Mechanistic insights into antifungal potential of Alexidine dihydrochloride and hexachlorophene in Candida albicans: a drug repurposing approach.

Candida albicans has been listed in the critical priority group by the WHO in 2022 depending upon its contribution in invasive candidiasis and increased resistance to conventional drugs. Drug repurposing offers an efficient, rapid, and cost-effective solution to develop alternative therapeutics against pathogenic microbes. Alexidine dihydrochloride (AXD) and hexachlorophene (HCP) are FDA approved anti-cancer and anti-septic drugs, respectively. In this study, we have shown antifungal properties of AXD and HCP against the wild type (reference strain) and clinical isolates of C. albicans. The minimum inhibitory concentrations (MIC50) of AXD and HCP against C. albicans ranged between 0.34 and 0.69 µM and 19.66-24.58 µM, respectively. The biofilm inhibitory and eradication concentration of AXD was reported comparatively lower than that of HCP for the strains used in the study. Further investigations were performed to understand the antifungal mode of action of AXD and HCP by studying virulence features like cell surface hydrophobicity, adhesion, and yeast to hyphae transition, were also reduced upon exposure to both the drugs. Ergosterol content in cell membrane of the wild type strain was upregulated on exposure to AXD and HCP both. Biochemical analyses of the exposed biofilm indicated reduced contents of carbohydrate, protein, and e-DNA in the extracellular matrix of the biofilm when compared to the untreated control biofilm. AXD exposure downregulated activity of tissue invading enzyme, phospholipase in the reference strain. In wild type strain, ROS level, and activities of antioxidant enzymes were found elevated upon exposure to both drugs. FESEM analysis of the drug treated biofilms revealed degraded biofilm. This study has indicated mode of action of antifungal potential of alexidine dihydrochloride and hexachlorophene in C. albicans.

Deletion of the Candida albicans TLO gene family results in alterations in membrane sterol composition and fluconazole tolerance.

Development of resistance and tolerance to antifungal drugs in Candida albicans can compromise treatment of infections caused by this pathogenic yeast species. The uniquely expanded C. albicans TLO gene family is comprised of 14 paralogous genes which encode Med2, a subunit of the multiprotein Mediator complex which is involved in the global control of transcription. This study investigates the acquisition of fluconazole tolerance in a mutant in which the entire TLO gene family has been deleted. This phenotype was reversed to varying degrees upon reintroduction of representative members of the alpha- and beta-TLO clades (i.e. TLO1 and TLO2), but not by TLO11, a gamma-clade representative. Comparative RNA sequencing analysis revealed changes in the expression of genes involved in a range of cellular functions, including ergosterol biosynthesis, mitochondrial function, and redox homeostasis. This was supported by the results of mass spectrometry analysis, which revealed alterations in sterol composition of the mutant cell membrane. Our data suggest that members of the C. albicans TLO gene family are involved in the control of ergosterol biosynthesis and mitochondrial function and may play a role in the responses of C. albicans to azole antifungal agents.

Miconazole induces aneuploidy-mediated tolerance in Candida albicans that is dependent on Hsp90 and calcineurin.

Antifungal resistance and antifungal tolerance are two distinct terms that describe different cellular responses to drugs. Antifungal resistance describes the ability of a fungus to grow above the minimal inhibitory concentration (MIC) of a drug. Antifungal tolerance describes the ability of drug susceptible strains to grow slowly at inhibitory drug concentrations. Recent studies indicate antifungal resistance and tolerance have distinct evolutionary trajectories. Superficial candidiasis bothers millions of people yearly. Miconazole has been used for topical treatment of yeast infections for over 40 years. Yet, fungal resistance to miconazole remains relatively low. Here we found different clinical isolates of Candida albicans had different profile of tolerance to miconazole, and the tolerance was modulated by physiological factors including temperature and medium composition. Exposure of non-tolerant strains with different genetic backgrounds to miconazole mainly induced development of tolerance, not resistance, and the tolerance was mainly due to whole chromosomal or segmental amplification of chromosome R. The efflux gene CDR1 was required for maintenance of tolerance in wild type strains but not required for gain of aneuploidy-mediated tolerance. Heat shock protein Hsp90 and calcineurin were essential for maintenance as well as gain of tolerance. Our study indicates development of aneuploidy-mediated tolerance, not resistance, is the predominant mechanism of rapid adaptation to miconazole in C. albicans, and the clinical relevance of tolerance deserves further investigations.

