scholarly journals Sequencing of FKS Hot Spot 1 from Saprochaete capitata To Search for a Relationship to Reduced Echinocandin Susceptibility

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Inés Arrieta-Aguirre ◽  
Pilar Menéndez-Manjón ◽  
María Soledad Cuétara ◽  
Iñigo Fernández de Larrinoa ◽  
Juan Carlos García-Ruiz ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Fungal Pathogen ◽  
Hot Spot ◽  
Fungal Species ◽  
Catalytic Subunit ◽  
Glucan Synthase ◽  
Content Type ◽  
Oligonucleotide Primers ◽  
Geotrichum Capitatum ◽  
Flanking Regions

ABSTRACT Saprochaete capitata, formerly known as Geotrichum capitatum, is an emerging fungal pathogen with low susceptibility to echinocandins. Here, we report the nucleotide sequence of the S. capitata hot spot 1 region of the FKS gene (FKS HS1), which codifies for the catalytic subunit of β-1,3-d-glucan synthase, the target of echinocandins. For that purpose, we first designed degenerated oligonucleotide primers derived from conserved flanking regions of the FKS1 HS1 segment of 12 different fungal species. Interestingly, analysis of the translated FKS HS1 sequences of 12 isolates of S. capitata revealed that all of them exhibited the same F-to-L substitution in a position that is highly related to reduced echinocandin susceptibility.

scholarly journals Clathrin- and Arp2/3-Independent Endocytosis in the Fungal Pathogen Candida albicans

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Elias Epp ◽  
Elena Nazarova ◽  
Hannah Regan ◽  
Lois M. Douglas ◽  
James B. Konopka ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Membrane Proteins ◽  
Fungal Pathogen ◽  
Mammalian Cells ◽  
Fluid Phase ◽  
Fungal Species ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Content Type ◽  
Actin Cables

ABSTRACT Clathrin-mediated endocytosis (CME) is conserved among eukaryotes and has been extensively analyzed at a molecular level. Here, we present an analysis of CME in the human fungal pathogen Candida albicans that shows the same modular structure as those in other fungi and mammalian cells. Intriguingly, C. albicans is perfectly viable in the absence of Arp2/3, an essential component of CME in other systems. In C. albicans, Arp2/3 function remains essential for CME as all 15 proteins tested that participate in CME, including clathrin, lose their characteristic dynamics observed in wild-type (WT) cells. However, since arp2/3 cells are still able to endocytose lipids and fluid-phase markers, but not the Ste2 and Mup1 plasma membrane proteins, there must be an alternate clathrin-independent pathway we term Arp2/3-independent endocytosis (AIE). Characterization of AIE shows that endocytosis in arp2 mutants relies on actin cables and other Arp2/3-independent actin structures, as inhibition of actin functions prevented cargo uptake in arp2/3 mutants. Transmission electron microscopy (TEM) showed that arp2/3 mutants still formed invaginating tubules, cell structures whose proper functions are believed to heavily rely on Arp2/3. Finally, Prk1 and Sjl2, two proteins involved in patch disassembly during CME, were not correctly localized to sites of endocytosis in arp2 mutants, implying a role of Arp2/3 in CME patch disassembly. Overall, C. albicans contains an alternative endocytic pathway (AIE) that relies on actin cable function to permit clathrin-independent endocytosis (CIE) and provides a system to further explore alternate endocytic routes that likely exist in fungal species. IMPORTANCE There is a well-established process of endocytosis that is generally used by eukaryotic cells termed clathrin-mediated endocytosis (CME). Although the details are somewhat different between lower and higher eukaryotes, CME appears to be the dominant endocytic process in all eukaryotes. While fungi such as Saccharomyces cerevisiae have proven excellent models for dissecting the molecular details of endocytosis, loss of CME is so detrimental that it has been difficult to study alternate pathways functioning in its absence. Although the fungal pathogen Candida albicans has a CME pathway that functions similarly to that of S. cerevisiae, inactivation of this pathway does not compromise growth of yeast-form C. albicans. In these cells, lipids and fluid-phase molecules are still endocytosed in an actin-dependent manner, but membrane proteins are not. Thus, C. albicans provides a powerful model for the analysis of CME-independent endocytosis in lower eukaryotes.

scholarly journals Troponoids Can Inhibit Growth of the Human Fungal Pathogen Cryptococcus neoformans

