Candida albicans' inorganic phosphate transport and evolutionary adaptation to phosphate scarcity.

Phosphorus is essential in all cells' structural, metabolic and regulatory functions. For fungal cells that import inorganic phosphate (Pi) up a steep concentration gradient, surface Pi transporters are critical capacitators of growth. Fungi must deploy Pi transporters that enable optimal Pi uptake in pH and Pi concentration ranges prevalent in their environments. Single, triple and quadruple mutants were used to characterize the four Pi transporters we identified for the human fungal pathogen Candida albicans, which must adapt to alkaline conditions during invasion of the host bloodstream and deep organs. A high-affinity Pi transporter, Pho84, was most efficient across the widest pH range while another, Pho89, showed high-affinity characteristics only within one pH unit of neutral. Two low-affinity Pi transporters, Pho87 and Fgr2, were active only in acidic conditions. Only Pho84 among the Pi transporters was clearly required in previously identified Pi-related functions including Target of Rapamycin Complex 1 signaling and hyphal growth. We used in vitro evolution and whole genome sequencing as an unbiased forward genetic approach to probe adaptation to prolonged Pi scarcity of two quadruple mutant lineages lacking all 4 Pi transporters. Lineage-specific genomic changes corresponded to divergent success of the two lineages in fitness recovery during Pi limitation. In this process, initial, large-scale genomic alterations like aneuploidies and loss of heterozygosity were eventually lost as populations presumably gained small-scale mutations. Severity of some phenotypes linked to Pi starvation, like cell wall stress hypersensitivity, decreased in parallel to evolving populations' fitness recovery in Pi scarcity, while that of others like membrane stress responses diverged from these fitness phenotypes. C. albicans therefore has diverse options to reconfigure Pi management during prolonged scarcity. Since Pi homeostasis differs substantially between fungi and humans, adaptive processes to Pi deprivation may harbor small-molecule targets that impact fungal growth and virulence.

Glycerophosphocholine provision rescues Candida albicans growth and signaling phenotypes associated with phosphate limitation.

IMPORTANCE: Candida albicans is the most commonly isolated species from patients suffering from invasive fungal disease. C. albicans is most commonly a commensal organism colonizing a variety of niches in the human host. The fungus must compete for resources with the host flora to acquire essential nutrients such as phosphate. Phosphate acquisition and homeostasis have been shown to play a key role in C. albicans virulence, with several genes involved in these processes being required for normal virulence and several being upregulated during infection. In addition to inorganic phosphate (Pi), C. albicans can utilize the lipid-derived metabolite glycerophosphocholine (GPC) as a phosphate source. As GPC is available within the human host, we examined the role of GPC in phosphate homeostasis in C. albicans. We find that GPC can substitute for Pi by many though not all criteria and is likely a relevant physiological phosphate source for C. albicans.

Stress- and metabolic responses of Candida albicans require Tor1 kinase N-terminal HEAT repeats.

Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells that express a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing wild type TOR1 from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. Consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats additionally were incapable of utilizing non-fermentable carbon sources. They were also hypersensitive to inhibitors of specific complexes within the respiratory electron transport chain, suggesting that inefficient ATP generation and a resulting dearth of nucleotide sugar building blocks for cell wall polysaccharides causes cell wall integrity defects in these mutants. Genome-wide expression analysis of cells lacking N-terminal HEAT repeats showed dysregulation of carbon metabolism, cell wall biosynthetic enzymes, translational machinery biosynthesis, oxidative stress responses, and hyphal- as well as white-opaque cell type-associated genes. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might selectively abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

Platelet transfusions in a murine model of neonatal polymicrobial sepsis: Divergent effects on inflammation and mortality.

