Inactivation of Kex2p diminishes the virulence of Candida albicans

Deletion of the kexin gene (KEX2) in Candida albicans has a pleiotropic effect on phenotype and virulence due partly to a defect in the expression of two major virulence factors: the secretion of active aspartyl proteinases and the formation of hyphae. kex2/kex2 mutants are highly attenuated in a mouse systemic infection model and persist within cultured macrophages for at least 24 h without causing damage. Pathology is modest, with little disruption of kidney matrix. The infecting mutant cells are largely confined to glomeruli, and are aberrant in morphology. The complex phenotype of the deletion mutants reflects a role for kexin in a wide range of cellular processes. Taking advantage of the specificity of Kex2p cleavage, an algorithm we developed to scan the 9168 open reading frames in Assembly 6 of the C. albicans genome identified 147 potential substrates of Kex2p. These include all previously identified substrates, including eight secreted aspartyl proteinases, the exoglucanase Xog1p, the immunodominant antigen Mp65, and the adhesin Hwp1p. Other putative Kex2p substrates identified include several adhesins, cell wall proteins, and hydrolases previously not implicated in pathogenesis. Kexins also process fungal mating pheromones; a modification of the algorithm identified a putative mating pheromone with structural similarities to Saccharomyces cerevisiae alpha-factor.     

Low virulence of a morphological Candida albicans mutant

The DNA fragment encoding malonate decarboxylase, involved in malonate assimilation, was cloned from Pseudomonas putida. The 11-kb DNA fragment contained nine open reading frames, which were designated mdcABCDEGHLM in the given order. N-terminal protein sequencing established that the mdcA, mdcC, mdcD, mdcE and mdcH genes encoded subunits alpha, delta, beta, gamma and epsilon of the malonate decarboxylase, respectively. Malonate decarboxylase was functionally expressed in Escherichia coli from plasmid harboring the entire gene cluster or the mdc genes lacking the mdcL and mdcM genes. The mdcL and mdcM genes encode membrane proteins and disruption of the genes of P. putida by the insertion of a kanamycin resistance cassette reduced the malonate uptake activity of the organism. Thus, we conclude that MdcLM is a malonate transporter.     

Candida albicans protein kinase CaHsl1p regulates cell elongation and virulence

Saccharomyces cerevisiae Hsl1p is a Ser/Thr protein kinase that regulates cell morphology. We identified Candida albicans CaHSL1 and analysed its function in C. albicans. Cells lacking CaHsl1p exhibited filamentous growth under yeast growth conditions with the filaments elongating more quickly than did those of the wild type under hyphal growth conditions, suggesting that it plays a role in the suppression of cell elongation. Green fluorescent protein-tagged CaHsl1p colocalized with a septin complex to the bud neck during yeast growth or to a potent septation site during hyphal growth, as expected from the localization in S. cerevisiae. However, the localization of the septin complex did not change in DeltaCahsl1, suggesting that CaHsl1p does not participate in septin organization. CaHsl1p was expressed in a cell cycle-dependent manner and, except for the G1 phase, phosphorylated throughout the cell cycle. In DeltaCahsl1 cells, the phosphorylation of a possible CaHsl1p target CaSwe1p decreased, while that of CaCdc28p at tyrosine18 increased. Either an extra copy of the tyrosine18-mutated CaCdc28p or deletion of CaSWE1 suppressed the cell elongation phenotype caused by CaHSL1 deletion. Furthermore, DeltaCahsl1 exhibited reduced virulence in the mouse systemic candidiasis model. Thus, the CaHsl1p-CaSwe1p-CaCdc28p pathway appears important in the cell elongation of both the yeast and hyphal forms and to the virulence of C. albicans.     

Candida albicans SRR1, a putative two-component response regulator gene, is required for stress adaptation, morphogenesis, and virulence

We report here the identification and characterization of a previously uncharacterized, two-component response regulator gene (orf19.5843) from Candida albicans. Because of its apparent functions in stress adaptation, we have named this gene SRR1 (stress response regulator 1). Disruption of SRR1 causes defects in hyphal development, reduced resistance to stress, and severe virulence attenuation in the mouse model of disseminated candidiasis.     

