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Oral squamous cell carcinoma is associated with many known risk factors including tobacco smoking, chronic alcoholism, poor oral hygiene, unhealthy dietary habits and microbial infection. Previous studies have highlighted Candida albicans host tissue infection as a risk factor in the initiation and progression of oral cancer. C albicans invasion induces several cancerous hallmarks, such as activation of proto-oncogenes, induction of DNA damage and overexpression of inflammatory signalling pathways. However, the molecular mechanisms behind these responses remain unclear. A recently discovered fungal toxin peptide, candidalysin, has been reported as an essential molecule in epithelial damage and host recognition of C albicans infection. Candidalysin has a clear role in inflammasome activation and induction of cell damage. Several inflammatory molecules such as IL-6, IL-17, NLRP3 and GM-CSF have been linked to carcinogenesis. Candidalysin is encoded by the ECE1 gene, which has been linked to virulence factors of C albicans such as adhesion, biofilm formation and filamentation properties. This review discusses the recent epidemiological burden of oral cancer and highlights the significance of the ECE1 gene and the ECE1 protein breakdown product, candidalysin in oral malignancy. The immunological and molecular mechanisms behind oral malignancy induced by inflammation and the role of the toxic fungal peptide candidalysin in oral carcinogenesis are explored. With increasing evidence associating C albicans with oral carcinoma, identifying the possible fungal pathogenicity factors including the role of candidalysin can assist in efforts to understand the link between C albicans infection and carcinogenesis, and pave the way for research into therapeutic potentials. [ABSTRACT FROM AUTHOR]

As human populaces develop, they are progressively squeezed into higher living densities. The same is true for horticulture and animals expected to bolster these communities. Despite the high potential for zoonotic transmission, connections among humans and cattle have been understudied; however, Candida albicans remains the most important medical mycosis. The genesis of the mycobiome can vary, and interactions between humans and cattle are progressively being perceived as a key interface for disease transmission. αINT1 is a unique gene from Candida albicans; hence, it has been used for detection as well as intraspecific and interspecific phylogenetic analysis of C. albicans collected from human patients and cattle with pulmonary distress in urban–rural populations. A total of 1,921 specimens were examined by direct microscopy and culture to recover yeast associated with human infection. Identification was performed by micromorphology using an API 20C AUX system. The fungal species identified in bovine nasal specimens were Alternaria species (15%), Penicillium species, and C. albicans (6.7%). Other fungal species, such as Aspergillus niger, Torulopsis species, Mucor species (5%), Aspergillus flavus, Fusarium species, Trichosporon species (3.3%), C. rugosa, C. tropical, and Saccharomyces species (1.7%), were also isolated. In human sputum specimens, C. albicans (20%) and C. parapsilosis (2.7%) were the only reported yeast species in our samples. The four identified C. albicans species (two human and two cattle) were subjected to αINT1 gene sequence analysis, which confirmed major phylogenetic relationships among human and cattle isolates. This finding highlights the public health importance of bovines as a potential source for C. albicans zoonotic transmission to humans in an urban–rural community. Additionally, the close relationship between circulating C. albicans strains recorded in Egypt and the United States indicates the possible cross-species transmission of C. albicans between imported foreign and native cattle breeds. [ABSTRACT FROM AUTHOR]

Objective The purpose of this study is to elucidate differences in the mechanism of the IL-1β release-inducing activity of Candida albicans toward dendritic cells and macrophages because IL-1β is one of the proinflammatory cytokines which is crucial in host defense against candidiasis. Design Two C. albicans strains were used in this study. One strain is uridine-auxotrophic (CAI4) that needs uridine to grow and form hyphae, and another is a strain without any specific auxotrophy (pACT1-GFP), which forms hyphae naturally by culturing with serum components. Murine macrophage and dendritic cell lines were primed with LPS and then stimulated with C. albicans CAI4 or pACT1-GFP. Results Both strains of C. albicans induced IL-1β release from dendritic cells, and C. albicans pACT1-GFP induced IL-1β release but CAI4 induced little amounts in macrophages. These differences were suggested to be due to the difference in the amount of extracellular ATP released in the cell culture supernatants induced by C. albicans CAI4 or pACT1-GFP. For induction of IL-1β release from both macrophages and dendritic cells by C. albicans , direct contacts of the microbes with cells were required. In addition, macrophages required morphological change of C. albicans from yeast to hyphae for induction of IL-1β release, whereas dendritic cells did not require it. Dead C. albicans could induce IL-1β release from dendritic cells, but could not from macrophages. Conclusions There are different mechanisms by which C. albicans induces IL-1β release from dendritic cells and macrophages. [ABSTRACT FROM AUTHOR]

Summary: Candida albicans can switch between yeast and hyphae growth forms, which is critical for its pathogenesis. Diluting from saturated cells into fresh medium at 37°C is routinely used to induce hyphae, which depends on the cAMP‐PKA pathway‐activated transcriptional down‐regulation of NRG1 and degradation of Nrg1 protein triggered by inoculation. It is reported that N‐acetylglucosamine (GlcNAc), serum or neutral pH could stimulate filamentation in log phase cells, whereas how C. albicans develops hyphae without inoculation remains unknown. Here, we show that NRG1 down‐regulation is necessary for hyphal growth under this condition. Instead of cAMP‐PKA pathway, GlcNAc sensor Ngs1 is responsible for the down‐regulation of NRG1 upon GlcNAc induction in log phase cells through its N‐acetyltransferase activity. From a genetic screen, Brg1 is found to be essential for hyphal development without inoculation. Ngs1 binds to BRG1 promoter to induce its expression in GlcNAc. Importantly, constitutively expressed BRG1 induces NRG1 down‐regulation even in the absence of GlcNAc or Ngs1. Serum or neutral pH‐induced filamentation in log phase cells is also through Brg1‐mediated NRG1 down‐regulation. Our study provides a molecular mechanism for how C. albicans forms hyphae in different cell states. This flexibility may facilitate C. albicans to adapt varied host environment during infection. [ABSTRACT FROM AUTHOR]

Normally, Candida albicans is a commensal microbe that resides in the human oral cavity, gut and vagina. However, the fungus can cause mucosal and systemic infections in immunocompromised individuals. The mechanism by which local mucosal infections progress to systemic candidiasis is poorly understood. Here, a murine model of gastrointestinal (GI) candidiasis was developed by inoculation of the oral cavity, followed by treatment with tetracycline (TC) and prednisolone (PSL). Temporal progression from a local infection of the oral cavity to a systemic infection was then monitored. Histological analysis of tissues from mice treated with both TC and PSL revealed massive infiltration of the tongue and stomach by hyphae. PSL increased the fungal burden in the tongue, stomach and small intestine, and facilitated dissemination to the spleen, kidney and liver within 3 days post-infection. Treatment with both TC and PSL supressed interferon (IFN)-γ and interleukin (IL)-17 (cytokines that play key roles in host defence against fungal infection) levels in the tongue, which were induced by C. albicans infection. In addition, the mucosal layer of the small intestine of mice treated with both TC and PSL was almost destroyed by the fungal infection; this may be a critical event that allows passage of the fungus across the mucosa and into the systemic circulation. Thus, this mouse model is useful for studying mechanisms underlying progression of C. albicans from a local infection of the oral cavity to a systemic infection in immunocompromised individuals. [ABSTRACT FROM AUTHOR]

