scholarly journals Crystal Structures of Full-Length Lanosterol 14α-Demethylases of Prominent Fungal Pathogens Candida albicans and Candida glabrata Provide Tools for Antifungal Discovery

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Mikhail V. Keniya ◽  
Manya Sabherwal ◽  
Rajni K. Wilson ◽  
Matthew A. Woods ◽  
Alia A. Sagatova ◽  
...  

ABSTRACT Targeting lanosterol 14α-demethylase (LDM) with azole drugs provides prophylaxis and treatments for superficial and disseminated fungal infections, but cure rates are not optimal for immunocompromised patients and individuals with comorbidities. The efficacy of azole drugs has also been reduced due to the emergence of drug-resistant fungal pathogens. We have addressed the need to improve the potency, spectrum, and specificity for azoles by expressing in Saccharomyces cerevisiae functional, recombinant, hexahistidine-tagged, full-length Candida albicans LDM (CaLDM6×His) and Candida glabrata LDM (CgLDM6×His) and determining their X-ray crystal structures. The crystal structures of CaLDM6×His, CgLDM6×His, and ScLDM6×His have the same fold and bind itraconazole in nearly identical conformations. The catalytic domains of the full-length LDMs have the same fold as the CaLDM6×His catalytic domain in complex with posaconazole, with minor structural differences within the ligand binding pocket. Our structures give insight into the LDM reaction mechanism and phenotypes of single-site CaLDM mutations. This study provides a practical basis for the structure-directed discovery of novel antifungals that target LDMs of fungal pathogens.

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Mikhail V. Keniya ◽  
Yasmeen N. Ruma ◽  
Joel D. A. Tyndall ◽  
Brian C. Monk

ABSTRACTTargeting lanosterol 14α-demethylase (LDM) with azole drugs provides prophylaxis and treatments for superficial and disseminated fungal infections, but cure rates are modest for immunocompromised patients and individuals with comorbidities. The efficacy of azole drugs has also been reduced due to the emergence of drug-resistant fungal pathogens. We have addressed these problems by expressing inSaccharomyces cerevisiaefunctional, hexahistidine-tagged, full-lengthCandida albicansLDM (CaLDM6×His) andCandida glabrataLDM (CgLDM6×His) for drug discovery purposes and determining their X-ray crystal structures. Compared withS. cerevisiaeoverexpressing LDM6×His (ScLDM6×His), the reduced susceptibility of CgLDM6×His to all azole drugs tested correlated with its level of overexpression. In contrast, the reduced susceptibility to short-tailed (fluconazole and voriconazole) but not medium-tailed (VT-1161) or long-tailed azoles (itraconazole and posaconazole) indicates CaLDM6×His works best when coexpressed with its cognate NADPH-cytochrome P450 reductase (CaNcp1A) rather than the host reductase (ScNcp1). Overexpression of LDM or Ncp1 modified the ergosterol content of yeast and affected growth inhibition by the polyene antibiotic amphotericin B. Affinity-purified recombinantCandidaLDMs bind carbon monoxide and show tight type II binding of a range of azole drugs, including itraconazole, posaconazole, fluconazole, and voriconazole. This study provides a practical basis for the phenotype-, biochemistry-, and structure-directed discovery of novel antifungals that target LDMs of fungal pathogens.


mSphere ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Lauren Wensing ◽  
Jehoshua Sharma ◽  
Deeva Uthayakumar ◽  
Yannic Proteau ◽  
Alejandro Chavez ◽  
...  

