Chromatin-mediated Candida albicans virulence

Candida albicans is the most prevalent human fungal pathogen. To successfully propagate an infection, this organism relies on the ability to change morphology, express virulence-associated genes and resist DNA damage caused by the host immune system. Many of these events involve chromatin alterations that are crucial for virulence. This review will focus on the studies that have been conducted on how chromatin function affects pathogenicity of C. albicans and other fungi. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.     

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.     

Dimorphism and virulence in Candida albicans

Two regulatory pathways govern filamentation in the pathogenic fungus Candida albicans. Recent virulence studies of filamentation regulatory mutants argue that both yeast and filamentous forms have roles in infection. Filamentation control pathways seem closely related in C. albicans and in Saccharomyces cerevisiae, thus permitting speculation about C. albicans filamentation genes not yet discovered.     

Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2

Elevated expression of the plasma membrane drug efflux pump proteins Cdr1p and Cdr2p was shown to accompany decreased azole susceptibility in Candida albicans clinical isolates. DNA sequence analysis revealed extensive allelic heterozygosity, particularly of CDR2. Cdr2p alleles showed different abilities to transport azoles when individually expressed in Saccharomyces cerevisiae. Loss of heterozygosity, however, did not accompany decreased azole sensitivity in isogenic clinical isolates. Two adjacent non-synonymous single nucleotide polymorphisms (NS-SNPs), G1473A and I1474V in the putative transmembrane (TM) helix 12 of CDR2, were found to be present in six strains including two isogenic pairs. Site-directed mutagenesis showed that the TM-12 NS-SNPs, and principally the G1473A NS-SNP, contributed to functional differences between the proteins encoded by the two Cdr2p alleles in a single strain. Allele-specific PCR revealed that both alleles were equally frequent among 69 clinical isolates and that the majority of isolates (81%) were heterozygous at the G1473A/I1474V locus, a significant (P < 0.001) deviation from the Hardy-Weinberg equilibrium. Phylogenetic analysis by maximum likelihood (Paml) identified 33 codons in CDR2 in which amino acid allelic changes showed a high probability of being selectively advantageous. In contrast, all codons in CDR1 were under purifying selection. Collectively, these results indicate that possession of two functionally different CDR2 alleles in individual strains may confer a selective advantage, but that this is not necessarily due to azole resistance.     

Chromosome 1 trisomy compromises the virulence of Candida albicans

Although increases in chromosome copy number typically have devastating developmental consequences in mammals, fungal cells such as Saccharomyces cerevisiae seem to tolerate trisomies without obvious impairment of growth. Here, we demonstrate that two commonly used laboratory strains of the yeast Candida albicans, CAI-4 and SGY-243, can carry three copies of chromosome 1. Although the trisomic strains grow well in the laboratory, Ura+ derivatives of CAI-4, carrying three copies of chromosome 1, are avirulent in the intravenously inoculated mouse model, unlike closely related strains carrying two copies of chromosome 1. Furthermore, changes in chromosome copy number occur during growth in an animal host and during growth in the presence of growth-inhibiting drugs. These results suggest that chromosome copy number variation provides a mechanism for genetic variation in this asexual organism.     

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.     

白假丝酵母CFL1基因通过转录调控参与氧化压力应答

【背景】CFL1基因是白假丝酵母高铁还原酶基因,介导胞外铁离子的还原,在白假丝酵母胞内铁稳态的维持方面发挥着重要作用。【目的】研究CFL1基因调节氧化压力应答的分子机制。【方法】采用液体培养及巨噬细胞模型,测定CFL1缺失对氧化压力耐受性和杀伤巨噬细胞能力的影响;使用羟基自由基清除剂二甲基亚砜(DMSO)分析其对缓解氧化压力敏感性的影响;采用实时荧光定量PCR分析CFL1缺失对氧化压力应答基因表达的影响;采用过氧化氢酶(CAT)活性测定方法研究CFL1缺失对CAT1基因表达的影响;通过构建WT-CAT1-GFP和cfl1Δ/Δ-CAT1-GFP菌株分析过氧化氢酶基因过表达对cfl1Δ/Δ氧化压力敏感性的影响。【结果】白假丝酵母CFL1基因的缺失会造成杀伤巨噬细胞能力的减弱,氧化压力应答基因表达的下降。过氧化氢酶基因的过表达则能恢复与野生型几乎一致的氧化压力水平。【结论】CFL1基因通过转录调控参与白假丝酵母氧化压力应答过程。     

Hemoglobin regulates expression of an activator of mating-type locus alpha genes in Candida albicans

