Purification and characterization of lysophospholipase-transacylase of pathogenic fungus Candida albicans

A lysophospholipase-transacylase was purified to homogeneity from the culture broth of Candida albicans by ammonium sulfate precipitation and chromatographs on DEAE-cellulose, Ultrogel AcA-44, first Mono Q, hydroxyapatite, TSKgel-3000 and second Mono Q columns. The purified protein was a single band (Mr 41,000) as inferred by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a specific activity of 78 mumol/min per mg protein for fatty acid release and 320 mumol/min per mg protein for phosphatidylcholine formation. Fatty acid release obeyed Michaelis-Menten kinetics and the apparent Km was 76 microM of 1-palmitoyl-sn-glycero-3-phosphatidylcholine, but Lineweaver-Burk plots of transacylase activity was parabolic. The ratio of hydrolase to transacylase activity of the purified enzyme was varied depending upon the concentration of lysophosphatidylcholine. Transacylation was prominent at high concentration of substrate and the ratio of hydrolase to transacylase was 0.24. Low concentration of palmitoylcarnitine (50 microM) inhibited markedly phosphatidylcholine formation but stimulated fatty acid release. The degree of esterification of 1-acyllysophosphatidylcholine was altered with mixtures of different molecular species of substrate, demonstrating acyl chain selectivity in the transfer process. These results suggest that C. albicans lysophospholipase-transacylase is different from the corresponding mammalian enzymes in enzymatic properties.     

Minor structural consequences of alternative CUG codon usage (Ser for Leu) in Candida albicans exoglucanase

In some species of Candida the CUG codon is encoded as serine and not leucine. In the case of the exo-beta-1,3-glucanase from the pathogenic fungus C. albicans there are two such translational events, one in the prepro-leader sequence and the other at residue 64. Overexpression of active mature enzyme in a yeast host indicated that these two positions are tolerant to substitution. By comparing the crystal structure of the recombinant protein with that of the native (presented here), it is seen how either serine or leucine can be accommodated at position 64. Examination of the relatively few solved protein structures from C. albicans indicates that other CUG encoded serines are also found at non-essential surface sites. However such codon usage is rare in C. albicans, in contrast to C. rugosa, with direct implications for respective recombinant protein production.     

A single MAPKKK regulates the Hog1 MAPK pathway in the pathogenic fungus Candida albicans

The Hog1 mitogen-activated protein kinase (MAPK) plays a central role in stress responses in the human pathogen Candida albicans. Here, we have investigated the MAPK kinase kinase (MAPKKK)-dependent regulation of the pathway. In contrast to the Hog1 pathway in Saccharomyces cerevisiae, which is regulated by three MAPKKKs (Ssk2, Ssk22, and Ste11), our results demonstrate that Hog1 in C. albicans is regulated by a single MAPKKK Ssk2. Deletion of SSK2 results in comparable stress and morphological phenotypes exhibited by hog1Delta cells, and Ssk2 is required for the stress-induced phosphorylation and nuclear accumulation of Hog1, and for Hog1-dependent gene expression. Furthermore, phenotypes associated with deletion of SSK2 can be circumvented by expression of a phosphomimetic mutant of the MAPKK Pbs2, indicating that Ssk2 regulates Hog1 via activation of Pbs2. In S. cerevisiae, the Hog1 pathway is also regulated by the MAPKKK Ste11. However, we can find no connection between Ste11 and the regulation of Hog1 in C. albicans. Furthermore, expression of a chimeric Pbs2 protein containing the Ste11-dependent regulatory region of S. cerevisiae Pbs2, fails to stimulate Ste11-dependent stress signaling in C. albicans. Collectively, our data show that Ssk2 is the sole MAPKKK to relay stress signals to Hog1 in C. albicans and that the MAPK signaling network in C. albicans has diverged significantly from the corresponding network in S. cerevisiae.     

Transfer RNA structural change is a key element in the reassignment of the CUG codon in Candida albicans.

