CAP1, an adenylate cyclase-associated protein gene, regulates bud-hypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans

In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.     

Increased high-affinity phosphodiesterase PDE2 gene expression in germ tubes counteracts CAP1-dependent synthesis of cyclic AMP, limits hypha production and promotes virulence of Candida albicans

Frequent interconversion between yeasts, pseudohyphae and true hyphae is a hallmark of Candida albicans growth in mammalian tissues. The requirement for transient CAP1-dependent pulses of cAMP for generating true hyphae, Hwp1 and virulence raises questions about the role of yeast and pseudohyphal forms in the pathogenesis of candidiasis. In this study, hyperfilamentous mutants, limited in their capacity to produce buds, were generated by disrupting the high-affinity phosphodiesterase gene PDE2. Degradation of cAMP by the PDE2 gene product was confirmed by higher basal cAMP levels in the pde2/pde2 mutant and by accumulation of cAMP to levels permitting germ tube formation upon disrupting PDE2 in the cap1/cap1 mutant. Similar phenotypes of the C. albicans and Saccharomyces cerevisiae pde2/pde2 mutants were found, including sensitivity to nutritional starvation and exogenous cAMP and defective entry into stationary phase. Importantly, the hyperfilamentous mutants were as avirulent as hypofilamentous mutants in a systemic model of candidiasis. Growth in a multiplicity of forms appears to be a virulence attribute that is controlled by tight coupling of cAMP synthesis and degradation. Delayed increases in PDE2 mRNA in cAMP-deficient cap1/cap1 mutants during germ tube-inducing conditions suggested a mechanism of control involving cAMP-dependent induction of PDE2 mRNA.     

The quorum-sensing molecules farnesol/homoserine lactone and dodecanol operate via distinct modes of action in Candida albicans

Living as a commensal, Candida albicans must adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects on C. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C(12)-homoserine lactone, a quorum-sensing molecule secreted by Pseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of the Candida adenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving the C. albicans hyphal repressor, Sfl1p. Deletion of SFL1 did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing in C. albicans is mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that the Burkholderia cenocepacia diffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C(12)-homoserine lactone, may be used by other quorum-sensing molecules.     

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.     

The 65 kDa mannoprotein gene of Candida albicans encodes a putative beta-glucanase adhesin required for hyphal morphogenesis and experimental pathogenicity

Mannoproteins are fungal cell wall components which play a main role in host-parasite relationship. Camp65p is a putative beta-glucanase mannoprotein of 65 kDa which has been characterized as a main target of human immune response against Candida albicans. However, nothing is known about its specific contribution to the biology and virulence of this fungus. We constructed CAMP65 knock-out mutants including null camp65/camp65 and CAMP65/camp65 heterozygous strains. The null strains had the same growth rate and morphology under yeast form as the wild-type strain but they were severely affected in hyphal morphogenesis both in vitro and in vivo. Hyphae formation was restored in revertant strains. The null mutants lost adherence to the plastic, and this was in keeping with the strong inhibition of fungal cell adherence to plastic exerted by anti-Camp65p antibodies. The null mutants were also significantly less virulent than the parental strains, and this loss of virulence was observed both in systemic and in mucosal C. albicans infection models. Nonetheless, the virulence in both infectious models was regained by the CAMP65 revertants. Thus, CAMP65 of C. albicans encodes a putative beta-glucanase, mannoprotein adhesin, which has a dual role (hyphal cell wall construction and virulence), accounting for the particular relevance of host immune response against this mannoprotein.     

