Search

In recent decades, there has been an increase in the occurrence of fungal infections; yet, the arsenal of drugs available to fight invasive infections remains very limited. The development of new antifungal agents is hindered by the restricted number of molecular targets that can be exploited, given the shared eukaryotic nature of fungi and their hosts which often leads to host toxicity. In this paper, we examine the riboflavin biosynthetic pathway as a potential novel drug target. Riboflavin is an essential nutrient for all living organisms. Its biosynthetic pathway does not exist in humans, who obtain riboflavin through their diet. Our findings demonstrate that all enzymes in the pathway are essential for Candida albicans , Candida glabrata, and Saccharomyces cerevisiae. Auxotrophic strains, which mimic a drug targeting the biosynthesis pathway, experience rapid mortality in the absence of supplemented riboflavin. Furthermore, RIB1 is essential for virulence in both C. albicans and C. glabrata in a systemic mouse model. The fungal burden of a RIB1 deletion strain is significantly reduced in the kidneys and brain of infected mice, and this reduction becomes more pronounced over time. Nevertheless, auxotrophic cells can still take up external riboflavin when supplemented. We identified Orf19.4337 as the riboflavin importer in C. albicans and named it Rut1. We found that Rut1 only facilitates growth at external riboflavin concentrations that exceed the physiological concentrations in the human body. This suggests that riboflavin uptake is unlikely to serve as a resistance mechanism against drugs targeting the biosynthesis pathway. Interestingly, the uptake system in S. cerevisiae is more effective than in C. albicans and C. glabrata, enabling an auxotrophic S. cerevisiae strain to outcompete an auxotrophic C. albicans strain in lower riboflavin concentrations., Importance: Candida species are a common cause of invasive fungal infections. Candida albicans , in particular, poses a significant threat to immunocompromised individuals. This opportunistic pathogen typically lives as a commensal on mucosal surfaces of healthy individuals but it can also cause invasive infections associated with high morbidity and mortality. Currently, there are only three major classes of antifungal drugs available to treat these infections. In addition, the efficacy of these antifungal agents is restricted by host toxicity, suboptimal pharmacokinetics, a narrow spectrum of activity, intrinsic resistance of fungal species, such as Candida glabrata , to certain drugs, and the acquisition of resistance over time. Therefore, it is crucial to identify new antifungal drug targets with novel modes of action to add to the limited armamentarium., Competing Interests: The authors declare no conflict of interest.

Science is challenging because we do not know what we do not know. Commercial chemicals are often marketed with >99% purity, but 0.5-1% impurity can impact results and cloud data interpretation. We recently developed an assay for farnesol and aromatic fusel alcohols from Candida albicans . During proof-of-concept experiments using RPMI-1640 growth media, the buffering compound was switched from MOPS obtained from Acros Organics to MOPS obtained from Sigma-Aldrich, both labeled 99% + purity. We observed a twofold decrease in growth, along with a three- to fivefold increase in farnesol production per cell upon the switch. ICP-MS showed that trace Mn(II) was present in Acros MOPS but absent in Sigma MOPS. Optimal growth was achieved by the addition of Mn(II), Zn(II), and Fe(II). We established upper and lower limits for Fe(II), Zn(II), Cu(II), and Mn(II) that allowed similar growth and then assessed 16 different mineral combinations in RPMI-1640 base media. The results show an increased production of farnesol and the aromatic fusel alcohols when Zn(II) is abundant, and a further increase in the aromatic fusel alcohols when both Fe(II) and Zn(II) are abundant. Finally, antifungal susceptibility testing displayed no significant difference between RPMI/MOPS with and without mineral supplementation. Supplemental Mn(II) was most needed for cell growth, while supplemental Zn(II) was most needed for the production of farnesol and the aromatic fusel alcohols. To avoid these artifacts due to metal contamination, we now use a modified RPMI supplemented with 1 mg/ L of Cu(II), Zn(II), Mn(II), and Fe(II)., Importance: The dimorphic fungus Candida albicans is a major opportunistic pathogen of humans. RPMI-1640 is a chemically defined growth medium commonly used with C. albicans . We identified over 32,000 publications with keywords RPMI and C. albicans . Additionally, Antifungal Susceptibility Testing (AFST) protocols in the United States (CLSI) and Europe (EUCAST) utilize RPMI as a base media to assess drug efficacy against clinical fungal isolates. RPMI contains many nutrients but no added trace metals. We found that the growth characteristics with RPMI were dependent on which MOPS buffer was chosen and the contamination of that buffer by trace levels of Mn(II) and Zn(II). Added Mn(II) was most needed for cell growth while added Zn(II) was most needed for secretion of farnesol and other signaling molecules., Competing Interests: The authors declare no conflict of interest.

Aims: Vulvovaginal candidiasis (VVC) is a prevalent condition affecting a significant proportion of women worldwide, with recurrent episodes leading to detrimental effects on quality of life. While treatment with clotrimazole is common, the specific alterations it evokes in the vaginal bacteriome and metabolome were previously underexplored., Methods and Results: In this prospective study, we enrolled reproductive-age women diagnosed with single VVC and conducted comprehensive analyses of vaginal fungi, bacteriome, and metabolome before and after local clotrimazole treatment. We observed a significant reduction in Candida albicans and notable improvements in vaginal cleanliness. Advanced sequencing revealed substantial shifts in the vaginal bacteriome, with an increase in Lactobacillus-dominant communities post-treatment. Our findings identified 17 differentially abundant bacterial species, including notable decreases in pathogenic anaerobes such as Gardnerella vaginalis, Dialister micraerophilus, and Aerococcus christensenii, suggesting a restoration of a healthier microbial balance. Furthermore, metabolomic analysis revealed significant changes in 230 metabolites, particularly within lipid metabolism pathways, with marked downregulation of lipid-related compounds linked to inflammation. Correlation studies indicated a strong interplay between lipid metabolites and specific bacterial species, emphasizing the influence of clotrimazole treatment on microbial and metabolic interactions. Importantly, predictive models using microbiota and metabolite signatures demonstrated high accuracy in distinguishing pre- and post-treatment states., Conclusions: This research highlights clotrimazole's dual role in effectively clearing Candida infection and promoting a healthier vaginal microenvironment, paving the way for novel microbial and metabolomic-based diagnostic approaches to enhance VVC management and understand its underlying mechanisms., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