ABSTRACT Fungal pathogens are emerging as an important cause of human disease, and Candida albicans is among the most common causative agents of fungal infections. Studying this fungal pathogen is of the utmost importance and necessitates the development of molecular technologies to perform comprehensive genetic and functional genomic analysis. Here, we designed and developed a novel clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for targeted genetic repression in C. albicans. We engineered a nuclease-dead Cas9 (dCas9) construct that, paired with a guide RNA targeted to the promoter of an endogenous gene, is capable of targeting that gene for transcriptional repression. We further optimized a favorable promoter locus to achieve repression and demonstrated that fusion of dCas9 to an Mxi1 repressor domain was able to further enhance transcriptional repression. Finally, we demonstrated the application of this CRISPRi system through genetic repression of the essential molecular chaperone HSP90. This is the first demonstration of a functional CRISPRi repression system in C. albicans, and this valuable technology will enable many future applications in this critical fungal pathogen. IMPORTANCE Fungal pathogens are an increasingly important cause of human disease and mortality, and Candida albicans is among the most common causes of fungal disease. Studying this important fungal pathogen requires a comprehensive genetic toolkit to establish how different genetic factors play roles in the biology and virulence of this pathogen. Here, we developed a CRISPR-based genetic regulation platform to achieve targeted repression of C. albicans genes. This CRISPR interference (CRISPRi) technology exploits a nuclease-dead Cas9 protein (dCas9) fused to transcriptional repressors. The dCas9 fusion proteins pair with a guide RNA to target genetic promoter regions and to repress expression from these genes. We demonstrated the functionality of this system for repression in C. albicans and show that we can apply this technology to repress essential genes. Taking the results together, this work presents a new technology for efficient genetic repression in C. albicans, with important applications for genetic analysis in this fungal pathogen.


mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Nicole M. Revie ◽  
Nicole Robbins ◽  
Luke Whitesell ◽  
John R. Frost ◽  
Solomon D. Appavoo ◽  
...  

ABSTRACT Opportunistic pathogens of the genus Candida reign as the leading cause of mycotic disease and are associated with mortality rates greater than 40%, even with antifungal intervention. This is in part due to the limited arsenal of antifungals available to treat systemic fungal infections. Azoles have been the most widely deployed class of antifungal drug for decades and function by targeting the biosynthesis of ergosterol, a key component of the fungal cell membrane. However, their utility is compromised by their fungistatic nature, which favors the development of resistance. Combination therapy has the potential to confer enhanced efficacy as well as mitigate the evolution of resistance. Previously, we described the generation of structurally diverse macrocyclic peptides with a 1,3,4-oxadiazole and an endocyclic amine grafted within the peptide backbone. Importantly, this noncanonical backbone displayed high membrane permeability, an important attribute for compounds that need to permeate across the fungal cell wall and membrane in order to reach their intracellular target. Here, we explored the bioactivity of this novel chemical scaffold on its own and in combination with the azole fluconazole. Although few of the oxadiazole-containing macrocyclic peptides displayed activity against Candida albicans on their own, many increased the efficacy of fluconazole, resulting in a synergistic combination that was independent of efflux inhibition. Interestingly, these molecules also enhanced azole activity against several non-albicans Candida species, including the azole-resistant pathogens Candida glabrata and Candida auris. This work characterizes a novel chemical scaffold that possesses azole-potentiating activity against clinically important Candida species. IMPORTANCE Fungal infections, such as those caused by pathogenic Candida species, pose a serious threat to human health. Treating these infections relies heavily on the use of azole antifungals; however, resistance to these drugs develops readily, demanding novel therapeutic strategies. This study characterized the antifungal activity of a series of molecules that possess unique chemical attributes and the ability to traverse cellular membranes. We observed that many of the compounds increased the activity of the azole fluconazole against Candida albicans, without blocking the action of drug efflux pumps. These molecules also increased the efficacy of azoles against other Candida species, including the emerging azole-resistant pathogen Candida auris. Thus, we describe a novel chemical scaffold with broad-spectrum bioactivity against clinically important fungal pathogens.


mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Irsa Shoukat ◽  
Corey Frazer ◽  
John S. Allingham

