic stress (KCl), oxidative stress (H₂O₂), and cell wall stress (Congo red) (Fig. 3B). One unresolved question is how the MAPK signaling pathway influences the survival of C. glabrata within the macrophages.

Fig 3.

The impact of MAPKs Fus3 and Kss1 on the survival of C. glabrata in macrophages. (A) RAW264.7 cells were cultured with wild-type, single mutants fus3 and kss1, and double mutant fus3 kss1. Non-phagocytosed C. glabrata cells were removed by washing with PBS after 3 h, and the CFUs of C. glabrata in RAW264.7 cells were determined after co-incubation for an additional 5 h. n = 3 biologically independent samples. (B) The MAPKs Fus3 and Kss1 are not implicated in stress responses in C. glabrata. Wild-type and indicated mutant strains were treated with stress agents, including 200  µg/mL Congo red, 1.5 M KCl, and 10 mM H₂O₂. Photographs were taken after growth at 30°C. (C) Gene Ontology (GO) biological process enrichment analysis of C. glabrata genes that are activated at both 24 h and 48 h upon invasive infection was performed using David (https://david.ncifcrf.gov/). (D) Heatmap showing the expression levels of genes for iron ion transport in (C), in z-score normalized to FPKM. UI, uninfected. (E) qRT-PCR analysis for the expression of SIT1, FTR1, and FET3 in WT C. glabrata cells under in vitro and in vivo conditions was performed as described in Fig. 1C. At 24 h post-infection, total RNA of the infected kidneys was extracted for the in vivo analysis. (F) qRT-PCR analysis for the expression of SIT1, FTR1, and FET3 in wild-type, single mutants fus3 and kss1, and double mutant fus3 kss1. The C. glabrata cells were subjected to male BALB/c mice by tail vein injection. Total RNA was extracted from the kidneys after 24 h of infection. (E and F) The mRNA levels of the indicated genes were normalized with ACT1. Mean data ± SD from three independent experiments were plotted. Significance was measured with an unpaired t-test in GraphPad Prism. ns, no significance; *, P  <  0.05; **, P  <  0.01; ***, P  <  0.001; ****, P  <  0.0001.

C. glabrata has developed effective strategies to sequester iron from host cells, which is critical for its pathogenicity (34). The siderophore-iron transporter Sit1 and the high-affinity iron permease Ftr1 have been reported to be required for Fe acquisition and survival of C. glabrata during macrophage infection (35–37). Indeed, we found that genes activated throughout the prompt response of C. glabrata upon infection (24 h and 48 h) were enriched for gene ontology (GO) terms associated with iron ion transport (P = 6.05 × 10⁻⁶), including SIT1 and FTR1 (Fig. 3C and D). Also, the activation of FET3 encoding the oxidase that oxidizes ferrous (Fe²⁺) to ferric iron (Fe³⁺) for subsequent cellular uptake by transmembrane permease Ftr1 was identified during the invasive infection of C. glabrata (Fig. 3D). The upregulation of SIT1, FTR1, and FET3 at 24 h post-invasive infection was then confirmed by qRT-PCR (Fig. 3E). Interestingly, the upregulation of SIT1 gene during invasive infection was dependent on Kss1 but less dependent on Fus3 (Fig. 3F). In contrast, both Fus3 and Kss1 were critical for the upregulation of FTR1 and FET3 (Fig. 3F). Therefore, MAPKs Fus3 and Kss1 regulate the expression of genes for iron ion transport in a coordinated and complementary manner.

MAPKs Fus3 and Kss1 regulate biofilm formation and adhesion to human epithelial cells in C. glabrata

