Antifungal activity of hypocrellin compounds and their synergistic effects with antimicrobial agents against Candida albicans

Candida albicans is a common human fungal pathogen. The previous study revealed that quinone compounds showed antimicrobial activity against C. albicans by inhibiting cell growth. However, it was unclear whether quinones have other antifungal effects against C. albicans in addition to fungicidal effects. In this study, we assessed the inhibitory activity of a total of 25 quinone compounds against C. albicans morphological transition, which is essential for the pathogenicity of C. albicans. Several quinones exhibited strong inhibition of mycelium formation by C. albicans SC5314. Three leading compounds, namely hypocrellins A, B and C, also exhibited marked attenuation of C. albicans SC5314 virulence in both human cell lines and mouse infection models. These three compounds significantly suppressed the proliferation of C. albicans SC5314 cells in a mouse mucosal infection model. Intriguingly, hypocrellins not only attenuated the cytotoxicity of a nystatin-resistant C. albicans strain but also showed excellent synergistic effects with antifungal agents against both wild-type C. albicans SC5314 and the drug-resistant mutant strains. In addition, hypocrellins A, B and C interfered with the biological functions and virulence of various clinical Candida species, suggesting the promising potential of these compounds for development as new therapeutic agents against infections caused by Candida pathogens.     

A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.     

Up-Regulation of Antimicrobial Peptides Gallerimycin and Galiomicin in <Emphasis Type="Italic">Galleria mellonella</Emphasis> Infected with <Emphasis Type="Italic">Candida</Emphasis> Yeasts Displaying Different Virulence Traits

Galleria mellonella has been described as a cheap and an easy-to-reproduce model for the study of fungal infections. We hypothesized that yeasts with higher virulence potential decrease survival and significantly trigger an immune response in G. mellonella through the regulation of innate immunity-related genes encoding antimicrobial peptides (AMPs) such as gallerimycin and galiomicin. Candida albicans SC5314 and Candida dubliniensis CBS 7987, selected because of their different virulence potential, were used for a killing assay followed by the determination of gene expression using qPCR. In vivo results confirmed a significantly (p?=?0.0321) lower pathogenicity for C. dubliniensis than for C. albicans. Accordingly, the induction of C. dubliniensis AMPs was lower at all the selected time points post-infection (1 h, 24 h, 48 h). Moreover, we observed an extremely high regulation of the galiomicin gene compared to the gallerimycin one, suggesting a different role of the tested AMPs in protecting G. mellonella from candidiasis.     

Niche-Specific Requirement for Hyphal Wall protein 1 in Virulence of Candida albicans

Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa.     

Effect of Tetrandrine against Candida albicans Biofilms

Candida albicans is the most common human fungal pathogen and has a high propensity to develop biofilms that are resistant to traditional antifungal agents. In this study, we investigated the effect of tetrandrine (TET) on growth, biofilm formation and yeast-to-hypha transition of C. albicans. We characterized the inhibitory effect of TET on hyphal growth and addressed its possible mechanism of action. Treatment of TET at a low concentration without affecting fungal growth inhibited hyphal growth in both liquid and solid Spider media. Real-time RT-PCR revealed that TET down-regulated the expression of hypha-specific genes ECE1, ALS3 and HWP1, and abrogated the induction of EFG1 and RAS1, regulators of hyphal growth. Addition of cAMP restored the normal phenotype of the SC5314 strain. These results indicate that TET may inhibit hyphal growth through the Ras1p-cAMP-PKA pathway. In vivo, at a range of concentrations from 4 mg/L to 32 mg/L, TET prolonged the survival of C. albicans-infected Caenorhabditis elegans significantly. This study provides useful information for the development of new strategies to reduce the incidence of C. albicans biofilm-associated infections.     

Anti-virulence activity of novel (1-heteroaryloxy-2-hydroxypropyl)- phenylpiperazine derivatives against both wild-type and clinical drug-resistant Candida albicans strains

Candida albicans is an important human fungal pathogen. Our previous study disclosed that aryloxy-phenylpiperazine skeleton was a promising molecule to suppress C. albicans virulence by inhibiting hypha formation and biofilm formation. In order to deeply understand the efficacy and mechanism of action of phenylpiperazine compounds, and obtain new derivatives with excellent activity against C. albicans, hence, we synthesized three series of (1-heteroaryloxy-2-hydroxypropyl)-phenylpiperazines and evaluated their inhibitory activity against C. albicans both in vitro and in vivo in this study. Compared with previously reported aryloxy-phenylpiperazines, part of these heteroaryloxy derivatives improved their activities by strongly suppressing hypha formation and biofilm formation in C. albicans SC5314. Especially, (9H-carbazol-4-yl)oxy derivatives 25 , 26 , 27 and 28 exhibited strong activity in reducing C. albicans virulence in both human cell lines in vitro and mouse infection models in vivo. The compound 27 attenuated the virulence of various clinical C. albicans strains, including clinical drug-resistant C. albicans strains. Moreover, additive effects of the compound 27 with antifungal drugs against drug-resistant C. albicans strains were also discussed. Furthermore, the compound 27 significantly improved the composition and richness of the faecal microbiota in mice infected by C. albicans. These findings indicate that these piperazine compounds have great potential to be developed as new therapeutic drugs against C. albicans infection.     