The human pathogenic yeast Candida albicans and a number of other Candida species translate the standard leucine CUG codon as serine. This is the latest addition to an increasing number of alterations to the standard genetic code which invalidate the theory that the code is frozen and universal. The unexpected finding that some organisms evolved alternative genetic codes raises two important questions: how have these alternative codes evolved and what evolutionary advantages could they create to allow for their selection? To address these questions in the context of serine CUG translation in C.albicans, we have searched for unique structural features in seryl-tRNA(CAG), which translates the leucine CUG codon as serine, and attempted to reconstruct the early stages of this genetic code switch in the closely related yeast species Saccharomyces cerevisiae. We show that a purine at position 33 (G33) in the C.albicans Ser-tRNA(CAG) anticodon loop, which replaces a conserved pyrimidine found in all other tRNAs, is a key structural element in the reassignment of the CUG codon from leucine to serine in that it decreases the decoding efficiency of the tRNA, thereby allowing cells to survive low level serine CUG translation. Expression of this tRNA in S.cerevisiae induces the stress response which allows cells to acquire thermotolerance. We argue that acquisition of thermotolerance may represent a positive selection for this genetic code change by allowing yeasts to adapt to sudden changes in environmental conditions and therefore colonize new ecological niches.     

Identification and characterization of the genes for N-acetylglucosamine kinase and N-acetylglucosamine-phosphate deacetylase in the pathogenic fungus Candida albicans.

Like bacteria and many fungi, the pathogenic fungus Candida albicans can utilize GlcNAc as a carbon source for growth. A cluster of six genes was identified in the C. albicans genome. One of the genes in the cluster was CaNAG1, which is responsible for GlcN6P deaminase and is therefore essential for GlcNAc-dependent growth. The other five genes were designated CaNAG2, CaNAG3, CaNAG4, CaNAG5 and CaNAG6. The mRNA levels of CaNAG1, CaNAG2 and CaNAG5 were significantly induced by GlcNAc, whereas those of CaNAG3, CaNAG4 and CaNAG6 were not. Neither CaNAG2 nor CaNAG5 was essential for growth, but disruption of CaNAG2 or CaNAG5 greatly retarded the growth of cells using GlcNAc as the sole carbon source. Although no homolog of CaNAG2 or CaNAG5 was found in the Saccharomyces cerevisiae genome, CaNag2p displayed sequence similarities to Escherichia coli nagA, and CaNag5p is homologous to a wide variety of hexose kinases. When expressed as a fusion protein with glutathione S-transferase (GST), CaNag5p produced GlcNAc-P from GlcNAc in the presence of ATP, whereas GST alone did not. Furthermore, the recombinant GST-CaNag2p fusion protein converted GlcNAcP, which was produced by CaNag5p, into GlcNP. These results clearly demonstrate that CaNAG2 and CaNAG5 encode GlcNAcP deacetylase and GlcNAc kinase, respectively. CaNag5p recognized glucose and mannose as substrates, whereas the recently identified human GlcNAc kinase was specific to GlcNAc. Deletion of CaNAG2 or CaNAG5 markedly, and that of CaNAG1 moderately, attenuated the virulence of C. albicans in a mouse systemic infection model. Thus, it appears that GlcNAc metabolism of C. albicans is closely associated with its virulence.     

Virulence genes in the pathogenic yeast Candida albicans

In recent years, the incidence of fungal infections has been rising all over the world. Although the amount of research in the field of pathogenic fungi has also increased, there is still a need for the identification of reliable determinants of virulence. In this review, we focus on identified Candida albicans genes whose deletant strains have been tested in experimental virulence assays. We discuss the putative relationship of these genes to virulence and also outline the use of new different systems to examine the precise effect in virulence of different genes.     