Transcriptome analysis of Candida albicans planktonic cells in response to plasma medicine.

Introduction. Cold plasma is frequently utilized for the purpose of eliminating microbial contaminants. Under optimal conditions, it can function as plasma medicine for treating various diseases, including infections caused by Candida albicans, an opportunistic pathogen that can overgrow in individuals with weakened immune system.Gap Statement. To date, there has been less molecular study on cold plasma-treated C. albicans.Research Aim. The study aims to fill the gap in understanding the molecular response of C. albicans to cold plasma treatment.Methodology. This project involved testing a cold plasma generator to determine its antimicrobial effectiveness on C. albicans' planktonic cells. Additionally, the cells' transcriptomics responses were investigated using RNA sequencing at various treatment durations (1, 3 and 5 min).Results. The results show that our cold plasma effectively eliminates C. albicans. Cold plasma treatment resulted in substantial downregulation of important pathways, such as 'nucleotide metabolism', 'DNA replication and repair', 'cell growth', 'carbohydrate metabolism' and 'amino acid metabolism'. This was an indication of cell cycle arrest of C. albicans to preserve energy consumption under unfavourable conditions. Nevertheless, C. albicans adapted its GSH antioxidant system to cope with the oxidative stress induced by reactive oxygen species, reactive nitrogen species and other free radicals. The treatment likely led to a decrease in cell pathogenicity as many virulence factors were downregulated.Conclusion. The study demonstrated the major affected pathways in cold plasma-treated C. albicans, providing valuable insights into the molecular response of C. albicans to cold plasma treatment. The findings contribute to the understanding of the antimicrobial efficiency of cold plasma and its potential applications in the field of microbiology.

A fungal metabolic regulator underlies infectious synergism during Candida albicans-Staphylococcus aureus intra-abdominal co-infection.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) demonstrates that synergistic lethality is driven by Candida-induced upregulation of functional S. aureus α-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken revealing that zcf13Δ/Δ fails to drive augmented α-toxin or lethal synergism during co-infection. A combination of transcriptional and phenotypic profiling approaches shows that ZCF13 regulates genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments reveal that ribose inhibits the staphylococcal agr quorum sensing system and concomitantly represses toxicity. Unlike wild-type C. albicans, zcf13Δ/Δ did not effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13Δ/Δ mutant fully restores pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

Strain variation in the Candida albicans iron limitation response.

