Biochemical characterization of recombinant Candida albicans mannosyltransferases Mnt1, Mnt2 and Mnt5 reveals new functions in O- and N-mannan biosynthesis

The cell surface of Candida albicans is enriched with highly glycosylated mannoproteins that are involved in the interaction with host tissues. N- and O-glycosylation are post-translational modifications that initiate in the endoplasmic reticulum, and finalize in the Golgi. The KRE2/MNT1 family encode a set of multifunctional mannosyltransferases that participate in O-, N- and phosphomannosylation. In order to gain insights into the substrate specificities of these enzymes, recombinant forms of Mnt1, Mnt2, and Mnt5 were expressed in Pichia pastoris and the enzyme activities characterized. Mnt1 and Mnt2 showed a high specificity for α-methylmannoside and α1,2-mannobiose as acceptor substrates. Notably, they also used Saccharomyces cerevisiaeO-mannans as acceptors and generated products with more than three mannose residues, suggesting than Mnt1 and Mnt2 could be the mannosyltransferases adding the fourth and fifth mannose residue to the O-mannans in C. albicans. Mnt5 only recognized α-methylmannoside as acceptor, suggesting that participates in the addition of the second mannose residues to the N-glycan outer chain.     

Structure of Kre2p/Mnt1p: a yeast alpha1,2-mannosyltransferase involved in mannoprotein biosynthesis

Kre2p/Mnt1p is a Golgi alpha1,2-mannosyltransferase involved in the biosynthesis of Saccharomyces cerevisiae cell wall glycoproteins. The protein belongs to glycosyltransferase family 15, a member of which has been implicated in virulence of Candida albicans. We present the 2.0 A crystal structures of the catalytic domain of Kre2p/Mnt1p and its binary and ternary complexes with GDP/Mn(2+) and GDP/Mn(2+)/acceptor methyl-alpha-mannoside. The protein has a mixed alpha/beta fold similar to the glycosyltransferase-A (GT-A) fold. Although the GDP-mannose donor was used in the crystallization experiments and the GDP moiety is bound tightly to the active site, the mannose is not visible in the electron density. The manganese is coordinated by a modified DXD motif (EPD), with only the first glutamate involved in a direct interaction. The position of the donor mannose was modeled using the binary and ternary complexes of other GT-A enzymes. The C1" of the modeled donor mannose is within hydrogen-bonding distance of both the hydroxyl of Tyr(220) and the O2 of the acceptor mannose. The O2 of the acceptor mannose is also within hydrogen bond distance of the hydroxyl of Tyr(220). The structures, modeling, site-directed mutagenesis, and kinetic analysis suggest two possible catalytic mechanisms. Either a double-displacement mechanism with the hydroxyl of Tyr(220) as the potential nucleophile or alternatively, an S(N)i-like mechanism with Tyr(220) positioning the substrates for catalysis. The importance of Tyr(220) in both mechanisms is highlighted by a 3000-fold reduction in k(cat) in the Y220F mutant.     

Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence

The cell surface of Candida albicans is the immediate point of contact with the host. The outer layer of the cell wall is enriched in highly glycosylated mannoproteins that are implicated in many aspects of the host-fungus interaction. Glycosylation of cell wall proteins is initiated in the endoplasmic reticulum and then elaborated in the Golgi as the protein passes through the secretory pathway. Golgi-bound mannosyltransferases require Mn(2+) as an essential cofactor. In Saccharomyces cerevisiae, the P-type ATPase Pmr1p transports Ca(2+) and Mn(2+) ions into the Golgi. To determine the effect of a gross defect in glycosylation on host-fungus interactions of C. albicans, we disrupted the PMR1 homolog, CaPMR1. This mutation would simultaneously inhibit many Golgi-located, Mn(2+)-dependent mannosyltransferases. The Capmr1Delta null mutant was viable in vitro and had no growth defect even on media containing low Ca(2+)/Mn(2+) ion concentrations. However, cells grown in these media progressively lost viability upon entering stationary phase. Phosphomannan was almost completely absent, and O-mannan was severely truncated in the null mutant. A defect in N-linked outer chain glycosylation was also apparent, demonstrated by the underglycosylation of surface acid phosphatase. Consistent with the glycosylation defect, the null mutant had a weakened cell wall, exemplified by hypersensitivity to Calcofluor white, Congo red, and hygromycin B and constitutive activation of the cell integrity pathway. In a murine model of systemic infection, the null mutant was severely attenuated in virulence. These results demonstrate the importance of glycosylation for cell wall structure and virulence of C. albicans.     