BACKGROUND: Platelet transfusions (PTxs) are often given to septic preterm neonates at high platelet count thresholds in an attempt to reduce bleeding risk. However, the largest randomized controlled trial (RCT) of neonatal transfusion thresholds found higher mortality and/or major bleeding in infants transfused at higher thresholds. Using a murine model, we investigated the effects of adult PTx on neonatal sepsis-induced mortality, systemic inflammation, and platelet consumption. STUDY DESIGN AND METHODS: Polymicrobial sepsis was induced via intraperitoneal injection of cecal slurry preparations (CS1, 2, 3) into P10 pups. Two hours after infection, pups were transfused with washed adult Green Flourescent Protein (GFP+) platelets or control. Weights, platelet counts, and GFP% were measured before 4 and 24 h post-infection. At 24 h, blood was collected for quantification of plasma cytokines. RESULTS: The CS batches varied in 24 h mortality (11%, 73%, and 30% in CS1, 2, and 3, respectively), due to differences in bacterial composition. PTx had differential effects on sepsis-induced mortality and systemic inflammatory cytokines, increasing both in mice infected with CS1 (low mortality) and decreasing both in mice infected with CS2 and 3. In a mathematical model of platelet kinetics, the consumption of transfused adult platelets was higher than that of endogenous neonatal platelets, regardless of CS batch. DISCUSSION: Our findings support the hypothesis that transfused adult platelets are consumed faster than endogenous neonatal platelets in sepsis and demonstrate that PTx can enhance or attenuate neonatal inflammation and mortality in a model of murine polymicrobial sepsis, depending on the composition of the inoculum and/or the severity of sepsis.

Inflammasome-mediated GSDMD activation facilitates escape of Candida albicans from macrophages.

Candida albicans is the most common cause of fungal sepsis. Inhibition of inflammasome activity confers resistance to polymicrobial and LPS-induced sepsis; however, inflammasome signaling appears to protect against C. albicans infection, so inflammasome inhibitors are not clinically useful for candidiasis. Here we show disruption of GSDMD, a known inflammasome target and key pyroptotic cell death mediator, paradoxically alleviates candidiasis, improving outcomes and survival of Candida-infected mice. Mechanistically, C. albicans hijacked the canonical inflammasome-GSDMD axis-mediated pyroptosis to promote their escape from macrophages, deploying hyphae and candidalysin, a pore-forming toxin expressed by hyphae. GSDMD inhibition alleviated candidiasis by preventing C. albicans escape from macrophages while maintaining inflammasome-dependent but GSDMD-independent IL-1ß production for anti-fungal host defenses. This study demonstrates key functions for GSDMD in Candida's escape from host immunity in vitro and in vivo and suggests that GSDMD may be a potential therapeutic target in C. albicans-induced sepsis.

Reverse Genetics System for Rabbit vesivirus.

Most caliciviruses are refractory to replication in cell culture and only a few members of the family propagate in vitro. Rabbit vesivirus (RaV) is unique due to its ability to grow to high titers in several animal and human cell lines. This outstanding feature makes RaV an ideal candidate for reverse genetics studies, an invaluable tool to understand the molecular basis of virus replication, the biological functions of viral genes and their roles in pathogenesis. The recovery of viruses from a cDNA clone is a prerequisite for reverse genetics studies. In this work, we constructed a RaV infectious cDNA clone using a plasmid expression vector, under the control of bacteriophage T7 RNA-polymerase promoter. The transfection of permissive cells with this plasmid DNA in the presence of T7 RNA-polymerase, provided in trans by a helper recombinant poxvirus, led to de novo synthesis of RNA transcripts that emulated the viral genome. The RaV progeny virus produced the typical virus-induced cytopathic effect after several passages of cell culture supernatants. Similarly, infectious RaV was recovered when the transcription step was performed in vitro, prior to transfection, provided that a 5'-cap structure was added to the 5' end of synthetic genome-length RNAs. In this work, we report an efficient and consistent RaV rescue system based on a cDNA transcription vector, as a tool to investigate calicivirus biology through reverse genetics.

Phosphate in Virulence of Candida albicans and Candida glabrata.

