Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells’ oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells’ hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.
Candida albicans is the most common invasive human fungal pathogen, whose infections carry a high mortality rate [1]. It is also a widespread commensal, colonizing gastrointestinal mucous membranes of around half of healthy humans [2] and competing with myriad bacteria for nutrients shed by the host or extractable from the food stream [3,4]. Sources of the macronutrients carbon, nitrogen and phosphate must be distributed between the host and its bacterial and fungal colonizers. During invasive disease, C. albicans uses the human as its source of nutrients and must withstand the host immune system [1]. Availability of inorganic phosphate (Pi) is critical for cells metabolizing carbon and nitrogen sources, synthesizing ribosomes and membranes, and preparing for DNA replication. Bacteria devote a Pi signalling and acquisition system, the PHO regulon, to Pi homeostasis. In many pathogenic bacteria, the PHO regulon has been linked to virulence, though definition of the perturbed pathogenic mechanisms has often remained elusive [5]. In some bacteria like Vibrio cholerae, the PHO regulon’s transcriptional regulator also controls or co-regulates hundreds of genes related to virulence and unrelated to Pi homeostasis [6,7] so that the role of the PHO regulon may be to inform the bacterial cell of the presence of host signals in its environment. In fungi and plants, a more complex PHO regulon monitors the availability of intracellular as well as extracellular Pi and is integrated with a large signalling network responding to nutrient availability and the absence of stressors [8]. Unicellular parasitic human pathogens, evolved to invade the bloodstream and infect vital tissues, have Pi import systems whose importance has recently come into focus [9]. Significantly, Leishmania infantum, an intracellular parasitic agent of human visceral leishmaniasis which is a neglected tropical disease of 12 million people [10], expresses a Pi transporter homologous to the Saccharomyces cerevisiae high-affinity H+-Pi symporter Pho84 as its major mode of Pi acquisition [11]. Insect stages of the related kinetoplastid parasite Trypanosoma cruzi, the agent of Chagas disease estimated to chronically infect 8 million people while leading to inexorable cardiac death in 20–30% of the infected [12,13], are Pi-dependent for their cellular differentiation [14]. T. cruzi insect stages utilize homologs of the Saccharomyces cerevisiae high- and low-affinity Pi importers to acquire the Pi quantities that permit their development and proliferation [14]. A high-affinity Pi transporter of C. albicans, Pho84, is required for normal Target of Rapamycin (TOR) signalling and hyphal morphogenesis [8]. Pharmacologic inhibition of Pho84 potentiates the activity of two major antifungal classes [8]. Pho84 is one of four predicted C. albicans plasma membrane Pi transporters, so that redundancy of its activity would be expected, and a role of Pho84 in virulence could not be assumed a priori. We previously observed failure of pho84 deletion mutants to appropriately induce hyphal growth in response to several in vitro conditions [8]. Since hyphal growth is a known virulence determinant in C. albicans, we examined virulence of pho84 mutant cells first in a wild type Drosophila melanogaster model, then in two murine models. Despite its redundancy as a Pi transporter, mutants in Pho84 exhibited attenuated virulence in these models, which may partially be attributable to their hyphal morphogenesis defect, observable in one of the murine models. Finding a requirement for Pho84 in resistance of C. albicans cells to whole human blood exposure, we then focused on isolating a molecular mechanism of this role of Pho84. Pho84 mutants were hypersensitive to killing by human neutrophils, the major cellular blood component that controls invading fungi. Our analysis led to the complex C. albicans superoxide dismutase (SOD) system [15], and demonstrated a novel requirement for active TOR complex 1 (TORC1) in maintaining appropriate cytosolic SOD expression. Generation of the superoxide anion and other reactive oxygen species (ROS) is a major defense mechanism of host phagocytes against the fungus [16]. In addition to its role in hyphal growth, the link between a cytosolic SOD and TORC1 activity provides a rationale for the contribution of Pho84, a redundant member of the cell surface Pi importers, to virulence of C. albicans.
Null mutants in PHO84 are defective in TORC1 signaling and in hyphal growth [8]. As C. albicans hyphal growth is a virulence determinant, we examined pho84 null mutant (-/-, pho84/pho84) cells’ virulence in a Drosophila melanogaster model [17]. In the wild type OregonR strain we used, Drosophila immune responses are intact, so that the fungus confronts the full complement of innate immunity. By 5 days after infection, 30% of flies injected with PHO84 wild type, and 8% of those injected with pho84 null mutant cells had died (p<0. 001 by Kaplan-Meier analysis). Virulence behavior of reintegrant (-/-/+, pho84/pho84: : PHO84) cells was statistically indistinguishable from wild type (p = 0. 