Genomic evolution towards azole resistance in Candida glabrata clinical isolates unveils the importance of CgHxt4/6/7 in azole accumulation.

The increasing prevalence of candidosis caused by Candida glabrata is related to its ability to acquire azole resistance. Although azole resistance mechanisms are well known, the mechanisms for azole import into fungal cells have remained obscure. In this work, we have characterized two hexose transporters in C. glabrata and further investigate their role as potential azole importers. Three azole susceptible C. glabrata clinical isolates were evolved towards azole resistance and the acquired resistance phenotype was found to be independent of CgPDR1 or CgERG11 mutations. Through whole-genome sequencing, CgHXT4/6/7 was found to be mutated in the three evolved strains, when compared to their susceptible parents. CgHxt4/6/7 and the 96% identical CgHxt6/7 were found to confer azole susceptibility and increase azole accumulation in C. glabrata cells, strikingly rescuing the susceptibility phenotype imposed by CgPDR1 deletion, while the identified loss-of-function mutation in CgHXT4/6/7, leads to increased azole resistance. In silico docking analysis shows that azoles display a strong predicted affinity for the glucose binding site of CgHxt4/6/7. Altogether, we hypothesize that hexose transporters, such as CgHxt4/6/7 and CgHxt6/7, may constitute a family of azole importers, involved in clinical drug resistance in fungal pathogens, and constituting promising targets for improved antifungal therapy.

From the first touch to biofilm establishment by the human pathogen Candida glabrata: a genome-wide to nanoscale view.

Candida glabrata is an opportunistic pathogen that adheres to human epithelial mucosa and forms biofilm to cause persistent infections. In this work, Single-cell Force Spectroscopy (SCFS) was used to glimpse at the adhesive properties of C. glabrata as it interacts with clinically relevant surfaces, the first step towards biofilm formation. Following a genetic screening, RNA-sequencing revealed that half of the entire transcriptome of C. glabrata is remodeled upon biofilm formation, around 40% of which under the control of the transcription factors CgEfg1 and CgTec1. Using SCFS, it was possible to observe that CgEfg1, but not CgTec1, is necessary for the initial interaction of C. glabrata cells with both abiotic surfaces and epithelial cells, while both transcription factors orchestrate biofilm maturation. Overall, this study characterizes the network of transcription factors controlling massive transcriptional remodelling occurring from the initial cell-surface interaction to mature biofilm formation.

Role of CgTpo4 in Polyamine and Antimicrobial Peptide Resistance: Determining Virulence in Candida glabrata.

Candida glabrata is an emerging fungal pathogen whose success depends on its ability to resist antifungal drugs but also to thrive against host defenses. In this study, the predicted multidrug transporter CgTpo4 (encoded by ORF CAGL0L10912g) is described as a new determinant of virulence in C. glabrata, using the infection model Galleria mellonella. The CgTPO4 gene was found to be required for the C. glabrata ability to kill G. mellonella. The transporter encoded by this gene is also necessary for antimicrobial peptide (AMP) resistance, specifically against histatin-5. Interestingly, G. mellonella's AMP expression was found to be strongly activated in response to C. glabrata infection, suggesting AMPs are a key antifungal defense. CgTpo4 was also found to be a plasma membrane exporter of polyamines, especially spermidine, suggesting that CgTpo4 is able to export polyamines and AMPs, thus conferring resistance to both stress agents. Altogether, this study presents the polyamine exporter CgTpo4 as a determinant of C. glabrata virulence, which acts by protecting the yeast cells from the overexpression of AMPs, deployed as a host defense mechanism.

Development of Polythiourethane/ZnO-Based Anti-Fouling Materials and Evaluation of the Adhesion of Staphylococcus aureus and Candida glabrata Using Single-Cell Force Spectroscopy.

The attachment of bacteria and other microbes to natural and artificial surfaces leads to the development of biofilms, which can further cause nosocomial infections. Thus, an important field of research is the development of new materials capable of preventing the initial adhesion of pathogenic microorganisms. In this work, novel polymer/particle composite materials, based on a polythiourethane (PTU) matrix and either spherical (s-ZnO) or tetrapodal (t-ZnO) shaped ZnO fillers, were developed and characterized with respect to their mechanical, chemical and surface properties. To then evaluate their potential as anti-fouling surfaces, the adhesion of two different pathogenic microorganism species, Staphylococcus aureus and Candida glabrata, was studied using atomic force microscopy (AFM). Our results show that the adhesion of both S. aureus and C. glabrata to PTU and PTU/ZnO is decreased compared to a model surface polydimethylsiloxane (PDMS). It was furthermore found that the amount of both s-ZnO and t-ZnO filler had a direct influence on the adhesion of S. aureus, as increasing amounts of ZnO particles resulted in reduced adhesion of the cells. For both microorganisms, material composites with 5 wt.% of t-ZnO particles showed the greatest potential for anti-fouling with significantly decreased adhesion of cells. Altogether, both pathogens exhibit a reduced capacity to adhere to the newly developed nanomaterials used in this study, thus showing their potential for bio-medical applications.