Tetracycline-regulated expression of genes is often used for functional analysis of Candida albicans genes. However, the widely used Tet-On system has certain limitations such as prolonged lag time (up to 8 h) for induction and non-uniform expression among the cells. We speculated that poor expression of tetracycline-controlled transactivator (Tet-transactivator) from CaADH1 promoter could be responsible for this, and thus compared the effect of expressing this protein under the control of CaADH1, CaTDH3 and CaRP10 promoters on the expression of GFP from the TET promoter. Only CaRP10 promoter facilitated a more uniform and rapid induction of GFP. However, a high concentration of doxycycline was needed for induction, which is not desirable for assessing certain phenotypes. Tet-Off systems are known to require a low concentration of doxycycline, but a limitation of the widely used Tet-Off system for C. albicans is the use of CaENO1 promoter, which is known to be repressed in the presence of gluconeogenic carbon source, for expression of transactivator. Thus, we have converted the above-mentioned Tet-On systems to Tet-Off systems by site-directed mutagenesis of the Tet-transactivator. Compared to the Tet-On systems, the Tet-Off systems required about 200-fold less concentration of doxycycline for modulation of gene expression. Only the Tet-Off system with CaRP10 promoter driving the expression of transactivator allowed rapid and high level expression of GFP compared to those with CaADH1 or CaTDH3 promoters. The utility of CaRP10 based Tet-On and Tet-Off systems was further validated by the conditional expression of the CaTUP1 gene. We have also adapted these systems for use with Candida tropicalis and find that the Tet-Off system is functional in this species. The Tet systems reported here will be useful for conditional expression of genes in C. albicans as well as C. tropicalis. [ABSTRACT FROM AUTHOR]

Candida albicans is the most prevalent opportunistic fungus of humans and animals. While most studies focus on human isolates, they rarely focus on poultry isolates. In this study, C. albicans strains were recovered from poultry in the southern Hebei Province (China) and identified. Molecular typing and analyses were performed to understand the molecular epidemiology and genetic relatedness of the strains. The fungi were isolated from live birds with presumed candidiasis or their corpses. The isolates were identified based on morphology, differential medium culture, and rDNA internal transcribed spacer sequencing. The identified C. albicans strains were analyzed by ABC genotyping and multilocus sequence typing. Clonal groups were identified using the eBURST (version 3.0) software, and an UPGMA phylogenetic tree was constructed using the MEGA (version 6.06) software. Overall, 72 isolates were divided into three genotypes (A, B, and C), 48 novel sequence types (STs), five groups with 10 singletons, and four clades. Results indicated that candidiasis is common in poultry in the southern Hebei Province, and that the genetic composition of the C. albicans poultry population from the area is relatively complicated. Based on the eBURST analysis for the STs in this study and others, we suggest that C. albicans poultry isolates were relatively independent but not completely separated from human isolates. The strains with the same or closely related genotypes but recovered from both birds and humans could have transferred and evolved between the two types of host. [ABSTRACT FROM AUTHOR]

The pathogenic fungus Candida albicans can undergo phenotypic switching between two heritable states: white and opaque. This phenotypic plasticity facilitates its colonization in distinct host niches. The master regulator WOR1 is exclusively expressed in opaque phase cells. Positive feedback regulation by Wor1 on the WOR1 promoter is essential for opaque formation, however the underlying mechanism of how Wor1 functions is not clear. Here, we use tandem affinity purification coupled with mass spectrometry to identify Wor1-interacting proteins. Tup1 and its associated complex proteins are found as the major factors associated with Wor1. Tup1 occupies the same regions of the WOR1 promoter as Wor1 preferentially in opaque cells. Loss of Tup1 is sufficient to induce the opaque phase, even in the absence of Wor1. This is the first such report of a bypass of Wor1 in opaque formation. These genetic analyses suggest that Tup1 is a key repressor of the opaque state, and Wor1 functions via alleviating Tup1 repression at the WOR1 promoter. Opaque cells convert to white en masse at 37°C. We show that this conversion occurs only in the presence of glycolytic carbon sources. The opaque state is stabilized when cells are cultured on non-glycolytic carbon sources, even in a MTLa/α background. We further show that temperature and carbon source affect opaque stability by altering the levels of Wor1 and Tup1 at the WOR1 promoter. We propose that Wor1 and Tup1 form the core regulatory circuit controlling the opaque transcriptional program. This model provides molecular insights on how C. albicans adapts to different host signals to undergo phenotypic switching for colonization in distinct host niches. [ABSTRACT FROM AUTHOR]

Monitoring fungal ecology and resistance to antifungal agents within intensive care units ( ICU) is essential for the management of invasive fungal infections. Therefore, a retrospective descriptive study was carried in the ICU of Nimes University Hospital, France, from 2007 to 2016. As the majority of invasive fungal infections in ICU are caused by Candida species, the study objectives were to describe Candida species distribution, to assess candidaemia incidence and to monitor the antifungal drug susceptibility of Candida isolates and the consumption of antifungal agents. Among the recorded invasive Candida infections (n=244), 43% were intra-abdominal and 22% bloodstream infections. Candida albicans was the most frequent species (55.8%), followed by Candida glabrata (14.1%), Candida tropicalis (10%), Candida parapsilosis (8%) and Candida krusei (5.3%). Candidaemia incidence was 4.49 per 1000 admissions. The mean consumption of antifungal agents was of 170.5 defined daily doses ( DDD) for 1000 hospital days ( HD) per year. Changes in antifungal drug consumption were observed, with an increased use of echinocandins (from 17.96 DDD/1000 HD in 2007 to 48.76 DDD/1000 HD in 2016), and the total treatment cost tripled during the study period. No significant change in fungal ecology or in the emergence of resistant species was observed; indeed, only 1.1% of isolates presented an unusual resistance to antifungal agents. [ABSTRACT FROM AUTHOR]

The regulatory networks governing morphogenesis of a pleomorphic fungus, Candida albicans are extremely complex and remain to be completely elucidated. This study investigated the function of C. albicans yeast casein kinase 2 (CaYck2p). The yck2Δ/yck2Δ strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the yck2Δ/yck2Δ strain which showed significant upregulation of UME6, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes ALS3, HWP1, and SUN41, all of which are associated with morphogenesis and biofilm architecture. The yck2Δ/yck2Δ strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, CHS2, CHS3, and CHS8. Absence of CaYck2p also affected fungal-host interaction; the yck2Δ/yck2Δ strain had significantly reduced ability to damage host cells. However, the yck2Δ/yck2Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in C. albicans, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the potential genes involved in drug resistance of Candida albicans (C. albicans) by performing microarray analysis. The gene expression profile of GSE65396 was downloaded from the Gene Expression Omnibus, including a control, 15‑min and 45‑min macrocyclic compound RF59‑treated group with three repeats for each. Following preprocessing using RAM, the differentially expressed genes (DEGs) were screened using the Limma package. Subsequently, the Kyoto Encyclopedia of Genes and Genomes pathways of these genes were analyzed using the Database for Annotation, Visualization and Integrated Discovery. Based on interactions estimated by the Search Tool for Retrieval of Interacting Gene, the protein‑protein interaction (PPI) network was visualized using Cytoscape. Subnetwork analysis was performed using ReactomeFI. A total of 154 upregulated and 27 downregulated DEGs were identified in the 15‑min treated group, compared with the control, and 235 upregulated and 233 downregulated DEGs were identified in the 45‑min treated group, compared with the control. The upregulated DEGs were significantly enriched in the ribosome pathway. Based on the PPI network, PRP5, RCL1, NOP13, NOP4 and MRT4 were the top five nodes in the 15‑min treated comparison. GIS2, URA3, NOP58, ELP3 and PLP7 were the top five nodes in the 45‑min treated comparison, and its subnetwork was significantly enriched in the ribosome pathway. The macrocyclic compound RF59 had a notable effect on the ribosome and its associated pathways of C. albicans. RCL1, NOP4, MRT4, GIS2 and NOP58 may be important in RF59‑resistance. [ABSTRACT FROM AUTHOR]

Objective: Recent studies into the antifungal activity of NK-cells against the Aspergillus fumigatus have presented differing accounts on their mode of antifungal activity. One of these mechanisms proposed that NK-cells may kill the fungus via the direct effects of exposure to Interferon gamma (IFN-γ). Results: In this study we investigated the direct antifungal effects of recombinant human IFN-γ against a range of pathogenic fungi by measuring cellular damage using an XTT-based assay and cell viability through plate counts. It was found that 32 pg/ml of IFN-γ exhibited a significant but small antifungal effect on A. fumigatus (p = 0.02), Aspergillus flavus (p = 0.04) and Saccharomyces cerevisiae (p = 0.03), inhibiting growth by 6, 11 and 17% respectively. No significant inhibitory effects were observed in Candida species (p > 0.05 for all species tested) or Cryptococus neoformans (p = 0.98). Short term exposure (3 h) to a combination of amphotericin B (1 μg/ml) and IFN-γ (32 pg/ml) increased the effectiveness of amphotericin B against A. fumigatus and S. cerevisiae but not Candida albicans. These data suggest that IFN-γ does not possess strong antifungal activity but can enhance the effect of amphotericin B under some testing conditions against Aspergillus species. [ABSTRACT FROM AUTHOR]