3) (Fig 1A). Hence, in a model of simple innate immunity, Pho84 was required for virulence. Given their attenuation in an insect model, we examined the virulence of pho84 null mutant cells in two murine models of infection. One path of natural C. albicans infection is invasion of the mucosa. We compared pho84 null and wild type cells for the ability to proliferate in a murine oropharyngeal candidiasis model [18]. Loss of Pho84 resulted in a decreased fungal burden of tongue tissue (Fig 1B), indicating that in this model, functions of Pho84 contribute to virulence during oropharyngeal infection. Loss of virulence of the pho84 null and pho84-/-/+ reintegrant cells in this model was similar. We previously observed haploinsufficiency of PHO84 heterozygous and reintegrant cells [8]. In fact, in one phenotype, hypersensitivity to oxidative stress, the degree of haploinsufficiency correlated with the intensity of the stress (S1 Fig. in S1 Data). Nevertheless, we cannot exclude that a second mutation in the null mutant strain diminished its virulence in this model. Once C. albicans cells have crossed the mucosa and entered the bloodstream, they disseminate to distant organs and initiate new foci of infection. We asked whether Pho84 is required for C. albicans virulence during hematogenous infection. Survival of mice injected intravenously with wild type, pho84 null mutant and pho84-/-/+ reintegrant cells was compared, and more than 70% of the mice infected with the wild type strain died within 4 days of infection (Fig 1C). Loss of Pho84 significantly extended survival in this model; p = 0. 017 for wild type versus pho84 null mutant cells, and p = 0. 76 for wild type versus reintegrant by Kaplan-Meier analysis. We assessed the morphogenetic state of infecting filamentous cells according to the criteria defined by Sudbery et al. [19]. Gomori-Methenamine Silver stained sections of kidneys from moribund mice showed wild type cells growing predominantly in the hyphal form with interspersed yeast cells (Fig 1D). In contrast, pho84 null mutant cells were a mixture of pseudohyphal filaments and yeast with fewer hyphae (Fig 1D), reflecting defective hyphal growth previously seen in vitro [8], while reintegrant cells grew as hyphae, pseudohyphae and yeast (Fig 1D). Hence, in a model of disseminated disease, loss of Pho84 correlated with prolonged survival of infected animals, and a hyphal growth defect of null mutant cells may have contributed to their virulence defect. During bloodstream infection, invading C. albicans cells encounter host blood components. We incubated heparinized whole blood from healthy human volunteers with C. albicans and found survival of pho84 null mutant cells to be significantly decreased after 5 hours, compared with the wild type and reintegrant (Fig 2A). This finding indicates that Pho84 has a role in C. albicans’ tolerance of whole blood candidacidal activity. Neutrophils are the major contributors to C. albicans’ transcriptional responses to whole blood exposure, and are critical components of cellular innate immunity against invasive candidiasis [20–22]. We therefore tested the ability of pho84 null mutant cells to survive the attack of human neutrophils, using the HL-60 human promyelocytic leukemia cell line which can differentiate into neutrophil-like cells [23]. Null mutants in PHO84 were significantly more sensitive to killing by these phagocytic cells than wild type or reintegrant cells (S2A Fig. in S1 Data). We confirmed this finding in primary neutrophils isolated from healthy human donors (Fig 2B). To distinguish hypersensitivity of pho84 null mutant cells to neutrophil cidal activity from their putatively increased phagocytic uptake, we performed phagocytosis assays and found that pho84 null and wild type cells were equally taken up by the neutrophils (S2B Fig. in S1 Data). Similarly, we examined whether pho84 null mutants may stimulate neutrophil ROS production more effectively, since ROS are a major neutrophil candidacidal mechanism [24]. We found no difference in intracellular or extracellular ROS production between neutrophils interacting with wild type or reintegrant cells, and those interacting with pho84 null mutant cells (S2C and S2D Figs. in S1 Data). These findings, obtained with neutrophils from identified healthy volunteers and from random, unidentifiable blood bank donors, indicate that Pho84 contributes to protection of C. albicans from neutrophil killing. To examine whether in fact ROS are responsible for pho84 null mutant cells’ increased susceptibility to neutrophils’ cidal activity, we treated Candida-ingesting neutrophils with the ROS-scavenging compound N-acetyl cysteine (NAC) [25]. In a dose-dependent manner, NAC rescued hypersensitivity of pho84 null mutant cells to neutrophil killing (Fig 2C). To block ROS production a priori, we then inhibited neutrophil NADPH oxidase (NOX), the enzyme complex responsible for generating the ROS oxidative burst, by preincubating neutrophils with diphenyliodonium (DPI) [26,27] and followed survival of C. albicans cells. Inhibition of neutrophil NOX with DPI abolished hypersensitivity of pho84 null mutant cells to neutrophil killing (Fig 2D). Chronic granulomatous disease (CGD) is caused by mutations that disrupt NOX function. pho84 null mutant cells were equally resistant to killing by neutrophils isolated from a CGD patient as wild type cells, while in the same experiment, as previously, they were hypersensitive to killing by neutrophils from a control healthy volunteer (Fig 2E). These results suggest that Pho84 is required for resistance specifically to ROS-mediated neutrophil candidacidal activity [24]. These findings raised the question whether pho84 null mutants are simply hypersensitive to ROS. Exposing wild type, pho84 null, and reintegrant cells to inducers of superoxide anion, plumbagin and menadione, as well as to hydrogen peroxide (H2O2), we found that each of these compounds inhibited growth of the mutant more strongly than that of the wild type (Fig 3A). Hypersensitivity of pho84 null mutant cells to neutrophil killing may therefore be due to