The biofilm represents a major form of resistance to host defense machinery during fungal infection, we therefore placed C. glabrata cells in a 48-well plate to allow the biofilm formation. Similar biofilm formation was observed among the wild-type, fus3 mutant, and kss1 mutant on the plastic surface of the wells (Fig. 4A). However, the number of adherent cells in the fus3 kss1 double mutant was approximately half of that observed in wild-type strain, suggesting that deletion of MAPKs Fus3 and Kss1 resulted in a decreased adhesion to the plastic surface in C. glabrata (Fig. 4A). Furthermore, the adherence assay on the human epithelial cells A549 revealed that fus3 kss1 double mutant exhibited significantly reduced (~35%) attachment compared with the wild-type (Fig. 4B). Therefore, our results provide evidence for the overlapping role of Fus3 and Kss1 on the regulation of adherence to materials or cellular surfaces in C. glabrata. The Epithelial Adhesin (EPA) family has been found to enable C. glabrata to attach to epithelial cells (12, 15). However, the in vivo infection did not trigger a significant change in the expression of these EPA adhesins based on our transcription profiling data. Instead, 10 genes involved in the fungal-type cell wall organization (P = 0.0493) showed a dramatically increased expression at both 24 h and 48 h post-infection (Fig. 3C and 4C). Among them, the YPS-family aspartyl protease Yps1 plays a role in remodeling C. glabrata cell wall by removal of Epa1, which is largely responsible for the in vitro adherence to epithelial cells (16). In fact, a specific requirement for Yps1 in biofilm formation has been identified in C. glabrata (38). As shown in Fig. 4D, the induced expression of YPS1 during invasive infection of C. glabrata was confirmed by qRT-PCR. Remarkably, we found that Fus3 and Kss1 are both involved in the transcriptional activation of YPS1 during in vivo infection (Fig. 4E). Thus, our data suggested that Fus3 and Kss1 may regulate the organization of cell wall proteins, such as the adhesin Epa1, to promote adherence of C. glabrata.

Fig 4.

Deletion of MAPKs Fus3 and Kss1 caused a reduction in capacity for adhesion of C. glabrata. (A) Cells of wild-type, single mutants fus3 and kss1, and double mutant fus3 kss1 were inoculated onto 48-well plates with 500 µL SDB medium at 37°C for 48 h. The formation of biofilm was analyzed by the number of adherent cells. Values are the means ± SD from three independent experiments. (B) Adhesion assay on epithelial cell monolayers. C. glabrata cells of wild-type and indicated mutant strains were inoculated with A549 cells in DMEM medium supplemented with 10% serum. After incubation for 4 h, non-adherent Candida cells were removed by washing with PBS. The numbers of adherent Candida cells were represented as means ± SD from three independent experiments. (C) Heatmap showing the expression levels of genes for fungal-type cell wall organization in Fig. 3C, in z-score normalized to FPKM. UI, uninfected. (D) qRT-PCR analysis for the YPS1 expression in WT C. glabrata cells under in vitro and in vivo conditions was performed as described in Fig. 1C. At 24 h post-infection, total RNA of the infected kidneys was extracted for the in vivo analysis. (E) qRT-PCR analysis for the expression of YPS1 in wild-type, single mutants fus3 and kss1, and double mutant fus3 kss1 during invasive infection. Total RNA was extracted from the kidneys after 24 h of infection. (D and E) The mRNA levels of YPS1 were normalized with ACT1. Mean data ± SD from three independent experiments were plotted. Significance was measured with an unpaired t-test in GraphPad Prism. ns, no significance; *, P  <  0.05; **, P  <  0.01; ***, P  <  0.001; ****, P  <  0.0001.

We next wanted to determine whether constitutive expression of FUS3 or KSS1 can lead to an increase in the adherence capacity of C. glabrata. As shown in Fig. S4A, the biofilm formation was not activated upon FUS3 or KSS1 overexpression. Also, overexpression of FUS3 or KSS1 under the TEF1 promoter failed to promote the adhesion to human epithelial cells (Fig. S4B) and was unable to increase the fungal burden in murine kidneys during invasive infection (Fig. S4C). Our data indicated that the transcriptional upregulation of FUS3 and KSS1 is insufficient to promote pathogenicity. Rather, we suggest that the activation of these two MAPKs relies on the phosphorylation event, although this hypothesis needs further study.