GLN3 encodes a global regulator of nitrogen metabolism and virulence of C. albicans

The function of GLN3, a GATA factor encoding gene, in nitrogen metabolism of Candida albicans was examined. GLN3 null mutants had reduced growth rates on multiple nitrogen sources. More severe growth defects were observed in mutants lacking both GLN3 and GAT1, a second GATA factor gene. GLN3 was an activator of two genes involved in ammonium assimilation, GDH3, encoding NADP-dependent glutamate dehydrogenase, and MEP2, which encodes an ammonium permease. GAT1 contributed to MEP2 expression, but not that of GDH3. A putative general amino acid permease gene, GAP2, was also activated by both GLN3 and GAT1, but activation by GLN3 was nitrogen source dependent. GLN3 was constitutively expressed, but GAT1 expression varied with nitrogen source and was reduced 2- to 3-fold in gln3 mutants. Both gln3 and gat1 mutants exhibited reduced sensitivity to rapamycin, suggesting they function downstream of TOR kinase. Hyphae formation by gln3 and gat1 mutants differed in relation to nitrogen source. The gln3 mutants formed hyphae on several nitrogen sources, but not ammonium or urea, suggesting a defect in ammonium assimilation. Virulence of gln3 mutants was reduced in a murine model of disseminated disease. We conclude that GLN3 has a broad role in nitrogen metabolism, partially overlapping, but distinct from that of GAT1, and that its function is important for the ability of C. albicans to survive within the host environment.     

URA3 as a selectable marker for disruption and virulence assessment of Candida albicans genes

The ability to generate isogenic sets of strains with mutations in a gene of interest but not in other genes by repeated use of the URA3 marker (Ura-blaster methodology) has advanced our understanding of the relationships between gene structure and function in Candida albicans. Common applications of Ura-blaster technology result in different genomic positions for the URA3 gene in strains complemented for the gene of interest compared with mutant strains. Studies using animal models of systemic candidiasis pointed to possible differences in URA3 gene expression, depending on its genomic location, which confounded interpretation of the role of the gene of interest in lethality. Positional effects on URA3 expression can be avoided by placement at a common locus in all strains used for comparison.     

GLN3 encodes a global regulator of nitrogen metabolism and virulence of C. albicans.

The function of GLN3, a GATA factor encoding gene, in nitrogen metabolism of Candida albicans was examined. GLN3 null mutants had reduced growth rates on multiple nitrogen sources. More severe growth defects were observed in mutants lacking both GLN3 and GAT1, a second GATA factor gene. GLN3 was an activator of two genes involved in ammonium assimilation, GDH3, encoding NADP-dependent glutamate dehydrogenase, and MEP2, which encodes an ammonium permease. GAT1 contributed to MEP2 expression, but not that of GDH3. A putative general amino acid permease gene, GAP2, was also activated by both GLN3 and GAT1, but activation by GLN3 was nitrogen source dependent. GLN3 was constitutively expressed, but GAT1 expression varied with nitrogen source and was reduced 2- to 3-fold in gln3 mutants. Both gln3 and gat1 mutants exhibited reduced sensitivity to rapamycin, suggesting they function downstream of TOR kinase. Hyphae formation by gln3 and gat1 mutants differed in relation to nitrogen source. The gln3 mutants formed hyphae on several nitrogen sources, but not ammonium or urea, suggesting a defect in ammonium assimilation. Virulence of gln3 mutants was reduced in a murine model of disseminated disease. We conclude that GLN3 has a broad role in nitrogen metabolism, partially overlapping, but distinct from that of GAT1, and that its function is important for the ability of C. albicans to survive within the host environment.     

Female gametophytic mutants of Arabidopsis thaliana identified in a gene trap insertional mutagenesis screen

In plants, the male and female gametophytes represent the haploid generation that alternates with the diploid sporophytic generation. Male and female gametophytes develop from haploid micro- and megaspores, respectively. In flowering plants (angiosperms), the spores themselves arise from the sporophyte through meiotic divisions of sporogenous cells in the reproductive organs of the flower. Male and female gametophytes contain two pairs of gametes that participate in double fertilization, a distinctive feature of angiosperms. In this paper, we describe the employment of a transposon-based gene trap system to identify mutations affecting the gametophytic phase of the plant life cycle. Mutants affecting female gametogenesis were identified in a two-step screen for (i) reduced fertility (seed abortion or undeveloped ovules) and (ii) segregation ratio distortion. Non-functional female gametophytes do not initiate seed development, leading to semi-sterility such that causal or linked alleles are transmitted at reduced frequency to the progeny (non-Mendelian segregation). From a population of 2,511 transposants, we identified 54 lines with reduced seed set (2%). Examination of their distorted segregation ratios and seed phenotypes led to the isolation of 12 gametophytic mutants, six of which are described herein. Chromosomal sequences flanking the transposon insertions were identified and physically mapped onto the genome sequence of Arabidopsis thaliana. Surprisingly, the insertion sites were often associated with chromosomal rearrangements, making it difficult to assign the mutant phenotypes to a specific gene. The mutants were classified according to the process affected at the time of arrest, i.e. showing mitotic, karyogamic, maternal or degenerative phenotypes.     