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Maureen J. Donlin ◽  
Anthony Zunica ◽  
Ashlyn Lipnicky ◽  
Aswin K. Garimallaprabhakaran ◽  
Alex J. Berkowitz ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Fungal Pathogen ◽  
Antimicrobial Activities ◽  
Fungal Species ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Red Cedar ◽  
Immunosuppressed Patients ◽  
Western Red Cedar

ABSTRACT Cryptococcus neoformans is a pathogen that is common in immunosuppressed patients. It can be treated with amphotericin B and fluconazole, but the mortality rate remains 15 to 30%. Thus, novel and more effective anticryptococcal therapies are needed. The troponoids are based on natural products isolated from western red cedar, and have a broad range of antimicrobial activities. Extracts of western red cedar inhibit the growth of several fungal species, but neither western red cedar extracts nor troponoid derivatives have been tested against C. neoformans. We screened 56 troponoids for their ability to inhibit C. neoformans growth and to assess whether they may be attractive candidates for development into anticryptococcal drugs. We determined MICs at which the compounds inhibited 80% of cryptococcal growth relative to vehicle-treated controls and identified 12 compounds with MICs ranging from 0.2 to 15 μM. We screened compounds with MICs of ≤20 μM for cytotoxicity in liver hepatoma cells. Fifty percent cytotoxicity values (CC50s) ranged from 4 to >100 μM. The therapeutic indexes (TI, CC50/MIC) for most of the troponoids were fairly low, with most being <8. However, two compounds had TI values that were >8, including a tropone with a TI of >300. These tropones are fungicidal and are not antagonistic when used in combination with fluconazole or amphotericin B. Inhibition by these two tropones remains unchanged under conditions favoring cryptococcal capsule formation. These data support the hypothesis that troponoids may be a productive scaffold for the development of novel anticryptococcal therapies.

scholarly journals Genome-Wide Analysis of Experimentally Evolved Candida auris Reveals Multiple Novel Mechanisms of Multidrug Resistance

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Hans Carolus ◽  
Siebe Pierson ◽  
José F. Muñoz ◽  
Ana Subotić ◽  
Rita B. Cruz ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Multidrug Resistance ◽  
Fungal Pathogen ◽  
Hot Spot ◽  
Novel Mutations ◽  
Candida Auris ◽  
Content Type ◽  
Genome Wide ◽  
High Concern

ABSTRACT Candida auris is globally recognized as an opportunistic fungal pathogen of high concern, due to its extensive multidrug resistance (MDR). Still, molecular mechanisms of MDR are largely unexplored. This is the first account of genome-wide evolution of MDR in C. auris obtained through serial in vitro exposure to azoles, polyenes, and echinocandins. We show the stepwise accumulation of copy number variations and novel mutations in genes both known and unknown in antifungal drug resistance. Echinocandin resistance was accompanied by a codon deletion in FKS1 hot spot 1 and a substitution in FKS1 “novel” hot spot 3. Mutations in ERG3 and CIS2 further increased the echinocandin MIC. Decreased azole susceptibility was linked to a mutation in transcription factor TAC1b and overexpression of the drug efflux pump Cdr1, a segmental duplication of chromosome 1 containing ERG11, and a whole chromosome 5 duplication, which contains TAC1b. The latter was associated with increased expression of ERG11, TAC1b, and CDR2 but not CDR1. The simultaneous emergence of nonsense mutations in ERG3 and ERG11 was shown to decrease amphotericin B susceptibility, accompanied with fluconazole cross-resistance. A mutation in MEC3, a gene mainly known for its role in DNA damage homeostasis, further increased the polyene MIC. Overall, this study shows the alarming potential for and diversity of MDR development in C. auris, even in a clade until now not associated with MDR (clade II), stressing its clinical importance and the urge for future research. IMPORTANCE Candida auris is a recently discovered human fungal pathogen and has shown an alarming potential for developing multi- and pan-resistance toward all classes of antifungals most commonly used in the clinic. Currently, C. auris has been globally recognized as a nosocomial pathogen of high concern due to this evolutionary potential. So far, this is the first study in which the stepwise progression of multidrug resistance (MDR) in C. auris is monitored in vitro. Multiple novel mutations in known resistance genes and genes previously not or vaguely associated with drug resistance reveal rapid MDR evolution in a C. auris clade II isolate. Additionally, this study shows that in vitro experimental evolution can be a powerful tool to discover new drug resistance mechanisms, although it has its limitations.