Downstream transcription factors of MAPK signaling pathway are implicated in C. glabrata pathogenicity

The downstream targets of MAPKs include the transcription factors Ste12 and Tec1, both of which are required for the expression of filamentation genes in S. cerevisiae (26). Although only one copy of STE12 or TEC1 is presented in S. cerevisiae genome, they are both duplicated in C. glabrata (39). We therefore deleted each of them to obtain four single mutant strains, including tec1 (CAGL0M01716g), tec2 (CAGL0F04081g), ste12(1) (CAGL0M01254g), and ste12(2) (CAGL0H02145g). Considering the evolutionary relationship of the homologous genes, we also constructed the tec1 tec2 and the ste12(1)(2) double mutant strains. These single mutant strains tec1, tec2, ste12 (1), and ste12 (2), and double mutant strains tec1 tec2 and ste12 (1) ste12 (2) grew well on rich medium (YPD medium), similar to the WT strain (Fig. S5). Also, these mutant cells displayed no obvious growth defect in fermentable carbon source maltose and nonfermentable carbon sources glycerol, ethanol, and lactate (Fig. 5A).

Fig 5.

Ste12 and Tec1 homologs impact the virulence of C. glabrata during disseminated infection in mice. (A) Dilutions of wild type and indicated mutant strains were spotted onto YNB solid medium containing 2% glucose, maltose, glycerol, ethanol, or lactate and incubated at 30°C. (B) The fungal loads of wild type, single mutants tec1, tec2, ste12 (1), and ste12 (2), and double mutants tec1 tec2 and ste12(1)(2) in immunosuppressed mice were determined as in Fig. 2B. n = 5 mice. Significance was measured with an unpaired t-test in GraphPad Prism. ns, no significance; *, P  <  0.05; **, P  <  0.01; ***, P  <  0.001.

The contribution of Ste12 and Tec1 to the C. glabrata virulence was then determined using the model of hematogenously disseminated candidiasis. As shown in Fig. 5B, the single mutant strain ste12 (1) and double mutant strains tec1 tec2 and ste12 (1) ste12 (2) showed severely attenuated fungal burden in the kidney during the invasive infection. Also, tec1 tec2 double mutant showed a notable decrease in the persistence within the liver. Although we could not detect a statistically significant difference in fungal burden of infected livers between the strains deleting STE12 homologs and the WT strain, the ste12 (1) single mutant and ste12 (1) ste12 (2) double mutant exhibited an observable reduction in survival ability in the liver compared with the WT strain (Fig. 5B). In fact, Ste12 (1) has been identified to be required to maintain wild-type levels of virulence in a reported murine model of C. glabrata systemic disease (22). Here, we found that the ste12(1)(2) double mutant displayed a more severe survival deficiency in infected kidneys than the ste12 (1) single mutant, suggesting that Ste12 homologs, Ste12 (1) and Ste12 (2), may have distinct functions during the invasive infection of C. glabrata. Moreover, mice infected with the tec1 tec2 double mutant strain had significantly lower fungal burdens compared with those infected with the WT strain or single mutants, tec1 and tec2 (Fig. 5B), indicating the overlapping functions of Tec1 and Tec2 in modulating C. glabrata virulence. Interestingly, only ste12 (1) single mutant and ste12 (1) ste12 (2) double mutant exhibited significantly lower fungal loads in the spleen (Fig. 5B), suggesting that C. glabrata MAPKs Fus3 and Kss1 may employ distinct downstream transcription factors to adapt and survive in different host niches during infection.

The regulation of Ste12 and Tec1 homologs in the adhesion and iron transport of C. glabrata

The biofilm formation assay was performed in mutant strains deleting STE12 or TEC1 homologs and revealed that ste12 (1) single mutant and ste12 (1) ste12 (2) double mutant exhibited reduced attachment compared with the wild-type (Fig. 6A), which is consistent with a previous study (23). Correspondingly, a reduction in the expression of YPS1 was observed in these mutants compared with WT strain during invasive infection (Fig. 6B). Our result suggested that the activation of YPS1 by MAPKs Fus3 and Kss1 might be mediated through Ste12 homologs. However, both Ste12 and Tec1 homologs were implicated in the regulation of iron acquisition as a lower SIT1 and FTR1 expression was observed in the ste12 (1) ste12 (2) or tec1 tec2 double mutant than that in WT strain during in vivo infection (Fig. 6C). We next investigated the fungistatic activity of macrophages toward mutant strains lacking STE12 or TEC1 homologs. Unexpectedly, no significant difference in fungal burden was observed in these mutant strains compared with the wild-type after exposure to macrophages for 8 h (Fig. 6D), suggesting the involvement of other downstream effectors of MAPKs Fus3 and Kss1 in the C. glabrata resistance to intracellular killing by phagocytic cells.

