ABSTRACT The majority of eukaryotes encode some intron-containing pre-tRNAs. Splicing of these pre-tRNAs requires a dedicated tRNA splicing machinery. The fungal and trypanosome tRNA ligase, Trl1, and the human RNA ligase, RTCB, catalyze an essential step in tRNA splicing. However, Trl1 and RTCB are nonhomologous and biochemically and structurally distinct from each other. Therefore, Trl1 could serve as a broad-spectrum antifungal and anti-trypanosomal target. While the functions and requirements of the three catalytic Trl1 domains have been extensively characterized in the model yeast Saccharomyces cerevisiae , the roles of Trl1 orthologs in pathogenic fungi remain unexplored. Here, we validate Trl1 as one of the few promising novel drug targets for the development of antifungal therapeutics. Functional analyses of the three Trl1 domains show that only the “sealing” domain is essential for growth and viability in Candida albicans and Aspergillus fumigatus . In contrast, the two “healing” domains are dispensable in these pathogenic fungi, suggesting the presence of redundant healing enzymes, unlike in S. cerevisiae . These findings indicate that only the sealing domain is a good drug target. Our analysis also shows that the Mucor enzyme, which only contains the sealing domain, is essential. Using a Caenorhabditis elegans infection model of C. albicans , we further demonstrated that inhibiting Trl1 expression protects worms during an established infection. In contrast to these fungal pathogens, we show that all three domains of Trl1 are essential in Trypanosoma brucei. Our findings show that the essentiality of the Trl1 sealing is conserved in important human pathogens and provides an impetus for future drug development. SIGNIFICANCE Fungal infections are an important cause of human disease and death and difficult to treat and there is an urgent need to develop additional drugs. Based on studies in yeast, one promising target for antifungal drug development is the tRNA splicing pathway. Human tRNA ligase is fundamentally distinct from the fungal one. To investigate the possibility of developing tRNA ligase-targeting drugs, we investigated the function of the catalytic domains of fungal tRNA ligase in different fungal pathogens. Surprisingly, only the first domain is essential in these pathogens and yeast is not a good model fungus. In contrast, all three domains of Trypanosome tRNA ligase are essential. These findings provide an impetus for future drug development.

Candida is one of the most frequent causes of bloodstream infections, and our first line of defense against these invasive infections is the innate immune system. The early immune response is critical in controlling Candida albicans infection, but C. albicans has several strategies to evade host immune attack. Phagocytosis of C. albicans blocks hyphal growth, limiting host damage and virulence, but how C. albicans limits early recruitment and phagocytosis in vertebrate infection is poorly understood. To study innate immune evasion by intravital imaging, we utilized the transparent larval zebrafish infection model to screen 131 C. albicans mutants for altered virulence and phagocyte response. Infections with each of the seven hypovirulent mutants led to altered phagocyte recruitment and/or phagocytosis, falling into four categories. Of particular interest among these is NMD5, a predicted β-importin and newly identified virulence factor. The nmd5∆/∆ mutant fails to limit phagocytosis, and its virulence defects are eliminated when phagocyte activity is compromised, suggesting that its role in virulence is limited to immune evasion. These quantitative intravital imaging experiments are the first to document altered Candida-phagocyte interactions for several additional mutants and clearly distinguish recruitment from phagocytic uptake, suggesting that Candida modulates both events. This initial large-scale screen of individual C. albicans mutants in a vertebrate, coupled with high-resolution imaging of Candida-phagocyte interactions, provides a more nuanced view of how diverse mutations can lead to more effective phagocytosis, a key immune process that blocks germination and drives anti-fungal immunity. IMPORTANCE:Candida albicans is part of the human microbial community and is a dangerous opportunistic pathogen, able to prevent its elimination by the host immune system. Although Candida avoids immune attack through several strategies, we still understand little about how it regulates when immune phagocytes get recruited to the infection site and when they engulf fungal cells. We tested over 130 selected Candida mutants for their ability to cause lethal infection and found several hypovirulent mutants, which provoked altered innate immune responses, resulting in lower overall inflammation and greater host survival. Of particular interest is NMD5, which acts to limit fungal phagocytosis and is predicted to regulate the activity of stress-associated transcription factors. Our high-content screening was enabled by modeling Candida infection in transparent vertebrate zebrafish larva. Our findings help us understand how Candida survives immune attack during commensal and pathogenic growth, and may eventually inform new strategies for controlling disease.