Iron acquisition is critical for pathogens to proliferate during invasive infection, and the human fungal pathogen Candida albicans is no exception. The iron regulatory network, established in reference strain SC5314 and derivatives, includes the central player Sef1, a transcription factor that activates iron acquisition genes in response to iron limitation. Here, we explored potential variation in this network among five diverse C. albicans strains through mutant analysis, Nanostring gene expression profiling, and, for two strains, RNA-Seq. Our findings highlight four features that may inform future studies of natural variation and iron acquisition in this species. (i) Conformity: In all strains, major iron acquisition genes are upregulated during iron limitation, and a sef1Δ/Δ mutation impairs that response and growth during iron limitation. (ii) Response variation: Some aspects of the iron limitation response vary among strains, notably the activation of hypha-associated genes. As this gene set is tied to tissue damage and virulence, variation may impact the progression of infection. (iii) Genotype-phenotype variation: The impact of a sef1Δ/Δ mutation on cell wall integrity varies, and for the two strains examined the phenotype correlated with sef1Δ/Δ impact on several cell wall integrity genes. (iv) Phenotype discovery: DNA repair genes were induced modestly by iron limitation in sef1Δ/Δ mutants, with fold changes we would usually ignore. However, the response occurred in both strains tested and was reminiscent of a much stronger response described in Cryptococcus neoformans, a suggestion that it may have biological meaning. In fact, we observed that the iron limitation of a sef1Δ/Δ mutant caused recessive phenotypes to emerge at two heterozygous loci. Overall, our results show that a network that is critical for pathogen proliferation presents variation outside of its core functions.IMPORTANCEA key virulence factor of Candida albicans is the ability to maintain iron homeostasis in the host where iron is scarce. We focused on a central iron regulator, SEF1. We found that iron regulator Sef1 is required for growth, cell wall integrity, and genome integrity during iron limitation. The novel aspect of this work is the characterization of strain variation in a circuit that is required for survival in the host and the connection of iron acquisition to genome integrity in C. albicans.

Erg251 has complex and pleiotropic effects on sterol composition, azole susceptibility, filamentation, and stress response phenotypes.

Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans. Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Homozygous deletions of ERG251 resulted in accumulation of ergosterol intermediates consistent with the fitness defect in rich medium. Dysfunction of ERG251, together with FLC exposure, resulted in decreased accumulation of the toxic sterol (14-ɑ-methylergosta-8,24(28)-dien-3ß,6α-diol) and increased accumulation of non-toxic alternative sterols. The altered sterol composition of the ERG251 mutants had pleiotropic effects on transcription, filamentation, and stress responses including cell membrane, osmotic and oxidative stress. Interestingly, while dysfunction of ERG251 resulted in azole tolerance, it also led to transcriptional upregulation of ZRT2, a membrane-bound Zinc transporter, in the presence of FLC, and overexpression of ZRT2 is sufficient to increase azole tolerance in wild-type C. albicans. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study demonstrates that single allele dysfunction of ERG251 is a recurrent and effective mechanism of acquired azole tolerance. We propose that altered sterol composition resulting from ERG251 dysfunction mediates azole tolerance as well as pleiotropic effects on stress response, filamentation and virulence.

Identification of Anticancer Peptides from the Genome of Candida albicans: in Silico Screening, in Vitro and in Vivo Validations.

Anticancer peptides (ACPs) are promising future therapeutics, but their experimental discovery remains time-consuming and costly. To accelerate the discovery process, we propose a computational screening workflow to identify, filter, and prioritize peptide sequences based on predicted class probability, antitumor activity, and toxicity. The workflow was applied to identify novel ACPs with potent activity against colorectal cancer from the genome sequences of Candida albicans. As a result, four candidates were identified and validated in the HCT116 colon cancer cell line. Among them, PCa1 and PCa2 emerged as the most potent, displaying IC50 values of 3.75 and 56.06 µM, respectively, and demonstrating a 4-fold selectivity for cancer cells over normal cells. In the colon xenograft nude mice model, the administration of both peptides resulted in substantial inhibition of tumor growth without causing significant adverse effects. In conclusion, this work not only contributes a proven computational workflow for ACP discovery but also introduces two peptides, PCa1 and PCa2, as promising candidates poised for further development as targeted therapies for colon cancer. The method as a web service is available at https://app.cbbio.online/acpep/home and the source code at https://github.com/cartercheong/AcPEP_classification.git.

Regulatory complexity of cellular differentiation in Candida albicans revealed through systematic screening of protein kinase mutants.