Fig 6.

Functional characterization of Ste12 and Tec1 homologs in C. glabrata. (A) The formation of biofilm for wild-type, single mutants tec1, tec2, ste12 (1), and ste12 (2), and double mutants tec1 tec2 and ste12(1)(2) was analyzed as described in Fig. 4A. Values are the means ± SD from three independent experiments. qRT-PCR analysis for the expression of YPS1 (B), FTR1 (C), and SIT1 (C) in wild-type and indicated mutant strains. C. glabrata cells were introduced into male BALB/c mice through tail vein injection. After 24 h of infection, total RNA was extracted from the kidneys. The mRNA levels of the indicated genes were normalized with ACT1. (D) The survival of mutant cells with the deletion of STE12 or TEC1 homologs was similar to that of wild-type after exposure to macrophages. The quantification of C. glabrata cells that survived within macrophages was carried out as detailed in Fig. 3A. n = 3 biologically independent samples. (A-D) Significance was measured with an unpaired t-test in GraphPad Prism. ns, no significance; *, P  <  0.05; **, P  <  0.01; ***, P  <  0.001; ****, P  <  0.0001.

Deletion of MAPKs Fus3 and Kss1 enhanced caspofungin efficacy against C. glabrata.

The echinocandins, including caspofungin, represent the first-line drugs for the treatment of systemic candidiasis, exerting their antifungal effect by targeting β−1,3-glucan synthase Fks1. It has been reported that the S. cerevisiae Fus3 homolog in C. albicans (Cek1) is activated in response to caspofungin treatment (40). We therefore determined the impact of MAPKs and downstream transcription factors in the susceptibility to caspofungin in YPD liquid medium containing caspofungin with different concentrations. As shown in Fig. 7A, only fus3 kss1 double mutant displayed a slightly increased susceptibility to caspofungin compared with that of WT C. glabrata. Fus3 and Kss1 may act in a synergistic manner for caspofungin tolerance as the fus3 and kss1 single mutants grew similarly to WT cells in a caspofungin-containing medium. The tec1 tec2 and ste12(1)(2) double mutant strains displayed no obvious defects in caspofungin response (Fig. 7A), implying that other downstream factors of MAPKs are responsible for caspofungin tolerance. In contrast, deletion of FUS3 and KSS1 had little effect on the sensitivity to fluconazole (Fig. 7B), suggesting that Fus3 and Kss1 MAPKs are specifically required for echinocandin tolerance. Consistently, the growth rate of tec1 tec2 and ste12 (1) ste12 (2) double mutant strains was comparable with that of WT strain in a fluconazole-containing medium (Fig. 7B).

Fig 7.

Caspofungin (A) and fluconazole (B) susceptibility assays were conducted in the YPD medium for wild-type and indicated mutant strains. Growth was measured by absorbance at 600  nm after 24  h at 30°C. Optical densities were normalized relative to those of antifungal drug-free controls. Data are quantitatively displayed in heat map format (see color bar).

DISCUSSION

The mitogen-activated protein (MAP) kinase pathways are important in mediating responses to diverse extracellular signals in fungi and other eukaryotic organisms. A classical MAP kinase cascade comprises a MAP kinase (MAPK), a MAPK kinase (MEK), and a MEK kinase (MEKK) (41). The MAPKs target downstream proteins, influencing transcriptional events and cellular behaviors (42). There are five MAPK genes (FUS3, KSS1, SLT2, HOG1, and SMK1) in the model organism S. cerevisiae. Two of them (FUS3 and KSS1) are closely related and have overlapping functions in pheromone response. In addition, Kss1 contributes to filamentation and invasive growth into agar (31). Slt2 and Hog1 MAPKs predominantly not only govern cell wall integrity and osmoregulation, respectively, but also participate in regulating responses to other stresses (43, 44). Recently, it has been found that surplus-extracellular iron activates the MAPK Hog1, resulting in the transcriptional activation of the adhesin Epa1 in C. glabrata (45). Smk1 is a meiosis-specific MAPK regulating ascospore wall assembly (46).