The ubiquitous fungal pathogen Candida albicans has the potential to either asymptomatically colonize the gastrointestinal (GI) tract or become an invasive pathogen through mechanisms that remain incompletely understood. Here we explored the fungal, host, and environmental factors that influence the ability of C. albicans to colonize the mouse GI tract using a representative clinical strain, CLCA10. After a single gavage challenge (5 × 106 CFU C. albicans), specific pathogen-free (SPF) mice remained colonized with C. albicans strain CLCA10, but not other Candida species, for at least 58 days with the fungus confined largely to the gut luminal contents. Colonized mice exhibited no weight loss or other signs of active infection, and CLCA10 did not disrupt the gut microbiome. Moreover, C. albicans colonization with CLCA10 was not substantially affected by the mouse commercial source or the method used to cultivate the fungus prior to gavage. Although some genetically manipulated C. albicans strains were unable to robustly colonize, strain SC5314 also colonized the mouse gut despite having enhanced pathogenicity. C. albicans CLCA10 gut colonization in part depended on the hypha-associated adhesins Als3 and Hwp1 and the peptide toxin candidalysin and could not be eradicated by potent antifungal therapy. Thus, this study concludes that C. albicans gut colonization in the mouse is critically dependent on fungal hyphal factors, the targeting of which could enhance strategies to reduce C. albicans gut colonization and the intractable threat to human health it represents.IMPORTANCECandida albicans is an important human fungal pathogen and a ubiquitous colonizer of the gastrointestinal (GI) tract. However, it is not understood how C. albicans persists within the GI tract and from which it may disperse to cause disease. Here, we demonstrated that multiple strains of C. albicans, including the widely used SC5314 strain, robustly colonize the mouse GI tract for at least 2 months. This colonization caused no disruption to the host tissue or bacterial microbiome and was resistant to clearance by antifungal drugs. Importantly, colonization was mediated by proteins expressed by C. albicans that are known to be involved in fungal virulence and unrelated to experimental conditions. Overall, this work identifies mechanisms by which C. albicans persists in the GI tract, enhancing our knowledge of host-fungal interactions during commensal colonization and potentially how to reduce such colonization.

The pathogenic yeast Candida auris represents a global threat of the utmost clinical relevance. This emerging fungal species is remarkable in its resistance to commonly used antifungal agents and its persistence in the nosocomial settings. The innate immune system is one the first lines of defense preventing the dissemination of pathogens in the host. C. auris is susceptible to circulating phagocytes, and understanding the molecular details of these interactions may suggest routes to improved therapies. In this work, we examined the interactions of this yeast with macrophages. We found that macrophages avidly phagocytose C. auris; however, intracellular replication is not inhibited, indicating that C. auris resists the killing mechanisms imposed by the phagocyte. Unlike Candida albicans, phagocytosis of C. auris does not induce macrophage lysis. The transcriptional response of C. auris to macrophage phagocytosis is very similar to other members of the CUG clade (C. albicans, C. tropicalis, C. parapsilosis, C. lusitaniae), i.e., downregulation of transcription/translation and upregulation of alternative carbon metabolism pathways, transporters, and induction of oxidative stress response and proteolysis. Gene family expansions are common in this yeast, and we found that many of these genes are induced in response to macrophage co-incubation. Among these, amino acid and oligopeptide transporters, as well as lipases and proteases, are upregulated. Thus, C. auris shares key transcriptional signatures shared with other fungal pathogens and capitalizes on the expansion of gene families coding for potential virulence attributes that allow its survival, persistence, and evasion of the innate immune system.

Fungal pathogens are a continuing challenge due to few effective antifungals and a rise in resistance. In previous work, we described the inhibition of Candida albicans virulence following exposure to the 68 amino acid bacteriocin, EntV, secreted by Enterococcus faecalis. Here, to optimize EntV as a potential therapeutic and better understand its antifungal features, an X-ray structure is obtained. The structure consists of six alpha helices enclosing a seventh 16 amino acid helix (α7). The individual helices are tested for antifungal activity using in vitro and nematode infection assays. Interestingly, α7 retains antifungal, but not antibacterial activity and is also effective against Candida auris and Cryptococcus neoformans. Further reduction of α7 to 12 amino acids retains full antifungal activity, and excellent efficacy is observed in rodent models of C. albicans oropharyngeal, systemic, and venous catheter infections. Together, these results showcase EntV-derived peptides as promising candidates for antifungal therapeutic development.