their hypersensitivity to oxidative stress. The HOG pathway is a major signaling system by which C. albicans induces survival responses to oxidative stress [28]. We questioned whether defective HOG pathway signaling might be responsible for the hypersensitivity of pho84 null mutant cells to neutrophil killing [29]. The phosphorylation state of the central kinase of the pathway, Hog1, was examined as a readout of HOG activation in response to oxidative stress [30]. Rejecting our idea, pho84 null mutant cells showed prolonged and hyperintense Hog1 phosphorylation during a time course of peroxide-mediated induction (Fig 3B). This phenotype was apparent only upon exposure to extrinsic peroxide. Growing in rich medium without extrinsic oxidative stressors, pho84 mutant, like wild type cells, exhibited a minimal signal from phosphorylated Hog1 (S3 Fig. in S1 Data) and we saw no difference between these strains; the Western blot may be too insensitive an assay to detect differences when signals are low. The concurrent findings of Hog1 pathway hyperactivation of pho84 null mutant cells, and their increased susceptibility to extrinsic ROS, suggested these cells might be unable to manage intrinsic intracellular ROS. We compared the ROS content of unstressed pho84 null and wild type cells, and of those exposed to an oxidative stressor. In fact, cells devoid of Pho84 contained more 2’, 7’-dichlorodihydrofluorescein diacetate (DCFDA) -detectable ROS, compared to the wild type, when unexposed or exposed to menadione (Fig 4A and 4B). Their inability to manage ROS was not due to starvation for inorganic phosphate (Pi), because in Pi-replete media, pho84 null mutant cells still contained significantly more ROS (Fig 4A). We concluded that Pho84 is required for ROS management in C. albicans. Superoxide dismutases (SODs) contribute to ROS management by disproportionating the superoxide anion into H2O2 and oxygen, using a redox-active metal [15]. In C. albicans pho4 null mutants, lacking the DNA binding protein that controls the PHO regulon, mRNA expression of intracellular copper-using superoxide dismutase SOD1 [15] is upregulated while its activity is decreased, and mRNA of manganese-using SOD3 is decreased [31]. We questioned whether increased ROS content in pho84 null mutant cells might be due to decreased SOD protein content or -activity. pho84 null mutant cells showed a subtle decrease of Sod1 activity during growth in standard media (Fig 4C), as assayed by nitroblue tetrazolium (NBT) reduction in non-denaturing protein gels, which display activity of the 2 abundant intracellular SODs, Sod1 and 2 [15]. During oxidative stress with menadione, variable activities of Sod1 and 2 were seen between experimental replicates, ruling out specific conclusions. Exposure to the copper chelator bathocuproine disulfonic acid (BCS) was used as a control for the identity of the bands since sparse ambient copper is known to decrease Sod1 activity [32] (Fig 4C). Since under most conditions, Sod3 activity is not detectable on these gels, we examined its protein abundance by Western blot. Sod3 protein concentration was markedly reduced in pho84 null mutant cells (Fig 4D) in the absence of extrinsic oxidative stress. In the presence of high ambient manganese, Sod3 concentrations in the pho84 mutant appeared as robust as those in the wild type (Fig 4D). Sod3 expression dropped below the detectable limit in wild type cells in high ambient copper (Fig 4D) as expected [32], as did that of the pho84 null mutant. Hence, Sod3 expression is diminished in standard culture conditions in cells lacking Pho84, but can be induced in conditions of high abundance of its metal co-factor manganese. Expression of SODs varies with metal availability in C. albicans [32]. Since in S. cerevisiae, Pho84 transports manganese in addition to Pi under certain conditions [33], we questioned whether lack of PHO84 might deplete C. albicans of manganese, resulting in Sod3 downmodulation. But upon measuring the intracellular manganese and copper concentrations, we found the opposite: both metals’ concentrations were increased in pho84 null mutant cells growing in standard synthetic complete medium, compared to wild type cells (S4A and S4B Figs. in S1 Data). Manganese concentration in pho84 null mutant cells supplemented with copper or manganese in the medium was like that of the wild type, while copper was increased in these cells under the same conditions (S4A and S4B Figs. in S1 Data). Consequently, lack of metal co-factors for SODs does not account for decreased Sod3 expression, or for slightly decreased Sod1 activity, in pho84 null mutant cells. Since their Pi transport defect was not sufficient to explain the ROS hypersensitivity of C. albicans cells lacking Pho84, and since they did not exhibit lack of metal co-factors for SODs, we turned to another of their phenotypes in order to understand their ROS management defect. These cells exhibit decreased TORC1 signalling [8]. This phenotype can be suppressed by overexpression of one, but not the other, of two small GTPases known to activate C. albicans TORC1 [8]. The small GTPase Gtr1, a component of the EGO complex, which we hypothesize to participate in transmitting a Pi signal to TORC1, suppresses some pho84 phenotypes when overexpressed [8]. Not all pho84 mutant cells’ phenotypes were suppressible in this way; e. g. GTR1 overexpression did not suppress the hyphal morphogenesis defect of cells lacking Pho84 (S5A Fig. in S1 Data). We asked whether ROS management of these cells might be improved by activating TORC1 downstream of overexpressed GTR1. In the absence of exogenous oxidative stressors, GTR1 overexpression decreased the