Here, we found that the expression of FUS3 and KSS1 underwent a significant increase throughout the prompt response during C. glabrata infection. Interestingly, the induction patterns of FUS3 and KSS1 differ in response to host environmental signals (Fig. 1D). Both serum and high CO₂ contributed to FUS3 induction, but neither alone was sufficient. However, the activation of KSS1 expression could be observed in either serum or high CO₂. The mechanisms underlying environmental cues that activate the MAPK signaling pathway need to be further investigated in C. glabrata. In addition, an increase in the SLT2 expression was identified in C. glabrata cells at 24 h post-infection in our transcription profiling data, although no significant difference was detected at 48 h post-infection (Zhang and Lu, submitted for publication). Indeed, the increased SLT2 expression has been found to facilitate the survival of C. glabrata cells in host tissues, perhaps due to increased tolerance to stressful conditions that affect cell wall integrity (47). It is worth noting that the activation of MAPK signaling pathway might be not only mediated by inducing the expression of MAPKs during C. glabrata disseminated infection since the constitutively overexpressed FUS3 and KSS1 have no effect on the fungal survival in the infected kidneys (Fig. S4C). In fact, phosphorylation is a common event for signal transduction in MAPK signaling pathway.

MAPKs Slt2 and Hog1 have been extensively studied in C. glabrata. Slt2 MAPK pathway is found to be implicated in the heat-induced expression of YPS1, which is essential for cell wall integrity and virulence in C. glabrata (16, 48). Although Hog1 is not required for virulence in a murine model of systemic infection, it plays an important role in the confrontation of C. glabrata with the common vaginal flora (49). Because most fungal pathogens have more complicated lifestyles and differentiation processes, it is reasonable to hypothesize that these MAP kinases may play more diverse roles in pathogenic fungi compared with their counterparts in S. cerevisiae. Several lines of evidence provided in our study support the role of MAPKs Fus3 and Kss1 as important regulators for the C. glabrata pathogenicity. First, the deletion of both FUS3 and KSS1 resulted in a profound defect in the fungal survival of C. glabrata during the invasive infection, suggesting the redundant role of Fus3 and Kss1 on C. glabrata infection. However, Fus3 and Kss1 also have different functions regarding C. glabrata physiology, as we noticed that Kss1 was required for the survival of C. glabrata within the macrophages, whereas Fus3 played a minor role in it (Fig. 3A). How Fus3 and Kss1 differentially regulate this process needs further study. Such overlapping and divergent roles of Fus3 and Kss1 were also found in S. cerevisiae in the regulation of pheromone response. Second, Fus3 and Kss1 contributed to the persistence of C. glabrata within macrophages, at least partially through the activation of genes involved in iron transport. Third, fus3 kss1 double mutant exhibited reduced adherence to the abiotic surfaces and human epithelial cells. Importantly, the colonization was diminished in the fus3 kss1 double mutant compared with the WT strain in a murine model of DSS-induced colitis (Fig. 2C). Fourth, Fus3 and Kss1 activate the expression of the aspartyl protease Yps1, which has been found to play an important role in remodeling C. glabrata cell wall (16). Whether and how Fus3 and Kss1 impact cell wall remodeling during C. glabrata invasive infection deserves further investigation. Interestingly, the cAMP/PKA pathway, rather than the MAPK pathway, plays a major role in the pathogenesis of C. albicans (50). For another important human pathogenic fungus C. neoformans, the Fus3 homolog does not contribute to the virulence (28). Nevertheless, we clearly demonstrate that the Fus3 and Kss1 MAP kinases, which play a minor role in the virulence of some pathogenic fungi, significantly impact the pathogenicity of C. glabrata. Our study provides an example of how a conserved signaling pathway rewires to regulate fungal pathogenesis.