Roles of three histidine kinase genes in hyphal development and virulence of the pathogenic fungus Candida albicans

The pathogenic fungus Candida albicans harbors three histidine kinase genes called CaSLN1, CaNIK1, and CaHK1. The disruption of any one of these three genes impaired the hyphal formation and attenuated the virulence of C. albicans in a mouse systemic candidiasis model. The effects of the disruption on hyphal formation and virulence were most severe in the cahk1Delta null mutants. Although the double disruption of CaSLN1 and CaNIK1 was impossible, further deletion of CaSLN1 or CaNIK1 in the cahk1Delta null mutants partially restored the serum-induced hypha-forming ability and virulence. When incubated with radiolabelled ATP, the recombinant CaSln1 and CaNik1 proteins, which contained their own kinase and response regulator domains, were autophosphorylated, whereas CaHk1p was not. These results imply that in C. albicans, CaSLN1 and CaNIK1 function upstream of CaHK1 but are in distinct signal transmission pathways.     

A molecular genetic system for the pathogenic yeast Candida dubliniensis

Candida dubliniensis is a recently described pathogenic yeast of the genus Candida that is closely related to Candida albicans but differs from it in several phenotypic and genotypic characteristics, including putative virulence traits, which may explain differences in the spectrum of diseases caused by the two species. In contrast to C. albicans, a molecular genetic system to study virulence of C. dubliniensis is lacking. We have developed a system for the genetic transformation of C. dubliniensis that is based on the use of the dominant selection marker MPA(R) from C. albicans that confers resistance to mycophenolic acid (MPA). Using this transformation system, a GFP (green fluorescent protein) reporter gene that was genetically engineered for functional expression in C. albicans and placed under control of the inducible C. albicans SAP2 (secreted aspartic proteinase) promoter was integrated into the C. dubliniensis genome. MPA-resistant transformants containing the SAP2P-GFP fusion fluoresced under SAP2-inducing conditions but not under SAP2-repressing conditions. These results demonstrate that the MPA(R) selection marker is useful for transformation of C. dubliniensis wild-type strains, that the GFP reporter gene is functionally expressed in C. dubliniensis, and that the C. albicans SAP2 promoter can be used for controlled gene expression in C. dubliniensis. These genetic tools will allow the dissection of the differences in virulence characteristics between the two pathogenic yeast species at the molecular level.     

The mitogen-activated protein kinase homolog HOG1 gene controls glycerol accumulation in the pathogenic fungus Candida albicans.

The Candida albicans HOG1 gene (HOG1CA) was cloned by functional complementation of the osmosensitive phenotype associated with Saccharomyces cerevisiae hog1 delta mutants. HOG1CA codes for a 377-amino-acid protein, 78% identical to S. cerevisiae Hog1p. A C. albicans hog1 null mutant was found to be sensitive to osmotic stress and failed to accumulate glycerol on high-osmolarity media.     

Gpr1, a putative G-protein-coupled receptor, regulates morphogenesis and hypha formation in the pathogenic fungus Candida albicans

In response to various extracellular signals, the morphology of the human fungal pathogen Candida albicans switches from yeast to hypha form. Here, we report that GPR1 encoding a putative G-protein-coupled receptor and GPA2 encoding a Galpha subunit are required for hypha formation and morphogenesis in C. albicans. Mutants lacking Gpr1 (gpr1/gpr1) or Gpa2 (gpa2/gpa2) are defective in hypha formation and morphogenesis on solid hypha-inducing media. These phenotypic defects in solid cultures are suppressed by exogenously added dibutyryl-cyclic AMP (dibutyryl-cAMP). Biochemical studies also reveal that GPR1 and GPA2 are required for a glucose-dependent increase in cellular cAMP. An epistasis analysis indicates that Gpr1 functions upstream of Gpa2 in the same signaling pathway, and a two-hybrid assay reveals that the carboxyl-terminal tail of Gpr1 interacts with Gpa2. Moreover, expression levels of HWP1 and ECE1, which are cAMP-dependent hypha-specific genes, are reduced in both mutant strains. These findings support a model that Gpr1, as well as Gpa2, regulates hypha formation and morphogenesis in a cAMP-dependent manner. In contrast, GPR1 and GPA2 are not required for hypha formation in liquid fetal bovine serum (FBS) medium. Furthermore, the gpr1 and the gpa2 mutant strains are fully virulent in a mouse infection. These findings suggest that Gpr1 and Gpa2 are involved in the glucose-sensing machinery that regulates morphogenesis and hypha formation in solid media via a cAMP-dependent mechanism, but they are not required for hypha formation in liquid medium or during invasive candidiasis.     