A recent study in mBio reports the construction and preliminary screening of a library containing mutants of 99 of the 119 predicted protein kinases in Candida albicans (the majority of the remaining 20 are probably essential) (J. Kramara, M.-J. Kim, T. L. Ollinger, L. C. Ristow, et al., mBio e01249-24, 2024, https://doi.org/10.1128/mbio.01249-24). Using a quantitative competition assay in 10 conditions that represent nutritional, osmotic, cell wall, and pH stresses that are considered to model various aspects of the host environment allowed them to phenotypically cluster kinases, which highlight both the integration and specialization of signaling pathways, suggesting novel functions for many kinases. In addition, they tackle two complex and partially overlapping differentiation events, hyphal morphogenesis and biofilm formation. They find that a remarkable 88% of the viable kinase mutants in C. albicans affect hyphal growth, illustrating how integrated morphogenesis is in the overall biology of this organism, and begin to dissect the regulatory relationships that control this key virulence trait.

The spliceosome impacts morphogenesis in the human fungal pathogen Candida albicans.

At human body temperature, the fungal pathogen Candida albicans can transition from yeast to filamentous morphologies in response to host-relevant cues. Additionally, elevated temperatures encountered during febrile episodes can independently induce C. albicans filamentation. However, the underlying genetic pathways governing this developmental transition in response to elevated temperatures remain largely unexplored. Here, we conducted a functional genomic screen to unravel the genetic mechanisms orchestrating C. albicans filamentation specifically in response to elevated temperature, implicating 45% of genes associated with the spliceosome or pre-mRNA splicing in this process. Employing RNA-Seq to elucidate the relationship between mRNA splicing and filamentation, we identified greater levels of intron retention in filaments compared to yeast, which correlated with reduced expression of the affected genes. Intriguingly, homozygous deletion of a gene encoding a spliceosome component important for filamentation (PRP19) caused even greater levels of intron retention compared with wild type and displayed globally dysregulated gene expression. This suggests that intron retention is a mechanism for fine-tuning gene expression during filamentation, with perturbations of the spliceosome exacerbating this process and blocking filamentation. Overall, this study unveils a novel biological process governing C. albicans filamentation, providing new insights into the complex regulation of this key virulence trait.IMPORTANCEFungal pathogens such as Candida albicans can cause serious infections with high mortality rates in immunocompromised individuals. When C. albicans is grown at temperatures encountered during human febrile episodes, yeast cells undergo a transition to filamentous cells, and this process is key to its virulence. Here, we expanded our understanding of how C. albicans undergoes filamentation in response to elevated temperature and identified many genes involved in mRNA splicing that positively regulate filamentation. Through transcriptome analyses, we found that intron retention is a mechanism for fine-tuning gene expression in filaments, and perturbation of the spliceosome exacerbates intron retention and alters gene expression substantially, causing a block in filamentation. This work adds to the growing body of knowledge on the role of introns in fungi and provides new insights into the cellular processes that regulate a key virulence trait in C. albicans.

Systematic analysis of the Candida albicans kinome reveals environmentally contingent protein kinase-mediated regulation of filamentation and biofilm formation in vitro and in vivo.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans. An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCECandida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.

Variations in candidalysin amino acid sequence influence toxicity and host responses.