ABSTRACT Mitotic spindles assume a bipolar architecture through the concerted actions of microtubules, motors, and cross-linking proteins. In most eukaryotes, kinesin-5 motors are essential to this process, and cells will fail to form a bipolar spindle without kinesin-5 activity. Remarkably, inactivation of kinesin-14 motors can rescue this kinesin-5 deficiency by reestablishing the balance of antagonistic forces needed to drive spindle pole separation and spindle assembly. We show that the yeast form of the opportunistic fungus Candida albicans assembles bipolar spindles in the absence of its sole kinesin-5, CaKip1, even though this motor exhibits stereotypical cell-cycle-dependent localization patterns within the mitotic spindle. However, cells lacking CaKip1 function have shorter metaphase spindles and longer and more numerous astral microtubules. They also show defective hyphal development. Interestingly, a small population of CaKip1-deficient spindles break apart and reform two bipolar spindles in a single nucleus. These spindles then separate, dividing the nucleus, and then elongate simultaneously in the mother and bud or across the bud neck, resulting in multinucleate cells. These data suggest that kinesin-5-independent mechanisms drive assembly and elongation of the mitotic spindle in C. albicans and that CaKip1 is important for bipolar spindle integrity. We also found that simultaneous loss of kinesin-5 and kinesin-14 (CaKar3Cik1) activity is lethal. This implies a divergence from the antagonistic force paradigm that has been ascribed to these motors, which could be linked to the high mitotic error rate that C. albicans experiences and often exploits as a generator of diversity. IMPORTANCE Candida albicans is one of the most prevalent fungal pathogens of humans and can infect a broad range of niches within its host. This organism frequently acquires resistance to antifungal agents through rapid generation of genetic diversity, with aneuploidy serving as a particularly important adaptive mechanism. This paper describes an investigation of the sole kinesin-5 in C. albicans, which is a major regulator of chromosome segregation. Contrary to other eukaryotes studied thus far, C. albicans does not require kinesin-5 function for bipolar spindle assembly or spindle elongation. Rather, this motor protein associates with the spindle throughout mitosis to maintain spindle integrity. Furthermore, kinesin-5 loss is synthetically lethal with loss of kinesin-14—canonically an opposing force producer to kinesin-5 in spindle assembly and anaphase. These results suggest a significant evolutionary rewiring of microtubule motor functions in the C. albicans mitotic spindle, which may have implications in the genetic instability of this pathogen.


2002 ◽  
Vol 46 (4) ◽  
pp. 947-957 ◽  
Author(s):  
N. Jia ◽  
B. Arthington-Skaggs ◽  
W. Lee ◽  
C. A. Pierson ◽  
N. D. Lees ◽  
...  

ABSTRACT The incidence of fungal infections has increased dramatically, which has necessitated additional and prolonged use of the available antifungal agents. Increased resistance to the commonly used antifungal agents, primarily the azoles, has been reported, thus necessitating the discovery and development of compounds that would be effective against the major human fungal pathogens. The sterol biosynthetic pathway has proved to be a fertile area for antifungal development, and steps which might provide good targets for novel antifungal development remain. The sterol C-14 reductase, encoded by the ERG24 gene, could be an effective target for drug development since the morpholine antifungals, inhibitors of Erg24p, have been successful in agricultural applications. The ERG24 gene of Candida albicans has been isolated by complementation of a Saccharomyces cerevisiae erg24 mutant. Both copies of the C. albicans ERG24 gene have been disrupted by using short homologous regions of the ERG24 gene flanking a selectable marker. Unlike S. cerevisiae, the C. albicans ERG24 gene was not required for growth, but erg24 mutants showed several altered phenotypes. They were demonstrated to be slowly growing, with doubling times at least twice that of the wild type. They were also shown to be significantly more sensitive to an allylamine antifungal and to selected cellular inhibitors including cycloheximide, cerulenin, fluphenazine, and brefeldin A. The erg24 mutants were also slightly resistant to the azoles. Most importantly, erg24 mutants were shown to be significantly less pathogenic in a mouse model system and failed to produce germ tubes upon incubation in human serum. On the basis of these characteristics, inhibitors of Erg24p would be effective against C. albicans.