The human gastrointestinal microbiome encompasses bacteria, fungi, and viruses forming complex bionetworks which, for organismal health, must be in a state of homeostasis. An important homeostatic mechanism derives from microbial competition, which maintains the relative abundance of microbial species in a healthy balance. Microbes compete for nutrients and secrete metabolites that inhibit other microbes. Short-chain fatty acids (SCFAs) are one such class of metabolites made by gut bacteria to very high levels. SCFAs are metabolised by microbes and host cells and have multiple roles in regulating cell physiology. Here, we review the mechanisms by which SCFAs regulate the fungal gut commensal Candida albicans. We discuss SCFA's ability to inhibit fungal growth, limit invasive behaviours and modulate cell surface antigens recognised by immune cells. We review the mechanisms underlying these roles: regulation of gene expression, metabolism, signalling and SCFA-driven post-translational protein modifications by acylation, which contribute to changes in acylome dynamics of C. albicans with potentially large consequences for cell physiology. Given that the gut mycobiome is a reservoir for systemic disease and has also been implicated in inflammatory bowel disease, understanding the mechanisms by which bacterial metabolites, such as SCFAs, control the mycobiome might provide therapeutic avenues., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To adapt to various host microenvironments, the human fungal pathogen Candida albicans possesses the capacity to accumulate and store glycogen as an internal carbohydrate source. In the model yeast Saccharomyces cerevisiae , Sc Glc7p and Sc Gac1p are the serine/threonine type 1 protein phosphatase catalytic and regulatory subunits that control glycogen synthesis by altering the phosphorylation state of the glycogen synthase Gsy2p. Despite recent delineation of the glycogen synthesis pathway in C. albicans , the molecular events driving synthase activation are currently undefined. In this study, using a combination of microbiologic and genetic techniques, we determined that the protein encoded by uncharacterized gene C1_01140C , and not the currently annotated C. albicans Gac1p, is the major regulatory subunit involved in glycogen synthesis. C1_01140Cp contains a conserved GVNK motif observed across multiple starch/glycogen-binding proteins in various species, and alanine substitution of each residue in this motif significantly impaired glycogen accumulation in C. albicans . Fluorescent protein tagging and microscopy indicated that C1_01140Cp-GFPy colocalized with Ca Glc7p-tdTomato and Ca Gsy1p-tdTomato accordingly. Co-immunoprecipitation assays further confirmed that C1_01140Cp associates with Ca Glc7p and Ca Gsy1p during glycogen synthesis. Lastly, c1_01140c Δ/Δ exhibited colonization defects in a murine model of vulvovaginal candidiasis. Collectively, our data indicate that uncharacterized C1_01140Cp is the functional ortholog of the PPP1R subunit Sc Gac1p in C. albicans .IMPORTANCEThe capacity to synthesize glycogen offers microbes metabolic flexibility, including the fungal pathogen Candida albicans . In Saccharomyces cerevisiae , dephosphorylation of glycogen synthase by the Sc Glc7p-containing phosphatase is a critical rate-limiting step in glycogen synthesis. Subunits, including Sc Gac1p, target Sc Glc7p to α-1,4-glucosyl primers for efficient Sc Gsy2p synthase activation. However, this process in C. albicans had not been delineated. Here, we show that the C. albicans genome encodes for two homologous phosphatase-binding subunits, annotated Ca Gac1p and uncharacterized C1_01140Cp, both containing a GVNK motif required for polysaccharide affinity. Surprisingly, loss of Ca Gac1p only moderately reduced glycogen accumulation, whereas loss of C1_01140Cp ablated it. Fluorescence microscopy and co-immunoprecipitation approaches revealed that C1_01140Cp associates with Ca Glc7p and Ca Gsy1p during glycogen synthesis. Moreover, C1_01140Cp contributed to fungal fitness at the vaginal mucosa during murine vaginitis. Therefore, this work demonstrates that glycogen synthase regulation is conserved in C. albicans and C1_01140Cp is the functional ortholog of Sc Gac1p., Competing Interests: The authors declare no conflict of interest.

Human fungal pathogens must survive diverse reactive oxygen species (ROS) produced by host immune cells that can oxidize a range of cellular molecules including proteins, lipids, and DNA. Formation of lipid radicals can be especially damaging, as it leads to a chain reaction of lipid peroxidation that causes widespread damage to the plasma membrane. Most previous studies on antioxidant pathways in fungal pathogens have been conducted with hydrogen peroxide, so the pathways used to combat organic peroxides and lipid peroxidation are not well understood. The most well-known peroxidase in Candida albicans, catalase, can only act on hydrogen peroxide. We therefore characterized a family of four glutathione peroxidases (GPxs) that were predicted to play an important role in reducing organic peroxides. One of the GPxs, Gpx3 is also known to activate the Cap1 transcription factor that plays the major role in inducing antioxidant genes in response to ROS. Surprisingly, we found that the only measurable role of the GPxs is activation of Cap1 and did not find a significant role for GPxs in the direct detoxification of peroxides. Furthermore, a CAP1 deletion mutant strain was highly sensitive to organic peroxides and oxidized lipids, indicating an important role for antioxidant genes upregulated by Cap1 in protecting cells from organic peroxides. We identified GLR1 (Glutathione reductase), a gene upregulated by Cap1, as important for protecting cells from oxidized lipids, implicating glutathione utilizing enzymes in the protection against lipid peroxidation. Furthermore, an RNA-sequencing study in C. albicans showed upregulation of a diverse set of antioxidant genes and protein damage pathways in response to organic peroxides. Overall, our results identify novel mechanisms by which C. albicans responds to oxidative stress resistance which open new avenues for understanding how fungal pathogens resist ROS in the host., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Swenson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pentatricopeptide repeat (PPR) proteins bind RNA and are present in mitochondria and chloroplasts of Eukaryota. In fungi, they are responsible for controlling mitochondrial genome expression, mainly on the posttranscriptional level. Candida albicans is a human opportunistic pathogen with a facultative anaerobic metabolism which, unlike the model yeast Saccharomyces cerevisiae, possesses mitochondrially encoded respiratory Complex I (CI) subunits and does not tolerate loss of mtDNA. We characterized the function of 4 PPR proteins of C. albicans that lack orthologs in S. cerevisiae and found that they are required for the expression of mitochondrially encoded CI subunits. We demonstrated that these proteins localize to mitochondria and are essential to maintain the respiratory capacity of cells. Deletion of genes encoding these PPR proteins results in changes in steady-state levels of mitochondrial RNAs and proteins. We demonstrated that C. albicans cells lacking CaPpr4, CaPpr11, and CaPpr13 proteins show no CI assembly, whereas the lack of CaPpr7p results in a decreased CI activity. CaPpr13p is required to maintain the bicistronic NAD4L-NAD5 mRNA, whereas the other 3 PPR proteins are likely involved in translation-related assembly of mitochondrially encoded CI subunits. In addition, we show that CaAep3p, which is an ortholog of ScAep3p, performs the evolutionary conserved function of controlling expression of the ATP8-ATP6 mRNA. We also show that C. albicans cells lacking PPR proteins express a higher level of the inducible alternative oxidase (AOX2) which likely rescues respiratory defects and compensates for oxidative stress., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Pdr5 is the most abundant ABC transporter in Saccharomyces cerevisiae and plays a major role in the pleiotropic drug resistance (PDR) network, which actively prevents cell entry of a large number of structurally unrelated compounds. Due to a high level of asymmetry in one of its nucleotide binding sites (NBS), Pdr5 serves as a perfect model system for asymmetric ABC transporter such as its medical relevant homologue Cdr1 from Candida albicans. In the past 30 years, this ABC transporter was intensively studied in vivo and in plasma membrane vesicles. Nevertheless, these studies were limited since it was not possible to isolate and reconstitute Pdr5 in a synthetic membrane system while maintaining its activity. Here, the functional reconstitution of Pdr5 in a native-like environment in an almost unidirectional inside-out orientation is described. We demonstrate that reconstituted Pdr5 is capable of translocating short-chain fluorescent NBD lipids from the outer to the inner leaflet of the proteoliposomes. Moreover, this transporter revealed its ability to utilize other nucleotides to accomplish transport of substrates in a reconstituted system. Besides, we were also able to estimate the NTPase activity of reconstituted Pdr5 and determine the kinetic parameters for ATP, GTP, CTP, and UTP., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Candidalysin, a cytolytic peptide produced by the fungal pathogen Candida albicans, is a key virulence factor. However, its host cell targets remain elusive. Here we performed a genome-wide loss-of-function CRISPR screen in the TR146 human oral epithelial cell line and identified that disruption of genes (XYLT2, B3GALT6 and B3GAT3) in glycosaminoglycan (GAG) biosynthesis conferred resistance to damage induced by candidalysin and live C. albicans. Surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin binds to sulfated GAGs, facilitating its enrichment on the host cell surface. Adding exogenous sulfated GAGs or the analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate also inhibited C. albicans invasion and fungal-induced epithelial cell cytokine production. In mice with vulvovaginal candidiasis, topical dextran sulfate administration reduced intravaginal tissue damage and inflammation. Collectively, sulfated GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The uridine derivatives UDP-glucose and UDP- N -acetylglucosamine are important for cell wall construction as they are the precursors for the synthesis of β-1,3-glucan and chitin, respectively. Previous studies have demonstrated attenuated virulence of uridine auxotrophs in mice, which has been attributed to insufficient uridine levels for growth in the host. We have discovered that uridine deprivation in the uridine auxotroph ura3 ΔΔ disrupts cell wall architecture by increasing surface mannans, exposing β-1,3-glucan and chitin, and decreasing UDP-sugar levels. Cell wall architecture and UDP-sugars can be rescued with uridine supplementation. The cell wall architectural disruptions in the ura3 ΔΔ mutant also impact immune activation since the mutant elicited greater TNFα secretion from RAW264.7 macrophages than wild type. To determine if cell wall defects contributed to decreased virulence in the ura3 ΔΔ mutant, we used a murine model of systemic infection. Mice infected with the ura3 ΔΔ mutant exhibited increased survival and reduced kidney fungal burden compared with mice infected with wild type. However, suppression of the immune response with cyclophosphamide did not rescue virulence in mice infected with the ura3 ΔΔ mutant, indicating the attenuation in virulence of uridine auxotrophs can be attributed to decreased growth in the host but not increased exposure of β-1,3-glucan. Moreover, the ura3 ΔΔ mutant is unable to grow on ex vivo kidney agar, which demonstrates its inability to colonize the kidneys due to poor growth. Thus, although uridine auxotrophy elicits changes to cell wall architecture that increase the exposure of immunogenic polymers, metabolic fitness costs more strongly drive the observed virulence attenuation.IMPORTANCE Candida albicans is a common cause of bloodstream infections (candidemia). Treatment of these bloodstream infections is made difficult because of increasing antifungal resistance and drug toxicity. Thus, new tactics are needed for antifungal drug development, with immunotherapy being of particular interest. The cell wall of C. albicans is composed of highly immunogenic polymers, particularly β-1,3-glucan. However, β-1,3-glucan is naturally masked by an outer layer of mannoproteins, which hampers the detection of the fungus by the host immune system. Alteration in cell wall components has been shown to increase β-1,3-glucan exposure; however, it is unknown how the inability to synthesize precursors to cell wall components affects unmasking. Here, we demonstrate how cell wall architecture is altered in response to a deficit in precursors for cell wall synthesis and how uridine is a crucial component of these precursors., Competing Interests: The authors declare no conflict of interest.