Candida albicans is responsible for many of the infections affecting immunocompromised individuals. Although most C. albicans are susceptible to antifungal drugs, uncontrolled use of these drugs has promoted the development of resistance to current antifungals. The clinical implication of resistant strains has led to the search for safer and more effective drugs as well as alternative approaches, such as controlled drug release using liposomes and photodynamic inactivation (PDI), to eliminate pathogens by combining light and photosensitizers. In this study, we used layer-by-layer (LBL) assembly to immobilize triclosan and acridine orange encapsulated in liposomes and investigated the possibility of controlled release using light. Experiments were carried out to examine the susceptibility of C. albicans to PDI. The effects of laser irradiation were investigated by fluorescence microscopy, atomic force microscopy, and release kinetics. Liposomes were successfully prepared and immobilized using the self-assembly LBL technique. Triclosan was released more quickly when the LBL film was irradiated. The release rate was approximately 40% higher in irradiated films (fluence of 15 J/cm 2 ) than in non-irradiated films. The results of the susceptibility experiments and surface morphological analysis indicated that C. albicans cell death is caused by photodynamic inactivation. Liposomes containing triclosan and acridine orange may be useful for inactivating C. albicans using light. Our results lay the foundation for the development of new clinical strategies to control resistant strains. [ABSTRACT FROM AUTHOR]

Efficient assimilation of alternative carbon sources in glucose-limited host niches is critical for colonization of Candida albicans, a commensal yeast that frequently causes opportunistic infection in human. C. albicans evolved mechanistically to regulate alternative carbon assimilation for the promotion of fungal growth and commensalism in mammalian hosts. However, this highly adaptive mechanism that C. albicans employs to cope with alternative carbon assimilation has yet to be clearly understood. Here we identified a novel role of C. albicans mitochondrial complex I (CI) in regulating assimilation of alternative carbon sources such as mannitol. Our data demonstrate that CI dysfunction by deleting the subunit Nuo2 decreases the level of NAD+, downregulates the NAD+-dependent mannitol dehydrogenase activity, and consequently inhibits hyphal growth and biofilm formation in conditions when the carbon source is mannitol, but not fermentative sugars like glucose. Mannitol-dependent morphogenesis is controlled by a ROS-induced signaling pathway involving Hog1 activation and Brg1 repression. In vivo studies show that nuo2Δ/Δ mutant cells are severely compromised in gastrointestinal colonization and the defect can be rescued by a glucose-rich diet. Thus, our findings unravel a mechanism by which C. albicans regulates carbon flexibility and commensalism. Alternative carbon assimilation might represent a fitness advantage for commensal fungi in successful colonization of host niches. [ABSTRACT FROM AUTHOR]

This study evaluated the effects of antimicrobial photodynamic therapy (aPDT) mediated by Photodithazine® (PDZ) and LED light on the virulence factors of fluconazole-susceptible (CaS) and fluconazole-resistant (CaR) Candida albicans. Standardized suspensions of strains were prepared (107), and after 48 h of biofilm formation, these strains were incubated with PDZ (100 mg/L) for 20 min and exposed to LED light (660 nm, 37.5 J/cm2). Additional samples were treated with PDZ or light only, and the control consisted of biofilms that received no treatment. After aPDT, the cells were recovered and the virulence factors were evaluated. To analyze the capacity of adhesion, cells were recovered after aPDT and submitted to the adhesion process in the bottom of a 96-well plate. After this, metabolic activity tests (XTT assay) and cell viability (colony forming units per milliliter, CFU/mL) were applied. To evaluate the biofilm-forming ability after aPDT, the cells recovered were submitted to biofilm formation procedures, and the biofilm formed was evaluated by XTT, CFU/mL, and total biomass (crystal violet) tests. Lastly, the capacity for synthesizing protease and phospholipase enzymes after aPDT was evaluated by fluorimetric tests. Data were analyzed by two- or three-way ANOVA tests (p ≤ 0.05). It was verified that aPDT reduced the viability of both strains, fluconazole-susceptible and fluconazole-resistant C. albicans. It was also observed that the CaR strain had lower susceptibility to the aPDT when compared with the CaS strain. However, regarding the virulence factors evaluated, it was demonstrated that aPDT did not alter the adherence and biofilm formation ability and enzymatic production. [ABSTRACT FROM AUTHOR]

Objectives: Sphingosine 1-phosphate (S1P) is a bioactive lipid that binds to cell surface receptors (S1P). In this study, we examined the effect of S1P agonist, ONO-W061, on murine Candida albicans water-soluble fraction (CAWS)-induced vasculitis. Methods: Mice were administered ONO-W061, and the number of peripheral blood cells was counted. Vasculitis was induced by an intraperitoneal injection of CAWS. Expression of S1P receptors and CXCL1 was analyzed by quantitative RT-PCR. ONO-W061 was orally administered, and vasculitis was evaluated histologically. Number of neutrophils, macrophages and T cells in the vasculitis tissue was counted using flow cytometry. Production of chemokines from S1P-stimulated human umbilical vein endothelial cells (HUVECs) was measured by ELISA. Results: Number of peripheral blood lymphocytes was decreased by ONO-W061. Expression of CXCL1 and S1P was enhanced in CAWS-induced vasculitis tissue. Vasculitis score, CXCL1 and number of neutrophils in the vasculitis tissue were lower in ONO-W061-treated mice. Treatment of HUVECs with S1P upregulated the production of CXCL1 and IL-8 in vitro, and this was inhibited by ONO-W061. Conclusions: ONO-W061 significantly improved CAWS-induced vasculitis. This effect may be partly exerted through the inhibited production of chemokines by endothelial cells, which in turn could induce neutrophil recruitment into inflamed vessels. [ABSTRACT FROM AUTHOR]

One of the most critical but still poorly understood aspects of eukaryotic cell proliferation is the basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans. In all species, a critical cell size threshold coordinates cell growth with cell division and thereby establishes a homeostatic cell size. While a comprehensive survey of cell size genetic determinism has been performed in the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, very little is known in pathogenic fungi. As a number of critical Start regulators are haploinsufficient for cell size, we applied a quantitative analysis of the size phenome, using elutriation-barcode sequencing methodology, to 5639 barcoded heterozygous deletion strains of the opportunistic yeast Candida albicans. Our screen identified conserved known regulators and biological processes required to maintain size homeostasis in the opportunistic yeast C. albicans. We also identified novel C. albicans-specific size genes and provided a conceptual framework for future mechanistic studies. Interestingly, some of the size genes identified were required for fungal pathogenicity suggesting that cell size homeostasis may be elemental to C. albicans fitness or virulence inside the host. [ABSTRACT FROM AUTHOR]

Candida albicans is a major human fungal pathogen responsible for both systemic and mucosal infections in a wide variety of immunocompromised individuals. Because the ability of C. albicans to undergo a reversible morphological transition from yeast to filaments is important for virulence, significant research efforts have focused on mechanisms that control this transition. While transcriptional and post-translational mechanisms have been well-studied, considerably less is known about the role of post-transcriptional mechanisms. However, in recent years several discoveries have begun to shed light on this important, but understudied, area. Here, I will review a variety of post-transcriptional mechanisms that have recently been shown to control C. albicans morphology, virulence and/or virulence-related processes, including those involving alternative transcript localization, mRNA stability and translation. I will also discuss the role that these mechanisms play in other pathogens as well as the potential they may hold to serve as targets for new antifungal strategies. Ultimately, gaining a better understanding of C. albicans post-transcriptional mechanisms will significantly improve our knowledge of how morphogenesis and virulence are controlled in fungal pathogens and open new avenues for the development of novel and more effective antifungals. [ABSTRACT FROM AUTHOR]