The signal transduction pathways mediated by MAPKs Fus3 and Kss1 for mating and invasive growth in S. cerevisiae converge on the transcription factor Ste12. For invasive growth, both Ste12 and its cofactor Tec1 are indispensable (51–53). Interestingly, only one copy of STE12 and TEC1 are presented in S. cerevisiae genome, but they are doubled in C. glabrata, indicating that the transcriptional regulatory network in C. glabrata is more complex than in the model yeast S. cerevisiae (39). Indeed, the distinct contributions to virulence from Ste12 (1) and Ste12 (2) were observed in C. glabrata. Ste12 (1) seems to play a more significant role compared with Ste12 (2) in C. glabrata virulence since deletion of STE12 (1) alone is sufficient to cause a decrease in the fungal survival during invasive infection (Fig. 5B). Although there was no significant difference in fungal loads between wild-type and ste12 (2) single mutant strain during invasive infection, deleting STE12 (2) in the ste12 (1) mutant strain exacerbated the defect in virulence. This suggested that Ste12 (1) and Ste12 (2) may have partially overlapping functions in modulating the virulence of C. glabrata. In contrast, the attenuated survival of C. glabrata in infected organs was only detected when TEC1 and TEC2 were both deleted, indicating a synergistic effect between these two Tec1 homologs on C. glabrata virulence. Interestingly, no defect was observed in the ste12 (1) ste12 (2) or tec1 tec2 double mutant in response to intracellular killing by phagocytic cells (Fig. 6D), in contrast to the lower burden of the kss1 fus3 mutant in macrophages (Fig. 3A). In addition, unlike the kss1 fus3 mutant, which was more susceptible to caspofungin, the susceptibility of ste12 (1) ste12 (2) and tec1 tec2 mutant to caspofungin was comparable with WT (Fig. 7B). These results suggest that other factors are involved in these processes and this requires further research.

In addition to regulating virulence attributes in C. glabrata, Fus3 and Kss1 impact the tolerance to caspofungin, an anti-fungal drug of echinocandin class. In fact, deletion of the Fus3 ortholog MpkB has been reported to increase the susceptibility to caspofungin in Aspergillus fumigatus (54). Since echinocandins exert their antifungal effect by compromising the integrity of the fungal cell wall, these results suggested that homologs for MAPKs Fus3 and Kss1 may play a role in cell wall biosynthesis. The function of other MAPKs in drug tolerance, such as Slt2 and Hog1, has been investigated in C. glabrata in previous studies. Slt2 was found to play an important role in response to echinocandins, including caspofungin and micafungin (47, 55, 56). Hog1 alters the susceptibility to fluconazole, another anti-fungal drug commonly used to treat Candida infections (57). It would be intriguing to investigate how these diverse MAPK pathways orchestrate responses to antifungal drugs.

MATERIALS AND METHODS

Media and growth conditions

C. glabrata strains were routinely grown at 30°C in YPD medium (2% Bacto peptone, 2% glucose, 1% yeast extract). Transformants were selected on YPD plates supplemented with 80 µg/mL nourseothricin or synthetic medium (0.17% Difco yeast nitrogen base w/o ammonium sulfate, 0.5% ammonium sulfate, and auxotrophic supplements) with 2% glucose. The ability of C. glabrata cells to grow was tested by spotting dilutions of cells onto YNB (0.17% Difco yeast nitrogen base w/o ammonium sulfate, 0.5% ammonium sulfate) solid media with 2% of different sugars followed by incubation at 30°C.

To determine the stress response of C. glabrata, freshly grown cells were serially diluted 10-fold, spotted onto YPD plates with or without 200 µg/mL Congo red, 1.5 M KCl, or 10 mM H₂O₂, and incubated at 30°C.

Plasmid and strain construction

CBS138 genomic DNA was used as the template for all PCR amplifications of C. glabrata genes. C. glabrata strains used in this study are listed in Table 1. The primers used for PCR amplifications are listed in Table S2.

TABLE 1.

