Small but crucial: the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans

Small heat shock proteins (sHsps) have multiple cellular functions. However, the biological function of sHsps in pathogenic microorganisms is largely unknown. In the present study we identified and characterized the novel sHsp Hsp21 of the human fungal pathogen Candida albicans. Using a reverse genetics approach we demonstrate the importance of Hsp21 for resistance of C. albicans to specific stresses, including thermal and oxidative stress. Furthermore, a hsp21Δ/Δ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filament-inducing conditions. Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21Δ/Δ mutant exhibited a strongly reduced capacity to damage both cell lines. Furthermore, Hsp21 was required for resisting killing by human neutrophils. Measurements of intracellular levels of stress protective molecules demonstrated that Hsp21 is involved in both glycerol and glycogen regulation and plays a major role in trehalose homeostasis in response to elevated temperatures. Mutants defective in trehalose and, to a lesser extent, glycerol synthesis phenocopied HSP21 deletion in terms of increased susceptibility to environmental stress, strongly impaired capacity to damage epithelial cells and increased sensitivity to the killing activities of human primary neutrophils. Via systematic analysis of the three main C. albicans stress-responsive kinases (Mkc1, Cek1, Hog1) under a range of stressors, we demonstrate Hsp21-dependent phosphorylation of Cek1 in response to elevated temperatures. Finally, the hsp21Δ/Δ mutant displayed strongly attenuated virulence in two in vivo infection models. Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C.?albicans pathogenicity. Hsp21 therefore represents the first reported example of a small heat shock protein functioning as a virulence factor in a eukaryotic pathogen.     

Host cell invasion and virulence mediated by Candida albicans Ssa1

Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1Δ/Δ mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2Δ/Δ mutant did not. In vitro studies revealed that the ssa1Δ/Δ mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1Δ/Δ mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease.     

Characterization of a hyperactive Cyr1 mutant reveals new regulatory mechanisms for cellular cAMP levels in Candida albicans

The adenylyl cyclase Cyr1 plays a pivotal role in regulating virulence traits in the human fungal pathogen Candida albicans. Although a diverse range of signals are known to activate Cyr1, it remains unclear how low activity is maintained in the absence of stimuli. To uncover negative regulatory elements, we designed a genetic screen to identify mutations in Cyr1 that increase its catalytic activity. We found such a mutant carrying a single Glu1541 to Lys substitution in a conserved motif C-terminal to the catalytic domain. This E1541K mutation caused constitutive filamentous growth, hypersensitivity to stress, resistance to farnesol and overproduction of riboflavin. The mutant phenotype depends on Cap1 and Ras1, two known positive regulators of Cyr1, and the filamentous growth requires Hgc1, a key promoter of hyphal growth. Strikingly, expressing a truncated version of the mutant protein lacking the entire region N-terminal to the catalytic domain in cyr1Δ cells caused a fivefold increase in the cellular cAMP level. Such cells exhibited dramatic enlargement, cytokinetic defects, G1 arrest and impaired hyphal development. Thus, our studies have revealed novel regulatory elements in Cyr1 that normally repress Cyr1 activity to prevent the toxicity of unregulated high cAMP levels.     

Role of CaECM25 in cell morphogenesis, cell growth and virulence in Candida albicans

Candida albicans is the most prominent opportunistic fungal pathogen in humans. Multiple factors are associated with the virulence of C. albicans, including morphogenesis, cell wall organization and growth rate. Here, we describe the identification and functional characterization of CaECM25, a gene that has not been reported before. We constructed Caecm25?/? mutants and investigated the role of the gene in morphogenesis, cell wall organization and virulence. CaECM25 deletion resulted in defects in cell separation, a slower growth rate, reduced filamentous growth and attenuated adherence to plastic surfaces. The Caecm25?/? mutant was also significantly less virulent than wild type when tested for systemic infection in mice. Therefore, CaECM25 plays important roles in morphogenesis, cell wall organization and virulence.     