Microorganisms rarely exist as single species in natural environments. The opportunistic fungal pathogen Candida albicans and lactic acid bacteria (LAB) are common members of the microbiota of several human niches such as the mouth, gut and vagina. Lactic acid bacteria are known to suppress filamentation, a key virulence feature of C. albicans, through the production of lactic acid and other metabolites. Here we report that C. albicans cells switch between two heritable cell types, white and opaque, to undergo filamentation to adapt to diversified environments. We show that acidic pH conditions caused by LAB and low temperatures support opaque cell filamentation, while neutral pH conditions and high temperatures promote white cell filamentation. The cAMP signalling pathway and the Rfg1 transcription factor play major roles in regulating the responses to these conditions. This cell type-specific response of C. albicans to different environmental conditions reflects its elaborate regulatory control of phenotypic plasticity. [ABSTRACT FROM AUTHOR]

To better understand the association between the ERG11 gene and drug resistance in Candida krusei, C. krusei strains were isolated from patients from January 2010 to May 2013. Susceptibility to 5-fluorocytosine (5-FC), amphotericin B (AMB), voriconazole (VRC), fluconazole (FLC), and itraconazole (ITR) were tested by broth microdilution method. Mutations were detected using PCR amplification and gene sequencing. Expression levels of ERG11 were measured by real-time PCR and compared by a 2-tailed Student's t test between ITR-susceptible strains and ITR-resistant strains. In total, 15 C. krusei strains were obtained, of which 20.00%, 53.33%, and 40.00% were resistant to 5-FC, FLC, and ITR, respectively, whereas all isolates were susceptible to AMB and VRC. Three synonymous codon substitutions were found in ERG11, including T939C, T642C, and A756T. However, T939C was found in both resistant and susceptible C. krusei strains. The expression level of ERG11 was significantly higher in resistant C. krusei strains (1.34 ± 0.08) than in susceptible C. krusei strains (0.94 ± 0.14) ( t = 3.74, p < 0.05). Our study demonstrates that point mutations (T642C and A756T) accompanied with the overexpression of ERG11 might be involved in the molecular mechanisms of drug resistance in C. krusei. [ABSTRACT FROM AUTHOR]

The capacity to transition between distinct morphological forms is a key virulence trait for diverse fungal pathogens. A poignant example of a leading opportunistic fungal pathogen of humans for which an environmentally responsive developmental program underpins virulence is Candida albicans. C. albicans mutants that are defective in the transition between yeast and filamentous forms typically have reduced virulence. Although many positive regulators of C. albicans filamentation have been defined, there are fewer negative regulators that have been implicated in repression of filamentation in the absence of inducing cues. To discover novel negative regulators of filamentation, we screened a collection of 1,248 C. albicans homozygous transposon insertion mutants to identify those that were filamentous in the absence of inducing cues. We identified the Rho1 GAP Lrg1, which represses filamentous growth by stimulating Rho1 GTPase activity and converting Rho1 to its inactive, GDP-bound form. Deletion of LRG1 or introduction of a RHO1 mutation that locks Rho1 in constitutively active, GTP-bound state, leads to filamentation in the absence of inducing cues. Deletion of the Rho1 downstream effector PKC1 results in defective filamentation in response to diverse host-relevant inducing cues, including serum. We further established that Pkc1 is not required to sense filament-inducing cues, but its kinase activity is critical for the initiation of filamentous growth. Our genetic analyses revealed that Pkc1 regulates filamentation independent of the canonical MAP kinase cascade. Further, although Ras1 activation is not impaired in a pkc1Δ/pkc1Δ mutant, adenylyl cyclase activity is reduced, consistent with a model in which Pkc1 functions in parallel with Ras1 in regulating Cyr1 activation. Thus, our findings delineate a signaling pathway comprised of Lrg1, Rho1 and Pkc1 with a core role in C. albicans morphogenesis, and illuminate functional relationships that govern activation of a central transducer of signals that control environmental response and virulence programs. [ABSTRACT FROM AUTHOR]

Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the activation domain of ‘free’ Tlo protein competes with DNA bound transcription factors for targets that regulate key aspects of C. albicans cell physiology. [ABSTRACT FROM AUTHOR]

Assembly of kinetochore complexes, involving greater than one hundred proteins, is essential for chromosome segregation and genome stability. Neocentromeres, or new centromeres, occur when kinetochores assemble de novo, at DNA loci not previously associated with kinetochore proteins, and they restore chromosome segregation to chromosomes lacking a functional centromere. Neocentromeres have been observed in a number of diseases and may play an evolutionary role in adaptation or speciation. However, the consequences of neocentromere formation on chromosome missegregation rates, gene expression, and three-dimensional (3D) nuclear structure are not well understood. Here, we used Candida albicans, an organism with small, epigenetically-inherited centromeres, as a model system to study the functions of twenty different neocentromere loci along a single chromosome, chromosome 5. Comparison of neocentromere properties relative to native centromere functions revealed that all twenty neocentromeres mediated chromosome segregation, albeit to different degrees. Some neocentromeres also caused reduced levels of transcription from genes found within the neocentromere region. Furthermore, like native centromeres, neocentromeres clustered in 3D with active/functional centromeres, indicating that formation of a new centromere mediates the reorganization of 3D nuclear architecture. This demonstrates that centromere clustering depends on epigenetically defined function and not on the primary DNA sequence, and that neocentromere function is independent of its distance from the native centromere position. Together, the results show that a neocentromere can form at many loci along a chromosome and can support the assembly of a functional kinetochore that exhibits native centromere functions including chromosome segregation accuracy and centromere clustering within the nucleus. [ABSTRACT FROM AUTHOR]

LYS21 and LYS22 genes from Candida albicans encoding isoforms of homocitrate synthase (HCS), an enzyme catalyzing the first committed step in the l -lysine biosynthetic pathway, were cloned and expressed as N-oligoHistagged fusion proteins in Escherichia coli . The purified gene products revealed HCS activity, i.e . catalyzed the condensation of α-ketoglutarate with acetyl-coenzyme A to yield homocitrate. The recombinant enzymes were purified to homogeneity and characterized for their physical properties and substrate specificities. As determined by size-exclusion chromatography (SEC) and native page electrophoresis, both isoenzymes adopt multiple quaternary structures, with the homotetrameric one being the most abundant. The K M (acetyl-CoA) = 0.8 ± 0.15 mM and K M (α-ketoglutarate) = 0.113 ± 0.02 mM for His 6 Ca Lys21p and K M (acetyl-CoA) = 0.48 ± 0.09 mM and K M (α-ketoglutarate) = 0.152 ± 0.03 mM values for His 6 Ca Lys22p were determined. Both enzyme versions were inhibited by l -Lys, i.e . the end product of the α-aminoadipate pathway but Lys22p was more sensitive than Lys21p, with K i (L-Lys) = 128 ± 8 μM for His 6 Ca Lys21p and K i (L-Lys) = 4.37 ± 0.68 μM for His 6 Ca Lys22p. The isoforms of C. albicans HCS exhibited differential sensitivity to several l -Lys analogues. Most notably, dl -α-difluoromethyllysine strongly inhibited His 6 Ca Lys22p (IC 50 32 ± 3 μM) but was not inhibitory at all towards His 6 Ca Lys21p. Differential sensitivity of recombinant C. albicans Δlys21/LYS22 , LYS21/Δlys22 and Δlys21/Δlys22 mutant strains to lysine analog, 2-aminoethyl- l -cysteine and biochemical properties of homocitrate synthase isoforms suggest different roles of two HCS isoenzymes in α-aminoadipate pathway. [ABSTRACT FROM AUTHOR]