C. glabrata strains used in this study

Strains	Genotype	Source
CBS138	Wild-type	ATCC collection
YLC113	fus3Δ	This study
YLC114	kss1Δ	This study
YLC115	fus3Δ kss1Δ	This study
YLC116	tec1Δ	This study
YLC117	tec2Δ	This study
YLC118	tec1Δ tec2Δ	This study
YLC119	ste12 (1)Δ	This study
YLC120	ste12 (2)Δ	This study
YLC121	ste12 (1)Δ ste12 (2)Δ	This study
YLC111	ura3Δ::SAT1	This study

Deletion of FUS3 (CAGL0J04290g), KSS1 (CAGL0K04169g), TEC1 (CAGL0M01716g), TEC2 (CAGL0F04081g), STE12(1) (CAGL0M01254g), and STE12(2) (CAGL0H02145g) was performed using CRISPR-Cas9 strategy as follows. The single-guide RNA (sgRNA) was annealed to insert into the pV1382 vector (58). The resulting plasmid was transformed into C. glabrata cells with the repair template. The mutants were verified by sequencing.

The coding sequences for FUS3 (Primers 37 and 38) and KSS1 (Primers 39 and 40) were amplified from C. glabrata genomic DNA. The PCR product was then inserted into the NotI-SacII site of pY26TEF-GPD, generating the overexpressing plasmid to express FUS3 or KSS1 under the control of TEF1p in C. glabrata.

In vivo RNA sequencing and analysis

For RNA-seq assay during C. glabrata infection, 6- to 8-week-old male BALB/c mice were inoculated with 4.5 × 10⁸ wild-type cells (CBS138) in a 200 µL volume of sterile PBS via tail vein injection. At 24 and 48 h post-infection, animals were euthanized. The infected kidneys from two mice were randomly picked from a certain group and combined as a sample for RNA extraction, with uninfected C. glabrata cells (UI) incubated in the YPD medium at 30°C as the control. DNA-depleted RNA samples were then depleted of ribosomal RNA using the Ribo-Zero rRNA Removal Kit (Epicentre) according to the manufacturer’s protocol. Sequencing was performed using the Illumina nova6000 platform. Clean reads were selected from raw reads by removing reads with adapter and low quality. Q30 and GC content of clean data were calculated. The sequencing reads were then aligned to C. glabrata reference genome (http://www.candidagenome.org/) using HISAT2 with default parameters, and the aligned reads were assembled and quantified. Genes with zero counts were excluded. Differentially expressed genes were defined by fold change ≥2 and a false discovery rate (FDR) < 0.05 was found by DESeq2. KEGG analysis was performed using KOBAS (http://bioinfo.org/kobas/). Gene Ontology (GO) biological process enrichment analysis was performed using David (https://david.ncifcrf.gov/).

Biofilm assay

Biofilm growth assays in vitro were conducted following a modified version of a previously described protocol (15). In brief, C. glabrata strains were cultured overnight in liquid YPD medium at 30°C. The cultures were washed twice using phosphate-buffered saline (PBS) and subsequently diluted in 500 µL of SDB medium (1% Bacto peptone, 4% glucose) to an optical density at OD₆₀₀ of 0.1. This mixture was placed in the 48-well polystyrene plate and incubated at 37°C with a shaking speed of 70 rpm to allow the adhesion. After 48 h of incubation, the non-adherent cells were removed by washing twice with PBS, and the adherent cells were treated with proteinase K for 1 h. The quantification of adherent cells was performed through direct microscopic counting.

Infection of macrophages

RAW264.7 cells were challenged with C. glabrata at a MOI of 1:1 (macrophage:Candida). Nonphagocytosed Candida cells were removed by washing with PBS after co-incubation for 3 h. To determine the growth of intracellular Candida cells, RAW264.7 cells were lysed with 0.1% Triton after incubation for an additional 5 h. After resuspension, serial dilution, and plating onto YPD plates, the phagocytized fungal cells were counted. The results are represented as means ± SD from three independent experiments.

Adhesion of C. glabrata to human epithelial cells

Adhesion assays were performed by following a modified protocol described previously (59) and by using the human epithelial cell line A549. A549 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO₂. For the adhesion assay, C. glabrata cells suspended in DMEM medium containing 10% FBS were added to the 24-well plate where A549 cell monolayers had been prepared, at a MOI of 1:4 (macrophage: Candida). After incubation at 37°C for 4 h, nonadherent C. glabrata cells were carefully removed by washing with PBS. Epithelial cells and adherent C. glabrata cells were visualized by Giemsa staining.