Biosynthesis of glycoproteins in the pathogenic fungus Candida albicans: activation of dolichol phosphate mannose synthase by cAMP-mediated protein phosphorylation

Following incubation with ATP and a cAMP-dependent protein kinase under optimal conditions of lipid acceptor, phospholipid and metal ion requirements, the transfer activity of partially purified dolichol phosphate mannose synthase (DPMS) increased about 60% and this activation correlated with a 50% increase in V(max) with no alteration in the apparent K(m) for GDP-Manose. Phosphorylation with [gamma-(32)P]ATP resulted in the labeling of several polypeptides, one of which exhibited the molecular weight of the enzyme (30 kDa) and was also recognized using a specific anti-DPMS monoclonal antibody. This and the fact that the phosphate label could be removed by an alkaline phosphatase indicate that Candida DPMS may be regulated by phosphorylation-dephosphorylation, a mechanism that has been proposed for the enzyme in other organisms.     

Biosynthesis of glycoproteins in the pathogenic fungus Candida albicans: Activation of dolichol phosphate mannose synthase by cAMP-mediated protein phosphorylation

Following incubation with ATP and a cAMP-dependent protein kinase under optimal conditions of lipid acceptor, phospholipid and metal ion requirements, the transfer activity of partially purified dolichol phosphate mannose synthase (DPMS) increased about 60% and this activation correlated with a 50% increase in V_max with no alteration in the apparent K_m for GDP-Manose. Phosphorylation with [γ-³²P]ATP resulted in the labeling of several polypeptides, one of which exhibited the molecular weight of the enzyme (30 kDa) and was also recognized using a specific anti-DPMS monoclonal antibody. This and the fact that the phosphate label could be removed by an alkaline phosphatase indicate that Candida DPMS may be regulated by phosphorylation–dephosphorylation, a mechanism that has been proposed for the enzyme in other organisms.     

A system for studying genetic changes in Candida albicans during infection

Candida albicans is a diploid yeast with a dimorphic life history. It exists commensally in many healthy humans but becomes a potent pathogen in immunocompromised hosts. The underlying genetic mechanisms by which C. albicans switches from a commensal to a pathogenic form in the host are not well understood. To study the evolution of virulence in mammalian hosts, we used GAL1 as selectable marker system that allows for both positive and negative selection in selective media. We show that the deletion of one or both copies of GAL1 in the C. albicans genome does not change virulence in a systemic mouse model. We obtained estimates for the frequency of mitotic recombination at the GAL1 locus during systemic infection. Our observations suggest that genetic changes such as mitotic recombination and gene conversion occur at a high enough frequency to be important in the transition of C. albicans from a commensal to a pathogenic organism.     

The appearance and characterization of cyanide-resistant respiration in the fungus Candida albicans

The respiration of yeast-form cells of the dimorphic fungus Candida albicans became resistant to cyanide during aging treatment in the resting state. An alternative, cyanide-resistant respiratory pathway was found to develop fully in cells aged at a concentration of 0.75 X 10(9)/ml or more at 25 C, but did not appear at 5 C. Chloramphenicol did not prevent the appearance of the alternative respiratory pathway. The effects of inhibitors, salicylhydroxamic acid (SHAM) and disulfiram (tetraethylthiuram disulfide), on respiration of aged cells were examined, and results indicated that SHAM binds at a site on the alternative respiratory pathway whereas disulfiram binds at two sites, one on the conventional respiratory pathway and the other on the alternative pathway. Thus, SHAM is a more selective inhibitor of the alternative respiration of C. albicans cells. SHAM-titration of the alternative respiration revealed that less than 10% of the maximal activity of the alternative respiratory pathway was utilized under normal conditions, indicating that the alternative respiratory pathway makes a small contribution to the total respiration. It was therefore concluded that the alternative, cyanide-resistant respiratory pathway operates fully when the cyanide-sensitive, cytochrome pathway is blocked although aged cells possess both respiratory pathways.