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants

In Candida albicans wild-type cells, the beta1, 6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via beta1,6-glucan to beta1,3-glucan. The remaining GPI-CWPs are linked through beta1,6-glucan to chitin. The beta1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to beta1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Delta and pmt1Delta mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through beta1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a beta1, 6-glucan-deficient hemizygous kre6Delta mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.     

Candida albicans Ecm33p is important for normal cell wall architecture and interactions with host cells

Candida albicans ECM33 encodes a glycosylphosphatidylinositol-linked cell wall protein that is important for cell wall integrity. It is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis. To identify potential mechanisms through which Ecm33p contributes to virulence, we investigated the interactions of C. albicans ecm33Delta mutants with endothelial cells and the FaDu oral epithelial cell line in vitro. The growth rate of blastospores of strains containing either one or no intact copies of ECM33 was 50% slower than that of strains containing two intact copies of ECM33. However, all strains germinated at the same rate, forming similar-length hyphae on endothelial cells and oral epithelial cells. Strains containing either one or no intact copies of ECM33 had modestly reduced adherence to both types of host cells, and a markedly reduced capacity to invade and damage these cells. Saccharomyces cerevisiae expressing C. albicans ECM33 did not adhere to or invade epithelial cells, suggesting that Ecm33p by itself does not act as an adhesin or invasin. Examination of ecm33Delta mutants by transmission electron microscopy revealed that the cell wall of these strains had an abnormally electron-dense outer mannoprotein layer, which may represent a compensatory response to reduced cell wall integrity. The hyphae of these mutants also had aberrant surface localization of the adhesin Als1p. Collectively, these results suggest that Ecm33p is required for normal cell wall architecture as well as normal function and expression of cell surface proteins in C. albicans.     

Loss of cell wall mannosylphosphate in Candida albicans does not influence macrophage recognition

The outer layer of the cell wall of the human pathogenic fungus Candida albicans is enriched with heavily mannosylated glycoproteins that are the immediate point of contact between the fungus and cells of the host, including phagocytes. Previous work had identified components of the acid-labile fraction of N-linked mannan, comprising beta-1,2-linked mannose residues attached via a phosphodiester bond, as potential ligands for macrophage receptors and modulators of macrophage function. We therefore isolated and disrupted the CaMNN4 gene, which is required for mannosyl phosphate transfer and hence the attachment of beta-1,2 mannose oligosaccharides to the acid-labile N-mannan side chains. With the mannosylphosphate eliminated, the mnn4Delta null mutant was unable to bind the charged cationic dye Alcian Blue and was devoid of acid-labile beta-1,2-linked oligomannosaccharides. The mnn4Delta mutant was unaffected in cell growth and morphogenesis in vitro and in virulence in a murine model of systemic C. albicans infection. The null mutant was also not affected in its interaction with macrophages. Mannosylphosphate is therefore not required for macrophage interactions or for virulence of C. albicans.     