The human fungal pathogen Candida albicans can reversibly switch between two cell types named "white" and "opaque," each of which is stable through many cell divisions. These two cell types differ in their ability to mate, their metabolic preferences and their interactions with the mammalian innate immune system. A highly interconnected network of eight transcriptional regulators has been shown to control switching between these two cell types. To identify additional regulators of the switch, we systematically and quantitatively measured white-opaque switching rates of 196 strains, each deleted for a specific transcriptional regulator. We identified 19 new regulators with at least a 10-fold effect on switching rates and an additional 14 new regulators with more subtle effects. To investigate how these regulators affect switching rates, we examined several criteria, including the binding of the eight known regulators of switching to the control region of each new regulatory gene, differential expression of the newly found genes between cell types, and the growth rate of each mutant strain. This study highlights the complexity of the transcriptional network that regulates the white-opaque switch and the extent to which switching is linked to a variety of metabolic processes, including respiration and carbon utilization. In addition to revealing specific insights, the information reported here provides a foundation to understand the highly complex coupling of white-opaque switching to cellular physiology. [ABSTRACT FROM AUTHOR]

Our aims of the research were to study the antimicrobial effect of dimethylaminododecyl methacrylate (DMADDM) modified denture base resin on multi-species biofilms and the biocompatibility of this modified dental material. Candida albicans (C. albicans), Streptococcus mutans (S. mutans), Streptococcus sanguinis (S. sanguinis), as well as Actinomyces naeslundii (A. naeslundii) were used for biofilm formation on denture base resin. Colony forming unit (CFU) counts, microbial viability staining, and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) array were used to evaluate the antimicrobial effect of DMADDM. C. albicans staining and Real-time PCR were used to analyze the morphology and expression of virulence genes of C. albicans in biofilm. Lactate dehydrogenase (LDH) array and Real-time PCR were conducted to examine the results after biofilm co-cultured with epithelial cell. Hematoxylin and eosin (HE) staining followed by histological evaluation were used to study the biocompatibility of this modified material. We found that DMADDM containing groups reduced both biomass and metabolic activity of the biofilm significantly. DMADDM can also inhibit the virulence of C. albicans by means of inhibiting the hyphal development and downregulation of two virulence related genes. DMADDM significantly reduced the cell damage caused by multi-species biofilm according to the LDH activity and reduced the expression of IL-18 gene of the cells simultaneously. The in vivo histological evaluation proved that the addition of DMADDM less than 6.6% in denture material did not increase the inflammatory response (p > 0.05). Therefore, we proposed that the novel denture base resin containing DMADDM may be considered as a new promising therapeutic system against problems caused by microbes on denture base such as denture stomatitis. [ABSTRACT FROM AUTHOR]

The article discusses the study conducted by E. Román and colleagues on MAPK-dependent cell wall regulation in Candida albicans. The study examines the role of the immune system in Cek1-mediated cell wall control and whether mice showed increased fungal susceptibility in a Cek1-dependent manner.

Loss of immune control over opportunistic infections can occur at different stages of HIV-1 (HIV) disease, among which mucosal candidiasis caused by the fungal pathogen Candida albicans (C. albicans) is one of the early and common manifestations in HIV-infected human subjects. The underlying immunological basis is not well defined. We have previously shown that compared to cytomegalovirus (CMV)-specific CD4 cells, C. albicans-specific CD4 T cells are highly permissive to HIV in vitro. Here, based on an antiretroviral treatment (ART) naïve HIV infection cohort (RV21), we investigated longitudinally the impact of HIV on C. albicans- and CMV-specific CD4 T-cell immunity in vivo. We found a sequential dysfunction and preferential depletion for C. albicans-specific CD4 T cell response during progressive HIV infection. Compared to Th1 (IFN-γ, MIP-1β) functional subsets, the Th17 functional subsets (IL-17, IL-22) of C. albicans-specific CD4 T cells were more permissive to HIV in vitro and impaired earlier in HIV-infected subjects. Infection history analysis showed that C. albicans-specific CD4 T cells were more susceptible to HIV in vivo, harboring modestly but significantly higher levels of HIV DNA, than CMV-specific CD4 T cells. Longitudinal analysis of HIV-infected individuals with ongoing CD4 depletion demonstrated that C. albicans-specific CD4 T-cell response was preferentially and progressively depleted. Taken together, these data suggest a potential mechanism for earlier loss of immune control over mucosal candidiasis in HIV-infected patients and provide new insights into pathogen-specific immune failure in AIDS pathogenesis. [ABSTRACT FROM AUTHOR]

Candida albicans Gca1p is a putative glucoamylase enzyme which contains 946 amino acids, 11 putative sites for N-glycosylation and 9 for O-glycosylation. Gca1p was identified in ß-mercaptoethanol extracts from isolated cell walls of strain C. albicans SC5314 and it is involved in carbohydrate metabolism. The significance and the role of this protein within the cell wall structure were studied in the corresponding mutants. The homozygous mutant showed that GCA1 was not an essential gene for cell viability. Subsequent phenotypic analysis performed in the mutants obtained did not show significant difference in the behavior of mutant when compared with the wild strain SC5314. Zymoliase, Calcofluor White, Congo red, SDS, caffeine or inorganic compounds did not affect the integrity of the cell wall. No differences were observed when hyphal formation assays were carried out. However, an enzyme assay in the presence of substrate p-nitrophenyl-α-D-glucopyranoside enabled us to detect a significant decrease in glycosidase activity in the mutants compared with the parental strain, revealing the function of Gca1. [ABSTRACT FROM AUTHOR]

Among the several mechanisms that contribute to MDR (multidrug resistance), the overexpression of drug-efflux pumps belonging to the ABC (ATP-binding cassette) superfamily is the most frequent cause of resistance to antifungal agents. The multidrug transporter proteins Cdr1p and Cdr2p of the ABCG subfamily are major players in the development of MDR in Candida albicans.Because several genes coding forABCproteins exist in the genome of C. albicans, but only Cdr1p and Cdr2p have established roles in MDR, it is implicit that the other members of the ABC family also have alternative physiological roles. The present study focuses on an ABC transporter of C. albicans, Mlt1p, which is localized in the vacuolar membrane and specifically transports PC (phosphatidylcholine) into the vacuolar lumen. Transcriptional profiling of the mlt1Δ/Δ mutant revealed a down-regulation of the genes involved in endocytosis, oxidoreductase activity, virulence and hyphal development. Highthroughput MS-based lipidome analysis revealed that the Mlt1p levels affect lipid homoeostasis and thus lead to a plethora of physiological perturbations. These include a delay in endocytosis, inefficient sequestering of reactive oxygen species (ROS), defects in hyphal development and attenuated virulence. The present study is an emerging example where new and unconventional roles of an ABC transporter are being identified. [ABSTRACT FROM AUTHOR]

In Candida albicans the transcription factor Efg1, which is differentially expressed in the white phase of the white-opaque transition, is essential for expression of the white phenotype. It is one of six transcription factors included in a proposed interactive transcription network regulating white-opaque switching and maintenance of the alternative phenotypes. Ten sites were identified in the EFG1 promoter that differentially bind one or more of the network transcription factors in the white and/or opaque phase. To explore the functionality of these binding sites in the differential expression of EFG1, we generated targeted deletions of each of the 10 binding sites, combinatorial deletions, and regional deletions using a Renilla reniformis luciferase reporter system. Individually targeted deletion of only four of the 10 sites had minor effects consistent with differential expression of EFG1, and only in the opaque phase. Alternative explanations are considered. [ABSTRACT FROM AUTHOR]