scholarly journals Breakthrough Candidemia Due to Multidrug-Resistant Candida glabrata during Prophylaxis with a Low Dose of Micafungin

2014 ◽  
Vol 58 (4) ◽  
pp. 2438-2440 ◽  
Author(s):  
Fernando César Bizerra ◽  
Cristina Jimenez-Ortigosa ◽  
Ana Carolina R. Souza ◽  
Giovanni Luis Breda ◽  
Flávio Queiroz-Telles ◽  
...  
Keyword(s):  
Candida Glabrata ◽  
Low Dose ◽  
Hot Spot ◽  
Multidrug Resistant ◽  
Blood Cultures ◽  
Glucan Synthase ◽  
Content Type ◽  
High Mics

ABSTRACTWe identified a case of breakthrough candidemia in a 25-year-old patient receiving micafungin prophylaxis (50 mg/day). FiveCandida glabrataisolates were obtained from blood cultures and were classified as multidrug-resistant isolates, since all of them exhibited high MICs for echinocandin and azole drugs. A mutation (S663F) in hot spot 1 of theFKS2 gene was found in all five isolates. This mutation yielded a 1,3-β-d-glucan synthase enzyme with highly reduced sensitivities to echinocandin drugs.

scholarly journals FKS2andFKS3Genes of Opportunistic Human PathogenCandida albicansInfluence Echinocandin Susceptibility

2018 ◽  
Vol 62 (4) ◽  
pp. e02299-17 ◽  
Author(s):  
Sumanun Suwunnakorn ◽  
Hironao Wakabayashi ◽  
Milena Kordalewska ◽  
David S. Perlin ◽  
Elena Rustchenko
Keyword(s):  
Cell Wall ◽  
Fungal Pathogen ◽  
Essential Gene ◽  
Negative Regulators ◽  
Glucan Synthase ◽  
Content Type ◽  
Evolutionarily Conserved ◽  
Opportunistic Fungal Pathogen ◽  
A Site ◽  
Encoding Genes

ABSTRACTCandida albicans, a prevailing opportunistic fungal pathogen of humans, has a diploid genome containing three homologousFKSgenes that are evolutionarily conserved. One of these, the essential geneFKS1, encodes the catalytic subunit of glucan synthase, which is the target of echinocandin drugs and also serves as a site of drug resistance. The other two glucan synthase-encoding genes,FKS2andFKS3, are also expressed, but their roles in resistance are considered unimportant. However, we report here that expression ofFKS1is upregulated in strains lacking eitherFKS2orFKS3. Furthermore, in contrast to what is observed in heterozygousFKS1deletion strains, cells lackingFKS2orFKS3contain increased amounts of cell wall glucan, are more resistant to echinocandin drugs, and consistently are tolerant to cell wall-damaging agents. Our data indicate thatC. albicansFKS2andFKS3can act as negative regulators ofFKS1, thereby influencing echinocandin susceptibility.

scholarly journals Positions and Numbers ofFKSMutations in Candida albicans Selectively InfluenceIn VitroandIn VivoSusceptibilities to Echinocandin Treatment

2014 ◽  
Vol 58 (7) ◽  
pp. 3626-3635 ◽  
Author(s):  
M. Lackner ◽  
M. Tscherner ◽  
M. Schaller ◽  
K. Kuchler ◽  
C. Mair ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Hot Spot ◽  
Fungal Species ◽  
Double Mutation ◽  
Content Type ◽  
Substance Dependent ◽  
Isogenic Mutants ◽  
First Time ◽  
Selection Of