2019 ◽  
Vol 63 (4) ◽  
Author(s):  
G. Tetz ◽  
M. Collins ◽  
D. Vikina ◽  
V. Tetz

ABSTRACT An urgent need exists for new antifungal compounds to treat fungal infections in immunocompromised patients. The aim of the current study was to investigate the potency of a novel antifungal compound, MYC-053, against the emerging yeast and yeast-like pathogens Candida glabrata, Candida auris, Cryptococcus neoformans, and Pneumocystis species. MYC-053 was equally effective against the susceptible control strains, clinical isolates, and resistant strains, with MICs of 0.125 to 4.0 μg/ml. Notably, unlike other antifungals such as azoles, polyenes, and echinocandins, MYC-053 was effective against Pneumocystis isolates, therefore being the only synthetic antifungal that may potentially be used against Pneumocystis spp., Candida spp., and Cryptococcus spp. MYC-053 was highly effective against preformed 48-h-old C. glabrata and C. neoformans biofilms, with minimal biofilm eradication concentrations equal to 1 to 4 times the MIC. Together, these data indicated that MYC-053 may be developed into a promising antifungal agent for the treatment and prevention of invasive fungal infections caused by yeasts and yeast-like fungi.


2019 ◽  
Vol 7 (7) ◽  
pp. 1067-1070 ◽  
Author(s):  
Sedigheh Bakhtiari ◽  
Soudeh Jafari ◽  
Jamileh Bigom Taheri ◽  
Tahereh Sadat Jafarzadeh Kashi ◽  
Zahra Namazi ◽  
...  

BACKGROUND: Candida species are the most common opportunistic fungal infections. Today, cinnamon plants have been considered for anti-Candida properties. AIM: This study aimed to investigate the effectiveness of cinnamaldehyde extract (from cinnamon derivatives) on Candida albicans and Candida glabrata species and comparison with nystatin. MATERIAL AND METHODS: In this study, cinnamaldehyde and nystatin were used. The specimens included Candida albicans and Candida glabrata. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were measured for each one by the microdilution method. This experiment was repeated three times. RESULTS: Cinnamaldehyde extract at a concentration of 62.5 μl/ml was able to prevent the growth of Candida albicans, at a concentration of 93.7 μl/ml, causing Candida albicans to disappear, at 48.8 μl/ml, to prevent the growth of Candida glabrata, and in the concentration of 62.5 μl/ml, causes the loss of Candida glabrata. In comparison, nystatin at 0.5 μg/ml concentration prevented the growth of Candida albicans, at concentrations of 1 μg/ml causing Candida albicans to be destroyed, at 4 μg/ml concentration to prevent the growth of Candida glabrata, and at a concentration of 8 μg/ml causes the loss of Candida glabrata. The results were the same every three times. CONCLUSIONS: Although cinnamaldehyde extract had an effect on fungal growth in both Candida albicans and Candida glabrata with a fatal effect; the effect on these two species was lower than nystatin.


2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Ioana Onac ◽  
Saadia Ali ◽  
Arti Mahto ◽  
Andrew Rutherford ◽  
James Galloway ◽  
...  