In Candida albicans, Cdr1 pumps azole drugs out of the cells to reduce intracellular accumulation at detrimental concentrations, leading to azole-drug resistance. Milbemycin oxime, a veterinary anti-parasitic drug, strongly and specifically inhibits Cdr1. However, how Cdr1 recognizes and exports azole drugs, and how milbemycin oxime inhibits Cdr1 remain unclear. Here, we report three cryo-EM structures of Cdr1 in distinct states: the apo state (Cdr1 Apo ), fluconazole-bound state (Cdr1 Flu ), and milbemycin oxime-inhibited state (Cdr1 Mil ). Both the fluconazole substrate and the milbemycin oxime inhibitor are primarily recognized within the central cavity of Cdr1 through hydrophobic interactions. The fluconazole is suggested to be exported from the binding site into the environment through a lateral pathway driven by TM2, TM5, TM8 and TM11. Our findings uncover the inhibitory mechanism of milbemycin oxime, which inhibits Cdr1 through competition, hindering export, and obstructing substrate entry. These discoveries advance our understanding of Cdr1-mediated azole resistance in C. albicans and provide the foundation for the development of innovative antifungal drugs targeting Cdr1 to combat azole-drug resistance., (© 2024. The Author(s).)

Candida albicans is a diploid pathogen known for its ability to live as a commensal fungus in healthy individuals but causing both superficial infections and disseminated candidiasis in immunocompromised patients where it is associated with high morbidity and mortality. Its success in colonizing the human host is attributed to a wide range of virulence traits that modulate interactions between the host and the pathogen, such as optimal growth rate at 37 °C, the ability to switch between yeast and hyphal forms, and a remarkable genomic and phenotypic plasticity. A fascinating aspect of its biology is a prominent heterogeneous proteome that arises from frequent genomic rearrangements, high allelic variation, and high levels of amino acid misincorporations in proteins. This leads to increased morphological and physiological phenotypic diversity of high adaptive potential, but the scope of such protein mistranslation is poorly understood due to technical difficulties in detecting and quantifying amino acid misincorporation events in complex protein samples. We have developed and optimized mass spectrometry and bioinformatics pipelines capable of identifying rare amino acid misincorporation events at the proteome level. We have also analyzed the proteomic profile of an engineered C. albicans strain that exhibits high level of leucine misincorporation at protein CUG sites and employed an in vivo quantitative gain-of-function fluorescence reporter system to validate our LC-MS/MS data. C. albicans misincorporates amino acids above the background level at protein sites of diverse codons, particularly at CUG, confirming our previous data on the quantification of leucine incorporation at single CUG sites of recombinant reporter proteins, but increasing misincorporation of Leucine at these sites does not alter the translational fidelity of the other codons. These findings indicate that the C. albicans statistical proteome exceeds prior estimates, suggesting that its highly plastic phenome may also be modulated by environmental factors due to translational ambiguity., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In recent years, epigenetics has been revealed as a mechanism able to modulate the expression of virulence traits in diverse pathogens, including Candida albicans. Indeed, epigenetic regulation can sense environmental changes, leading to the rapid and reversible modulation of gene expression with consequent adaptation to novel environments. How epigenetic changes can impact expression and signalling output, including events associated with mechanisms of morphological transition and virulence, is still poorly studied. Here, using nicotinamide as a sirtuin inhibitor, we explored how the accumulation of the H3K56 acetylation, the most prominent histone acetylation in C. albicans, might affect its interaction with the host. Our experiments demonstrate that H3K56 acetylation profoundly affects the production and/or secretion of soluble factors compromising actin remodelling and cytokine production. ChIP- and RNA-seq analyses highlighted a direct impact of H3K56 acetylation on genes related to phenotypic switching, biofilm formation and cell aggregation. Direct and indirect regulation also involves genes related to cell wall protein biosynthesis, β-glucan and mannan exposure, and hydrolytic secreted enzymes, supporting the hypothesis that the fluctuations of H3K56 acetylation in C. albicans might impair the macrophage response to the yeast and thus promote the host-immune escaping., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work described in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Candida albicans is an opportunistic fungal pathogen known for surviving in various nutrient-limited conditions within the host and causing infections. Our prior research revealed that Hfl1p, an archaeal histone-like or Hap5-like protein, is linked to mitochondrial ATP generation and yeast-hyphae morphogenesis. However, the specific roles of Hfl1p in these virulence behaviours, through its function in the CBF/NF-Y complex or as a DNA polymerase II subunit, remain unclear. This study explores Hfl1p's diverse functions in energy metabolism and morphogenesis. By combining proteomic analysis and phenotypic evaluations of the hfl1Δ/hfl1Δ mutant with ChIP data, we found that Hfl1p significantly impacts mitochondrial DNA-encoded CI subunits, the tricarboxylic acid (TCA) cycle, and morphogenetic pathways. This influence occurs either independently or alongside other transcription factors recognizing a conserved DNA motif (TAXXTAATTA). These findings emphasize Hfl1p's critical role in linking carbon metabolism and mitochondrial respiration to the yeast-to-filamentous form transition, enhancing our understanding of C. albicans ' metabolic adaptability during morphological transition, an important pathogenic trait of this fungus. This could help identify therapeutic targets by disrupting the relationship between energy metabolism and cell morphology in C. albicans.