Candida albicans dimorphism and virulence: Role of copper

Previously reported observations that Candida albicans grows in the yeast phase at 30° C and the mycelial phase at 37° C and that the former phase is more virulent than the latter were confirmed. A novel factor, copper, was discovered to suppress filamentation. Injection of copper into mice permitted the filamentous phase to be as virulent as the yeast phase. In subsequent studies on candidosis, copper assays should be performed on relevant body fluids to determine if there might be a correlation between elevated copper and heightened susceptibility to the fungus.     

Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans

Candida dubliniensis is a pathogenic yeast species that was first identified as a distinct taxon in 1995. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and that it is primarily associated with oral carriage and oropharyngeal infections in human immunodeficiency virus (HIV)-infected and acquired immune deficiency syndrome (AIDS) patients. However, unlike Candida albicans, C. dubliniensis is rarely found in the oral microflora of normal healthy individuals and is responsible for as few as 2% of cases of candidemia (compared to approximately 65% for C. albicans). The vast majority of C. dubliniensis isolates identified to date are susceptible to all of the commonly used antifungal agents, however, reduced susceptibility to azole drugs has been observed in clinical isolates and can be readily induced in vitro. The primary mechanism of fluconazole resistance in C. dubliniensis has been shown to be overexpression of the major facilitator efflux pump Mdr1p. It has also been observed that a large number of C. dubliniensis strains express a non-functional truncated form of Cdr1p, and it has been demonstrated that this protein does not play a significant role in fluconazole resistance in the majority of strains examined to date. Data from a limited number of infection models reflect findings from epidemiological studies and suggest that C. dubliniensis is less pathogenic than C. albicans. The reasons for the reduced virulence of C. dubliniensis are not clear as it has been shown that the two species express a similar range of virulence factors. However, although C. dubliniensis produces hyphae, it appears that the conditions and dynamics of induction may differ from those in C. albicans. In addition, C. dubliniensis is less tolerant of environmental stresses such as elevated temperature and NaCl and H(2)O(2) concentration, suggesting that C. albicans may have a competitive advantage when colonising and causing infection in the human body. It is our hypothesis that a genomic comparison between these two closely-related species will help to identify virulence factors responsible for the far greater virulence of C. albicans and possibly identify factors that are specifically implicated in either superficial or systemic candidal infections.     

Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans

The relevance of the mitogen-activated protein (MAP) kinase Hog1p in Candida albicans was addressed through the characterization of C. albicans strains without a functional HOG1 gene. Analysis of the phenotype of hog1 mutants under osmostressing conditions revealed that this mutant displays a set of morphological alterations as the result of a failure to complete the final stages of cytokinesis, with parallel defects in the budding pattern. Even under permissive conditions, hog1 mutants displayed a different susceptibility to some compounds such as nikkomycin Z or Congo red, which interfere with cell wall functionality. In addition, the hog1 mutant displayed a colony morphology different from that of the wild-type strain on some media which promote morphological transitions in C. albicans. We show that C. albicans hog1 mutants are derepressed in the serum-induced hyphal formation and, consistently with this behavior, that HOG1 overexpression in Saccharomyces cerevisiae represses the pseudodimorphic transition. Most interestingly, deletion of HOG1 resulted in a drastic increase in the mean survival time of systemically infected mice, supporting a role for this MAP kinase pathway in virulence of pathogenic fungi. This finding has potential implications in antifungal therapy.     

Isolates of Candida albicans that differ in virulence for mice elicit strain-specific antibody-mediated protective responses

Three distinct isolates of Candida albicans were used to establish systemic and oral infections in inbred mice that are genetically resistant or susceptible to tissue damage. Patterns of infection differed significantly between both yeasts and mouse strains. Systemic infection conferred significant protection against re-challenge with the homologous, but not the heterologous yeast; however, the protective effect was more evident in the tissue-susceptible CBA/CaH mice than in the resistant BALB/c strain. In contrast, oral infection induced protection against both homologous and heterologous oral challenge, although this was significant only in the CBA/CaH mice. CBA/CaH mice produced antibodies of both IgG1 and IgG2a subclasses, whereas BALB/c mice produced predominantly IgG1. Western blotting demonstrated considerable differences between epitopes recognised by serum antibodies from mice of both strains after immunisation with each of the three yeasts. Thus, different strains of yeast show considerable specificity in antibody responses elicited by either systemic or oral infection.