Sulfone derivatives reduce growth,adhesion and aspartic protease SAP2 gene expression

Fungal virulence factors represent a strategy for the design of new compounds with effective activities against Candida spp. Dichloromethyl-4-chloro-3-nitrophenylsulfone (named Compound 1) and chlorodibromomethyl-4-hydrazino-3-nitrophenylsulfone (Compound 2) versus Candida albicans virulence factors (SAP2 expression and adhesion to Caco-2 cell line) were investigated. Candida albicans SC5314 and its mutants: Δsap9, Δsap10, Δsap9/10 were used. MICs of the Compounds (concentrated at 0.0313–16 µg/ml) were determined using M27-A3. Percentage of cell inhibition was assessed spectrophotometrically (OD₄₀₅) after 48 h at 35 °C. The SAP2 expression was analyzed with the use of RT-PCR; relative quantification was normalized against ACT1 in cells grown in YEPD and on Caco-2. Adherence assay of C. albicans to Caco-2 was performed in a 24-well-plate. Compound 1 showed higher activity (% = 100 at 4 µg/ml) than Compound 2 (MIC₉₀ = 16 µg/ml). Dichloromethyl at the para position of the phenyl ring exerted anti-Candidal potential. Under Compound 1, SAP2 was down-regulated in all the strains (P ≤ 0.05). Conversely, SAP2 was over-expressed in Δsap9-10 (untreated cells) compared with the wild-type. The Compounds significantly affected adherence to epithelium (P ≤ 0.05). The tested sulfones interfered with the adhesion of C. albicans cells to the epithelial tissues without affecting their viability after 90-min of incubation. The Compounds’ mode of action was attributed to the reduced adhesiveness and the lower SAP2 expression. Saps9-10 play a role in C. albicans adhesion and they can be involved in the sulfone resistance mechanisms.     

Proteomics Analysis of Candida albicans dnm1 Haploid Mutant Unraveled the Association between Mitochondrial Fission and Antifungal Susceptibility

Candida albicans is a major fungal pathogen, accounting for approximately 15% of healthcare infections with associated mortality as high as 40% in the case of systemic candidiasis. Antifungal agents for C. albicans infections are limited, and rising resistance is an inevitable problem. Therefore, understanding the mechanism behind antifungal responses is among the top research focuses in combating Candida infections. Herein, the recently developed C. albicans haploid model is employed to examine the association between mitochondrial fission, regulated by Dnm1, and the pathogen's response to antifungals. Proteomic analysis of dnm1Δ and its wild‐type haploid parent, GZY803, reveal changes in proteins associated with mitochondrial structures and functions, cell wall, and plasma membrane. Antifungal susceptibility testing revealed that dnm1Δ is more susceptible to SM21, a novel antifungal, than GZY803. Analyses of reactive oxygen species release, antioxidant response, lipid peroxidation, and membrane damages uncover an association between dnm1Δ and the susceptibility to SM21. Dynasore‐induced mitochondrial inhibition in SC5314 diploids corroborate the findings. Interestingly, Dynasore‐primed SC5314 cultures exhibit increased susceptibility to all antifungals tested. These data suggest an important contribution of mitochondrial fission in antifungal susceptibility of C. albicans. Hence, mitochondrial fission can be a potential target for combined therapy in anti‐C. albicans treatment.     

Soybean Toxin (SBTX) Impairs Fungal Growth by Interfering with Molecular Transport,Carbohydrate/Amino Acid Metabolism and Drug/Stress Responses

Soybean toxin (SBTX) is an antifungal protein from soybeans with broad inhibitory activity against the growth and filamentation of many fungi, including human and plant pathogenic species such as Candida albicans, Candida parapsilosis, Aspergillus niger, Penicillium herquei, Cercospora sojina and Cercospora kikuchii. Understanding the mechanism by which SBTX acts on fungi and yeasts may contribute to the design of novel antifungal drugs and/or the development of transgenic plants resistant to pathogens. To this end, the polymorphic yeast C. albicans was chosen as a model organism and changes in the gene expression profile of strain SC5314 upon exposure to SBTX were examined. Genes that were differentially regulated in the presence of SBTX were involved in glucose transport and starvation-associated stress responses as well as in the control of both the induction and repression of C. albicans hyphal formation. Transmission electron microscopy showed that C. albicans cells exposed to SBTX displayed severe signs of starvation and were heavily granulated. Our data were indicative of C. albicans cell starvation despite sufficient nutrient availability in the medium; therefore, it can be speculated that SBTX blocks nutrient uptake systems. Because neither the starvation signal nor the alkaline response pathway lead to the induction of hyphae, we hypothesise that conflicting signals are transmitted to the complex regulatory network controlling morphogenesis, eventually preventing the filamentation signal from reaching a significant threshold.     