Ketoconazole is a classical antifungal drug commonly used in the clinic. With the increased use of ketoconazole in recent years, an increasing number of drug-resistant strains have emerged during clinical treatment. It is well known that fungi acquire drug resistance in multiple ways, while the molecular mechanisms underlying ketoconazole resistance remain for comprehensive exploration. In this study, we found that the expression of the small plasma membrane protein-encoding gene PMP3 was significantly down-regulated in several clinically isolated ketoconazole-resistant strains, indicating the relationship between PMP3 expression and ketoconazole resistance. By knocking out the PMP3, we found that the absence of the Pmp3 resulted in a significant increase in resistance of Candida albicans to ketoconazole, which was also confirmed in a systemic infection model in mice. We further demonstrated that various physiological properties, such as cell membrane fluidity, plasma membrane potential, permeability and ergosterol distribution were altered in the pmp3Δ/Δ mutant, which is associated with the enhanced cellular resistance to ketoconazole. In addition, overexpression rather than deletion of PMP3 alters the hyphal development and biofilm formation capacity in C. albicans. This study reveals the contribution of Pmp3 to alteration of drug resistance in fungal pathogens, which may guide the development of novel antifungal strategies., Competing Interests: Conflict of Interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Candida albicans , a part of normal flora, is an opportunistic fungal pathogen and causes severe health issues in immunocompromised patients. Its pathogenicity is intricately linked to the transcriptional regulation of its metabolic pathways. Paf1 complex (Paf1C) is a crucial transcriptional regulator that is highly conserved in eukaryotes. The objective of this study was to explore the role of Paf1C in the metabolic pathways and how it influences the pathogenicity of C. albicans . Paf1C knockout mutant strains of C. albicans ( ctr9 Δ/Δ, leo1 Δ/Δ, and cdc73 Δ/Δ) were generated using the CRISPR-Cas9 system. To investigate the effect of Paf1C on pathogenicity, macrophage interaction assays and mouse survival tests were conducted. The growth patterns of the Paf1C knockout mutants were analyzed through spotting assays and growth curve measurements. Transcriptome analysis was conducted under yeast conditions (30°C without serum) and hyphal conditions (37°C with 10% FBS), to further elucidate the role of Paf1C in the pathogenicity of C. albicans . CTR9 deletion resulted in the attenuation of C. albicans virulence, in macrophage and mouse models. Furthermore, we confirmed that the reduced virulence of the ctr9 Δ/Δ mutant can be attributed to a decrease in C. albicans cell abundance. Moreover, transcriptome analysis revealed that metabolic processes required for cell proliferation are impaired in ctr9 Δ/Δ mutant. Notably, CTR9 deletion led to the downregulation of methionine biosynthetic genes and the cAMP-PKA signaling pathway-related hypha essential genes, which are pivotal for virulence. Our results suggest that Ctr9-regulated methionine metabolism is a crucial factor for determining C. albicans pathogenicity.

Geosmithia pallida (KU693285) was isolated from Brucea mollis an endangered medicinal plant of North-East India. The secondary metabolites, produced by the endophytic fungi, extracted by ethyl acetate were screened for antimicrobial activity. G. pallida extract displayed the highest antimicrobial activity against Candida albicans with a minimum inhibitory concentration of 80.5 ± 1.25 µg/mL. G. pallida also showed the highest antioxidant activity which differed insignificantly from Penicillium sp. ( P  > 0.05). The G. pallida extract also exhibited the highest cellulase activity and also amylase and protease activities. The cytotoxicity assay of the ethyl acetate extract of this endophyte showed negligible effect (1.93 ± 0.42%) on chromosomal aberration as compared to the control (cyclophosphamide monohydrate) (7.20 ± 1.51%). The internal transcribed spacer rDNA sequence of G. pallida was submitted to the NCBI (Accession number KU693285) from India for the first time. The FT-IR spectrophotometry of the bioactive metabolite of G. pallida showed the presence of different functional groups such as alcohol, carboxylic acids, amines, aromatics, alkyl halides, aliphatic amines and alkynes. The GC-MS analysis revealed the presence of acetic acid, 2-phenylethyl ester; tetracosane; cyclooctasiloxane hexadecamethyl; cyclononasiloxane octadecamethyl; octadecanoic acid; phthalic acid, di(2-propylpentyl) ester and nonadecane, 2,6,10,14,18-pentamethyl as the major compounds in the metabolite. The findings of the present work indicated G. pallida as a potential source of important biomolecules without mammalian cytotoxic effects, which can be utilized for pharmaceutical purposes.

Our immune system possesses sophisticated mechanisms to cope with invading microorganisms, while pathogens evolve strategies to deal with threats imposed by host immunity. Human plasma protein α1-antitrypsin (AAT) exhibits pleiotropic immune-modulating properties by both preventing immunopathology and improving antimicrobial host defence. Genetic associations suggested a role for AAT in candidemia, the most frequent fungal blood stream infection in intensive care units, yet little is known about how AAT influences interactions between Candida albicans and the immune system. Here, we show that AAT differentially impacts fungal killing by innate phagocytes. We observed that AAT induces fungal transcriptional reprogramming, associated with cell wall remodelling and downregulation of filamentation repressors. At low concentrations, the cell-wall remodelling induced by AAT increased immunogenic β-glucan exposure and consequently improved fungal clearance by monocytes. Contrastingly, higher AAT concentrations led to excessive C. albicans filamentation and thus promoted fungal immune escape from monocytes and macrophages. This underscores that fungal adaptations to the host protein AAT can differentially define the outcome of encounters with innate immune cells, either contributing to improved immune recognition or fungal immune escape.

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 ., Competing Interests: L.E.C. is a co-founder and shareholder in Bright Angel Therapeutics, a platform company for the development of novel antifungal therapeutics. L.E.C. is a science advisor for Kapoose Creek, a company that harnesses the therapeutic potential of fungi. All other authors have no conflicts of interest to declare.

Candida albicans, an opportunistic fungal pathogen, produces the quorum-sensing molecule farnesol, which we have shown alters the transcriptional response and phenotype of human monocyte-derived dendritic cells (DCs), including their cytokine secretion and ability to prime T cells. This is partially dependent on the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ), which has numerous ligands, including the sphingolipid metabolite sphingosine 1-phosphate. Sphingolipids are a vital component of membranes that affect membrane protein arrangement and phagocytosis of C. albicans by DCs. Thus, we quantified sphingolipid metabolites in monocytes differentiating into DCs by High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Farnesol increased the activity of serine palmitoyltransferase, leading to increased levels of 3-keto-dihydrosphingosine, dihydrosphingosine, and dihydrosphingosine 1-phosphate and inhibited dihydroceramide desaturase by inducing oxidative stress, leading to increased levels of dihydroceramide and dihydrosphingomyelin species and reduced ceramide levels. Accumulation of dihydroceramides can inhibit mitochondrial function; accordingly, farnesol reduced mitochondrial respiration. Dihydroceramide desaturase inhibition increases lipid droplet formation, which we observed in farnesol-treated cells, coupled with an increase in intracellular triacylglycerol species. Furthermore, inhibition of dihydroceramide desaturase with either farnesol or specific inhibitors impaired the ability of DCs to prime interferon-γ-producing T cells. The effect of farnesol on sphingolipid metabolism, triacylglycerol synthesis, and mitochondrial respiration was not dependent on PPAR-γ. In summary, our data reveal novel effects of farnesol on sphingolipid metabolism, neutral lipid synthesis, and mitochondrial function in DCs that affect their instruction of T cell cytokine secretion, indicating that C. albicans can manipulate host cell metabolism via farnesol secretion.IMPORTANCE Candida albicans is a common commensal yeast, but it is also an opportunistic pathogen which is one of the leading causes of potentially lethal hospital-acquired infections. There is growing evidence that its overgrowth in the gut can influence diseases as diverse as alcohol-associated liver disease and COVID-19. Previously, we found that its quorum-sensing molecule, farnesol, alters the phenotype of dendritic cells differentiating from monocytes, impairing their ability to drive protective T cell responses. Here, we demonstrate that farnesol alters the metabolism of sphingolipids, important structural components of the membrane that also act as signaling molecules. In monocytes differentiating to dendritic cells, farnesol inhibited dihydroceramide desaturase, resulting in the accumulation of dihydroceramides and a reduction in ceramide levels. Farnesol impaired mitochondrial respiration, known to occur with an accumulation of dihydroceramides, and induced the accumulation of triacylglycerol and oil bodies. Inhibition of dihydroceramide desaturase resulted in the impaired ability of DCs to induce interferon-γ production by T cells. Thus, farnesol production by C. albicans could manipulate the function of dendritic cells by altering the sphingolipidome., Competing Interests: The authors declare no conflict of interest.

Antibiotic-induced loss of intestinal hypoxia boosts the growth of C. albicans in mice.