Candida albicans causes millions of mucosal infections in humans annually. Hyphal overgrowth on mucosal surfaces is frequently associated with tissue damage caused by candidalysin, a secreted peptide toxin that destabilizes the plasma membrane of host cells thereby promoting disease and immunopathology. Candidalysin was first identified in C. albicans strain SC5314, but recent investigations have revealed candidalysin "variants" of differing amino acid sequence in isolates of C. albicans, and the related species C. dubliniensis, and C tropicalis, suggesting that sequence variation among candidalysins may be widespread in natural populations of these Candida species. Here, we analyzed ECE1 gene sequences from 182 C. albicans isolates, 10 C. dubliniensis isolates, and 78 C. tropicalis isolates and identified 10, 3, and 2 candidalysin variants in these species, respectively. Application of candidalysin variants to epithelial cells revealed differences in the ability to cause cellular damage, changes in metabolic activity, calcium influx, MAPK signalling, and cytokine secretion, while biophysical analyses indicated that variants exhibited differences in their ability to interact with and permeabilize a membrane. This study identifies candidalysin variants with differences in biological activity that are present in medically relevant Candida species. IMPORTANCE: Fungal infections are a significant burden to health. Candidalysin is a toxin produced by Candida albicans that damages host tissues, facilitating infection. Previously, we demonstrated that candidalysins exist in the related species C. dubliniensis and C. tropicalis, thereby identifying these molecules as a toxin family. Recent genomic analyses have highlighted the presence of a small number of candidalysin "variant" toxins, which have different amino acid sequences to those originally identified. Here, we screened genome sequences of isolates of C. albicans, C. dubliniensis, and C. tropicalis and identified candidalysin variants in all three species. When applied to epithelial cells, candidalysin variants differed in their ability to cause damage, activate intracellular signaling pathways, and induce innate immune responses, while biophysical analysis revealed differences in the ability of candidalysin variants to interact with lipid bilayers. These findings suggest that intraspecies variation in candidalysin amino acid sequence may influence fungal pathogenicity.

The zinc cluster transcription factor Znc1 regulates Rta3-dependent miltefosine resistance in Candida albicans.

Zinc cluster transcription factors (ZCFs) are a family of transcription regulators that are almost exclusively found in the fungal kingdom. Activating mutations in the ZCFs Mrr1, Tac1, and Upc2 frequently cause acquired resistance to the widely used antifungal drug fluconazole in the pathogenic yeast Candida albicans. Similar to a hyperactive Tac1, a constitutively active form of the ZCF Znc1 causes increased fluconazole resistance by upregulating the multidrug efflux pump-encoding gene CDR1. Hyperactive forms of both Tac1 and Znc1 also cause overexpression of RTA3, which encodes a seven-transmembrane receptor protein involved in the regulation of asymmetric lipid distribution in the plasma membrane. RTA3 expression is also upregulated by miltefosine, an antiparasitic drug that is active against fungal pathogens and considered for treatment of invasive candidiasis, and rta3Δ mutants are hypersensitive to miltefosine. We found that activated forms of both Tac1 and Znc1 confer increased miltefosine resistance, which was dependent on RTA3 whereas CDR1 was dispensable. Intriguingly, the induction of RTA3 expression by miltefosine depended on Znc1, but not Tac1, in contrast to the known Tac1-dependent RTA3 upregulation by fluphenazine. In line with this observation, znc1Δ mutants were hypersensitive to miltefosine, whereas tac1Δ mutants showed wild-type tolerance. Forced expression of RTA3 reverted the hypersensitivity of znc1Δ mutants, demonstrating that the hypersensitivity was caused by the inability of the mutants to upregulate RTA3 in response to the drug. These findings establish Znc1 as a key regulator of miltefosine-induced RTA3 expression that is important for wild-type miltefosine tolerance. IMPORTANCE: Transcription factors are central regulators of gene expression, and knowledge about which transcription factor regulates specific genes in response to a certain signal is important to understand the behavior of organisms. In the pathogenic yeast Candida albicans, the RTA3 gene is required for wild-type tolerance of miltefosine, an antiparasitic drug that is considered for treatment of invasive candidiasis. Activated forms of the transcription factors Tac1 and Znc1 cause constitutive overexpression of RTA3 and thereby increased miltefosine resistance, but only Tac1 mediates upregulation of RTA3 in response to the known inducer fluphenazine. RTA3 expression is also induced by miltefosine, and we found that this response depends on Znc1, whereas Tac1 is dispensable. Consequently, znc1Δ mutants were hypersensitive to miltefosine, whereas tac1Δ mutants showed wild-type tolerance. These findings demonstrate that Znc1 is the key regulator of RTA3 expression in response to miltefosine that is important for wild-type miltefosine tolerance.

Metabolic reprogramming during Candida albicans planktonic-biofilm transition is modulated by the transcription factors Zcf15 and Zcf26.