Abstract Case report - Introduction Bacterial and fungal infections are recognised complications of viral pneumonia, particularly in patients who are critically ill. We describe a case of fungal sacroiliitis complicating severe COVID-19 pneumonia following a prolonged intensive care unit (ICU) admission. Candida albicans sacroilitis is a rarely reported infection with few case reports in the literature. Candida osteoarticular infections can present as septic arthritis, with knee involvement in 75% of cases, or osteomyelitis. The latter presentation differs based on age - vertebral involvement (51%) is more common in adults while children are more likely to present with infection in the long bones, ribs, or sternum. Case report - Case description A 48-year-old Afro-Caribbean gentleman with a history of hypertension and obesity was admitted to the ICU with clinical, laboratory and radiographic features of COVID-19 infection despite persistently negative swabs. Whilst in ICU he required mechanical ventilation. His stay was further complicated by multiple infections, pulmonary emboli, and the presence of a cavitating lesion in the left lung. Cultures from bronchoalveolar lavage and a central venous catheter line grew Serratia Mascense, candida glabrata and pseudomonas were isolated from his urine. He was treated with multiple antibiotics including meropenem, tazocin, ceftazidime and avibactam. After 61 days in the ICU he was transferred to the ward. He developed severe pain in his right hip which was worse on movement. This was followed by urinary incontinence and sensory deficit in the right L2/L3 dermatome. He underwent magnetic resonance imaging (MRI) of his spine and sacroiliac joints which showed right sided sacroiliitis and oedema around the iliopsoas muscle. He was started on vancomycin, later changed to ceftazidime avibactam and metronidazole. An echocardiogram did not show any vegetations. He underwent a biopsy of his sacroiliac joints which confirmed the presence of leucocytes, extended cultures yielded candida albicans in one out of two biopsy specimens. Considering ongoing pyrexia, pain and inflammatory markers, intravenous fluconazole was added to his antibiotic regimen which resulted in a marked improvement in mobility. After four weeks, ceftazidime, metronidazole and avibactam were stopped, and fluconazole was administered as oral tablets. 6 days later he became febrile and IV fluconazole was restarted. A repeat chest CT showed resolution of the cavity but ongoing changes suggestive of organising pneumonia. A repeat MRI of the sacroiliac joints revealed minor improvement. Intravenous Fluconazole was continued for a total of 8 weeks and was changed to tablets for complete a total of 12 weeks. Case report - Discussion This is a severe case of COVID-19 infection who despite 9 negative PCR tests, on day 53, had positive IgG for SARS-CoV-2 infection, confirming our clinical suspicion. Particularly in the ICU setting, individuals are approximately ten times more likely to have secondary bacterial/fungal infections with more frequent detection of multidrug-resistant Gram- negative pathogens. This case highlights several difficulties. Urine cultures had confirmed candida albicans, likely to be related to catheter related urinary tract infections, and a possible source for our patient but also a resistant pseudomonas aeruginosa species. Furthermore, cultures were positive for Serratia Mascense, candida glabrata. He had also already been treated with prolonged, broad spectrum antimicrobial treatment. Considering this, establishing the aetiology of the septic sacroiliitis was challenging. The rarity of candida sacroiliitis and presence of the organism in just one specimen made this more difficult. This led to the decision of a repeat sacroiliac biopsy to supply sufficient samples for further microbial analyses such as 16S, 18S and mycobacteria culture, all of which were negative. He became febrile after the discontinuation of antimicrobials and a switch to oral fluconazole therapy. He was extensively re-investigated and despite resolution of the lung cavity, there were changes which could have been consistent with an organising pneumonia. At this point he was neutropenic, mildly eosinophilic, and therefore a drug reaction was also considered. Repeat MRI revealed resolving muscle inflammation and minimal change at the bone site, with erosions and possible reactive bone marrow oedema. Following discussion with microbiology the decision was made to persist with intravenous Fluconazole. He continued to improve, and his inflammatory markers normalised after 8 weeks of treatment. Prednisolone was started for COVID-19 related pneumonitis. Long-term antifungal treatment is advisable, and we aim to complete 12 weeks of treatment. Case report - Key learning points  Patients with SARS-CoV-2 infection, particularly those requiring ICU admission were at risk of developing superinfections with multidrug-resistant Gram-negative bacteria or fungal infections.Candida albicans sacroiliitis is rare therefore early aspiration/biopsy is essential for the management.Longer treatment is needed in osteoarticular candida infections, even up to 6 or 12 months, therefor long-term close monitoring of this patients is essential.The utility and timing of reimaging patients following such infections is still unclearClose multidisciplinary and interdisciplinary team collaboration is essential in the management of this complex patients


mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Liesbeth Demuyser ◽  
Erwin Swinnen ◽  
Alessandro Fiori ◽  
Beatriz Herrera-Malaver ◽  
Kevin Verstrepen ◽  
...  