Candida glabrata Transcription Factor Rpn4 Mediates Fluconazole Resistance through Regulation of Ergosterol Biosynthesis and Plasma Membrane Permeability.

The ability to acquire azole resistance is an emblematic trait of the fungal pathogen Candida glabrata Understanding the molecular basis of azole resistance in this pathogen is crucial for designing more suitable therapeutic strategies. This study shows that the C. glabrata transcription factor (TF) CgRpn4 is a determinant of azole drug resistance. RNA sequencing during fluconazole exposure revealed that CgRpn4 regulates the expression of 212 genes, activating 80 genes and repressing, likely in an indirect fashion, 132 genes. Targets comprise several proteasome and ergosterol biosynthesis genes, including ERG1, ERG2, ERG3, and ERG11 The localization of CgRpn4 to the nucleus increases upon fluconazole stress. Consistent with a role in ergosterol and plasma membrane homeostasis, CgRpn4 is required for the maintenance of ergosterol levels upon fluconazole stress, which is associated with a role in the upkeep of cell permeability and decreased intracellular fluconazole accumulation. We provide evidence that CgRpn4 directly regulates ERG11 expression through the TTGCAAA binding motif, reinforcing the relevance of this regulatory network in azole resistance. In summary, CgRpn4 is a new regulator of the ergosterol biosynthesis pathway in C. glabrata, contributing to plasma membrane homeostasis and, thus, decreasing azole drug accumulation.

Screening the Drug:H+ Antiporter Family for a Role in Biofilm Formation in Candida glabrata.

Biofilm formation and drug resistance are two key pathogenesis traits exhibited by Candida glabrata as a human pathogen. Interestingly, specific pathways appear to be in the crossroad between the two phenomena, making them promising targets for drug development. In this study, the 10 multidrug resistance transporters of the Drug:H+ Antiporter family of C. glabrata were screened for a role in biofilm formation. Besides previously identified players in this process, namely CgTpo1_2 and CgQdr2, two others are shown to contribute to biofilm formation: CgDtr1 and CgTpo4. The deletion of each of these genes was found to lead to lower biofilm formation, in both SDB and RPMI media, while their expression was found to increase during biofilm development and to be controlled by the transcription factor CgTec1, a predicted key regulator of biofilm formation. Additionally, the deletion of CgDTR1, CgTPO4, or even CgQDR2 was found to increase plasma membrane potential and lead to decreased expression of adhesin encoding genes, particularly CgALS1 and CgEPA1, during biofilm formation. Although the exact role of these drug transporters in biofilm formation remains elusive, our current model suggests that their control over membrane potential by the transport of charged molecules, may affect the perception of nutrient availability, which in turn may delay the triggering of adhesion and biofilm formation.

YEASTRACT+: a portal for cross-species comparative genomics of transcription regulation in yeasts.

The YEASTRACT+ information system (http://YEASTRACT-PLUS.org/) is a wide-scope tool for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in yeasts of biotechnological or human health relevance. YEASTRACT+ is a new portal that integrates the previously existing YEASTRACT (http://www.yeastract.com/) and PathoYeastract (http://pathoyeastract.org/) databases and introduces the NCYeastract (Non-Conventional Yeastract) database (http://ncyeastract.org/), focused on the so-called non-conventional yeasts. The information in the YEASTRACT database, focused on Saccharomyces cerevisiae, was updated. PathoYeastract was extended to include two additional pathogenic yeast species: Candida parapsilosis and Candida tropicalis. Furthermore, the NCYeastract database was created, including five biotechnologically relevant yeast species: Zygosaccharomyces baillii, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica and Komagataella phaffii. The YEASTRACT+ portal gathers 289 706 unique documented regulatory associations between transcription factors (TF) and target genes and 420 DNA binding sites, considering 247 TFs from 10 yeast species. YEASTRACT+ continues to make available tools for the prediction of the TFs involved in the regulation of gene/genomic expression. In this release, these tools were upgraded to enable predictions based on orthologous regulatory associations described for other yeast species, including two new tools for cross-species transcription regulation comparison, based on multi-species promoter and TF regulatory network analyses.