Candida albicans is a commensal of the human microbiota that can form biofilms on implanted medical devices. These biofilms are tolerant to antifungals and to the host immune system. To identify novel genes modulating C. albicans biofilm formation, we performed a large-scale screen with 2,454 C. albicans doxycycline-dependent overexpression strains and identified 16 genes whose overexpression significantly hampered biofilm formation. Among those, overexpression of the ZCF15 and ZCF26 paralogs that encode transcription factors and have orthologs only in biofilm-forming species of the Candida clade, caused impaired biofilm formation both in vitro and in vivo. Interestingly, overexpression of ZCF15 impeded biofilm formation without any defect in hyphal growth. Transcript profiling, transcription factor binding, and phenotypic microarray analyses conducted upon overexpression of ZCF15 and ZCF26 demonstrated their role in reprogramming cellular metabolism by regulating central metabolism including glyoxylate and tricarboxylic acid cycle genes. Taken together, this study has identified a new set of biofilm regulators, including ZCF15 and ZCF26, that appear to control biofilm development through their specific role in metabolic remodeling.

Absence of farnesol salvage in Candida albicans and probably in other fungi.

Farnesol salvage, a two-step pathway converting farnesol to farnesyl pyrophosphate (FPP), occurs in bacteria, plants, and animals. This paper investigates the presence of this pathway in fungi. Through bioinformatics, biochemistry, and physiological analyses, we demonstrate its absence in the yeasts Saccharomyces cerevisiae and Candida albicans, suggesting a likely absence across fungi. We screened 1,053 fungal genomes, including 34 from C. albicans, for potential homologs to four genes (Arabidopsis thaliana AtFOLK, AtVTE5, AtVTE6, and Plasmodium falciparum PfPOLK) known to accomplish farnesol/prenol salvage in other organisms. Additionally, we showed that 3H-farnesol was not converted to FPP or any other phosphorylated prenol, and exogenous farnesol was not metabolized within 90 minutes at any phase of growth and did not rescue cells from the toxic effects of atorvastatin, but it did elevate the levels of intracellular farnesol (Fi). All these experiments were conducted with C. albicans. In sum, we found no evidence for farnesol salvage in fungi. IMPORTANCE: The absence of farnesol salvage constitutes a major difference in the metabolic capabilities of fungi. In terms of fungal physiology, the lack of farnesol salvage pathways relates to how farnesol acts as a quorum-sensing molecule in Candida albicans and why farnesol should be investigated for use in combination with other known antifungal antibiotics. Its absence is essential for a model (K. W. Nickerson et al., Microbiol Mol Biol Rev 88:e00081-22, 2024), wherein protein farnesylation, protein chaperones, and the unfolded protein response are combined under the unifying umbrella of a cell's intracellular farnesol (Fi). In terms of human health, farnesol should have at least two different modes of action depending on whether those cells have farnesol salvage. Because animals have farnesol salvage, we can now see the importance of dietary prenols as well as the potential importance of farnesol in treating neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis.

The atypical antipsychotic aripiprazole alters the outcome of disseminated Candida albicans infections.

Invasive fungal infections impose an enormous clinical, social, and economic burden on humankind. One of the most common species responsible for invasive fungal infections is Candida albicans. More than 30% of patients with disseminated candidiasis fail therapy with existing antifungal drugs, including the widely used azole class. We previously identified a collection of 13 medications that antagonize the activity of the azoles on C. albicans. Although gain-of-function mutations responsible for antifungal resistance are often associated with reduced fitness and virulence, it is currently unknown how exposure to azole antagonistic drugs impacts C. albicans physiology, fitness, or virulence. In this study, we examined how exposure to seven azole antagonists affects C. albicans phenotype and capacity to cause disease. Most of the azole antagonists appear to have little impact on fungal growth, morphology, stress tolerance, or gene transcription. However, aripiprazole had a modest impact on C. albicans hyphal growth and increased cell wall chitin content. It also aggravated the disseminated C. albicans infections in mice. This effect was abrogated in immunosuppressed mice, indicating that it is at least in part dependent upon host immune responses. Collectively, these data provide proof of principle that unanticipated drug-fungus interactions have the potential to influence the incidence and outcomes of invasive fungal disease.