Objective The present study aimed to evaluate the interactions between the species Candida albicans , Candida krusei and Candida glabrata in monotypic and mixed biofilm models formed in vitro as well as the relative expression of the ALS1 , ALS3 , HWP1 , BCR1 , EFG1 , TEC1 , SAP5 , PLB2 and LIP9 genes. Material and methods Mixed ( C. albicans / C. krusei and C. albicans/C. glabrata ) and monotypic biofilms were cultured for 0, 12 and 24 h. Gene expression was analyzed in the same biofilm model in which the number of CFU/mL was counted. Results The C. albicans CFU/mL values were lower at the 12 and 24 h time points in the mixed biofilms compared with the monotypic biofilms, and decreases of 56.23% and 64.4% in C. albicans were observed when this species was associated with C. glabrata and C. krusei , respectively. In the presence of C. krusei , the expression of the ALS3 , HWP1 , BCR1 , EFG1 and TEC1 genes of C. albicans was completely inhibited, indicating both transcriptome and the phenotypic antagonism between these two species, but genes related to the secretion of enzymes were stimulated. In the presence of C. glabrata , C. albicans showed a similar gene expression profile to that obtained in association with C. krusei , though it was altered to a lesser degree. Conclusion We conclude that C. krusei and C. glabrata may alter or inhibit the mechanisms involved in the in vitro adherence and formation of C. albicans biofilms, influencing the pathogenicity of this species and suggesting a competitive interaction with C. krusei and C. glabrata in biofilm formation. [ABSTRACT FROM AUTHOR]

Background: The fungal pathogen Candida albicans colonizes the gastrointestinal (GI) tract of mammalian hosts as a benign commensal. However, in an immunocompromised host, the fungus is capable of causing life-threatening infection. We previously showed that the major transcription factor Efg1p is differentially expressed in GI-colonizing C. albicans cells dependent on the host immune status. To understand the mechanisms that underlie this hostdependent differential gene expression, we utilized mathematical modeling to dissect host-pathogen interactions. Specifically, we used principles of evolutionary game theory to study the mechanism that governs dynamics of EFG1 expression during C. albicans colonization. Results: Mathematical modeling predicted that down-regulation of EFG1 expression within individual fungal cells occurred at different average rates in different hosts. Rather than using relatively transient signaling pathways to adapt to a new environment, we demonstrate that C. albicans overcomes the host defense strategy by modulating the activity of diverse fungal histone modifying enzymes that control EFG1 expression. Conclusion: Based on our modeling and experimental results we conclude that C. albicans cells sense the local environment of the GI tract and respond to differences by altering EFG1 expression to establish optimal survival strategies. We show that the overall process is governed via modulation of epigenetic regulators of chromatin structure. [ABSTRACT FROM AUTHOR]

Chitin is an important structural constituent of fungal cell walls composed of N-acetylglucosamine ( GlcNAc) monosaccharides, but catabolism of GlcNAc has not been studied in filamentous fungi so far. In the yeast C andida albicans, the genes encoding the three enzymes responsible for stepwise conversion of GlcNAc to fructose-6-phosphate are clustered. In this work, we analysed GlcNAc catabolism in ascomycete filamentous fungi and found that the respective genes are also clustered in these fungi. In contrast to C . albicans, the cluster often contains a gene for an Ndt80-like transcription factor, which we named RON1 ( regulator of N-acetylglucosamine catabolism 1). Further, a gene for a glycoside hydrolase 3 protein related to bacterial N-acetylglucosaminidases can be found in the GlcNAc gene cluster in filamentous fungi. Functional analysis in T richoderma reesei showed that the transcription factor RON1 is a key activator of the GlcNAc gene cluster and essential for GlcNAc catabolism. Furthermore, we present an evolutionary analysis of Ndt80-like proteins in Ascomycota. All GlcNAc cluster genes, as well as the GlcNAc transporter gene ngt1, and an additional transcriptional regulator gene, csp2, encoding the homolog of N eurospora crassa CSP2/ GRHL, were functionally characterised by gene expression analysis and phenotypic characterisation of knockout strains in T . reesei. [ABSTRACT FROM AUTHOR]

This study was intended to isolate and identification of Candida albicans which causing vaginitis in women. Atotal 204 samples of vaginal tract swabs were collected from patients women from April to November, 2015 in Dyala/Iraq. Standard microbiological methods were used to isolate and identify candida from vaginal swabs obtained from study object, sabouroud dextrose agar used for isolation of candida, CHROMagar of candida used to identification of C. albicans, germ tube test done. The final identification done by using VITEK Yeast Biochemical Card. From 204 infected women 75 have vaginal candidiasis about (36.76%), C. albicans was causative agent of 54 isolates about (72%). The genetic diversity of recent clinical isolates of Candida albicans was studied basis on amplified DNA bands fragment that spans the side of the transposable intron in the 25s rDNA by using CA-INT-L and CA-INT-R primers. Our analysis of 54 C. albicans isolates showed that genotype-A with (450bp)band presented in 27 isolates about (50%) was a predominant C. albicans genotype, genotype-B with (840bp)band was found in16 isolates with (29.62) ratio, and finally genotype-C with both bands (450-840bp), was found in 11 isolates about (20.37%). [ABSTRACT FROM AUTHOR]

The human fungal pathogen Candida albicans can switch between two cell types, "white" and "opaque," each of which is heritable through many cell divisions. Switching between these two cell types is regulated by six transcriptional regulators that form a highly interconnected circuit with multiple feedback loops. Here, we identify a seventh regulator of white-opaque switching, which we have named Wor4. We show that ectopic expression of Wor4 is sufficient to drive switching from the white to the opaque cell type, and that deletion of Wor4 blocks switching from the white to the opaque cell type. A combination of ectopic expression and deletion experiments indicates that Wor4 is positioned upstream of Wor1, and that it is formally an activator of the opaque cell type. The combination of ectopic expression and deletion phenotypes for Wor4 is unique; none of the other six white-opaque regulators show this pattern. We determined the pattern of Wor4 binding across the genome by ChIP-seq and found it is highly correlated with that of Wor1 and Wor2, indicating that Wor4 is tightly integrated into the existing white-opaque regulatory circuit. We previously proposed that white-to-opaque switching relies on the activation of a complex circuit of feedback loops that remains excited through many cell divisions. The identification of a new, central regulator of white-opaque switching supports this idea by indicating that the white-opaque switching mechanism is considerably more complex than those controlling conventional, nonheritable patterns of gene expression. [ABSTRACT FROM AUTHOR]

Dectin-1 (Clec7a) is a paradigmatic C-type lectin receptor that binds Syk through a hemITAM motif and couples sensing of pathogens such as fungi to induction of innate responses. Dectin-1 engagement triggers a plethora of activating events, but little is known about the modulation of such pathways. Trying to define a more precise picture of early Dectin-1 signaling, we explored the interactome of the intracellular tail of the receptor in mouse dendritic cells. We found unexpected binding of SHIP-1 phosphatase to the phosphorylated hemITAM. SHIP-1 colocalized with Dectin-1 during phagocytosis of zymosan in a hemITAM-dependent fashion. Moreover, endogenous SHIP-1 relocated to live or heat-killed Candida albicans-containing phagosomes in a Dectin-1-dependent manner in GM-CSF-derived bone marrow cells (GM-BM). However, SHIP-1 absence in GM-BM did not affect activation of MAPK or production of cytokines and readouts dependent on NF-κB and NFAT. Notably, ROS production was enhanced in SHIP-1-deficient GM-BM treated with heat-killed C. albicans, live C. albicans, or the specific Dectin-1 agonists curdlan or whole glucan particles. This increased oxidative burst was dependent on Dectin-1, Syk, PI3K, phosphoinositide-dependent protein kinase 1, and NADPH oxidase. GM-BM from CD11c∆SHIP-1 mice also showed increased killing activity against live C. albicans that was dependent on Dectin-1, Syk, and NADPH oxidase. These results illustrate the complexity of myeloid C-type lectin receptor signaling, and how an activating hemITAM can also couple to intracellular inositol phosphatases to modulate selected functional responses and tightly regulate processes such as ROS production that could be deleterious to the host. [ABSTRACT FROM AUTHOR]