The tight association of Candida albicans with the human host has driven the evolution of mechanisms that permit metabolic flexibility. Amino acids, present in a free or peptide-bound form, are abundant carbon and nitrogen sources in many host niches. In C. albicans, the capacity to utilize certain amino acids, like proline, is directly connected to fungal morphogenesis and virulence. Yet the precise nature of proline sensing and uptake in this pathogenic fungus has not been investigated. Since C. albicans encodes 10 putative orthologs of the four Saccharomyces cerevisiae proline transporters, we tested deletion strains of the respective genes and identified Gnp2 (CR_09920W) as the main C. albicans proline permease. In addition, we found that this specialization of Gnp2 was reflected in its transcriptional regulation and further assigned distinct substrate specificities for the other orthologs, indicating functional differences of the C. albicans amino acid permeases compared to the model yeast. The physiological relevance of proline uptake is exemplified by the findings that strains lacking GNP2 were unable to filament in response to extracellular proline and had a reduced capacity to damage macrophages and impaired survival following phagocytosis. Furthermore, GNP2 deletion rendered the cells more sensitive to oxidative stress, illustrating new connections between amino acid uptake and stress adaptation in C. albicans. IMPORTANCE The utilization of various nutrients is of paramount importance for the ability of Candida albicans to successfully colonize and infect diverse host niches. In this context, amino acids are of special interest due to their ubiquitous availability, relevance for fungal growth, and direct influence on virulence traits like filamentation. In this study, we identify a specialized proline transporter in C. albicans encoded by GNP2. The corresponding amino acid permease is essential for proline-induced filamentation, oxidative stress resistance, and fungal survival following interaction with macrophages. Altogether, this work highlights the importance of amino acid uptake for metabolic and stress adaptation in this fungus.

Purpose: To evaluate the effectiveness and ease of N95 respirator decontamination methods in a clinic setting and to identify the extent of microbial colonization on respirators associated with reuse. Methods: In a prospective fashion, N95 respirators (n = 15) were randomized to a decontamination process (time, dry heat, or ultraviolet C light [UVC]) in outpatient clinics. Each respirator was re-used up to 5 separate clinic sessions. Swabs on each respirator for SARS-CoV-2, bacteria, and fungi were obtained before clinic, after clinic and post-treatment. Mask integrity was checked after each treatment (n = 68). Statistical analyses were performed to determine factors for positive samples. Results: All three decontamination processes reduced bacteria counts similarly. On multivariate mixed model analysis, there were an additional 8.1 colonies of bacteria (95% CI 5.7 to 10.5; p < 0.01) on the inside compared to the outside surface of the respirators. Treatment resulted in a decrease of bacterial load by 8.6 colonies (95% CI-11.6 to-5.5; p < 0.01). Although no decontamination treatment affected the respirator filtration efficiency, heat treatments were associated with the breakdown of thermoplastic elastomer straps. Contamination with fungal and SARS-CoV-2 viral particles were minimal to non-existent. Conclusions: Time, heat and UVC all reduced bacterial load on reused N95 respirators. Fungal contamination was minimal. Heat could permanently damage some elastic straps making the respirators nonfunctional. Given its effectiveness against microbes, lack of damage to re-treated respirators and logistical ease, UVC represents an optimal decontamination method for individual N95 respirators when reuse is necessary.

The tight association of Candida albicans with the human host has driven the evolution of mechanisms that permit metabolic flexibility. Amino acids, present in free form or peptide bound, are an abundant carbon and nitrogen source in many host niches. Further,the capacity to sense and utilize certain amino acids, like proline, is directly linked to virulence. The C. albicans genome encodes for at least 24 amino acid permeases (AAPs), highlighting the importance of flexible amino acid uptake for fungal growth and virulence. Although the substrate specificity and role of certain AAPs has been investigated, a comprehensive characterization was missing. Therefore, we assembled a library of AAP deletion strains, which was tested for resistance to toxic amino acid analogs. Most striking was the specific resistance of gnp2Δ to the proline analog 3,4-dehydroproline. Subsequent tests validated that Gnp2 is a specific proline permease in C. albicans, which is contrary to the model yeast Saccharomyces cerevisiae where proline transport is mediated by four permeases. Furthermore, the induction of GNP2 appears to be independent of the SPS (Ssy1-Ptr3-Ssy5) regulatory pathway that controls proline utilization in the model yeast, pointing towards rewired proline uptake in C. albicans. Additionally, strains lacking GNP2were unable to respond to proline-induced filamentation, displayed decreased cytotoxicity to macrophages and showed increased sensitivity to oxidative stress, underlining the importance of proline uptake for fungal virulence. Taken together, the role of Gnp2-mediated proline uptake illustrates the importance of metabolism-driven virulence in C. albicans.