Divergent Approaches to Virulence in C. albicans and C. glabrata: Two Sides of the Same Coin.

Candida albicans and Candida glabrata are the two most prevalent etiologic agents of candidiasis worldwide. Although both are recognized as pathogenic, their choice of virulence traits is highly divergent. Indeed, it appears that these different approaches to fungal virulence may be equally successful in causing human candidiasis. In this review, the virulence mechanisms employed by C. albicans and C. glabrata are analyzed, with emphasis on the differences between the two systems. Pathogenesis features considered in this paper include dimorphic growth, secreted enzymes and signaling molecules, and stress resistance mechanisms. The consequences of these traits in tissue invasion, biofilm formation, immune system evasion, and macrophage escape, in a species dependent manner, are discussed. This review highlights the observation that C. albicans and C. glabrata follow different paths leading to a similar outcome. It also highlights the lack of knowledge on some of the specific mechanisms underlying C. glabrata pathogenesis, which deserve future scrutiny.

Microevolution of the pathogenic yeasts Candida albicans and Candida glabrata during antifungal therapy and host infection.

Infections by the pathogenic yeasts Candida albicans and Candida glabrata are among the most common fungal diseases. The success of these species as human pathogens is contingent on their ability to resist antifungal therapy and thrive within the human host. C. glabrata is especially resilient to azole antifungal treatment, while C. albicans is best known for its wide array of virulence features. The core mechanisms that underlie antifungal resistance and virulence in these pathogens has been continuously addressed, but the investigation on how such mechanisms evolve according to each environment is scarcer. This review aims to explore current knowledge on micro-evolution experiments to several treatment and host-associated conditions in C. albicans and C. glabrata. The analysis of adaptation strategies that evolve over time will allow to better understand the mechanisms by which Candida species are able to achieve stable phenotypes in real-life scenarios, which are the ones that should constitute the most interesting drug targets.

A Transcriptomics Approach To Unveiling the Mechanisms of In Vitro Evolution towards Fluconazole Resistance of a Candida glabrata Clinical Isolate.

Candida glabrata is an emerging fungal pathogen. Its increased prevalence is associated with its ability to rapidly develop antifungal drug resistance, particularly to azoles. In order to unravel new molecular mechanisms behind azole resistance, a transcriptomics analysis of the evolution of a C. glabrata clinical isolate (isolate 044) from azole susceptibility to posaconazole resistance (21st day), clotrimazole resistance (31st day), and fluconazole and voriconazole resistance (45th day), induced by longstanding incubation with fluconazole, was carried out. All the evolved strains were found to accumulate lower concentrations of azole drugs than the parental strain, while the ergosterol concentration remained mostly constant. However, only the population displaying resistance to all azoles was found to have a gain-of-function mutation in the C. glabrataPDR1 gene, leading to the upregulation of genes encoding multidrug resistance transporters. Intermediate strains, exhibiting posaconazole/clotrimazole resistance and increased fluconazole/voriconazole MIC levels, were found to display alternative ways to resist azole drugs. Particularly, posaconazole/clotrimazole resistance after 31 days was correlated with increased expression of adhesin genes. This finding led us to identify the Epa3 adhesin as a new determinant of azole resistance. Besides being required for biofilm formation, Epa3 expression was found to decrease the intracellular accumulation of azole antifungal drugs. Altogether, this work provides a glimpse of the transcriptomics evolution of a C. glabrata population toward multiazole resistance, highlighting the multifactorial nature of the acquisition of azole resistance and pointing out a new player in azole resistance.

Host-Pathogen Interactions Mediated by MDR Transporters in Fungi: As Pleiotropic as it Gets!

Fungal infections caused by Candida, Aspergillus, and Cryptococcus species are an increasing problem worldwide, associated with very high mortality rates. The successful prevalence of these human pathogens is due to their ability to thrive in stressful host niche colonization sites, to tolerate host immune system-induced stress, and to resist antifungal drugs. This review focuses on the key role played by multidrug resistance (MDR) transporters, belonging to the ATP-binding cassette (ABC), and the major facilitator superfamilies (MFS), in mediating fungal resistance to pathogenesis-related stresses. These clearly include the extrusion of antifungal drugs, with C. albicans CDR1 and MDR1 genes, and corresponding homologs in other fungal pathogens, playing a key role in this phenomenon. More recently, however, clues on the transcriptional regulation and physiological roles of MDR transporters, including the transport of lipids, ions, and small metabolites, have emerged, linking these transporters to important pathogenesis features, such as resistance to host niche environments, biofilm formation, immune system evasion, and virulence. The wider view of the activity of MDR transporters provided in this review highlights their relevance beyond drug resistance and the need to develop therapeutic strategies that successfully face the challenges posed by the pleiotropic nature of these transporters.