Murine model of C. glabrata infection

BALB/c and c57 mice were purchased from Beijing Vital River Laboratory Animal Technology Company. Mice were housed in a temperature-constant animal room (22°C) with a reversed dark/light cycle (7:00 a.m. on and 7:00 p.m. off) and 40%–70% humidity.

Neutropenia was induced in 19–21 g male BALB/c mice by administering cyclophosphamide intraperitoneally (150 mg/kg of body weight per day) 3 days before challenge with C. glabrata infection and on the day of infection. For the infection, C. glabrata cells were delivered into the immunosuppressed mice through a tail vein injection at a dosage of 5 × 10⁷ cells. On day 4 post-infection, the infected kidneys, spleens, and livers from these mice were harvested, homogenized, and cultured on agar plates supplemented with streptomycin (100 µg/mL) and ampicillin (50 µg/mL). Fungal colony forming units (CFUs) were counted after incubation at 30°C.

The DSS (Dextran Sulfate Sodium)-induced colitis experiment was conducted on 8- to 10-week-old female c57 mice, following previously described methods with some modifications (18, 33). To induce intestinal inflammation, the mice were given 2% DSS (36–50 kDa; MP Biomedicals) in their drinking water for a period of two weeks (from day 1 to day 14). On day 1, mice were inoculated with 10⁸ C. glabrata cells via oral gavage. After 14 days, the mice were euthanized, and their stomachs and colons were collected. These gastrointestinal (GI) tract segments were longitudinally dissected, and the intestinal contents were removed. Subsequently, the tissue samples were thoroughly washed in PBS to minimize contamination from C. glabrata within the lumen. The tissue homogenates were serially diluted and plated on agar plates containing streptomycin (100 µg/mL) and ampicillin (50 µg/mL). Fungal colonies were counted, and the results were expressed as C. glabrata CFU per gram of tissue.

Quantitative PCR analysis

Total RNA was extracted and purified from C. glabrata cells incubated under the in vitro conditions using the RNeasy Mini kit. To remove DNA contamination, the RNA samples were treated with RNase-free DNase Set (Qiagen) for 15 min at room temperature. To determine the expression of C. glabrata genes during invasive infection, 19–21 g male BABL/c mice were inoculated with 7 × 10⁸ live C. glabrata cells by tail vein injection, and infected organs were taken at 24 h post-infection. Total RNA of the infected tissues of mice was extracted using the RNAprep Pure Tissue Kit (Tiangen). cDNA was synthesized using the Maxima H Minus cDNA Synthesis Master Mix with dsDNase (Thermo). For qRT-PCR analysis, iQ SYBR Green Supermix (Bio-Rad) was used in 96-well plates. The primers for qRT-PCR are listed in Table S1. Signals obtained from ACT1 mRNA were used for normalization. All data are shown as the means of three independent experiments, with error bars representing the SD.

Antifungal susceptibility testing

Susceptibility to fluconazole or caspofungin was assayed in a total volume of 0.1 mL/well with various concentrations of each drug in a liquid YPD medium. The 96-well plates were incubated in the dark at 30°C for 24 h before the optical density at 600 nm (OD₆₀₀) was determined using a spectrophotometer (BioTek Instruments). Data were displayed as heat maps. Caspofungin (Selleck) was dissolved in DMSO. Fluconazole (Selleck) was dissolved in ethanol.

Statistical and reproducibility

All experiments were performed with at least three biological repeats. No data were excluded from analyses. Analyses were conducted using GraphPad Prism. The results are expressed as the mean ± standard deviation (SD) except as indicated in the figure legends. All data were analyzed using unpaired Student’s t-tests. P values of less than 0.05 were considered statistically significant. Sample allocation was random in all experiments. No blinding was performed because none of the analyses reported involved procedures that could be influenced by investigator bias.

DATA AVAILABILITY

RNA-Seq data that support the findings of this study have been deposited into GEO under the accession code GSE279281.

ETHICS APPROVAL

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Wuhan University and performed as outlined in the guide for the care and use of laboratory animals issued by the Ministry of Science and Technology of the People’s Republic of China.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/msphere.00715-24.

Supplemental material. msphere.00715-24-s0001.pdf.

Figures S1 to S5; Table S2.

Table S1. msphere.00715-24-s0002.xlsx.

In vivo RNA-seq data.

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