Nutrient acquisition is a central challenge for all organisms. For the fungal pathogen Candida albicans , utilization of amino acids has been shown to be critical for survival, immune evasion, and escape, while the importance of catabolism of host-derived proteins and peptides in vivo is less well understood. Stp1 and Stp2 are paralogous transcription factors (TFs) regulated by the S sy1- P tr3- S sy5 (SPS) amino acid sensing system and have been proposed to have distinct, if uncertain, roles in protein and amino acid utilization.

Introduction Candida albicans is an opportunistic fungus that can cause both mucosal and invasive infections. As a commensal, C. albicans is well adapted to the rapidly changing environments within the host, where availability of many nutrients is limited. Any organism trying to colonize these environments must adapt to the nutrients that are available in order to thrive and also must cope with mechanisms of immune surveillance. This Pearl will discuss recent findings highlighting the remarkable ability of C. albicans to utilize a wide array of nutrients and how this ability has been channeled to promote both commensal and pathogenic interactions.

Candida albicans is well adapted to its host and is able to sense and respond to the nutrients available within. We have shown that C. albicans avidly utilizes amino acids as a carbon source, which allows this opportunistic pathogen to neutralize acidic environments, including the macrophage phagosome. The transcription factor Stp2 is a key regulator of this phenomenon, and we sought to understand the mechanism of activation of Stp2, focusing on the SPS sensor system previously characterized for its role in nitrogen acquisition. We generated deletion mutants of the three components, SSY1, PTR3 and SSY5 and demonstrated that these strains utilize amino acids poorly as carbon source, cannot neutralize the medium in response to these nutrients, and have reduced ammonia release. Exogenous amino acids rapidly induce proteolytic processing of Stp2 and nuclear translocation in an SPS-dependent manner. A truncated version of Stp2, lacking the amino terminal nuclear exclusion domain, could suppress the growth and pH neutralization defects of the SPS mutants. We showed that the SPS system is required for normal resistance of C. albicans to macrophages and that mutants defective in this system reside in more acidic phagosomes compared with wild type cells; however, a more equivocal contribution was observed in the murine model of disseminated candidiasis. Taken together, these results indicate that the SPS system is activated under carbon starvation conditions resembling host environments, regulating Stp2 functions necessary for amino acid catabolism and normal interactions with innate immune cells.

Vulvovaginal candidiasis, a superficial infection caused predominantly by the pathogenic fungus Candida albicans, is frequently treated with clotrimazole. Some drug formulations contain lactate for improved solubility. Lactate may modify C. albicans physiology and drug sensitivity by serving as a carbon source for the fungus and/or affecting local pH. Here, we explored the effects of lactate, in combination with pH changes, on C. albicans proliferation, morphology and clotrimazole sensitivity. Moreover, we determined the influence of growth phase and morphology per se on drug sensitivity. We showed that utilization of lactate as a carbon source did not promote fast fungal proliferation or filamentation. Lactate had no influence on clotrimazole-mediated killing of C. albicans in standard fungal cultivation medium but had an additive effect on the fungicidal clotrimazole action under in vitro vagina-simulative conditions. Moreover, clotrimazole-mediated killing was growth-phase and morphology dependent. Post-exponential cells were resistant to the fungicidal action of clotrimazole, whilst logarithmic cells were sensitive, and hyphae showed the highest susceptibility. Finally, we showed that treatment of pre-formed C. albicans hyphae with sublethal concentrations of clotrimazole induced a reversion to yeast-phase growth. As C. albicans hyphae are considered the pathogenic morphology during mucosal infections, these data suggest that elevated fungicidal activity of clotrimazole against hyphae plus clotrimazole-induced hyphae-to-yeast reversion may help to dampen acute vaginal infections by reducing the relative proportion of hyphae and thus shifting to a non-invasive commensal-like population. In addition, lactate as an ingredient of clotrimazole formulations may potentiate clotrimazole killing of C. albicans in the vaginal microenvironment.