Mechanisms of Candida albicans trafficking to the brain

During hematogenously disseminated disease, Candida albicans infects most organs, including the brain. We discovered that a C. albicans vps51Δ/Δ mutant had significantly increased tropism for the brain in the mouse model of disseminated disease. To investigate the mechanisms of this enhanced trafficking to the brain, we studied the interactions of wild-type C. albicans and the vps51Δ/Δ mutant with brain microvascular endothelial cells in vitro. These studies revealed that C. albicans invasion of brain endothelial cells is mediated by the fungal invasins, Als3 and Ssa1. Als3 binds to the gp96 heat shock protein, which is expressed on the surface of brain endothelial cells, but not human umbilical vein endothelial cells, whereas Ssa1 binds to a brain endothelial cell receptor other than gp96. The vps51Δ/Δ mutant has increased surface expression of Als3, which is a major cause of the increased capacity of this mutant to both invade brain endothelial cells in vitro and traffic to the brain in mice. Therefore, during disseminated disease, C. albicans traffics to and infects the brain by binding to gp96, a unique receptor that is expressed specifically on the surface of brain endothelial cells.     

Phosphatidylserine synthase and phosphatidylserine decarboxylase are essential for cell wall integrity and virulence in Candida albicans

Phospholipid biosynthetic pathways play crucial roles in the virulence of several pathogens; however, little is known about how phospholipid synthesis affects pathogenesis in fungi such as Candida albicans. A C. albicans phosphatidylserine (PS) synthase mutant, cho1Δ/Δ, lacks PS, has decreased phosphatidylethanolamine (PE), and is avirulent in a mouse model of systemic candidiasis. The cho1Δ/Δ mutant exhibits defects in cell wall integrity, mitochondrial function, filamentous growth, and is auxotrophic for ethanolamine. PS is a precursor for de novo PE biosynthesis. A psd1Δ/Δ psd2Δ/Δ double mutant, which lacks the PS decarboxylase enzymes that convert PS to PE in the de novo pathway, has diminished PE levels like those of the cho1Δ/Δ mutant. The psd1Δ/Δ psd2Δ/Δ mutant exhibits phenotypes similar to those of the cho1Δ/Δ mutant; however, it is slightly more virulent and has less of a cell wall defect. The virulence losses exhibited by the cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ mutants appear to be related to their cell wall defects which are due to loss of de novo PE biosynthesis, but are exacerbated by loss of PS itself. Cho1p is conserved in fungi, but not mammals, so fungal PS synthase is a potential novel antifungal drug target.     

The Tlo proteins are stoichiometric components of Candida albicans mediator anchored via the Med3 subunit

The amplification of the TLO (for telomere-associated) genes in Candida albicans, compared to its less pathogenic, close relative Candida dubliniensis, suggests a role in virulence. Little, however, is known about the function of the Tlo proteins. We have purified the Mediator coactivator complex from C. albicans (caMediator) and found that Tlo proteins are a stoichiometric component of caMediator. Many members of the Tlo family are expressed, and each is a unique member of caMediator. Protein expression analysis of individual Tlo proteins, as well as the purification of tagged Tlo proteins, demonstrate that there is a large free population of Tlo proteins in addition to the Mediator-associated population. Coexpression and copurification of Tloα12 and caMed3 in Escherichia coli established a direct physical interaction between the two proteins. We have also made a C. albicans med3Δ/Δ strain and purified an intact Mediator from this strain. The analysis of the composition of the med3Δ Mediator shows that it lacks a Tlo subunit. Regarding Mediator function, the med3Δ/Δ strain serves as a substitute for the difficult-to-make tloΔ/Δ C. albicans strain. A potential role of the TLO and MED3 genes in virulence is supported by the inability of the med3Δ/Δ strain to form normal germ tubes. This study of caMediator structure provides initial clues to the mechanism of action of the Tlo genes and a platform for further mechanistic studies of caMediator's involvement in gene regulatory patterns that underlie pathogenesis.     