Phosphorus is essential in all cells' structural, metabolic and regulatory functions. For fungal cells that import inorganic phosphate (Pi) up a steep concentration gradient, surface Pi transporters are critical capacitators of growth. Fungi must deploy Pi transporters that enable optimal Pi uptake in pH and Pi concentration ranges prevalent in their environments. Single, triple and quadruple mutants were used to characterize the four Pi transporters we identified for the human fungal pathogen Candida albicans, which must adapt to alkaline conditions during invasion of the host bloodstream and deep organs. A high-affinity Pi transporter, Pho84, was most efficient across the widest pH range while another, Pho89, showed high-affinity characteristics only within one pH unit of neutral. Two low-affinity Pi transporters, Pho87 and Fgr2, were active only in acidic conditions. Only Pho84 among the Pi transporters was clearly required in previously identified Pi-related functions including Target of Rapamycin Complex 1 signaling, oxidative stress resistance and hyphal growth. We used in vitro evolution and whole genome sequencing as an unbiased forward genetic approach to probe adaptation to prolonged Pi scarcity of two quadruple mutant lineages lacking all 4 Pi transporters. Lineage-specific genomic changes corresponded to divergent success of the two lineages in fitness recovery during Pi limitation. Initial, large-scale genomic alterations like aneuploidies and loss of heterozygosity eventually resolved, as populations gained small-scale mutations. Severity of some phenotypes linked to Pi starvation, like cell wall stress hypersensitivity, decreased in parallel to evolving populations' fitness recovery in Pi scarcity, while severity of others like membrane stress responses diverged from Pi scarcity fitness. Among preliminary candidate genes for contributors to fitness recovery, those with links to TORC1 were overrepresented. Since Pi homeostasis differs substantially between fungi and humans, adaptive processes to Pi deprivation may harbor small-molecule targets that impact fungal growth, stress resistance and virulence., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Acosta-Zaldívar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 O’Connor-Moneley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Because of the decreasing supply of new antibiotics, recent outbreaks of infectious diseases, and the emergence of antibiotic-resistant microorganisms, it is imperative to develop new effective strategies for deactivating a broad spectrum of microorganisms and viruses. We have implemented electrically polarized nanoscale metallic (ENM) coatings that deactivate a wide range of microorganisms including Gram-negative and Gram-positive bacteria with greater than 6-log reduction in less than 10 minutes of treatment. The electrically polarized devices were also effective in deactivating lentivirus and Candida albicans . The key to the high deactivation effectiveness of ENM devices is electrochemical production of micromolar cuprous ions, which mediated reduction of oxygen to hydrogen peroxide. Formation of highly damaging species, hydroxyl radicals and hypochlorous acid, from hydrogen peroxide contributed to antimicrobial properties of the ENM devices. The electric polarization of nanoscale coatings represents an unconventional tool for deactivating a broad spectrum of microorganisms through in situ production of reactive oxygenated and chlorinated species.

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 sef1 Δ/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 sef1 Δ/Δ mutation on cell wall integrity varies, and for the two strains examined the phenotype correlated with sef1 Δ/Δ impact on several cell wall integrity genes. (iv) Phenotype discovery: DNA repair genes were induced modestly by iron limitation in sef1 Δ/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 sef1 Δ/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 ., Competing Interests: The authors declare no conflict of interest.

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-3β,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., Competing Interests: Dr. MDP, M.D., is a Co-Founder and Chief Scientific Officer (CSO) of MicroRid Technologies Inc. The goal of MicroRid Technologies Inc. is to develop new antifungal agents for therapeutic use. All other authors declare no competing interests., (Copyright: © 2024 Zhou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 3 H-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 (F i ). 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 (F i ). 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., Competing Interests: The authors declare no conflict of interest.

Energy metabolism is crucial for all living cells, especially during fast growth or stress scenarios. Many cancer and activated immune cells (Warburg effect) or yeasts (Crabtree effect) mostly rely on aerobic glucose fermentation leading to lactate or ethanol, respectively, to generate ATP. In recent years, several mathematical models have been proposed to explain the Warburg effect on theoretical grounds. Besides glucose, glutamine is a very important substrate for eukaryotic cells-not only for biosynthesis, but also for energy metabolism. Here, we present a minimal constraint-based stoichiometric model for explaining both the classical Warburg effect and the experimentally observed respirofermentation of glutamine (WarburQ effect). We consider glucose and glutamine respiration as well as the respective fermentation pathways. Our resource allocation model calculates the ATP production rate, taking into account enzyme masses and, therefore, pathway costs. While our calculation predicts glucose fermentation to be a superior energy-generating pathway in human cells, different enzyme characteristics in yeasts reduce this advantage, in some cases to such an extent that glucose respiration is preferred. The latter is observed for the fungal pathogen Candida albicans, which is a known Crabtree-negative yeast. Further, optimization results show that glutamine is a valuable energy source and important substrate under glucose limitation, in addition to its role as a carbon and nitrogen source of biomass in eukaryotic cells. In conclusion, our model provides insights that glutamine is an underestimated fuel for eukaryotic cells during fast growth and infection scenarios and explains well the observed parallel respirofermentation of glucose and glutamine in several cell types., (© 2024. The Author(s).)

Candida albicans, a significant human pathogenic fungus, employs hydrolytic proteases for host invasion. Conventional antifungal agents are reported with resistance issues from around the world. This study investigates the role of Bacillus licheniformis extracellular proteins (ECP) as effective antifungal peptides (AFPs). The aim was to identify and characterize the ECP of B. licheniformis through LC-MS/MS and bioinformatics analysis. LC-MS/MS analysis identified 326 proteins with 69 putative ECP, further analyzed in silico. Of these, 21 peptides exhibited antifungal properties revealed by classAMP tool and are predominantly anionic. Peptide-protein docking revealed interactions between AFPs like Peptide chain release factor 1 (Q65DV1_Seq1: SASEQLSDAK) and Putative carboxy peptidase (Q65IF0_Seq7: SDSSLEDQDFILESK) with C. albicans virulent SAP5 proteins (PDB ID 2QZX), forming hydrogen bonds and significant Pi-Pi interactions. The identification of B. licheniformis ECP is the novelty of the study that sheds light on their antifungal potential. The identified AFPs, particularly those interacting with bonafide pharmaceutical targets SAP5 of C. albicans represent promising avenues for the development of antifungal treatments with AFPs that could be the pursuit of a novel therapeutic strategy against C. albicans. SIGNIFICANCE OF STUDY: The purpose of this work was to carry out proteomic profiling of the secretome of B. licheniformis. Previously, the efficacy of Bacillus licheniformis extracellular proteins against Candida albicans was investigated and documented in a recently communicated manuscript, showcasing the antifungal activity of these proteins. In order to achieve high-throughput identification of ES (Excretory-secretory) proteins, the utilization of liquid chromatography tandem mass spectrometry (LC-MS) was utilized. There was a lack of comprehensive research on AFPs in B. licheniformis, nevertheless. The proteins secreted by B. licheniformis in liquid medium were initially discovered using liquid chromatography-tandem mass spectrometry (LC-MS) analysis and identification in order to immediately characterize the unidentified active metabolites in fermentation broth., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript., (Copyright © 2024. Published by Elsevier B.V.)

In human pathogenic fungi, receiver domains from hybrid histidine kinases (hHK) have to recognize one HPt. To understand the recognition mechanism, we have assessed phosphorelay from receiver domains of five hHKs of group III, IV, V, VI, and XI to HPt from Chaetomium thermophilum and obtained the structures of Ct_HPt alone and in complex with the receiver domain of hHK group VI. Our data indicate that receiver domains phosphotransfer to Ct_HPt, show a low affinity for complex formation, and prevent a Leu-Thr switch to stabilize phosphoryl groups, also derived from the structures of the receiver domains of hHK group III and Candida albicans Sln1. Moreover, we have elucidated the envelope structure of C. albicans Ypd1 using small-angle X-ray scattering which reveals an extended flexible conformation of the long loop αD-αE which is not involved in phosphotransfer. Finally, we have analyzed the role of salt bridges in the structure of Ct_HPt alone., (© 2024. The Author(s).)