Antifungal potential of multi-drug-resistant Pseudomonas aeruginosa: harnessing pyocyanin for candida growth inhibition.

Introduction: Pseudomonas aeruginosa is notorious for its multidrug resistance and its involvement in hospital-acquired infections. In this study, 20 bacterial strains isolated from soil samples near the Hindan River in Ghaziabad, India, were investigated for their biochemical and morphological characteristics, with a focus on identifying strains with exceptional drug resistance and pyocyanin production. Methods: The isolated bacterial strains were subjected to biochemical and morphological analyses to characterize their properties, with a particular emphasis on exopolysaccharide production. Strain GZB16/CEES1, exhibiting remarkable drug resistance and pyocyanin production. Biochemical and molecular analyses, including sequencing of its 16S rRNA gene (accession number LN735036.1), plasmid-curing assays, and estimation of plasmid size, were conducted to elucidate its drug resistance mechanisms and further pyocynin based target the Candida albicans Strain GZB16/CEES1 demonstrated 100% resistance to various antibiotics used in the investigation, with plasmid-curing assays, suggesting plasmid-based resistance gene transmission. The plasmid in GZB16/CEES1 was estimated to be approximately 24 kb in size. The study focused on P. aeruginosa's pyocyanin production, revealing its association with anticandidal activity. The minimum inhibitory concentration (MIC) of the bacterial extract against Candida albicans was 50 µg/ml, with a slightly lower pyocyanin-based MIC of 38.5 µg/ml. Scanning electron microscopy illustrated direct interactions between P. aeruginosa strains and Candida albicans cells, leading to the destruction of the latter. Discussion: These findings underscore the potential of P. aeruginosa in understanding microbial interactions and developing strategies to combat fungal infections. The study highlights the importance of investigating bacterial-fungal interactions and the role of pyocyanin in antimicrobial activity. Further research in this area could lead to the development of novel therapeutic approaches for combating multidrug-resistant infections.

Mechanism and bioinformatics analysis of the effect of berberine-enhanced fluconazole against drug-resistant Candida albicans.

Biofilms produced by Candida albicans present a challenge in treatment with antifungal drug. Enhancing the sensitivity to fluconazole (FLC) is a reasonable method for treating FLC-resistant species. Moreover, several lines of evidence have demonstrated that berberine (BBR) can have antimicrobial effects. The aim of this study was to clarify the underlying mechanism of these effects. We conducted a comparative study of the inhibition of FLC-resistant strain growth by FLC treatment alone, BBR treatment alone, and the synergistic effect of combined FLC and BBR treatment. Twenty-four isolated strains showed distinct biofilm formation capabilities. The antifungal effect of combined FLC and BBR treatment in terms of the growth and biofilm formation of Candida albicans species was determined via checkerboard, time-kill, and fluorescence microscopy assays. The synergistic effect of BBR and FLC downregulated the expression of the efflux pump genes CDR1 and MDR, the hyphal gene HWP1, and the adhesion gene ALS3; however, the gene expression of the transcriptional repressor TUP1 was upregulated following treatment with this drug combination. Furthermore, the addition of BBR led to a marked reduction in cell surface hydrophobicity. To identify resistance-related genes and virulence factors through genome-wide sequencing analysis, we investigated the inhibition of related resistance gene expression by the combination of BBR and FLC, as well as the associated signaling pathways and metabolic pathways. The KEGG metabolic map showed that the metabolic genes in this strain are mainly involved in amino acid and carbon metabolism. The metabolic pathway map showed that several ergosterol (ERG) genes were involved in the synthesis of cell membrane sterols, which may be related to drug resistance. In this study, BBR + FLC combination treatment upregulated the expression of the ERG1, ERG3, ERG4, ERG5, ERG24, and ERG25 genes and downregulated the expression of the ERG6 and ERG9 genes compared with fluconazole treatment alone (p < 0.05).