The White Cell Response to Pheromone Is a General Characteristic of Candida albicans Strains

For Candida albicans, evidence has suggested that the mating pheromones activate not only the mating response in mating-competent opaque cells but also a unique response in mating-incompetent white cells that includes increased cohesion and adhesion, enhanced biofilm formation, and expression of select mating-related and white cell-specific genes. On the basis of a recent microarray analysis comparing changes in the global expression patterns of white cells in two strains in response to α-pheromone, however, skepticism concerning the validity and generality of the white cell response has been voiced. Here, we present evidence that the response occurs in all tested media (Lee's, RPMI, SpiderM, yeast extract-peptone-dextrose, and a synthetic medium) and in all of the 27 tested strains, including a/a and α/α strains, derivatives of the common laboratory strain SC5314, and representatives from all of the five major clades. The white cell response to pheromone is therefore a general characteristic of MTL-homozygous strains of C. albicans.     

Posaconazole Exhibits In Vitro and In Vivo Synergistic Antifungal Activity with Caspofungin or FK506 against Candida albicans

The object of this study was to test whether posaconazole, a broad-spectrum antifungal agent inhibiting ergosterol biosynthesis, exhibits synergy with the β-1,3 glucan synthase inhibitor caspofungin or the calcineurin inhibitor FK506 against the human fungal pathogen Candida albicans. Although current drug treatments for Candida infection are often efficacious, the available antifungal armamentarium may not be keeping pace with the increasing incidence of drug resistant strains. The development of drug combinations or novel antifungal drugs to address emerging drug resistance is therefore of general importance. Combination drug therapies are employed to treat patients with HIV, cancer, or tuberculosis, and has considerable promise in the treatment of fungal infections like cryptococcal meningitis and C. albicans infections. Our studies reported here demonstrate that posaconazole exhibits in vitro synergy with caspofungin or FK506 against drug susceptible or resistant C. albicans strains. Furthermore, these combinations also show in vivo synergy against C. albicans strain SC5314 and its derived echinocandin-resistant mutants, which harbor an S645Y mutation in the CaFks1 β-1,3 glucan synthase drug target, suggesting potential therapeutic applicability for these combinations in the future.     

Virulence phenotypes result from interactions between pathogen ploidy and genetic background

Studying fungal virulence is often challenging and frequently depends on many contexts, including host immune status and pathogen genetic background. However, the role of ploidy has often been overlooked when studying virulence in eukaryotic pathogens. Since fungal pathogens, including the human opportunistic pathogen Candida albicans, can display extensive ploidy variation, assessing how ploidy impacts virulence has important clinical relevance. As an opportunistic pathogen, C. albicans causes nonlethal, superficial infections in healthy individuals, but life‐threatening bloodstream infections in individuals with compromised immune function. Here, we determined how both ploidy and genetic background of C. albicans impacts virulence phenotypes in healthy and immunocompromised nematode hosts by characterizing virulence phenotypes in four near‐isogenic diploid and tetraploid pairs of strains, which included both laboratory and clinical genetic backgrounds. We found that C. albicans infections decreased host survival and negatively impacted host reproduction, and we leveraged these two measures to survey both lethal and nonlethal virulence phenotypes across the multiple C. albicans strains. In this study, we found that regardless of pathogen ploidy or genetic background, immunocompromised hosts were susceptible to fungal infection compared to healthy hosts. Furthermore, for each host context, we found a significant interaction between C. albicans genetic background and ploidy on virulence phenotypes, but no global differences between diploid and tetraploid pathogens were observed.     

Ocimum sanctum essential oil inhibits virulence attributes in Candida albicans

Candida albicans is an opportunistic human fungal pathogen which causes disease mainly in immunocompromised patients. Activity of hydrolytic enzymes is essential for virulence of C. albicans and so is the capacity of these cells to undergo transition from yeast to mycelial form of growth. Ocimum sanctum is cultivated worldwide for its essential oil which exhibits medicinal properties. This work evaluates the anti-virulence activity of O. sanctum essential oil (OSEO) on 22 strains of C. albicans (including a standard strain ATCC 90028) isolated from both HIV positive and HIV negative patients. Candida isolates were exposed to sub-MICs of OSEO. In vitro secretion of proteinases and phospholipases was evaluated by plate assay containing BSA and egg yolk respectively. Morphological transition from yeast to filamentous form was monitored microscopically in LSM. For genetic analysis, respective genes associated with morphological transition (HWP1), proteinase (SAP1) and phospholipase (PLB2) were also investigated by Real Time PCR (qRT-PCR). Results were analyzed using Student's t-test. OSEO inhibits morphological transition in C. albicans and had a significant inhibitory effect on extracellular secretion of proteinases and phospholipases. Expression profile of respective selected genes associated with C. albicans virulence by qRT-PCR showed a reduced expression of HWP1, SAP1 and PLB2 genes in cells treated with sub-inhibitory concentrations of OSEO. This work suggests that OSEO inhibits morphological transition in C. albicans and decreases the secretion of hydrolytic enzymes involved in the early stage of infection as well as down regulates the associated genes. Further studies will assess the clinical application of OSEO and its constituents in the treatment of fungal infections.