Current microbiological methods for pneumonia diagnosis require invasive specimen collection and time-consuming analytical procedures. There is a need for less invasive and faster methods to detect lower respiratory tract infections. The analysis of volatile metabolites excreted by pathogenic microorganisms provides the basis for developing such a method. Given the synergistic role of Candida albicans in increasing the virulence of pathogenic bacteria causing pneumonia and the cross-kingdom metabolic interactions between microorganisms, we compare the emission of volatiles from Candida albicans yeasts and the bacteria Staphylococcus aureus using single and mixed co-cultures and apply that knowledge to human in vivo investigations. Gas chromatography-mass spectrometry (GC-MS) analysis resulted in the identification of sixty-eight volatiles that were found to have significantly different levels in cultures compared to reference medium samples. Certain volatiles were found in co-cultures that mainly originated from C. albicans metabolism (e.g., isobutyl acetate), whereas other volatiles primarily came from S. aureus (e.g., ethyl 2-methylbutyrate). Isopentyl valerate reflects synergic interactions of both microbes, as its level in co-cultures was found to be approximately three times higher than the sum of its amounts in monocultures. Hydrophilic-lipophilic-balanced (HLB) coated meshes for thin-film microextraction (TFME) were used to preconcentrate volatiles directly from bronchoalveolar lavage (BAL) specimens collected from patients suffering from ventilation-associated pneumonia (VAP), which was caused explicitly by C. albicans and S. aureus . GC-MS analyses confirmed the existence of in vitro-elucidated microbial VOCs in human specimens. Significant differences in BAL-extracted amounts respective to the pathogen-causing pneumonia were found. The model in vitro experiments provided evidence that cross-kingdom interactions between pathogenic microorganisms affect the synthesis of volatile compounds. The TFME meshes coated with HLB particles proved to be suitable for extracting VOCs from human material, enabling the translation of in vitro experiments on the microbial volatilome to the in vivo situation involving infected patients. This indicates the direction that should be taken for further clinical studies on VAP diagnosis based on volatile analysis.

Lovastatin has received interest for its potential therapeutic use in treating numerous diseases, for example, the blood cholesterol level by restraining hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The research utilized the fungal growth bioassay technique to disengage and evaluate filamentous organism for the lovastatin creation. The clever type of Aspergillus terreus (KF971363.1) was embraced for lovastatin creation by solid-state fermentation (SSF). Lovastatin production was optimized using physiological parameters such as pH and temperature at SSF. The addition of nitrogen source enhanced the production of lovastatin by the breakdown of lignocellulose that improved the production of lovastatin. The research verified a yeast growth inhibition bioassay approach, in addition to thin-layer chromatography and liquid chromatography-mass spectrometry (LC-MS). All of these techniques were used to confirm lovastatin production. The purified extract subjected to the TLC analysis showed retention factor (Rf) value of 0.73. Moreover, the inhibition bioassay method reassures the lovastatin production by comparing the zone of inhibition against C. albicans.

The human pathogenic fungus Candida albicans damages epithelial cells during superficial infections. Here we use three-dimensional-sequential-confocal Raman spectroscopic imaging and atomic force microscopy to investigate the interaction of C. albicans wild type cells, the secreted C. albicans peptide toxin candidalysin and mutant cells lacking candidalysin with epithelial cells. The candidalysin is responsible for epithelial cell damage and exhibits in its deuterated form an identifiable Raman signal in a frequency region distinct from the cellular frequency region. Vibration modes at 2100-2200 cm -1 attributed to carbon‑deuterium bending and at 477 cm -1 , attributed to the nitrogen‑deuterium out-of-plane bending, found around the nucleus, can be assigned to deuterated candidalysin. Atomic force microscopy visualized 100 nm deep lesions on the cell and force-distance curves indicate the higher adhesion on pore surrounding after incubation with candidalysin. Candidalysin targets the plasma membrane, but is also found inside of the cytosol of epithelial cells during C. albicans infection., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

The human fungal pathogen Candida albicans can cause lethal systemic infections due to its ability to resist stress from the host and to undergo invasive hyphal growth. Previous studies showed that plasma membrane MCC/eisosome domains were important for virulence by promoting the ability of Sur7 to mediate normal cell wall morphogenesis and stress resistance. The sur7Δ mutant displayed abnormal clusters of PI 4,5 P 2 , suggesting that misregulation of this lipid underlies the sur7Δ phenotype. To test this, we increased PI 4,5 P 2 levels by deleting combinations of the three PI 4,5 P 2 5' phosphatase genes ( INP51 , INP52 , and INP54 ) and found that some combinations, such as inp51Δ inp52Δ , gave phenotypes similar the sur7Δ mutant. In contrast, deleting one copy of MSS4 , the gene that encodes the 5' kinase needed to create PI 4,5 P 2 , reduced the abnormal PI 4,5 P 2 clusters and also decreased the abnormal cell wall and stress sensitive phenotypes of the sur7Δ mutant. Additional studies support a model that the abnormal PI 4,5 P 2 patches recruit septin proteins, which in turn promote aberrant cell wall growth. These results identify Sur7 as a novel regulator of PI 4,5 P 2 and highlight the critical role of PI 4,5 P 2 in the regulation of C. albicans virulence properties.