MNN5 encodes an iron-regulated alpha-1,2-mannosyltransferase important for protein glycosylation, cell wall integrity, morphogenesis, and virulence in Candida albicans

The cell walls of microbial pathogens mediate physical interactions with host cells and hence play a key role in infection. Mannosyltransferases have been shown to determine the cell wall properties and virulence of the pathogenic fungus Candida albicans. We previously identified a C. albicans alpha-1,2-mannosyltransferase, Mnn5, for its novel ability to enhance iron usage in Saccharomyces cerevisiae. Here we have studied the enzymatic properties of purified Mnn5 and characterized its function in its natural host. Mnn5 catalyzes the transfer of mannose to both alpha-1,2- and alpha-1,6-mannobiose, and this activity requires Mn2+ as a cofactor and is regulated by the Fe2+ concentration. An mnn5Delta mutant showed a lowered ability to extend O-linked, and possibly also N-linked, mannans, hypersensitivity to cell wall-damaging agents, and a reduction of cell wall mannosylphosphate content, phenotypes typical of many fungal mannosyltransferase mutants. The mnn5Delta mutant also exhibited some unique defects, such as impaired hyphal growth on solid media and attenuated virulence in mice. An unanticipated phenotype was the mnn5Delta mutant's resistance to killing by the iron-chelating protein lactoferrin, rendering it the first protein found that mediates lactoferrin killing of C. albicans. In summary, MNN5 deletion impairs a wide range of cellular events, most likely due to its broad substrate specificity. Of particular interest was the observed role of iron in regulating the enzymatic activity, suggesting an underlying relationship between Mnn5 activity and cellular iron homeostasis.     

Role of the fungal Ras-protein kinase A pathway in governing epithelial cell interactions during oropharyngeal candidiasis

Tpk1p, Tpk2p and Efg1p are members of the Ras-protein kinase A pathway that governs the yeast-to-hyphal transition in Candida albicans. We used tpk1Delta/tpk1Delta, tpk2Delta/tpk2Delta and efg1Delta/efg1Delta mutants to investigate the role of these proteins in regulating the interactions of C. albicans with oral epithelial cell lines in vitro and virulence in murine models of oropharyngeal candidiasis (OPC) and haematogenously disseminated candidiasis (HDC). The tpk1Delta/tpk1Delta strain adhered to, invaded and damaged oral epithelial cells in vitro similarly to the wild-type strain. In contrast, both the tpk2Delta/tpk2Delta and efg1Delta/efg1Delta strains had reduced capacity to invade and damage oral epithelial cells, and the efg1Delta/efg1Delta strain also exhibited decreased adherence to these cells. Consistent with these in vitro findings, the tpk2Delta/tpk2Delta and efg1Delta/efg1Delta strains also had significantly attenuated virulence during OPC. Therefore, Tpk2p and Efg1p both govern factors that enable C. albicans to invade and damage oral epithelial cells in vitro and cause OPC. These results also suggest that hyphal formation mediated by the Ras-protein kinase A pathway is a key virulence mechanism during OPC. Interestingly, the efg1Delta/efg1Delta strain, but not the tpk2Delta/tpk2Delta had reduced virulence during HDC. Thus, Tpk2p may be more important for governing virulence during OPC than HDC.     

Inactivation of CaMIT1 inhibits Candida albicans phospholipomannan beta-mannosylation, reduces virulence, and alters cell wall protein beta-mannosylation