Candida Biofilms: Threats, Challenges, and Promising Strategies.

Candida species are fungal pathogens known for their ability to cause superficial and systemic infections in the human host. These pathogens are able to persist inside the host due to the development of pathogenicity and multidrug resistance traits, often leading to the failure of therapeutic strategies. One specific feature of Candida species pathogenicity is their ability to form biofilms, which protects them from external factors such as host immune system defenses and antifungal drugs. This review focuses on the current threats and challenges when dealing with biofilms formed by Candida albicans, Candida glabrata, Candida tropicalis, and Candida parapsilosis, highlighting the differences between the four species. Biofilm characteristics depend on the ability of each species to produce extracellular polymeric substances (EPS) and display dimorphic growth, but also on the biofilm substratum, carbon source availability and other factors. Additionally, the transcriptional control over processes like adhesion, biofilm formation, filamentation, and EPS production displays great complexity and diversity within pathogenic yeasts of the Candida genus. These differences not only have implications in the persistence of colonization and infections but also on antifungal resistance typically found in Candida biofilm cells, potentiated by EPS, that functions as a barrier to drug diffusion, and by the overexpression of drug resistance transporters. The ability to interact with different species in in vivo Candida biofilms is also a key factor to consider when dealing with this problem. Despite many challenges, the most promising strategies that are currently available or under development to limit biofilm formation or to eradicate mature biofilms are discussed.

YEASTRACT: an upgraded database for the analysis of transcription regulatory networks in Saccharomyces cerevisiae.

The YEAst Search for Transcriptional Regulators And Consensus Tracking (YEASTRACT-www.yeastract.com) information system has been, for 11 years, a key tool for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in Saccharomyces cerevisiae. Since its last update in June 2017, YEASTRACT includes approximately 163000 regulatory associations between transcription factors (TF) and target genes in S. cerevisiae, based on more than 1600 bibliographic references; it also includes 247 specific DNA binding consensus recognized by 113 TFs. This release of the YEASTRACT database provides new visualization tools to visualize each regulatory network in an interactive fashion, enabling the user to select and observe subsets of the network such as: (i) considering only DNA binding evidence or both DNA binding and expression evidence; (ii) considering only either positive or negative regulatory associations; or (iii) considering only one set of related environmental conditions. A further tool to observe TF regulons is also offered, enabling a clear-cut understanding of the exact meaning of the available data. We believe that with this new version, YEASTRACT will improve its role as an open web resource instrumental for Yeast Biologists and Systems Biology researchers.

A New Determinant of Candida glabrata Virulence: The Acetate Exporter CgDtr1.

Persistence and virulence of Candida glabrata infections are multifactorial phenomena, whose understanding is crucial to design more suitable therapeutic strategies. In this study, the putative multidrug transporter CgDtr1, encoded by ORF CAGL0M06281g, is identified as a determinant of C. glabrata virulence in the infection model Galleria mellonella. CgDTR1 deletion is shown to decrease the ability to kill G. mellonella larvae by decreasing C. glabrata ability to proliferate in G. mellonella hemolymph, and to tolerate the action of hemocytes. The possible role of CgDtr1 in the resistance to several stress factors that underlie death induced by phagocytosis was assessed. CgDTR1 was found to confer resistance to oxidative and acetic acid stress. Consistently, CgDtr1 was found to be a plasma membrane acetic acid exporter, relieving the stress induced upon C. glabrata cells within hemocytes, and thus enabling increased proliferation and virulence against G. mellonella larvae.

Transcriptional Control of Drug Resistance, Virulence and Immune System Evasion in Pathogenic Fungi: A Cross-Species Comparison.