Candida albicans is an opportunistic fungal pathogen that can switch between yeast and hyphal morphologies depending on the environmental cues it receives. The switch to hyphal form is crucial for the establishment of invasive infections. The hyphal form is also characterized by the cell surface expression of hyphae-specific proteins, many of which are GPI-anchored and important determinants of its virulence. The coordination between hyphal morphogenesis and the expression of GPI-anchored proteins is made possible by an interesting cross-talk between GPI biosynthesis and the cAMP-PKA signaling cascade in the fungus; a parallel interaction is not found in its human host. On the other hand, in the nonpathogenic yeast, Saccharomyces cerevisiae, GPI biosynthesis is shut down when filamentation is activated and vice versa. This too is achieved by a cross-talk between GPI biosynthesis and cAMP-PKA signaling. How are diametrically opposite effects obtained from the cross-talk between two reasonably well-conserved pathways present ubiquitously across eukarya? This Review attempts to provide a model to explain these differences. In order to do so, it first provides an overview of the two pathways for the interested reader, highlighting the similarities and differences that are observed in C. albicans versus the well-studied S. cerevisiae model, before going on to explain how the different mechanisms of regulation are effected. While commonalities enable the development of generalized theories, it is hoped that a more nuanced approach, that takes into consideration species-specific differences, will enable organism-specific understanding of these processes and contribute to the development of targeted therapies., Competing Interests: Conflict of interest The author declares that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Guo, Zheng, Dong, Wang, Deng, Wang and Xu.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fungi and bacteria coexist in many polymicrobial communities, yet the molecular basis of their interactions remains poorly understood. Here, we show that the fungus Candida albicans sequesters essential magnesium ions from the bacterium Pseudomonas aeruginosa. To counteract fungal Mg2+ sequestration, P. aeruginosa expresses the Mg2+ transporter MgtA when Mg2+ levels are low. Thus, loss of MgtA specifically impairs P. aeruginosa in co-culture with C. albicans, but fitness can be restored by supplementing Mg2+. Using a panel of fungi and bacteria, we show that Mg2+ sequestration is a general mechanism of fungal antagonism against gram-negative bacteria. Mg2+ limitation enhances bacterial resistance to polymyxin antibiotics like colistin, which target gram-negative bacterial membranes. Indeed, experimental evolution reveals that P. aeruginosa evolves C. albicans-dependent colistin resistance via non-canonical means; antifungal treatment renders resistant bacteria colistin-sensitive. Our work suggests that fungal-bacterial competition could profoundly impact polymicrobial infection treatment with antibiotics of last resort., Competing Interests: HSM is a member of the PLOS Biology Editorial Board., (Copyright: © 2024 Hsieh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The gut microbiota is closely linked to atherosclerosis. However, the role of intestinal fungi, essential members of the complex microbial community, in atherosclerosis is poorly understood. Herein, we show that gut fungi dysbiosis is implicated in patients with dyslipidemia, characterized by higher levels of Candida albicans (C. albicans), which are positively correlated with plasma total cholesterol and low-density lipoprotein-cholesterol (LDL-C) levels. Furthermore, C. albicans colonization aggravates atherosclerosis progression in a mouse model of the disease. Through gain- and loss-of-function studies, we show that an intestinal hypoxia-inducible factor 2α (HIF-2α)-ceramide pathway mediates the effect of C. albicans. Mechanistically, formyl-methionine, a metabolite of C. albicans, activates intestinal HIF-2α signaling, which drives increased ceramide synthesis to accelerate atherosclerosis. Administration of the HIF-2α selective antagonist PT2385 alleviates atherosclerosis in mice by reducing ceramide levels. Our findings identify a role for intestinal fungi in atherosclerosis progression and highlight the intestinal HIF-2α-ceramide pathway as a target for atherosclerosis treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Candida albicans, a prominent member of the human microbiome, can make an opportunistic switch from commensal coexistence to pathogenicity accompanied by an epigenetic shift between the white and opaque cell states. This transcriptional switch is under precise regulation by a set of transcription factors (TFs), with Enhanced Filamentous Growth Protein 1 (Efg1) playing a central role. Previous research has emphasized the importance of Efg1's prion-like domain (PrLD) and the protein's ability to undergo phase separation for the white-to-opaque transition of C. albicans. However, the underlying molecular mechanisms of Efg1 phase separation have remained underexplored. In this study, we delved into the biophysical basis of Efg1 phase separation, revealing the significant contribution of both N-terminal (N) and C-terminal (C) PrLDs. Through NMR structural analysis, we found that Efg1 N-PrLD and C-PrLD are mostly disordered but have prominent partial α-helical secondary structures in both domains. NMR titration experiments suggest that the partially helical structures in N-PrLD act as hubs for self-interaction as well as Efg1 interaction with RNA. Using condensed-phase NMR spectroscopy, we uncovered diverse amino acid interactions underlying Efg1 phase separation. Particularly, we highlight the indispensable role of tyrosine residues within the transient α-helical structures of PrLDs particularly in the N-PrLD compared to the C-PrLD in stabilizing phase separation. Our study provides evidence that the transient α-helical structure is present in the phase-separated state and highlights the particular importance of aromatic residues within these structures for phase separation. Together, these results enhance the understanding of C. albicans transcription factor interactions that lead to virulence and provide a crucial foundation for potential antifungal therapies targeting the transcriptional switch., Competing Interests: Declaration of interests N.L.F. is a consultant for Dewpoint Therapeutics and Confluence Therapeutics., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Candida albicans ( C. albicans ) and Lactiplantibacillus plantarum subsp. plantarum ( L. plantarum ) are frequently identified in various niches, but their dual-species interaction, especially with C. albicans in yeast form, remains unclear. This study aimed to investigate the dual-species interaction of L. plantarum and C. albicans , including proliferation, morphology, and transcriptomes examined by selective agar plate counting, microscopy, and polymicrobial RNA-seq, respectively. Maintaining a stable and unchanged growth rate, L. plantarum inhibited C. albicans yeast cell proliferation but not hyphal growth. Combining optical microscopy and atomic force microscopy, cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed during dual-species interaction. Reduced C. albicans yeast cell proliferation in mixed culture was partially due to L. plantarum cell-free culture supernatant but not the acidic environment. Upon polymicrobial transcriptomics analysis, interesting changes were identified in both L. plantarum and C. albicans gene expression. First, two L. plantarum quorum-sensing systems showed contrary changes, with the activation of lamBDCA and repression of luxS . Second, the upregulation of stress response-related genes and downregulation of cell cycle, cell survival, and cell integrity-related pathways were identified in C. albicans , possibly connected to the stress posed by L. plantarum and the reduced yeast cell proliferation. Third, a large scale of pathogenesis and virulence factors were downregulated in C. albicans , indicating the potential interruption of pathogenic activities by L. plantarum . Fourth, partial metabolism and transport pathways were changed in L. plantarum and C. albicans . The information in this study might aid in understanding the behavior of L. plantarum and C. albicans in dual-species interaction.IMPORTANCEThe anti- Candida albicans activity of Lactiplantibacillus plantarum has been explored in the past decades. However, the importance of C. albicans yeast form and the effect of C. albicans on L. plantarum had also been omitted. In this study, the dual-species interaction of L. plantarum and C. albicans was investigated with a focus on the transcriptomes. Cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed. Upon polymicrobial transcriptomics analysis, interesting changes were identified, including contrary changes in two L. plantarum quorum-sensing systems and reduced cell survival-related pathways and pathogenesis determinants in C. albicans ., Competing Interests: The authors declare no conflict of interest.

Liver disease, a major health concern worldwide, is a serious and progressive disorder. Herein, we not only established a mouse model of DEN+CCl 4 -induced primary liver disease but also collected clinical human samples to investigate longitudinal alterations in the gut mycobiome. As liver disease advanced, gut integrity was disrupted, and the mycobiota was disturbed in the mouse models. The metabolites associated with hepatocellular carcinoma (HCC) differed from those associated with the cirrhotic phase as follows: levels of stercobilin and aflatoxin B1 dialcohol were reduced, while levels of triterpenoids, bafilomycin A1, and DHEA were increased in the HCC group. The abundance of the phylum Chytridiomycota increased as the chronic liver disease progressed and was then replaced by the phylum Ascomycota in HCC. Based on the results from clinical human samples, the genus Candida (Ascomycota) (in humans) and the genus Kazachstania (Ascomycota) (in mice) occupied a dominant position in the HCC group, while other fungi were depleted. The increased abundance of C. albicans and depletion of S. cerevisiae may be hallmarks of the progression of liver cirrhosis to early HCC. Moreover, the administration of C. albicans and S. cerevisiae in the LC-HCC progression could accelerate or retard the progression of HCC. Therefore, gut fungi have the potential to serve as a noninvasive clinical biomarker and even a treatment method., (© 2024. The Author(s).)

Candida albicans is a commensal fungus that can cause epithelial infections and life-threatening invasive candidiasis. The fungus secretes candidalysin (CL), a peptide that causes cell damage and immune activation by permeation of epithelial membranes. The mechanism of CL action involves strong peptide assembly into polymers in solution. The free ends of linear CL polymers can join, forming loops that become pores upon binding to membranes. CL polymers constitute a therapeutic target for candidiasis, but little is known about CL self-assembly in solution. Here, we examine the assembly mechanism of CL in the absence of membranes using complementary biophysical tools, including a new fluorescence polymerization assay, mass photometry, and atomic force microscopy. We observed that CL assembly is slow, as tracked with the fluorescent marker C-laurdan. Single-molecule methods showed that CL polymerization involves a convolution of four processes. Self-assembly begins with the formation of a basic subunit, thought to be a CL octamer that is the polymer seed. Polymerization proceeds via the addition of octamers, and as polymers grow they can curve and form loops. Alternatively, secondary polymerization can occur and cause branching. Interplay between the different rates determines the distribution of CL particle types, indicating a kinetic control mechanism. This work elucidates key physical attributes underlying CL self-assembly which may eventually evoke pharmaceutical development., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The human microbiome contains genetic information that regulates metabolic processes in response to host health and disease. While acidic vaginal pH is maintained in normal conditions, the pH level increases in infectious vaginitis. We propose that this change in the vaginal environment triggers the biosynthesis of anti-vaginitis metabolites. Gene expression levels of Chryseobacterium gleum, a vaginal symbiotic bacterium, were found to be affected by pH changes. The distinctive difference in the metabolic profiles between two C. gleum cultures incubated under acidic and neutral pH conditions was suggested to be an anti-vaginitis molecule, which was identified as phenylacetic acid (PAA) by spectroscopic data analysis. The antimicrobial activity of PAA was evaluated in vitro, showing greater toxicity toward Gardnerella vaginalis and Candida albicans, two major vaginal pathogens, relative to commensal Lactobacillus spp. The activation of myeloperoxidase, prostaglandin E 2 , and nuclear factor-κB, and the expression of cyclooxygenase-2 were reduced by an intravaginal administration of PAA in the vaginitis mouse model. In addition, PAA displayed the downregulation of mast cell activation. Therefore, PAA was suggested to be a messenger molecule that mediates interactions between the human microbiome and vaginal health., (© 2024. The Author(s).)