Background: Yeasts show remarkable variation in the organization of their mitochondrial genomes, yet there is little experimental data on organellar gene expression outside few model species. Candida albicans is interesting as a human pathogen, and as a representative of a clade that is distant from the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Unlike them, it encodes seven Complex I subunits in its mtDNA. No experimental data regarding organellar expression were available prior to this study. Methods: We used high-throughput RNA sequencing and traditional RNA biology techniques to study the mitochondrial transcriptome of C. albicans strains BWP17 and SN148. Results: The 14 protein-coding genes, two ribosomal RNA genes, and 24 tRNA genes are expressed as eight primary polycistronic transcription units. We also found transcriptional activity in the noncoding regions, and antisense transcripts that could be a part of a regulatory mechanism. The promoter sequence is a variant of the nonanucleotide identified in other yeast mtDNAs, but some of the active promoters show significant departures from the consensus. The primary transcripts are processed by a tRNA punctuation mechanism into the monocistronic and bicistronic mature RNAs. The steady state levels of various mature transcripts exhibit large differences that are a result of posttranscriptional regulation. Transcriptome analysis allowed to precisely annotate the positions of introns in the RNL (2), COB (2) and COX1 (4) genes, as well as to refine the annotation of tRNAs and rRNAs. Comparative study of the mitochondrial genome organization in various Candida species indicates that they undergo shuffling in blocks usually containing 2-3 genes, and that their arrangement in primary transcripts is not conserved. tRNA genes with their associated promoters, as well as GC-rich sequence elements play an important role in these evolutionary events. Conclusions: The main evolutionary force shaping the mitochondrial genomes of yeasts is the frequent recombination, constantly breaking apart and joining genes into novel primary transcription units. The mitochondrial transcription units are constantly rearranged in evolution shaping the features of gene expression, such as the presence of secondary promoter sites that are inactive, or act as "booster" promoters, simplified transcriptional regulation and reliance on posttranscriptional mechanisms. [ABSTRACT FROM AUTHOR]

Human fungal pathogens like Candida albicans respond to host immune surveillance by rapidly adapting their transcriptional programs. Chromatin assembly factors are involved in the regulation of stress genes by modulating the histone density at these loci. Here, we report a novel role for the chromatin assembly-associated histone acetyltransferase complex NuB4 in regulating oxidative stress resistance, antifungal drug tolerance and virulence in C. albicans. Strikingly, depletion of the NuB4 catalytic subunit, the histone acetyltransferase Hat1, markedly increases resistance to oxidative stress and tolerance to azole antifungals. Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment. Furthermore, hat1Δ/Δ cells, despite showing growth defects in vitro, display reduced susceptibility to reactive oxygen-mediated killing by innate immune cells. Thus, clearance from infected mice is delayed although cells lacking Hat1 are severely compromised in killing the host. Interestingly, increased oxidative stress resistance and azole tolerance are phenocopied by the loss of histone chaperone complexes CAF-1 and HIR, respectively, suggesting a central role for NuB4 in the delivery of histones destined for chromatin assembly via distinct pathways. Remarkably, the oxidative stress phenotype of hat1Δ/Δ cells is a species-specific trait only found in C. albicans and members of the CTG clade. The reduced azole susceptibility appears to be conserved in a wider range of fungi. Thus, our work demonstrates how highly conserved chromatin assembly pathways can acquire new functions in pathogenic fungi during coevolution with the host. [ABSTRACT FROM AUTHOR]

C andida albicans is the most common human fungal pathogen, yet is a normal commensal resident of the human gut. CO2 levels in the gut are much higher than in air, and it is known that elevated CO2 concentration promotes C . albicans cells to undergo a phenotypic switch from white to opaque phase. Wor1, the master regulator of opaque cell formation, is required for both the white to opaque transition and opaque maintenance. To elucidate the regulatory mechanism of Wor1, we set out to identify Wor1-interacting proteins using a yeast two-hybrid screen. A SUMO E3 ligase named Wos1 ( W or1 SUMO-ligase 1) was identified to interact with Wor1 and regulate Wor1 SUMOylation. WOS 1 expression is upregulated in response to high CO2, and the induction by CO2 is dependent on the transcription factor Flo8. Under high CO2 conditions, Wos1 is required for the white to opaque switch and acts downstream of Flo8. At atmospheric CO2 levels, overexpression of Wos1 enhances Wor1 SUMOylation and promotes the white to opaque switch. Wor1 is found to be SUMOylated at lysine 385, and loss of this mark by point mutation leads to a defect in CO2-mediated opaque cell induction. Together, our genetic and biological data show that Wos1-mediated Wor1 SUMOylation contributes to the regulation of CO2-induced white to opaque switching as well as heritable maintenance of the opaque cell type. [ABSTRACT FROM AUTHOR]

Background and objectives: More Candida albicans strains are reported resistant to fluconazole in patients with AIDS, cancer and organ recipients. Fluconazole resistance can be attributed to changes in pathways of sterol biosynthesis, mutation in or overexpression of ERG11 and the expression of CDR1, CDR2, and MDR1. This study aimed to compare the expression of CDR1, CDR2, and MDR1 in C. albicans resistant and susceptible to fluconazole. Methods: MIC testing for fluconazole was performed on C. albicans isolates isolated from patients with oral and vaginal candidiasis to determine resistant and susceptible strains. Then real time PCR was performed on the resistant and susceptible isolates and the expression of CDR1, CDR2, and MDR1 was compared in C. albicans. Results: Of 46 Candida albicans isolates, 20 susceptible isolates, 12 semi-susceptible isolates and 14 resistant isolates were identified by MIC. After real time PCR was performed, Candida albicans isolates susceptible to fluconazole showed moderate expression of CDR1, CDR2, and MDR1 genes, while resistant isolates showed slight or no expression. Conclusion: Increased expression of CDR1, CDR2, and MDR1 had less and insignificant role in resistance to fluconazole. [ABSTRACT FROM AUTHOR]

The opportunistic human fungal pathogen Candida albican s is a natural diploid that does not have a complete sexual cycle. The ability to switch between diverse cellular forms is important to its virulence. Here, we describe the characterization of the C. albican s DBF4 gene, a Saccharomyces cerevisiae homolog that encodes a regulatory subunit of Cdc7 kinase that is known to initiate DNA replication. We made a C. albicans strain, with one DBF4 allele deleted by the mini-Ura-blaster and the other controlled by a repressible promoter. We also found a third CaDBF4 copy that was later verified to be inducibly duplicated by targeted recombination with the min-Ura-blaster. Surprisingly, the strain deleted with the third CaDBF4 copy exhibited hyphal growth under repressed conditions. We conclude that the CaDBF4 gene is prone to being duplicated by the mini-Ura-blaster and that it suppresses hyphal growth in C. albicans . [ABSTRACT FROM AUTHOR]

Candida albicans is a common commensal in the human gut but in predisposed patients it can become an important human fungal pathogen. As a commensal, C. albicans adapts to low-oxygen conditions and represses its hyphal development by the transcription factor Efg1, which under normoxia activates filamentation. The repressive hypoxic but not the normoxic function of Efg1 required its unmodified N-terminus, was prevented by phosphomimetic residues at normoxic phosphorylation sites T179 and T206 and occurred only at temperatures ≤35°C. Genome-wide binding sites for native Efg1 identified 300 hypoxia-specific target genes, which overlapped partially with hypoxic binding sites for Ace2, a known positive regulator of hypoxic filamentation. Transcriptional analyses revealed that EFG1, ACE2 and their identified targets BCR1 and BRG1 encode an interconnected regulatory hub, in which Efg1/Bcr1 act as negative and Ace2/Brg1 act as positive regulators of gene expression under hypoxia. In this circuit, the hypoxic function of Ace2 was stimulated by elevated CO2 levels. The hyperfilamentous phenotype of efg1 and bcr1 mutants depended on Ace2/Brg1 regulators and required increased expression of genes encoding Cek1 MAP kinase and its downstream target Cph1. The intricate temperature-dependent regulatory mechanisms under hypoxia suggest that C. albicans restricts hyphal morphogenesis in oxygen-poor body niches, possibly to persist as a commensal in the human host. [ABSTRACT FROM AUTHOR]