Candida albicans is a well-adapted human commensal but is also a facultative pathogen that can cause superficial and systemic infections. Its remarkable capacity to thrive within the human host relies on its ability to adapt and respond to the local environment of different niches. C. albicans is able to cope with oxidative stress in a coordinated fashion via upregulation of different protective mechanisms. Here, we unravel the role of a family of glutathione peroxidase (GPx), designated Gpx31, Gpx32, and Gpx33, in oxidative stress resistance. We show that GPx activity in C. albicans is induced upon exposure to peroxides and that this enzymatic activity is required for full resistance to oxidative stress. The GPx activity relies on the presence of GPX31, with no apparent contribution from GPX32 and GPX33 during in vitro short-term (3 h) exposure to peroxides. However, a triple gpx31-33Δ/Δ mutant exhibited a more pronounced sensitivity than a single gpx31Δ/Δ mutant on solid media in the presence of oxidants, suggesting that GPX32 and GPX33 may be involved in long-term adaptation to oxidative stress. Interestingly, reintegration of a single allele of GPX31 was sufficient to restore the wild-type phenotype in both the single and triple mutants. We found that mutants lacking GPX31-33 were more susceptible to killing by phagocytic cells, suggesting that GPxs are required for full resistance to innate immune effector cells. Despite the sensitivity to oxidative stress and phagocytes, these mutants were not affected in their virulence in the chicken embryo model of candidiasis.

ABSTRACT Human fungal pathogens are distributed throughout their kingdom, suggesting that pathogenic potential evolved independently. Candida albicans is the most virulent member of the CUG clade of yeasts and a common cause of both superficial and invasive infections. We therefore hypothesized that C. albicans possesses distinct pathogenicity mechanisms. In silico genome subtraction and comparative transcriptional analysis identified a total of 65 C. albicans-specific genes (ASGs) expressed during infection. Phenotypic characterization of six ASG-null mutants demonstrated that these genes are dispensable for in vitro growth but play defined roles in host-pathogen interactions. Based on these analyses, we investigated two ASGs in greater detail. An orf19.6688Δ mutant was found to be fully virulent in a mouse model of disseminated candidiasis and to induce higher levels of the proinflammatory cytokine interleukin-1β (IL-1β) following incubation with murine macrophages. A pga16Δ mutant, on the other hand, exhibited attenuated virulence. Moreover, we provide evidence that secondary filamentation events (multiple hyphae emerging from a mother cell and hyphal branching) contribute to pathogenicity: PGA16 deletion did not influence primary hypha formation or extension following contact with epithelial cells; however, multiple hyphae and hyphal branching were strongly reduced. Significantly, these hyphae failed to damage host cells as effectively as the multiple hypha structures formed by wild-type C. albicans cells. Together, our data show that species-specific genes of a eukaryotic pathogen can play important roles in pathogenicity.

The pathology of vulvovaginal candidiasis (VVC) caused by Candida albicans is associated with a nonprotective inflammatory response and is frequently treated with clotrimazole. We investigated the mechanisms by which clotrimazole resolves VVC. Low levels of clotrimazole, which do not block fungal growth, inhibit expression of a "danger response" transcription factor, c-Fos, block production of proinflammatory cytokines, and inhibit neutrophil infiltration to the site of infection.

Certain Candida spp. (e.g. C. albicans, C. tropicalis, C. parapsilosis and C. glabrata) are not only well-adapted fungal commensals of humans, but are also able to cause superficial mucosal infections or even systemic disease. Professional phagocytes (neutrophils, macrophages and dendritic cells) constitute the first line of defence against Candida spp. Here, we review the interactions of phagocytes with pathogenic Candida spp., focusing on macrophages and neutrophils. We discuss the mechanisms involved in recognition, uptake and killing of these fungi. We go on to analyse the cellular responses of these yeasts towards phagocyte-imposed stresses, including metabolic flexibility, robust oxidative stress response and ability to cope with nitrosative stress. Finally, we address strategies that allow these opportunistic pathogens to thrive within the host, evading and escaping from the phagocyte attack.