Candida species are the most commonly isolated invasive human fungal pathogens. A role for phosphate acquisition in their growth, resistance against host immune cells, and tolerance of important antifungal medications is becoming apparent. Phosphorus is an essential element in vital components of the cell, including chromosomes and ribosomes. Producing the energy currency of the cell, ATP, requires abundant inorganic phosphate. A comparison of the network of regulators and effectors that controls phosphate acquisition and intracellular distribution, the PHO regulon, between the model yeast Saccharomyces cerevisiae, a plant saprobe, its evolutionarily close relative C. glabrata, and the more distantly related C. albicans, highlights the need to coordinate phosphate homeostasis with adenylate biosynthesis for ATP production. It also suggests that fungi that cope with phosphate starvation as they invade host tissues, may link phosphate acquisition to stress responses as an efficient mechanism of anticipatory regulation. Recent work indicates that connections among the PHO regulon, Target of Rapamycin Complex 1 signaling, oxidative stress management, and cell wall construction are based both in direct signaling links, and in the provision of phosphate for sufficient metabolic intermediates that are substrates in these processes. Fundamental differences in fungal and human phosphate homeostasis may offer novel drug targets.

Phosphoric Metabolites Link Phosphate Import and Polysaccharide Biosynthesis for Candida albicans Cell Wall Maintenance.

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin (TOR) signaling, oxidative stress resistance, and virulence of this fungal pathogen. It also contributes to C. albicans' tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild-type cells recovering from phosphate starvation. Nonphosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar, GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. We posit that low substrate concentrations of beta-d-glucan- and chitin synthases, together with pharmacologic inhibition of their activity, diminish enzymatic reaction rates as well as the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-d-glucans or chitin. Hence, inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.IMPORTANCECandida species cause hundreds of thousands of invasive infections with high mortality each year. Developing novel antifungal agents is challenging due to the many similarities between fungal and human cells. Maintaining phosphate balance is essential for all organisms but is achieved completely differently by fungi and humans. A protein that imports phosphate into fungal cells, Pho84, is not present in humans and is required for normal cell wall stress resistance and cell wall integrity signaling in C. albicans Nucleotide sugars, which are phosphate-containing building block molecules for construction of the cell wall, are diminished in cells lacking Pho84. Cell wall-constructing enzymes may be slowed by lack of these building blocks, in addition to being inhibited by drugs. Combined targeting of Pho84 and cell wall-constructing enzymes may provide a strategy for antifungal therapy by which two sequential steps of cell wall maintenance are blocked for greater potency.

Cytosolic Acidification Is the First Transduction Signal of Lactoferrin-induced Regulated Cell Death Pathway.

In yeast, we reported the critical role of K+-efflux for the progress of the regulated cell death (RCD) induced by human lactoferrin (hLf), an antimicrobial protein of the innate immune system that blocks Pma1p H+-ATPase. In the present study, the K+ channel Tok1p was identified as the K+ channel-mediating K+-efflux, as indicated by the protective effect of extracellular K+ (30 mM), K+-channel blockers, and the greater hLf-resistance of TOK1-disrupted strains. K+-depletion was necessary but not sufficient to induce RCD as inferred from the effects of valinomycin, NH4Cl or nigericin which released a percentage of K+ similar to that released by lactoferrin without affecting cell viability. Cytosolic pH of hLf-treated cells decreased transiently (0.3 pH units) and its inhibition prevented the RCD process, indicating that cytosolic acidification was a necessary and sufficient triggering signal. The blocking effect of lactoferrin on Pma1p H+-ATPase caused a transitory decrease of cytosolic pH, and the subsequent membrane depolarization activated the voltage-gated K+ channel, Tok1p, allowing an electrogenic K+-efflux. These ionic events, cytosolic accumulation of H+ followed by K+-efflux, constituted the initiating signals of this mitochondria-mediated cell death. These findings suggest, for the first time, the existence of an ionic signaling pathway in RCD.

Candida albicans Dispersed Cells Are Developmentally Distinct from Biofilm and Planktonic Cells.