Studies on Candida albicans phospholipomannan have suggested a novel biosynthetic pathway for yeast glycosphingolipids. This pathway is thought to diverge from the usual pathway at the mannose-inositol-phospho-ceramide (MIPC) step. To confirm this hypothesis, a C. albicans gene homologue for the Saccharomyces cerevisiae SUR1 gene was identified and named MIT1 as it coded for GDP-mannose:inositol-phospho-ceramide mannose transferase. Two copies of this gene were disrupted. Western blots of cell extracts revealed that strain mit1Delta contained no PLM. Thin layer chromatography and mass spectrometry confirmed that mit1Delta did not synthesize MIPC, demonstrating a role of MIT1 in the mannosylation of C. albicans IPCs. As MIT1 disruption prevented downstream beta-1,2 mannosylation, mit1Delta represents a new C. albicans mutant affected in the expression of these specific virulence attributes, which act as adhesins/immunomodulators. mit1Delta was less virulent during both the acute and chronic phases of systemic infection in mice (75 and 50% reduction in mortality, respectively). In vitro, mit1Delta was not able to escape macrophage lysis through down-regulation of the ERK1/2 phosphorylation pathway previously shown to be triggered by PLM. Phenotypic analysis also revealed pleiotropic effects of MIT1 disruption. The most striking observation was a reduced beta-mannosylation of phosphopeptidomannan. Increased beta-mannosylation of mannoproteins was observed under growth conditions that prevented the association of beta-oligomannosides with phosphopeptidomannan, but not with PLM. This suggests that C. albicans has strong regulatory mechanisms associating beta-oligomannoses with different cell wall carrier molecules. These mechanisms and the impact of the different presentations of beta-oligomannoses on the host response need to be defined.     

Mnt2p and Mnt3p of Saccharomyces cerevisiae are members of the Mnn1p family of alpha-1,3-mannosyltransferases responsible for adding the terminal mannose residues of O-linked oligosaccharides.

The genome of Saccharomyces cerevisiae contains five genes that encode type II transmembrane proteins with significant amino acid similarity to the alpha-1,3-mannosyltransferase Mnn1p. The roles of the three genes most closely related to MNN1 were examined in mutants carrying single and multiple combinations of the disrupted genes. Paper chromatographic analysis of [2-3H]mannose-labeled O-linked oligosaccharides released by beta-elimination showed that the MNT2 (YGL257c) and MNT3 (YIL014w) genes in combination with MNN1 have overlapping roles in the addition of the fourth and fifth alpha-1,3-linked mannose residues to form Man4 and Man5 oligosaccharides whereas MNT4 (YNR059w) does not appear to be required for O-glycan synthesis.     

Crh1p and Crh2p are required for the cross-linking of chitin to beta(1-6)glucan in the Saccharomyces cerevisiae cell wall

In budding yeast, chitin is found in three locations: at the primary septum, largely in free form, at the mother-bud neck, partially linked to beta(1-3)glucan, and in the lateral wall, attached in part to beta(1-6)glucan. By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to beta(1-6)glucan is diminished in mutants of the CRH1 or the CRH2/UTR2 gene and completely absent in a double mutant. This indicates that Crh1p and Crh2p, homologues of glycosyltransferases, ferry chitin chains from chitin synthase III to beta(1-6)glucan. Deletion of CRH1 and/or CRH2 aggravated the defects of fks1Delta and gas1Delta mutants, which are impaired in cell wall synthesis. A temperature shift from 30 degrees C to 38 degrees C increased the proportion of chitin attached to beta(1-6)glucan. The expression of CRH1, but not that of CRH2, was also higher at 38 degrees C in a manner dependent on the cell integrity pathway. Furthermore, the localization of both Crh1p and Crh2p at the cell cortex, the area where the chitin-beta(1-6)glucan complex is found, was greatly enhanced at 38 degrees C. Crh1p and Crh2p are the first proteins directly implicated in the formation of cross-links between cell wall components in fungi.     

Candida albicans protein kinase CK2 governs virulence during oropharyngeal candidiasis

To identify Candida albicans genes whose proteins are necessary for host cell interactions and virulence, a collection of C. albicans insertion mutants was screened for strains with reduced capacity to damage endothelial cells in vitro. This screen identified CKA2. CKA2 and its homologue CKA1 encode the catalytic subunits of the protein kinase CK2. cka2delta/cka2delta strains of C. albicans were constructed and found to have significantly reduced capacity to damage both endothelial cells and an oral epithelial cell line in vitro. Although these strains invaded endothelial cells similarly to the wild-type strain, they were defective in oral epithelial cell invasion. They were also hypersusceptible to hydrogen peroxide, but not to high salt or to cell wall damaging agents. A cka1delta/cka1delta mutant caused normal damage to both endothelial cells and oral epithelial cells, and it was not hypersusceptible to hydrogen peroxide. However, overexpression of CKA1 in a cka2delta/cka2delta strain restored wild-type phenotype. Although the cka2delta/cka2delta mutant had normal virulence in the mouse model of haematogenously disseminated candidiasis, it had significantly attenuated virulence in the mouse model of oropharyngeal candidiasis. Therefore, Cka2p governs the interactions of C. albicans with endothelial and oral epithelial cells in vitro and virulence during oropharyngeal candidiasis.     

Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae

The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Delta2Delta mutant and the quintuple yapsin mutant (5ypsDelta) undergo osmoremedial cell lysis at 37 degrees C. The cell walls of both 5ypsDelta and yps1Delta2Delta mutants have decreased amounts of 1,3- and 1,6-beta-glucan. Although there is decreased incorporation of both 1,3- and 1,6-beta-glucan in the 5ypsDelta mutant in vivo, in vitro specific activity of both 1,3- and 1,6-beta-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsDelta. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Delta mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.     

Peroxisomal fatty acid beta-oxidation is not essential for virulence of Candida albicans

Phagocytic cells form the first line of defense against infections by the human fungal pathogen Candida albicans. Recent in vitro gene expression data suggest that upon phagocytosis by macrophages, C. albicans reprograms its metabolism to convert fatty acids into glucose by inducing the enzymes of the glyoxylate cycle and fatty acid beta-oxidation pathway. Here, we asked whether fatty acid beta-oxidation, a metabolic pathway localized to peroxisomes, is essential for fungal virulence by constructing two C. albicans double deletion strains: a pex5Delta/pex5Delta mutant, which is disturbed in the import of most peroxisomal enzymes, and a fox2Delta/fox2Delta mutant, which lacks the second enzyme of the beta-oxidation pathway. Both mutant strains had strongly reduced beta-oxidation activity and, accordingly, were unable to grow on media with fatty acids as a sole carbon source. Surprisingly, only the fox2Delta/fox2Delta mutant, and not the pex5Delta/pex5Delta mutant, displayed strong growth defects on nonfermentable carbon sources other than fatty acids (e.g., acetate, ethanol, or lactate) and showed attenuated virulence in a mouse model for systemic candidiasis. The degree of virulence attenuation of the fox2Delta/fox2Delta mutant was comparable to that of the icl1Delta/icl1Delta mutant, which lacks a functional glyoxylate cycle and also fails to grow on nonfermentable carbon sources. Together, our data suggest that peroxisomal fatty acid beta-oxidation is not essential for virulence of C. albicans, implying that the attenuated virulence of the fox2Delta/fox2Delta mutant is largely due to a dysfunctional glyoxylate cycle.     

Interactions of Arg2 in the Mnt N-terminal arm with the central and flanking regions of the Mnt operator

Arg2, in the N-terminal arm of the Mnt repressor, plays an important role in determining operator-binding specificity. In the complex of the Mnt tetramer with the 21 base-pair mnt operator, there are four potential sites for Arg2 interactions, two in the central region of the operator and two on the outer flanks of the operator. Single-chain variants of the dimeric N-terminal domain of Mnt containing one Arg2 residue and one Lys2 or Met2 residue were constructed and interactions with operator DNA were probed using Fe. EDTA affinity cleavage. The results of these orientation studies show that the majority of the energetically significant interactions mediated by Arg2 occur in the central region of the mnt operator. The RK2, RA2, and RM2 mutations reduce the free energy of operator binding by 1.7 kcal/mol, 3.3 kcal/mol, and 4.9 kcal/mol, respectively. Double-mutant thermodynamic cycle analyses using the RA2, RM2, and operator variants also reveal interaction free energies between Arg2 and operator base-pairs 9, 10, 11, 12 and 13, which in aggregate account for most of the Arg2 contribution to operator binding.