Application of the systematic "DAmP" approach to create a partially defective C. albicans mutant

An understanding of gene function often relies upon creating multiple kinds of alleles. Functional analysis in Candida albicans, a major fungal pathogen, has generally included characterization of mutant strains with insertion or deletion alleles and over-expression alleles. Here we use in C. albicans another type of allele that has been employed effectively in the model yeast Saccharomyces cerevisiae, a "Decreased Abundance by mRNA Perturbation" (DAmP) allele (Yan et al., 2008). DAmP alleles are created systematically through replacement of 30 noncoding regions with nonfunctional heterologous sequences, and thus are broadly applicable. We used a DAmP allele to probe the function of Sun41, a surface protein with roles in cell wall integrity, cell-cell adherence, hyphal formation, and biofilm formation that has been suggested as a possible therapeutic target (Firon et al., 2007; Hiller et al., 2007; Norice et al., 2007). A SUN41-DAmP allele results in approximately 10-fold reduced levels of SUN41 RNA, and yields intermediate phenotypes in most assays. We report that a sun41Δ/Δ mutant is defective in biofilm formation in vivo, and that the SUN41-DAmP allele complements that defect. This finding argues that Sun41 may not be an ideal therapeutic target for biofilm inhibition, since a 90% decrease in activity has little effect on biofilm formation in vivo. We anticipate that DAmP alleles of C. albicans genes will be informative for analysis of other prospective drug targets, including essential genes.     

N-acetylglucosamine (GlcNAc) induction of hyphal morphogenesis and transcriptional responses in Candida albicans are not dependent on its metabolism

N-acetylglucosamine (GlcNAc) stimulates important signaling pathways in a wide range of organisms. In the human fungal pathogen Candida albicans, GlcNAc stimulates hyphal cell morphogenesis, virulence genes, and the genes needed to catabolize GlcNAc. Previous studies on the GlcNAc transporter (NGT1) indicated that GlcNAc has to be internalized to induce signaling. Therefore, the role of GlcNAc catabolism was examined by deleting the genes required to phosphorylate, deacetylate, and deaminate GlcNAc to convert it to fructose-6-PO(4) (HXK1, NAG1, and DAC1). As expected, the mutants failed to utilize GlcNAc. Surprisingly, GlcNAc inhibited the growth of the nag1Δ and dac1Δ mutants in the presence of other sugars, suggesting that excess GlcNAc-6-PO(4) is deleterious. Interestingly, both hxk1Δ and an hxk1Δ nag1Δ dac1Δ triple mutant could be efficiently stimulated by GlcNAc to form hyphae. These mutants could also be stimulated to express GlcNAc-regulated genes. Because GlcNAc must be phosphorylated by Hxk1 to be catabolized, and also for it to enter the anabolic pathways that form chitin, N-linked glycosylation, and glycosylphosphatidylinositol anchors, the mutant phenotypes indicate that GlcNAc metabolism is not needed to induce signaling in C. albicans. Thus, these studies in C. albicans reveal a novel role for GlcNAc in cell signaling that may also regulate critical pathways in other organisms.     

An Arabidopsis heat shock protein complements a thermotolerance defect in yeast.

The heat shock protein Hsp104 of the yeast Saccharomyces cerevisiae plays a key role in promoting survival at extreme temperatures. We found that when diverse higher plant species are exposed to high temperatures they accumulate proteins that are antigenically related to Hsp104. We isolated a cDNA corresponding to one of these proteins from Arabidopsis. The protein, AtHSP101, is 43% identical to yeast Hsp104. DNA gel blot analysis indicated that AtHSP101 is encoded by a single- or low-copy number gene. AtHsp101 mRNA was undetectable in the absence of stress but accumulated to high levels during exposure to high temperatures. When AtHSP101 was expressed in yeast, it complemented the thermotolerance defect caused by a deletion of the HSP104 gene. The ability of AtHSP101 to protect yeast from severe heat stress strongly suggests that this HSP plays an important role in thermotolerance in higher plants.     