ABSTRACT MGE1 encodes a yeast chaperone involved in Fe-S cluster metabolism and protein import into the mitochondria. In this study, we identified MGE1 as a multicopy suppressor of susceptibility to the antifungal fluconazole in the model yeast Saccharomyces cerevisiae. We demonstrate that this phenomenon is not exclusively dependent on the integrity of the mitochondrial DNA or on the presence of the drug efflux pump Pdr5. Instead, we show that the increased dosage of Mge1 plays a protective role by retaining increased amounts of ergosterol upon fluconazole treatment. Iron metabolism and, more particularly, Fe-S cluster formation are involved in regulating this process, since the responsible Hsp70 chaperone, Ssq1, is required. Additionally, we show the necessity but, by itself, insufficiency of activating the iron regulon in establishing the Mge1-related effect on drug susceptibility. Finally, we confirm a similar role for Mge1 in fluconazole susceptibility in the pathogenic fungi Candida glabrata and Candida albicans. IMPORTANCE Although they are mostly neglected compared to bacterial infections, fungal infections pose a serious threat to the human population. While some of them remain relatively harmless, infections that reach the bloodstream often become lethal. Only a few therapies are available, and resistance of the pathogen to these drugs is a frequently encountered problem. It is thus essential that more research is performed on how these pathogens cope with the treatment and cause recurrent infections. Baker’s yeast is often used as a model to study pathogenic fungi. We show here, by using this model, that iron metabolism and the formation of the important iron-sulfur clusters are involved in regulating susceptibility to fluconazole, the most commonly used antifungal drug. We show that the same process likely also occurs in two of the most regularly isolated pathogenic fungi, Candida glabrata and Candida albicans. IMPORTANCE Although they are mostly neglected compared to bacterial infections, fungal infections pose a serious threat to the human population. While some of them remain relatively harmless, infections that reach the bloodstream often become lethal. Only a few therapies are available, and resistance of the pathogen to these drugs is a frequently encountered problem. It is thus essential that more research is performed on how these pathogens cope with the treatment and cause recurrent infections. Baker’s yeast is often used as a model to study pathogenic fungi. We show here, by using this model, that iron metabolism and the formation of the important iron-sulfur clusters are involved in regulating susceptibility to fluconazole, the most commonly used antifungal drug. We show that the same process likely also occurs in two of the most regularly isolated pathogenic fungi, Candida glabrata and Candida albicans.


2015 ◽  
Vol 14 (10) ◽  
pp. 1054-1061 ◽  
Author(s):  
Fabien Cottier ◽  
Alrina Shin Min Tan ◽  
Xiaoli Xu ◽  
Yue Wang ◽  
Norman Pavelka

ABSTRACTCandida albicansis the leading cause of fungal infections; but it is also a member of the human microbiome, an ecosystem of thousands of microbial species potentially influencing the outcome of host-fungal interactions. Accordingly, antibacterial therapy raises the risk of candidiasis, yet the underlying mechanism is currently not fully understood. We hypothesize the existence of bacterial metabolites that normally controlC. albicansgrowth and of fungal resistance mechanisms against these metabolites. Among the most abundant microbiota-derived metabolites found on human mucosal surfaces are weak organic acids (WOAs), such as acetic, propionic, butyric, and lactic acid. Here, we used quantitative growth assays to investigate the dose-dependent fungistatic properties of WOAs onC. albicansgrowth and found inhibition of growth to occur at physiologically relevant concentrations and pH values. This effect was conserved across distantly related fungal species both inside and outside the CTG clade. We next screened a library of transcription factor mutants and identified several genes required for the resistance ofC. albicansto one or more WOAs. A single gene,MIG1, previously known for its role in glucose repression, conferred resistance against all four acids tested. Consistent with glucose being an upstream activator of Mig1p, the presence of this carbon source was required for WOA resistance in wild-typeC. albicans. Conversely, aMIG1-complemented strain completely restored the glucose-dependent resistance against WOAs. We conclude that Mig1p plays a central role in orchestrating a transcriptional program to fight against the fungistatic effect of this class of highly abundant metabolites produced by the gastrointestinal tract microbiota.


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