<Emphasis Type="Italic">Candida albicans VPS4</Emphasis> is Required for Secretion of Aspartyl Proteases and In Vivo Virulence

Candida albicans secretes aspartyl proteases (Saps) during infection. Although Saps are secretory proteins, little is known about the intracellular trafficking and secretion of these proteins. We previously cloned and analyzed the C. albicans pre-vacuolar protein sorting gene VPS4, and demonstrated that extracellular Sap2p is absent in the culture supernatants of the vps4delta null mutant. We therefore investigated the role of the C. albicans pre-vacuolar secretion pathway in the trafficking of Sap4-6p and in vivo virulence. The C. albicans vps4delta mutant failed to produce extracellular Sap4-6p. Next, when tested in a mouse model of disseminated candidiasis, the vps4delta mutant was greatly attenuated in virulence. Histopathological analysis indicated that infection with the vps4delta mutant did not cause renal microabscess formation, in contrast to the wild-type strain. Our results imply that VPS4 is required for extracellular secretion of Sap4-6p, and that C. albicans requires an intact pre-vacuolar secretory pathway for wild-type virulence in vivo.     

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.     

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.     

The moonlighting protein Tsa1p is implicated in oxidative stress response and in cell wall biogenesis in Candida albicans

Candida albicans is one of the most common fungal pathogens in humans. The cell wall is the first contact site between host and pathogen and thus is critical for colonization and infection of the host. We have identified Tsa1p, a protein that is differentially localized to the cell wall of C. albicans in hyphal cells but remains in the cytosol and nucleus in yeast-form cells. This is different from Saccharomyces cerevisiae, where the homologous protein solely has been found in the cytoplasm. We report here that TSA1 confers resistance towards oxidative stress as well as is involved in the correct composition of hyphal cell walls. However, no significant change of the cell wall composition was observed in a TSA1 deletion strain in yeast-form cells, which is in good agreement with the observation that Tsa1p is absent from the yeast-form cell wall. This indicates that Tsa1p of C. albicans might represent a moonlighting protein with specific functions correlating to its respective localization. Furthermore, the translocation of Tsa1p to the hyphal cell wall of C. albicans depends on Efg1p, suggesting a contribution of the cAMP/PKA pathway to the localization of this protein. In a strain deleted for TUP1 that filaments constitutively Tsa1p can be found in the cell wall under all conditions tested, confirming the result that Tsa1p localization to the cell wall is correlated to the morphology of C. albicans.     

白念珠菌生物被膜形成的遗传调控机制

白念珠菌是人体重要的条件性致病真菌。形态的多样性和可塑性是白念珠菌典型的生物学特征,这与它的致病性、宿主适应能力以及有性生殖过程密切相关。白念珠菌生物被膜(Biofilm)是由不同形态细胞(包括酵母型、菌丝和假菌丝)以及胞外基质组成的致密结构,也是毒性和耐药性形成的重要因子。生物被膜对抗真菌药物、宿主免疫系统和环境胁迫因子等都表现出较强的抵抗力和耐受性,是临床上病原真菌感染防治的重大挑战。随着基因表达谱和遗传操作技术的发展,白念珠菌生物被膜的形成及其耐药性的获得所依赖的遗传调控通路和分子调控机制越来越清楚。主要包括MAPK和cAMP介导的信号途径以及Bcr1和Tec1等因子介导的转录调控。此外,白念珠菌生物被膜的形成与形态转换和有性生殖之间存在密切的联系。文中综述了白念珠菌生物被膜形成的遗传调控机制,重点介绍了细胞壁相关蛋白、转录因子和交配型对该过程的调控以及生物被膜的耐药机制。     

Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae

Candida albicans is a dimorphic human pathogen in which the yeast to hyphal switch may be an important factor in virulence in mammals. This pathogen has recently been shown to also kill insects such as the Greater Wax Moth Galleria mellonella when injected into the haemocoel of the insect larvae. We have investigated the effect of previously characterised C. albicans mutations that influence the yeast to hyphal transition on virulence in G. mellonella larvae. There is a good correlation between the virulence of these mutants in the insect host and the virulence measured through systemic infection of mice. Although the predominant cellular species detected in G. mellonella infections is the yeast form of C. albicans, mutations that influence the hyphal transition also reduce pathogenicity in the insect. The correlation with virulence measured in the mouse infection system suggests that Galleria may provide a convenient and inexpensive model for the in vivo screening of mutants of C. albicans.