Phenotypic switching from the white to the opaque phase is a necessary step for mating in the pathogenic fungus Candida albicans. Suppressing switching during vascular dissemination of the organism may be advantageous, because opaque cells are more susceptible to host defenses. A repressor of white-opaque switching, HBR1 (hemoglobin response gene 1), was identified based on its specific induction following growth in the presence of exogenous hemoglobin. Deletion of a single HBR1 allele allowed opaque phase switching and mating competence, accompanied by a lack of detectable MTL alpha1 and alpha2 gene expression and enhanced MTLa1 gene expression. Conversely, overexpression of Hbr1p or exposure to hemoglobin increased MTLalpha gene expression. The a1/alpha2 repressed target gene CAG1 was derepressed in the same mutant in a hemoglobin-sensitive manner. Regulation of CAG1 by hemoglobin required an intact MTLa1 gene. Several additional Mtlp targets were perturbed in HBR1 mutants in a manner consistent with commitment to an a mating phenotype, including YEL007w, MFalpha, HST6, and RAM2. Therefore, Hbr1 is part of a host factor-regulated signaling pathway that controls white-opaque switching and mating in the absence of allelic deletion at the MTL locus.     

Candida albicans adhesins: Biochemical aspects and virulence

The recognition of host cells by the pathogenic yeast, Candida albicans, is probably an essential step in the pathogenesis of disease development. The interaction of yeast and hyphal mannoproteins with host cell receptors has been studied by a number of laboratories. C. albicans recognizes a variety of host cells as well as host cell extracellular matrix proteins. This observation is not unexpected given the number of sites within and on the body which can be colonized and infected by the organism. Indeed, it would appear that C. albicans has evolved a number of ways in which it recognizes the host. This statement is made with the qualification that the organism uses other processes to infect, such as morphogenesis, phenotypic switching and the production of invasive enzymes, including secreted aspartyl proteases and phosholipases. Recognition of epithelial cells is accomplished through cell surface mannoproteins (adhesins) which bind to carbohydrate-containing receptors. The number of such mannoproteins is not known; pro adhesins exist. The organism also binds to keratinocytes, endothelial cells and matrix proteins, such as fibronectin, laminin, collagen and entactin, and, as such, appears to have a integrin-like cell surface adhesin. In most cases, the adhesin for each of these host proteins is a mannoprotein. The biochemistry of the candidal adhesins has been extensively studied. However, molecular analyses of the encoding genes is only now being studied. Thus, until clean, genetic analyses are complete and strains lacking an adhesin function are constructed, a direct role for the adhesins in pathogenesis can only be inferred. At present, spontaneous, non-adhering strains of the organism have been described which are avirulent in animal models of candidiasis. However, these data only suggest a role for adherence; future studies should be directed towards resolving questions about the role of these proteins in pathogenesis.     

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.     

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.     

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.     

Modulation of Candida albicans virulence by bacterial biofilms on titanium surfaces

Whilst Candida albicans occurs in peri-implant biofilms, its role in peri-implantitis remains unclear. This study therefore examined the virulence of C. albicans in mixed-species biofilms on titanium surfaces. Biofilms of C. albicans (Ca), C. albicans with streptococci (Streptococcus sanguinis, S. mutans) (Ca-Ss-Sm) and those incorporating Porphyromonas gingivalis (Ca-Pg and Ca-Ss-Sm-Pg) were developed. Expression of C. albicans genes associated with adhesion (ALS1, ALS3, HWP1) and hydrolytic enzymes (SAP2, SAP4, SAP6, PLD1) was measured and hyphal production by C. albicans quantified. Compared with Ca biofilms, significant (p<0.05) up-regulation of ALS3, HWP1, SAP2 and SAP6, and hyphal production occurred in biofilms containing streptococci (Ca-Ss-Sm). In Ca-Pg biofilms, down-regulation of HWP1 and SAP4 expression, with reduced hyphal production occurred. Ca-Ss-Sm-Pg biofilms had increased hyphal proportions and up-regulation of ALS3, SAP2 and SAP6. In conclusion, C. albicans expressed virulence factors in biofilms that could contribute to peri-implantitis, but this was dependent on associated bacterial species.     

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.     

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.     

TOS9 regulates white-opaque switching in Candida albicans

In Candida albicans, the a1-alpha2 complex represses white-opaque switching, as well as mating. Based upon the assumption that the a1-alpha2 corepressor complex binds to the gene that regulates white-opaque switching, a chromatinimmunoprecipitation-microarray analysis strategy was used to identify 52 genes that bound to the complex. One of these genes, TOS9, exhibited an expression pattern consistent with a "master switch gene." TOS9 was only expressed in opaque cells, and its gene product, Tos9p, localized to the nucleus. Deletion of the gene blocked cells in the white phase, misexpression in the white phase caused stable mass conversion of cells to the opaque state, and misexpression blocked temperature-induced mass conversion from the opaque state to the white state. A model was developed for the regulation of spontaneous switching between the opaque state and the white state that includes stochastic changes of Tos9p levels above and below a threshold that induce changes in the chromatin state of an as-yet-unidentified switching locus. TOS9 has also been referred to as EAP2 and WOR1.