ABSTRACTCandidemia is the fourth most common kind of microbial bloodstream infection, withCandida albicansbeing the most common causative species. Echinocandins are employed as the first-line treatment for invasive candidiasis until the fungal species is determined and confirmed by clinical diagnosis. Echinocandins block theFKSglucan synthases responsible for embedding β-(1,3)-d-glucan in the cell wall. The increasing use of these drugs has led to the emergence of antifungal resistance, and elevated MICs have been associated with single-residue substitutions in specific hot spot regions ofFKS1andFKS2. Here, we show for the first time the caspofungin-mediatedin vivoselection of a double mutation within one allele of theFKS1hot spot 1 in a clinical isolate. We created a set of isogenic mutants and used a hematogenous murine model to evaluate thein vivooutcomes of echinocandin treatment. Heterozygous and homozygous double mutations significantly enhance thein vivoresistance ofC. albicanscompared with the resistance seen with heterozygous single mutations. The variousFKS1hot spot mutations differ in the degree of their MIC increase, substance-dependentin vivoresponse, and impact on virulence. Our results demonstrate that echinocandin EUCAST breakpoint definitions correlate with thein vivoresponse when a standard dosing regimen is used but cannot predict thein vivoresponse after a dose escalation. Moreover, patients colonized by aC. albicansstrain with multiple mutations inFKS1have a higher risk for therapeutic failure.

scholarly journals Cytochrome P450 Monooxygenase-Mediated Metabolic Utilization of Benzo[a]Pyrene by Aspergillus Species

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Erin M. Ostrem Loss ◽  
Mi-Kyung Lee ◽  
Ming-Yueh Wu ◽  
Julia Martien ◽  
Wanping Chen ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Environmental Pollutants ◽  
Energy Generation ◽  
Fungal Species ◽  
Metabolic Changes ◽  
Cytochrome P450 Monooxygenase ◽  
Cellular Growth ◽  
P450 Monooxygenase ◽  
Content Type ◽  
Fundamental Knowledge

ABSTRACT Soil-dwelling fungal species possess the versatile metabolic capability to degrade complex organic compounds that are toxic to humans, yet the mechanisms they employ remain largely unknown. Benzo[a]pyrene (BaP) is a pervasive carcinogenic contaminant, posing a significant concern for human health. Here, we report that several Aspergillus species are capable of degrading BaP. Exposing Aspergillus nidulans cells to BaP results in transcriptomic and metabolic changes associated with cellular growth and energy generation, implying that the fungus utilizes BaP as a growth substrate. Importantly, we identify and characterize the conserved bapA gene encoding a cytochrome P450 monooxygenase that is necessary for the metabolic utilization of BaP in Aspergillus. We further demonstrate that the fungal NF-κB-type velvet regulators VeA and VelB are required for proper expression of bapA in response to nutrient limitation and BaP degradation in A. nidulans. Our study illuminates fundamental knowledge of fungal BaP metabolism and provides novel insights into enhancing bioremediation potential. IMPORTANCE We are increasingly exposed to environmental pollutants, including the carcinogen benzo[a]pyrene (BaP), which has prompted extensive research into human metabolism of toxicants. However, little is known about metabolic mechanisms employed by fungi that are able to use some toxic pollutants as the substrates for growth, leaving innocuous by-products. This study systemically demonstrates that a common soil-dwelling fungus is able to use benzo[a]pyrene as food, which results in expression and metabolic changes associated with growth and energy generation. Importantly, this study reveals key components of the metabolic utilization of BaP, notably a cytochrome P450 monooxygenase and the fungal NF-κB-type transcriptional regulators. Our study advances fundamental knowledge of fungal BaP metabolism and provides novel insight into designing and implementing enhanced bioremediation strategies.

scholarly journals In Vitro Activity of Ibrexafungerp, a Novel Glucan Synthase Inhibitor against Candida glabrata Isolates with FKS Mutations

2019 ◽  
Vol 63 (11) ◽  
Author(s):  
Natalie S. Nunnally ◽  
Kizee A. Etienne ◽  
David Angulo ◽  
Shawn R. Lockhart ◽  
Elizabeth L. Berkow
Keyword(s):  
Candida Glabrata ◽  
Vitro Activity ◽  
Good Activity ◽  
In Vitro Activity ◽  
Broth Microdilution ◽  
Glucan Synthase ◽  
Content Type ◽  
Synthase Inhibitor

ABSTRACT Ibrexafungerp is a first-in-class glucan synthase inhibitor. In vitro activity was determined for 89 Candida glabrata isolates with molecularly identified FKS1 or FKS2 mutations conferring resistance to the echinocandins. All isolates were resistant to at least one echinocandin (i.e., anidulafungin, caspofungin, or micafungin) by broth microdilution. Results for ibrexafungerp were compared with those for each echinocandin. Ibrexafungerp had good activity against all echinocandin-resistant C. glabrata isolates.

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