The human fungal pathogen C andida albicans undergoes white-opaque phenotypic switching, which enhances its adaptation to host niches. Switching is controlled by a transcriptional regulatory network of interlocking feedback loops acting on the transcription of WOR 1, the master regulator of white-opaque switching, but regulation of the network on the translational level is not yet explored. Here, we show that the long 5′ untranslated region of WOR 1 regulates the white-opaque phenotype. Deletion of the WOR 1 5′ UTR promotes white-to-opaque switching and stabilizes the opaque state. The WOR 1 5′ UTR reduces translational efficiency and the association of the transcript with polysomes. Reduced polysome association was observed for additional key regulators of cell fate and morphology with long 5′ UTR as well. Overall, we find a novel regulatory step of white-opaque switching at the translational level. This translational regulation is implicated for many key regulators of cell fate and morphology in C . albicans. [ABSTRACT FROM AUTHOR]

The production of hydrolytic enzymes is considered a key virulence determinant of Candida albicans. Aminopeptidase 2 (Ape2) facilitates the penetration of C. albicans into the host tissue, by providing free amino acids to support fungal growth and proliferation. The objective of this study was to estimate the APE2 expression profile in C. albicans cells during invasion of the human epithelium. Sixty-one clinical fungal isolates and five reference strains were included in this study. The wild-type APE2 sequence was analyzed by polymerase chain reaction (PCR) amplification using genomic DNA from pathogenic isolates. Amplicons were verified in 1 % agarose gel and visualized by illumination with ultraviolet (UV) light. The APE2 expression levels were analyzed with reverse transcription quantitative real-time PCR (RT-qPCR) and APE2 quantification was normalized against the reference gene in C. albicans cells grown in YEPD and during Caco-2 invasion. The APE2-specific PCR product band was found in all C. albicans and C. dubliniensis strains, but not in other common pathogenic fungi. APE2 transcript abundance was elevated in the clinical isolates growing on the Caco-2 cell line in comparison to their counterparts grown in YEPD. Our data indicate a potential role for Ape2 in the invasion of epithelial cells. APE2 expression is also strain-specific, and it is not related to isolation site or disease entity. [ABSTRACT FROM AUTHOR]

Mouth is an important source of infections and oral infections such as Candida infections increase the risk of mortality. Our purpose was to investigate differences in proteolytic activity of non- albicans Candida albicans (non- albicans Candida) between clinical isolates and laboratory samples. The second aim was to assess the concentration of pro- and anti-inflammatory cytokine levels IL-1β, IL-10, and TNF-α in saliva of patients with the non- albicans Candida and Candida-negative saliva samples. Clinical yeast samples from our laboratory were used for analyses. Candida strains were grown in YPG at 37 °C for 24 h in water bath with shaking. The activity of Candida proteinases of cell and cell-free fractions were analyzed by MDPF-gelatin zymography. The levels of IL-1β, IL-10, and TNF-α were measured from saliva with ELISA. The study showed differences in the proteolytic activity among the non- albicans Candida strain s. C. tropicalis had higher proteolytic activity when compared to the other strains. Significant difference was found in salivary IL-1β levels between the non- albicans Candida and control strains ( P < 0.002). The present findings showed differences in proteolytic activity among the non- albicans Candida strains. The increased IL-1β concentration may be one of the host response components associated with non- albicans Candida infection. [ABSTRACT FROM AUTHOR]

Candida albicans remains the most common fungal pathogen. This species is closely related to 2 phenotypically similar cryptic species, Candida dubliniensis and Candida africana . This study aims to compare the antifungal activities of echinocandins against 7 C. albicans , 5 C. dubliniensis , and 2 C. africana strains by time–kill methodology. MIC values were similar for the 3 species; however, differences in killing activity were observed among species, isolates, and echinocandins. Echinocandins produced weak killing activity against the 3 species. In all drugs, the fungicidal endpoint (99.9% mortality) was reached at ≤31 h with ≥0.5 μg/mL for anidulafungin in 4 C. albicans and 1 C. dubliniensis , for caspofungin in 1 C. albicans and 2 C. dubliniensis , and for micafungin in 4 C. albicans and 1 C. dubliniensis . None of echinocandins showed lethality against C. africana . Identification of these new cryptic species and time–kill studies would be recommendable when echinocandin treatment fails. [ABSTRACT FROM AUTHOR]

The oestrogenised vagina is colonised by Candida species in at least 20% of women; in late pregnancy and in immunosuppressed patients, this increases to at least 30%. In most cases, Candida albicans is involved. Host factors, particularly local defence mechanisms, gene polymorphisms, allergies, serum glucose levels, antibiotics, psycho-social stress and oestrogens influence the risk of candidal vulvovaginitis. Non- albicans species, particularly Candida glabrata, and in rare cases also Saccharomyces cerevisiae, cause less than 10% of all cases of vulvovaginitis with some regional variation; these are generally associated with milder signs and symptoms than normally seen with a C. albicans-associated vaginitis. Typical symptoms include premenstrual itching, burning, redness and odourless discharge. Although itching and redness of the introitus and vagina are typical symptoms, only 35-40% of women reporting genital itching in fact suffer from vulvovaginal candidosis. Medical history, clinical examination and microscopic examination of vaginal content using 400× optical magnification, or preferably phase contrast microscopy, are essential for diagnosis. In clinically and microscopically unclear cases and in chronically recurring cases, a fungal culture for pathogen determination should be performed. In the event of non- C. albicans species, the minimum inhibitory concentration ( MIC) should also be determined. Chronic mucocutaneous candidosis, a rarer disorder which can occur in both sexes, has other causes and requires different diagnostic and treatment measures. Treatment with all antimycotic agents on the market (polyenes such as nystatin; imidazoles such as clotrimazole; and many others including ciclopirox olamine) is easy to administer in acute cases and is successful in more than 80% of cases. All vaginal preparations of polyenes, imidazoles and ciclopirox olamine and oral triazoles (fluconazole, itraconazole) are equally effective (Table [Antimycoti]); however, oral triazoles should not be administered during pregnancy according to the manufacturers. C. glabrata is not sufficiently sensitive to the usual dosages of antimycotic agents approved for gynaecological use. In other countries, vaginal suppositories of boric acid (600 mg, 1-2 times daily for 14 days) or flucytosine are recommended. Boric acid treatment is not allowed in Germany and flucytosine is not available. Eight hundred-milligram oral fluconazole per day for 2-3 weeks is therefore recommended in Germany. Due to the clinical persistence of C. glabrata despite treatment with high-dose fluconazole, oral posaconazole and, more recently, echinocandins such as micafungin are under discussion; echinocandins are very expensive, are not approved for this indication and are not supported by clinical evidence of their efficacy. In cases of vulvovaginal candidosis, resistance to C. albicans does not play a significant role in the use of polyenes or azoles. Candida krusei is resistant to the triazoles, fluconazole and itraconazole. For this reason, local imidazole, ciclopirox olamine or nystatin should be used. There are no studies to support this recommendation, however. Side effects, toxicity, embryotoxicity and allergies are not clinically significant. Vaginal treatment with clotrimazole in the first trimester of a pregnancy reduces the rate of premature births. Although it is not necessary to treat a vaginal colonisation of Candida in healthy women, vaginal administration of antimycotics is often recommended in the third trimester of pregnancy in Germany to reduce the rate of oral thrush and napkin dermatitis in healthy full-term newborns. Chronic recurrent vulvovaginal candidosis continues to be treated in intervals using suppressive therapy as long as immunological treatments are not available. The relapse rate associated with weekly or monthly oral fluconazole treatment over 6 months is approximately 50% after the conclusion of suppressive therapy according to current studies. Good results have been achieved with a fluconazole regimen using an initial 200 mg fluconazole per day on 3 days in the first week and a dosage-reduced maintenance therapy with 200 mg once a month for 1 year when the patient is free of symptoms and fungal infection (Table [Individual]). Future studies should include Candida autovaccination, antibodies to Candida virulence factors and other immunological experiments. Probiotics with appropriate lactobacillus strains should also be examined in future studies on the basis of encouraging initial results. Because of the high rate of false indications, OTC treatment (self-treatment by the patient) should be discouraged. [ABSTRACT FROM AUTHOR]