DCFDA-detectable ROS in cells with active TORC1, as well as in cells experiencing TORC1 inhibition by exposure to a low concentration of rapamycin (Fig 5A). Sod3 expression recovered in pho84 cells overexpressing GTR1 (Fig 5B), but resistance to killing by whole blood or neutrophils did not (S5B, C Fig. in S1 Data). Previously we had shown that inhibition of Pho84 with small molecules, phosphonoacetic acid (PAA) and phosphonoformic acid (foscarnet, Fos), leads to decreased TORC1 signaling [8]. To test whether small-molecule Pho84 inhibition also leads to defective ROS management, we exposed wild type cells to these compounds and measured their DCFDA-detectable ROS. Exposure to Pho84 inhibitors increased the ROS content of wild type cells unexposed to external oxidative stressors (Fig 5C), suggesting that the ROS detoxifying role of Pho84 can be targeted pharmacologically. Together, these results indicate that activating TORC1 downstream of Pho84 can suppress a specific ROS detoxification defect of pho84 cells, Sod3 expression, but that this is only one among the mechanisms that render these cells hypersensitive to neutrophil-imposed oxidative stress. We then tested whether heterologously controlled overexpression of SOD3 could directly suppress ROS hypersensitivity of cells lacking Pho84. In PHO84 wild type and pho84 null mutant backgrounds that express a repressible tTA [34], we replaced 50 bases of the native promoter of one SOD3 allele with a construct in which transcription is controlled by tetO. In the absence of doxycyline, this construct induces high-level transcription, while it results in substantial transcriptional repression during doxycycline exposure [35]. In pho84 null mutant cells, SOD3 overexpression largely, but not completely, suppressed in vitro sensitivity to the superoxide-inducing compound plumbagin, but it had no detectable effect in the wild type (Fig 6). Transcriptional repression of one SOD3 allele from tetO had a strong plumbagin-sensitizing effect on pho84 null mutant cells. To control for potential artifactual effects of doxycycline exposure, we also replaced the same SOD3 promoter sequence with a glucose-repressible MAL2 promoter and examined the response to plumbagin. Transcriptional repression from pMAL2 (in glucose) phenotypically resembled that from tetO (in doxycycline) (Fig 6), indicating that repression of SOD3 transcription, and not an off-target effect of doxycycline, was responsible for superoxide hypersensitivity of the cells lacking Pho84 under these conditions. Since ROS resistance was not completely recovered in pho84 null mutant cells overexpressing SOD3, we concluded that misregulation of SOD3 expression level is one, but not the only mechanism of ROS hypersensitivity of cells lacking Pho84. Cells lacking Pho84 that overexpressed SOD3 from tetO did not recover resistance to whole blood or to neutrophils (S6A, B Fig. in S1 Data), indicating that in these complex ex vivo environments, Pho84 is required for further functions beyond Sod3 expression, to confer resistance to host defenses on the fungal cell. Our working model is that one mechanism, among others, by which Pho84 contributes to oxidative stress management is by activating TORC1, which in turn induces Sod3 expression (Fig 7). This model raises several testable questions, including which downstream branch of TORC1 signaling impacts Sod3 levels, whether Sod3 is regulated at a transcriptional, translational or posttranslational level, and whether other C. albicans mechanisms for ROS management, affected by lack of Pho84, also depend on TORC1 signaling.
Virulence attenuation in mutants of a single macronutrient transporter like Pho84 (Fig 1), one of 4 predicted cell-surface Pi transporters of C. albicans, is unusual and has not been reported to our knowledge. In addition to their hyphal growth defect (Fig 1D), we posit that pho84 null mutant cells’ diminished virulence is due to their hypersensitivity to neutrophil killing (Fig 2B–2D). When a neutrophil encounters an invasive pathogen, a broad array of noxious molecules and hydrolytic enzymes are exocytosed, released into the phagosome [36] or onto microbial cells during formation of extracellular traps [21]. Among these, ROS are critical for killing C. albicans [16,37,38]. We found that pharmacologically scavenging neutrophil-generated ROS (Fig 2C), blocking neutrophil ROS production (Fig 2D), or exposing C. albicans to neutrophils from a patient with a genetic defect in ROS production (Fig 2E) rescued viability of pho84 null mutant cells in this interaction. These findings indicate that among the large neutrophil armamentarium [26], C. albicans cells without functional Pho84 are particularly sensitive to ROS. pho84 null mutant cells were hypersensitive in vitro to each of 3 distinct sources of exogenous ROS, plumbagin and menadione, which generate the superoxide anion, and to the peroxide source H2O2 (Fig 3A). In C. albicans, the HOG signaling pathway responds to oxidative stress to induce protective responses [28,39]. The paradox that pho84 null mutant cells with supranormal HOG signalling exhibited increased oxidative stress sensitivity (Fig 3) was resolved when these cells were found to harbor excessive ROS concentrations, including in unstressed normal growth conditions. This constellation of elevated intracellular ROS and hyperreactive HOG signaling suggests that pho84 mutant cells’ capacity for detoxifying ROS is at its limit during unperturbed growth, leading to unchecked oxidative stress and increased cell death during exposure to neutrophils. Lack of sufficient Pi for NADPH synthesis, required for the ROS-detoxifying activities of glutathione- and thioredoxin reductases [40], could also account for ROS hypersensitivity of pho84 mutant cells. However, in high ambient Pi, where the contribution of Pho84 to intracellular Pi stores is less critical [8], their ROS were still ~2-fold those of the wild type (Fig 4A), evincing an additional mechanism. The first line of defense against exogenous ROS released by host phagocytes onto a fungal cell are SODs bound to the cell surface [41,42]. Their importance for C. albicans is underscored by an unusually numerous complement of 3 extracellular SODs in this species [15]. Among extracellular SODs, hyphal-specific Sod5 was recently shown to participate in morphogenetic regulation by disproportionating superoxide anion generated by the newly discovered C. albicans NADPH oxidase Fre8, since the resulting peroxide stimulates hyphal extension [43]. Transcription of SOD5 is upregulated in cells lacking the PHO regulon transcription factor Pho4, which induces PHO84 expression [31], and whose nuclear localization is predicted to be induced in the absence of Pho84 [44]. Potential relationships between Pho4-regulated PHO84 and SOD5 expression and activities, and their possible effect on hyphal morphogenesis, await further experimental examination. If exogenous superoxide anion escapes detoxification by extracellular SODs and becomes protonated, it can cross the cytoplasmic membrane to damage intracellular macromolecules; cytosolic SODs are present to scavenge these ROS [15]. We found a sharp decrease in the protein level of the cytosolic superoxide dismutase Sod3 in pho84 null mutant cells. Sod3 is a singular manganese-requiring cytosolic SOD [45], one of an unusual pair of cytosolic SODs encoded only by some fungal genomes [32]. As during invasive infection C. albicans cells are exposed to drastic changes in copper concentrations, they switch between expression of cytosolic copper-Sod1 during copper repletion or manganese-Sod3 during copper starvation [32]. Expression of PHO84 is controlled by the Pho4 transcription factor, which regulates almost a thousand genes in response to Pi availability [31]. The contribution of C. albicans Pho4 to virulence is attributed to its role in metal homeostasis [31]. In contrast, we found Pho84 to be dispensable for metal repletion of the cell (S3 Fig. in S1 Data). Failing to find a defect in metal uptake of pho84 null mutant cells to account for decreased Sod3 levels (S3 Fig. in S1 Data), we looked to their defect in TORC1 signalling. The mitochondria are major sources of ROS which are byproducts of respiration. TORC1 and mitochondrial ROS both impact the yeast chronological lifespan and aging [46], and mitochondrial protein quality control affects TORC1 signaling and C. albicans virulence [47]. But a requirement for active TORC1 to induce cytosolic SOD expression and thereby manage innate or exogenous ROS (Fig 5) has not previously been described in any organism to our knowledge. Further work will have to determine whether the TORC1-activating function of Pho84 depends on Pi transport or whether it resides in a genetically or chemically separable signalling activity, like the transceptor activity of S. cerevisiae Pho84 towards PKA [48,49]. Our finding that Sod3 protein levels are decreased in cells lacking Pho84, but can be recovered when TORC1 is activated downstream of Pho84 (Fig 5), opens further areas of investigation. Among them is the relationship between TORC1 activity and that of other SODs, since one could speculate that active TORC1 corresponds to an active metabolic state, which gives rise to increased intrinsic ROS and requires increased SOD activity. TORC1 signaling integrates information and responses to a large number of parameters, most of which are likely to be involved in the interaction with competing members of the microbiome and with the host. Hence we do not expect that simply upregulating TORC1 by overexpressing GTR1 enhances C. albicans virulence. It is likely the flexibility, not the intensity of pro-anabolic TORC1 signaling that maximizes fungal survival and proliferation. Our findings nevertheless highlight the importance of Pi signaling as one input to TORC1 for the capacity of the fungus to withstand stressors imposed by the host and maximize its virulence potential. Resistance of C. albicans pho84 null mutant cells to in vitro ROS exposure was significantly restored by overexpression of SOD3 (Fig 6). Transcriptional repression of one SOD3 allele resulted in synthetic ROS hypersensitivity with deletion of PHO84 (Fig 6). However, resistance to ex vivo stressors, whole blood and neutrophil killing, was not restored to pho84 mutant cells by overexpression of SOD3 from the heterologous tetO construct. Hence, maintenance of Sod3 levels via TORC1 is only part of the mechanisms by which Pho84 contributes to resistance to host defenses. Other mechanisms, including provision of Pi for NADPH-dependent ROS management and interactions with other SODs, await experimental identification. In addition to reactive oxygen species detoxification, Pho84 also contributes to hyphal growth of C. albicans [8]. In the murine disseminated candidiasis model, we observed reduced hyphal growth of cells lacking Pho84 in vivo. A requirement for SOD activity in virulence was found each time it was examined [20,41,50]; this is not the case for hyphal growth [51–53]. Nevertheless, defective regulation of hyphal morphogenesis may also contribute to pho84 mutants’ virulence attenuation. This defect is not suppressed by GTR1 overexpression. Loss of Pho84 appears to affect virulence of C. albicans by further mechanisms beyond those we have defined so far. We previously showed that C. albicans Pho84 and its TORC1-activating