Candida albicans surface-attached biofilms such as those formed on intravenous catheters with direct access to the bloodstream often serve as a nidus for continuous release of cells capable of initiating new infectious foci. We previously reported that cells dispersed from a biofilm are yeast cells that originate from the top-most hyphal layers of the biofilm. Compared to their planktonic counterparts, these biofilm dispersal yeast cells displayed enhanced virulence-associated characteristics and drug resistance. However, little is known about their molecular properties. To address that issue, in this study we aimed to define the molecular characteristics of these biofilm dispersal cells. We found that the inducer of dispersal, PES1, genetically interacts with the repressor of filamentation, NRG1, in a manner consistent with the definition of dispersed cells as yeast cells. Further, using a flow biofilm model, we performed comprehensive comparative RNA sequencing on freshly dispersed cells in order to identify unique transcriptomic characteristics. Gene expression analysis demonstrated that dispersed cells largely inherit a biofilm-like mRNA profile. Strikingly, however, dispersed cells seemed transcriptionally reprogrammed to acquire nutrients such as zinc and amino acids and to metabolize alternative carbon sources, while their biofilm-associated parent cells did not induce the same high-affinity transporters or express gluconeogenetic genes, despite exposure to the same nutritional signals. Collectively, the findings from this study characterize cell dispersal as an intrinsic step of biofilm development which generates propagules more adept at colonizing distant host sites. This developmental step anticipates the need for virulence-associated gene expression before the cells experience the associated external signals.IMPORTANCECandida albicans surface-attached biofilms serve as a reservoir of cells to perpetuate and expand an infection; cells released from biofilms on catheters have direct access to the bloodstream. Biofilm dispersal yeast cells exhibit enhanced adhesion, invasion, and biofilm formation compared to their planktonic counterparts. Here, we show using transcriptome sequencing (RNA-seq) that dispersed yeast cells are developmentally distinct from the cells in their parent biofilms as well as from planktonic yeast cells. Dispersal cells possess an anticipatory expression pattern that primes them to infect new sites in the host, to survive in nutrient-starved niches, and to invade new sites. These studies identified dispersal cells as a unique proliferative cell type of the biofilm and showed that they could serve as targets for antibiofilm drug development in the future.

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence.

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.

Antifungal Mechanism of Action of Lactoferrin: Identification of H+-ATPase (P3A-Type) as a New Apoptotic-Cell Membrane Receptor.

Human lactoferrin (hLf) is a protein of the innate immune system which induces an apoptotic-like process in yeast. Determination of the susceptibility to lactoferrin of several yeast species under different metabolic conditions, respiratory activity, cytoplasmic ATP levels, and external medium acidification mediated by glucose assays suggested plasma membrane Pma1p (P3A-type ATPase) as the hLf molecular target. The inhibition of plasma membrane ATPase activity by hLf and the identification of Pma1p as the hLf-binding membrane protein confirmed the previous physiological evidence. Consistent with this, cytoplasmic ATP levels progressively increased in hLf-treated Candida albicans cells. However, oligomycin, a specific inhibitor of the mitochondrial F-type ATPase proton pump (mtATPase), abrogated the antifungal activity of hLf, indicating a crucial role for mtATPase in the apoptotic process. We suggest that lactoferrin targeted plasma membrane Pma1p H(+)-ATPase, perturbing the cytoplasmic ion homeostasis (i.e., cytoplasmic H(+) accumulation and subsequent K(+) efflux) and inducing a lethal mitochondrial dysfunction. This initial event involved a normal mitochondrial ATP synthase activity responsible for both the ATP increment and subsequent hypothetical mitochondrial proton flooding process. We conclude that human lactoferrin inhibited Pma1p H(+)-ATPase, inducing an apoptotic-like process in metabolically active yeast. Involvement of mitochondrial H(+)-ATPase (nonreverted) was essential for the progress of this programmed cell death in which the ionic homeostasis perturbation seems to precede classical nonionic apoptotic events.