The Hsp90 Co-Chaperone Sgt1 Governs Candida albicans Morphogenesis and Drug Resistance

The molecular chaperone Hsp90 orchestrates regulatory circuitry governing fungal morphogenesis, biofilm development, drug resistance, and virulence. Hsp90 functions in concert with co-chaperones to regulate stability and activation of client proteins, many of which are signal transducers. Here, we characterize the first Hsp90 co-chaperone in the leading human fungal pathogen, Candida albicans. We demonstrate that Sgt1 physically interacts with Hsp90, and that it governs C. albicans morphogenesis and drug resistance. Genetic depletion of Sgt1 phenocopies depletion of Hsp90, inducing yeast to filament morphogenesis and invasive growth. Sgt1 governs these traits by bridging two morphogenetic regulators: Hsp90 and the adenylyl cyclase of the cAMP-PKA signaling cascade, Cyr1. Sgt1 physically interacts with Cyr1, and depletion of either Sgt1 or Hsp90 activates cAMP-PKA signaling, revealing the elusive link between Hsp90 and the PKA signaling cascade. Sgt1 also mediates tolerance and resistance to the two most widely deployed classes of antifungal drugs, azoles and echinocandins. Depletion of Sgt1 abrogates basal tolerance and acquired resistance to azoles, which target the cell membrane. Depletion of Sgt1 also abrogates tolerance and resistance to echinocandins, which target the cell wall, and renders echinocandins fungicidal. Though Sgt1 and Hsp90 have a conserved impact on drug resistance, the underlying mechanisms are distinct. Depletion of Hsp90 destabilizes the client protein calcineurin, thereby blocking crucial responses to drug-induced stress; in contrast, depletion of Sgt1 does not destabilize calcineurin, but blocks calcineurin activation in response to drug-induced stress. Sgt1 influences not only morphogenesis and drug resistance, but also virulence, as genetic depletion of C. albicans Sgt1 leads to reduced kidney fungal burden in a murine model of systemic infection. Thus, our characterization of the first Hsp90 co-chaperone in a fungal pathogen establishes C. albicans Sgt1 as a global regulator of morphogenesis and drug resistance, providing a new target for treatment of life-threatening fungal infections.     

The Paf1 Complex Subunit Rtf1 Buffers Cells Against the Toxic Effects of [PSI+] and Defects in Rkr1-Dependent Protein Quality Control in Saccharomyces cerevisiae

The Rtf1 subunit of the Paf1 complex is required for specific histone modifications, including histone H2B lysine 123 monoubiquitylation. In Saccharomyces cerevisiae, deletion of RTF1 is lethal in the absence of Rkr1, a ubiquitin-protein ligase involved in the destruction of nonstop proteins, which arise from mRNAs lacking stop codons or translational readthrough into the poly(A) tail. We performed a transposon-based mutagenesis screen to identify suppressors of rtf1Δ rkr1Δ lethality and found that a mutation in the gene encoding the protein chaperone Hsp104 rescued viability. Hsp104 plays a role in prion propagation, including the maintenance of [PSI(+)], which contributes to the synthesis of nonstop proteins. We demonstrate that rtf1Δ and rkr1Δ are synthetically lethal only in the presence of [PSI(+)]. The deletion, inactivation, and overexpression of HSP104 or the overexpression of prion-encoding genes URE2 and LSM4 clear [PSI(+)] and rescue rtf1Δ rkr1Δ lethality. In addition, the presence of [PSI(+)] decreases the fitness of rkr1Δ strains. We investigated whether the loss of RTF1 exacerbates an overload in nonstop proteins in rkr1Δ [PSI(+)] strains but, using reporter plasmids, found that rtf1Δ decreases nonstop protein levels, indicating that excess nonstop proteins may not be the cause of synthetic lethality. Instead, our data suggest that the loss of Rtf1-dependent histone modifications increases the burden on quality control pathways in cells lacking Rkr1 and containing [PSI(+)].