function can be inhibited by small molecules [8], raising the possibility of identifying further inhibitors with favorable therapeutic indices. The compounds we investigated include an FDA-approved antiviral agent, which at concentrations achieved in plasma during therapeutic dosing inhibits hyphal morphogenesis and potentiates the in vitro activity of important medications representing 2 of the 3 classes of antifungal agents in clinical use [8]. Pho84 has no human homolog, so its inhibitors can spare human targets. We now find that these compounds also perturb ROS management (Fig 5C), so that inhibiting Pho84 with novel compounds could simultaneously potentiate antifungal drugs and impair C. albicans virulence. Pathogenic unicellular parasites require intact Pi homeostasis for development and virulence [9,11,14,54], as do bacterial pathogens [55]. Among fungi, representatives of all extant lineages possess core components of the PHO regulon, including high-affinity H+-Pi symporters like Pho84 [56]. In a basal fungal phylum, the water mold Blastocladiella emersonii’s complex life cycle [57] depends on Pi availability [56,58] as it progresses between flagellated zoospores, their germination, vegetative growth with production of a rhizoidal system, and sporulation [58]. Pi uptake and expression of PHO regulon components correspond to the life cycle stage of this fungus, indicating an ancient origin of the PHO system at or near the emergence of the fungal kingdom and suggesting its early connection to developmental regulation [56]. Links of Pi homeostasis with the complex developmental steps required in C. albicans pathogenesis hence have primordial precedents in its primitive fungal predecessors. Advancing analysis of Pi acquisition and homeostasis in fungal pathogens [31,59–63] to the level of understanding achieved in model fungi like S. cerevisiae, Neurospora crassa and mycorrhizal fungi [44,64–68], could elucidate further virulence determinants and, given fundamental differences between human and fungal phosphate homeostasis, potentially identify much-needed further targets of antifungal therapy.
For experiments utilizing human neutrophils whose donors were identifiable, written informed consent was obtained from study subjects, according to protocols approved by the Boston Children’s Hospital Institutional Review Board. For human neutrophils isolated from anonymized blood bank donation aliquots, no informed consent was obtained because the donors were not identifiable, and this procedure was approved by the Boston Children’s Hospital Institutional Review Board. The Blood Bank Laboratory at the Boston Children’s Hospital provided the anonymized blood samples. All animal experiments were performed in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Institutional Animal Care and Use Committee of Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA reviewed and approved the murine oropharyngeal candidiasis experimental protocol, IACUC Project No. 30842–01 (A3330-01). All animal experimentation of the intravenously infected, disseminated murine candidiasis model was conducted in an AAALAC-certified facility at The University of Texas at San Antonio (UTSA) following the National Institutes of Health guidelines for housing and care of laboratory animals and performed in accordance with institutional regulations after pertinent review and approval by the Institutional Animal Care and Use Committee at The University of Texas at San Antonio. UTSA Animal Welfare Assurance ID D16-00357 and UTSA IACUC Protocol Number MU018. Mice were allowed a 1-week acclimatisation period before experiments were started. C. albicans strains used are shown in Supporting Information (S) Table 1 in S1 Text. Strains were constructed as in [8]; introduced mutations were confirmed by PCR spanning the upstream and downstream homologous recombination junctions of transforming constructs, and sequencing. Plasmids and primers used for strain construction are listed in Table 2 and Table 3 in S1 Text, respectively. Prototrophs or strains with equal auxotrophies were compared. At least two strains of newly constructed genotypes were examined phenotypically. Only after confirming that their phenotypes were the same, was one strain of each genotype chosen for representation. Experiments with defined ambient Pi concentrations were performed in YNB 0 Pi (ForMedium Ltd, Norfolk, UK) with added KH2PO4 to stated concentrations. Low fluorescence medium for ROS measurement was made from YNB Loflo (ForMedium Ltd, Norfolk, UK). Other media were used as previously described [35]. Culture conditions of cells during growth before each experiment, e. g. overnight, were standardized for each set of experiments. Normal saline (0. 9% NaCl) was used for all wash steps to avoid uncontrolled exposure to Pi in PBS. Cells used for SOD3 repression or -overexpression experiments were maintained in conditions without or with 50 ng/ml doxycycline or using maltose or glucose as carbon source, throughout the course of the experiment, including during washes, beginning at the point of reviving frozen stocks. Nomenclature of strains used in text and figure legends is +/+ (PHO84/PHO84, wild type); -/- (pho84/pho84, null mutant) and -/-/+ (pho84/pho84: : PHO84, reintegrant of intact PHO84 at the native locus). For growth curves in liquid media, cells grown on a YPD plate for 2 days were washed once in 0. 9% NaCl and diluted to an OD600 of 0. 1 in 150 μl medium in flat bottom 96-well dishes. OD600 readings were obtained every 15 min in a BioTekSynergy 2 Multi-Mode Microplate Reader (Winooski, VT, USA). At least 3 biological replicates were obtained on different days. Standard deviations of 3 technical replicates, representing separate wells, were calculated and graphed in Graphpad Prism. Cells were revived from frozen stocks on solid YPD overnight, washed and resuspended in 0. 