Candida albicans (C. albicans) can behave as a commensal yeast colonizing the vaginal mucosa, and in this condition is tolerated by the epithelium. When the epithelial tolerance breaks down, due to C. albicans overgrowth and hyphae formation, the generated inflammatory response and cell damage lead to vulvovaginal candidiasis (VVC) symptoms. Here, we focused on the induction of mitochondrial reactive oxygen species (mtROS) in vaginal epithelial cells after C. albicans infection and the involvement of fungal burden, morphogenesis and candidalysin (CL) production in such induction. Bioluminescent (BLI) C. albicans, C. albicans PCA-2 and C. albicans 529L strains were employed in an in vitro infection model including reconstituted vaginal epithelium cells (RVE), produced starting from A-431 cell line. The production of mtROS was kinetically measured by using MitoSOX™ Red probe. The potency of C. albicans to induced cell damage to RVE and C. albicans proliferation have also been evaluated. C. albicans induces a rapid mtROS release from vaginal epithelial cells, in parallel with an increase of the fungal load and hyphal formation. Under the same experimental conditions, the 529L C. albicans strain, known to be defective in CL production, induced a minor mtROS release showing the key role of CL in causing epithelial mithocondrial activation. C. albicans PCA-2, unable to form hyphae, induced comparable but slower mtROS production as compared to BLI C. albicans yeasts. By reducing mtROS through a ROS scavenger, an increased fungal burden was observed during RVE infection but not in fungal cultures grown on abiotic surface. Collectively, we conclude that CL, more than fungal load and hyphae formation, seems to play a key role in the rapid activation of mtROS by epithelial cells and in the induction of cell-damage and that mtROS are key elements in the vaginal epithelial cells response to C. albicans., Competing Interests: The Authors have declared that no competing interests exist., (Copyright: © 2024 Spaggiari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The transition between yeast and hyphae is crucial for regulating the commensalism and pathogenicity in Candida albicans. The mechanisms that affect the invasion of hyphae in solid media, whose deficiency is more related to the pathogenicity of C. albicans, have not been elucidated. Here, we found that the disruption of VAM6 or VPS41 which are components of the homotypic vacuolar fusion and protein sorting (HOPS) complex, or the Rab GTPase YPT72, all responsible for vacuole fusion, led to defects in hyphal growth in both liquid and solid media, but more pronounced on solid agar. The phenotypes of vac8Δ/Δ and GTR1 OE -vam6Δ/Δ mutants indicated that these deficiencies are mainly caused by the reduced mechanical forces that drive agar and organs penetration, and confirmed that large vacuoles are required for hyphal mechanical penetration. In summary, our study revealed that large vacuoles generated by vacuolar fusion support hyphal penetration and provided a perspective to refocus attention on the role of solid agar in evaluating C. albicans invasion., (© 2024. The Author(s).)

Biofilm formation by the fungal pathogen Candida albicans is the basis for its ability to infect medical devices. The metabolic gene ERG251 has been identified as a target of biofilm transcriptional regulator Efg1, and here we report that ERG251 is required for biofilm formation but not conventional free-living planktonic growth. An erg251Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo catheter infection model. In both in vitro and in vivo biofilm contexts, cell number is reduced and hyphal length is limited. To determine whether the mutant defect is in growth or some other aspect of biofilm development, we examined planktonic cell features in a biofilm-like environment, which was approximated with sealed unshaken cultures. Under those conditions, the erg251Δ/Δ mutation causes defects in growth and hyphal extension. Overexpression in the erg251Δ/Δ mutant of the paralog ERG25, which is normally expressed more weakly than ERG251, partially improves biofilm formation and biofilm hyphal content, as well as growth and hyphal extension in a biofilm-like environment. GC-MS analysis shows that the erg251Δ/Δ mutation causes a defect in ergosterol accumulation when cells are cultivated under biofilm-like conditions, but not under conventional planktonic conditions. Overexpression of ERG25 in the erg251Δ/Δ mutant causes some increase in ergosterol levels. Finally, the hypersensitivity of efg1Δ/Δ mutants to the ergosterol inhibitor fluconazole is reversed by ERG251 overexpression, arguing that reduced ERG251 expression contributes to this efg1Δ/Δ phenotype. Our results indicate that ERG251 is required for biofilm formation because its high expression levels are necessary for ergosterol synthesis in a biofilm-like environment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Xiong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Microorganisms resist fluoride toxicity using fluoride export proteins from one of several different molecular families. Cariogenic species Streptococcus mutans and Candida albicans extrude intracellular fluoride using a CLC F F - /H + antiporter and FEX fluoride channel, respectively, whereas oral commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel. In this work, we examine how genetic knockout of fluoride export impacts pathogen fitness in single-species and three-species dental biofilm models. For biofilms generated using S. mutans with the genetic knockout of the CLC F transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans , increased the relative proportion of oral commensal S. gordonii , and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution. Biofilms prepared with C. albicans with genetic knockout of the FEX channel also exhibited reduced fitness in the presence of fluoride but to a lesser degree. Imaging studies indicate that S. mutans is highly sensitive to fluoride, with the knockout strain undergoing complete lysis when exposed to low fluoride for a moderate amount of time. Biochemical purification of the S. mutans CLC F transporter and functional reconstitution establishes that the functional protein is a dimer encoded by a single gene. Together, these findings suggest that fluoride export by oral pathogens can be targeted by specific inhibitors to restore biofilm symbiosis in dental biofilms and that S. mutans is especially susceptible to fluoride toxicity., Importance: Dental caries is a globally prevalent condition that occurs when pathogenic species, including Streptococcus mutans and Candida albicans , outcompete beneficial species, such as Streptococcus gordonii , in the dental biofilm. Fluoride is routinely used in oral hygiene to prevent dental caries. Fluoride also has antimicrobial properties, although most microbes possess fluoride exporters to resist its toxicity. This work shows that sensitization of cariogenic species S. mutans and C. albicans to fluoride by genetic knockout of fluoride exporters alters the microbial composition and pathogenic properties of dental biofilms. These results suggest that the development of drugs that inhibit fluoride exporters could potentiate the anticaries effect of fluoride in over-the-counter products like toothpaste and mouth rinses. This is a novel strategy to treat dental caries., Competing Interests: The authors declare no conflict of interest.

tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker's yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis , differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits species-specific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C-the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.IMPORTANCEFungal infections are on the rise worldwide, and their global burden on human life and health is frequently underestimated. Among them, the human commensal and opportunistic pathogen, Candida albicans, is one of the major causative agents of severe infections. Its virulence is closely linked to its ability to change morphologies from yeasts to hyphae. Here, this ability is linked-to our knowledge for the first time-to modifications of tRNA and translational efficiency. One tRNA-modifying enzyme, Hma1, plays a specific role in C. albicans and its ability to invade the host. This adds a so-far unknown layer of regulation to the fungal virulence program and offers new potential therapeutic targets to fight fungal infections., Competing Interests: The authors declare no conflict of interest.

The escalating prevalence of membrane drug transporters and drug efflux pumps in pathogenic yeast like Candida albicans necessitates a comprehensive understanding of their roles in MDR. The overexpression of drug transporter families, ABC and MFS, implicated in MDR through drug efflux and poses a significant challenge in the diagnosis and treatment of fungal infection. Various mechanisms have been proposed for MDR; however, the upregulation of ABC and MFS superfamily transporters is most noticeable in MDR. The direct inhibition of these transporters seems an efficient strategy to overcome this problem. The goal of the article is to present an overview of the prospect of utilizing these modulators of C. albicans drug transports as effective antifungal molecules against MDR addressing a critical gap in the field. The review tries to address to prevent drug extrusion by modulating the expression of drug transporters of C. albicans. The review discussed the progress in identifying potent, selective, and non-toxic modulators of these transporters to develop some effective antifungals and overcome MDR. We reviewed major studies in this area and found that recent work has shifted toward the exploration of natural compounds as potential modulators to restore drug sensitivity in MDR fungal cells. The focus of this review is to survey and interpret current research information on modulators of C. albicans drug transporters from natural sources emphasizing those compounds that are potent antifungal agents., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)