Transcription factors are key players in the control of the activation or repression of gene expression programs in response to environmental stimuli. The study of regulatory networks taking place in fungal pathogens is a promising research topic that can help in the fight against these pathogens by targeting specific fungal pathways as a whole, instead of targeting more specific effectors of virulence or drug resistance. This review is focused on the analysis of regulatory networks playing a central role in the referred mechanisms in the human fungal pathogens Aspergillus fumigatus, Cryptococcus neoformans, Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis. Current knowledge on the activity of the transcription factors characterized in each of these pathogenic fungal species will be addressed. Particular focus is given to their mechanisms of activation, regulatory targets and phenotypic outcome. The review further provides an evaluation on the conservation of transcriptional circuits among different fungal pathogens, highlighting the pathways that translate common or divergent traits among these species in what concerns their drug resistance, virulence and host immune evasion features. It becomes evident that the regulation of transcriptional networks is complex and presents significant variations among different fungal pathogens. Only the oxidative stress regulators Yap1 and Skn7 are conserved among all studied species; while some transcription factors, involved in nutrient homeostasis, pH adaptation, drug resistance and morphological switching are present in several, though not all species. Interestingly, in some cases not very homologous transcription factors display orthologous functions, whereas some homologous proteins have diverged in terms of their function in different species. A few cases of species specific transcription factors are also observed.

Clotrimazole Drug Resistance in Candida glabrata Clinical Isolates Correlates with Increased Expression of the Drug:H(+) Antiporters CgAqr1, CgTpo1_1, CgTpo3, and CgQdr2.

For years, antifungal drug resistance in Candida species has been associated to the expression of ATP-Binding Cassette (ABC) multidrug transporters. More recently, a few drug efflux pumps from the Drug:H(+) Antiporter (DHA) family have also been shown to play a role in this process, although to date only the Candida albicans Mdr1 transporter has been demonstrated to be relevant in the clinical acquisition of antifungal drug resistance. This work provides evidence to suggest the involvement of the C. glabrata DHA transporters CgAqr1, CgQdr2, CgTpo1_1, and CgTpo3 in the clinical acquisition of clotrimazole drug resistance. A screening for azole drug resistance in 138 C. glabrata clinical isolates, from patients attending two major Hospitals in Portugal, was performed. Based on this screening, 10 clotrimazole susceptible and 10 clotrimazole resistant isolates were selected for further analysis. The transcript levels of CgAQR1, CgQDR2, CgTPO1_1, and CgTPO3 were found to be significantly up-regulated in resistant isolates when compared to the susceptible ones, with a level of correlation that was found to be similar to that of CgCDR2, an ABC gene known to be involved in the clinical acquisition of resistance. As a proof-of-concept experiment, the CgTPO3 gene was deleted in an azole resistant C. glabrata isolate, exhibiting high levels of expression of this gene. The deletion of CgTPO3 in this isolate was found to lead to decreased resistance to clotrimazole and fluconazole, and increased accumulation of azole drugs, thus suggesting the involvement of this transporter in the manifestation of azole resistance.

New Mechanisms of Flucytosine Resistance in C. glabrata Unveiled by a Chemogenomics Analysis in S. cerevisiae.

5-Flucytosine is currently used as an antifungal drug in combination therapy, but fungal pathogens are rapidly able to develop resistance against this drug, compromising its therapeutic action. The understanding of the underlying resistance mechanisms is crucial to deal with this problem. In this work, the S. cerevisiae deletion mutant collection was screened for increased resistance to flucytosine. Through this chemogenomics analysis, 183 genes were found to confer resistance to this antifungal agent. Consistent with its known effect in DNA, RNA and protein synthesis, the most significant Gene Ontology terms over-represented in the list of 5-flucytosine resistance determinants are related to DNA repair, RNA and protein metabolism. Additional functional classes include carbohydrate and nitrogen-particularly arginine-metabolism, lipid metabolism and cell wall remodeling. Based on the results obtained for S. cerevisiae as a model system, further studies were conducted in the pathogenic yeast Candida glabrata. Arginine supplementation was found to relieve the inhibitory effect exerted by 5-flucytosine in C. glabrata. Lyticase susceptibility was found to increase within the first 30min of 5-flucytosine exposure, suggesting this antifungal drug to act as a cell wall damaging agent. Upon exponential growth resumption in the presence of 5-flucytosine, the cell wall exhibited higher resistance to lyticase, suggesting that cell wall remodeling occurs in response to 5-flucytosine. Additionally, the aquaglyceroporin encoding genes CgFPS1 and CgFPS2, from C. glabrata, were identified as determinants of 5-flucytosine resistance. CgFPS1 and CgFPS2 were found to mediate 5-flucytosine resistance, by decreasing 5-flucytosine accumulation in C. glabrata cells.