9% NaCl to OD600 0. 1. Variations between single colonies and colony density effects were minimized by spotting 3 μl cell suspension at 6 equidistant points, using a template, around the perimeter of an agar medium plate as in [35]. Spider medium and RPMI1640 with glutamine, without sodium bicarbonate (Gibco Cat. # 31800–022) were used, the latter buffered to pH 7 with 165 mM MOPS and 2 g/L sodium bicarbonate. All panels shown represent at least 3 biological replicates. Cell harvesting, lysis and western blotting were performed as described in [69]. Antibodies used are listed in Table 4 in S1 Text. At least three biological replicates were obtained on different days. For densitometry, ImageJ (imagej. net/welcome) software (opensource) was used to quantitate signals obtained on a KODAK Image Station 4000MM. The wild-type Drosophila melanogaster OrR strain obtained from the Bloomington stock center was maintained on standard cornmeal agar medium at 25°C. Single colonies of C. albicans strains on YPD were used to inoculate overnight cultures at 30°C. The cultures were diluted in fresh YPD to an OD600 of 0. 1 and incubated shaking at 30°C until an OD600 of 1. 0. A 1 ml aliquot of the culture was centrifuged at 16,000 x g, washed once in phosphate-buffered saline (PBS; pH 7) and re-suspended in 1 ml of PBS. For infection of flies, protocols were followed according to [17]. Male and female flies, 1–5 days old, were injected with approximately 50 nl fungal cell suspensions (approx. 500 cells/fly) using a fine glass capillary needle with a micro-injector (TriTech Research, Los Angeles, CA, USA). Cohorts of 30 flies were injected and maintained in separate vials. At least 3 (up to 7) biological replicates (independently prepared fungal preparations derived from individual colonies) and 6–15 technical replicates per strain were carried out. Infected flies were maintained at 29°C for up to five days after infection and the number of surviving flies was noted on daily basis. Mice were infected by inoculating with Candida albicans cells sublingually as in [18]. Eight mice were infected for each C. albicans strain. Fungal burden was calculated by counting CFU per gram tissue on agar medium after homogenizing tongue tissue. Cultures of C. albicans strains for injection were grown overnight in YPD medium and incubated at 30°C. C. albicans cells (4 × 105) were delivered by tail vein injection into mice, each consisting of eight 6-to-8-week-old female BALB/c mice. Days on which mice died were recorded, and moribund animals were euthanised and recorded as dying the following day. For histology, kidneys retrieved from sacrificed mice were fixed in 10% buffered formalin and embedded in paraffin, and thin tissue slices were obtained and stained with Grocott-Gomori Methenamine Silver (GMS) stain prior to microscopic evaluation. Peripheral blood was collected by venipuncture of healthy adults who had not taken any medications during the ten days prior to the experiment. Blood was gently drawn through a 21-gauge needle into heparinized glass vacutainers. Before exposure to blood or other stressors, the C. albicans cells were inoculated into 10 ml YPD at a density of OD600 = 0. 2, and incubated for 15 hours at 30°C with shaking at 200 rpm. Harvested cells were washed once with normal saline and diluted to a concentration of 5x104/ml counted by an Innovatis CASY Cell Counter (Roche). Fifteen μl cells were added into a 96-well plate first (VWR, Cat. # 62406–081) and incubated with 135 μl blood for indicated time points. To determine CFU/ml, cells were spread on Trypticase Soy Agar with 5% Sheep Blood (Thermo Fisher Scientific, Cat. # B21239X) or on laboratory-made YPD plates and colonies were counted after incubation at room temperature for 2 days. At least 3 biological replicates were performed on different days for each result shown. Human primary neutrophils were isolated from apheresis-derived buffy coats provided by the Blood Bank Laboratory at the Boston Children’s Hospital. Erythrocytes were sedimented by adding an equal volume of dextran/saline solution (3% dextran in PBS) at room temperature for 30 min. The erythrocyte-depleted supernatants were then layered on Lymphocyte Separation Medium (Lymphoprep, Axis Shield) and centrifuged at 400 g at room temperature for 30 min. Contaminating erythrocytes in the neutrophil pellets were lysed by a brief (<30 second) treatment with ice cold water and 0. 6 M KCl. The purity of neutrophils was >90% as determined by Wright—Giemsa staining. Peripheral blood was collected from healthy adult volunteers or from a CGD patient into heparinized glass vacutainers. Granulocytes were isolated using Polymorphoprep (Axis Shield) according to manufacturer instructions. Briefly, whole blood was loaded on top of a Polymorphoprep gradient and centrifuged at 500g for 30 min without brake. According to manufacturer instructions, the neutrophil containing band was harvested. Osmolarity was reestablished by adding an equal volume of 50% HBSS and neutrophils were pelleted. Contaminating red blood cells were lysed by treating the pellet twice with 9 ml of cell culture ready dH2O (Gibco A1287301) and reestablishing osmolarity with 1 ml of 10x PBS. Neutrophils were obtained at a purity >95% with the contaminating cells mainly being RBCs. Boston Children’s Hospital Institutional Review Board protocols were followed for all human blood samples. C. albicans strains were incubated with HL-60 derived neutrophils for 90 minutes at 37°C with 