Antifungal Activity of Mefloquine Against Candida albicans Growth and Virulence Factors: Insights Into Mode of Action.

The antimalarial drug Mefloquine has demonstrated antifungal activity against growth and virulence factors of Candida albicans. The current study focused on the identification of Mefloquine's mode of action in C. albicans by performing cell susceptibility assay, biofilm assay, live and dead assay, propidium iodide uptake assay, ergosterol quantification assay, cell cycle study, and gene expression studies by RT-PCR. Mefloquine inhibited the virulence factors in C. albicans, such as germ tube formation and biofilm formation at 0.125 and 1 mg/ml, respectively. Mefloquine-treated cells showed a decrease in the quantity of ergosterol content of cell membrane in a concentration-dependent manner. Mefloquine (0.25 mg/ml) arrested C. albicans cells at the G2/M phase and S phase of the cell cycle thereby preventing the progression of the normal yeast cell cycle. ROS level was measured to find out oxidative stress in C. albicans in the presence of mefloquine. The study revealed that, mefloquine was found to enhance the ROS level and subsequently oxidative stress. Gene expression studies revealed that mefloquine treatment upregulates the expressions of SOD1, SOD2, and CAT1 genes in C. albicans. In vivo, the antifungal efficacy of mefloquine was confirmed in mice for systemic candidiasis and it was found that there was a decrease in the pathogenesis of C. albicans after the treatment of mefloquine in mice. In conclusion, mefloquine can be used as a repurposed drug as an alternative drug against Candidiasis.

Probing gene function in Candida albicans wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.

The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast Candida albicans, because homozygous mutants of this diploid fungus can now be generated in a single step, allowing the rapid screening of candidate genes for their involvement in a phenotype of interest. However, the Cas9-mediated double-strand breaks at the target site may result in an undesired loss of heterozygosity (LOH) on the affected chromosome and cause phenotypic alterations that are not related to the function of the investigated gene. In our present study, we harnessed Cas9-facilitated gene deletion to probe a set of genes that are constitutively overexpressed in strains containing hyperactive forms of the transcription factor Mrr1 for a possible contribution to the fluconazole resistance of such strains. To this aim, we used gene deletion cassettes containing two different dominant selection markers, caSAT1 and HygB, which confer resistance to nourseothricin and hygromycin, respectively, for simultaneous genomic integration in a single step, hypothesizing that this would minimize undesired LOH events at the target locus. We found that selection for resistance to both nourseothricin and hygromycin strongly increased the proportion of homozygous deletion mutants among the transformants compared with selection on media containing only one of the antibiotics, but it did not avoid undesired LOH events. Our results demonstrate that LOH on the target chromosome is a significant problem when using Cas9 for the generation of C. albicans gene deletion mutants, which demands a thorough examination of recombination events at the target site. IMPORTANCE: Candida albicans is one of the medically most important fungi and a model organism to study fungal pathogenicity. Investigating gene function in this diploid yeast has been facilitated by the adaptation of gene deletion methods based on the bacterial CRISPR-Cas9 system, because they enable the generation of homozygous mutants in a single step. We found that, in addition to increasing the efficiency of gene replacement by selection markers, the Cas9-mediated double-strand breaks also result in frequent loss of heterozygosity on the same chromosome, even when two different selection markers were independently integrated into the two alleles of the target gene. Since loss of heterozygosity for other genes can result in phenotypic alterations that are not caused by the absence of the target gene, these findings show that it is important to thoroughly analyze recombination events at the target locus when using Cas9 to generate gene deletion mutants in C. albicans.