5% CO2 and quantitative culturing on Trypticase Soy Agar with 5% Sheep Blood plates (Thermo Fisher Scientific, Cat. # B21239X). Percent survival was calculated by dividing the number of CFU after co-culturing with HL-60 derived neutrophils by the number of CFU from C. albicans incubated with media without neutrophils. HL-60 derived neutrophils were tested at a 20: 1 phagocyte: fungus ratio in RPMI plus 10% pooled human serum. C. albicans strains were incubated with freshly isolated human neutrophils for the indicated time points at 37°C with 5% CO2. To determine CFU/ml, cells were spread on Trypticase Soy Agar with 5% Sheep Blood (Thermo Fisher Scientific, Cat. # B21239X) or on laboratory-made YPD plates and colonies were counted after incubation at room temperature for 2 days. Isolated human neutrophils were tested in HBSS medium (GIBCO, Cat. # 14025076). To scavenge ROS or inhibit ROS formation, neutrophils were treated with N-acetyl-l-cysteine (NAC) (Sigma) for 90 min at M. O. I. 2, or pretreated with 10μM Diphenyleneiodonium, DPI, (Tocris) for 5 minutes. At least 3 biological replicates were obtained for each experiment on different days. C. albicans cells were grown overnight for 15 hours as described above and washed once with HBSS. 1 x 105 Neutrophils were incubated with C. albicans yeast cells expressing GFP at M. O. I. 2 and 10 for 30 min. Phagocytosing neutrophils were quantified as CD11b+ GFP+ Cells. Two biological replicates were obtained on different days. Extracellular ROS production was determined as described in [70]. Briefly, neutrophils were stimulated with C. albicans yeast at M. O. I. 2 for 1 hour in the presence of 100 mM Cytochrome C. To measure superoxide release, the difference in absorbance of the oxidated form of Cytochrome C (550 nm) and the background absorbance (540 nm) was measured every two minutes in a FLUOstar Omega Microplate Reader. To measure intracellular ROS, neutrophils were stimulated with C. albicans yeast at M. O. I. 2 for 30 minutes and subsequently stained with the ROS sensitive dye chloromethyl-H2DCFDA (Thermo Fisher Scientific, Cat. # D399) for 30 min. Two biological replicates were obtained on different days. Yeast cells grown overnight in YPD for 15 hours were washed twice with normal saline and diluted into SC medium (Loflo) supplemented with different concentrations of KH2PO4 at OD600 0. 5. The fluorescent dye 2’, 7’-dichlorodihydrofluorescein diacetate (DCFDA) (Sigma, Cat# D6883) was added into the medium to a final concentration of 50 μM. After incubation for 50 min, cells were washed twice with normal saline. The intensity of fluorescence was read in a TE-CAN plate reader at excitation- 485 nm and emission wavelength 528 nm [71], and a ratio of intensity with OD600 of the culture was calculated. At least 3 biological replicates were obtained for each experiment on different days. Cells were grown for 13 hours in SC medium with starting OD600 0. 01. Harvested cells were washed with sterile MilliQ H2O once and lysed by bead beating. A 10% Tris-glycine gel (Invitrogen, Cat. # XP00100BOX) gel was used for running samples and stained with nitroblue tetrazolium as described in [72]. For Western blotting, the membrane was probed with primary antibodies: anti-Sod3 antibody [72] and anti-tubulin rat monoclonal Ab (Abcam, Cat. # ab6161) as loading control. Secondary antibodies were anti-rabbit secondary antibody (Santa Cruz Biotechnology, Cat. # 2370) and anti-rat secondary antibody (Santa Cruz Biotechnology, Cat. #97057). The blots were imaged using a KODAK Image Station 4000MM. At least 3 biological replicates were obtained for each experiment on different days. Cells were grown in SC medium with starting OD600 = 0. 01 for 13 hours. Harvested cell were washed twice with cold 1X TE (10 mM Tris, 1 mM EDTA pH 7. 5) and once with cold milliQ water. Cells were resuspended in 1 mL of deionised water and 10 OD600 cells were used for analysis of Cu and Mn by a PerkinElmer Life Sciences AAnalyst 600 graphite furnace atomic absorption spectrometer. Three biological replicates were obtained for each experiment on different days. Statistical analysis was performed by Kaplan-Meier analysis for survival curves, and unpaired Student’s t test for comparison of numerical values in Prism 7 Graphpad software (GraphPad Software, Inc. , CA, USA).
Candida albicans is the species most often isolated from patients with invasive fungal disease, and is also a common colonizer of healthy people. It is well equipped to compete for nutrients with bacteria co-inhabiting human gastrointestinal mucous membranes, since it possesses multiple transporters to internalize important nutrients like sugars, nitrogen sources, and phosphate. During infection, the fungus needs to withstand human defense cells that attack it with noxious chemicals, among which reactive oxygen species (ROS) are critical. We found that a high-affinity phosphate transporter, Pho84, is required for C. albicans’ ability to successfully invade animal hosts and to eliminate ROS. Levels of a fungal enzyme that breaks down ROS, Sod3, were decreased in cells lacking Pho84. A connection between this phosphate transporter and the ROS-detoxifying enzyme was identified in the Target of Rapamycin (TOR) pathway, to which Pho84 is known to provide activating signals when phosphate is abundant. Small molecules that block Pho84 activity impair the ability of C. albicans to detoxify ROS. Since humans manage phosphate differently than fungi and have no Pho84 homolog, a drug that inhibits Pho84 could disable the defense of the fungus against the host.
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Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence