scholarly journals Pseudohyphal growth of the emerging pathogen Candida auris is triggered by genotoxic stress through the S phase checkpoint

2019 ◽  
Author(s):  
Gustavo Bravo Ruiz ◽  
Zoe K. Ross ◽  
Neil A.R. Gow ◽  
Alexander Lorenz
Keyword(s):  
Fungal Pathogens ◽  
Genotoxic Stress ◽  
Multidrug Resistant ◽  
S Phase ◽  
Antifungal Drugs ◽  
Yeast Cells ◽  
Candida Auris ◽  
Pseudohyphal Growth ◽  
Cellular Feature ◽  
S Phase Checkpoint

ABSTRACTThe morphogenetic switching between yeast cells and filaments (true hyphae and pseudohyphae) is a key cellular feature required for full virulence in many polymorphic fungal pathogens, such as Candida albicans. In the recently emerged yeast pathogen Candida auris, occasional elongation of cells has been reported. However, environmental conditions and genetic triggers for filament formation have remained elusive. Here, we report that induction of DNA damage and perturbation of replication forks by treatment with genotoxins, such as hydroxyurea, methyl methanesulfonate, and the clinically relevant fungistatic 5-fluorocytosine, causes filamentation in C. auris. The filaments formed were characteristic of pseudohyphae and not parallel-sided true hyphae. Pseudohyphal growth is apparently signalled through the S phase checkpoint and, interestingly, is Tup1-independent in C. auris. Intriguingly, the morphogenetic switching capability is strain-specific in C. auris, highlighting the heterogenous nature of the species as a whole.IMPORTANCECandida auris is a newly emerged fungal pathogen of humans. This species was first reported in 2009, when it was identified in an ear infection of a patient in Japan. However, despite intense interest in this organism as an often multidrug-resistant fungus there is little knowledge about its cellular biology. During infection of human patients, fungi are able to change cell shape from ellipsoidal yeast cells to elongated filaments to adapt to various conditions within the host organism. There are different types of filaments, which are triggered by reactions to different cues. Candida auris fails to form filaments when exposed to triggers that stimulate yeast-filament morphogenesis in other fungi. Here, we show that it does form filaments when its DNA is damaged. These conditions might arise when Candida auris cells interact with host immune cells, or growing in certain host tissues (kidney, bladder), or during treatment with antifungal drugs.

scholarly journals Pseudohyphal Growth of the Emerging Pathogen Candida auris Is Triggered by Genotoxic Stress through the S Phase Checkpoint

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Gustavo Bravo Ruiz ◽  
Zoe K. Ross ◽  
Neil A. R. Gow ◽  
Alexander Lorenz
Keyword(s):  
Fungal Pathogens ◽  
Genotoxic Stress ◽  
Multidrug Resistant ◽  
S Phase ◽  
Antifungal Drugs ◽  
Yeast Cells ◽  
Candida Auris ◽  
Content Type ◽  
Pseudohyphal Growth ◽  
S Phase Checkpoint

ABSTRACT The morphogenetic switching between yeast cells and filaments (true hyphae and pseudohyphae) is a key cellular feature required for full virulence in many polymorphic fungal pathogens, such as Candida albicans. In the recently emerged yeast pathogen Candida auris, occasional elongation of cells has been reported. However, environmental conditions and genetic triggers for filament formation have remained elusive. Here, we report that induction of DNA damage and perturbation of replication forks by treatment with genotoxins, such as hydroxyurea, methyl methanesulfonate, and the clinically relevant fungistatic 5-fluorocytosine, cause filamentation in C. auris. The filaments formed were characteristic of pseudohyphae and not parallel-sided true hyphae. Pseudohyphal growth is apparently signaled through the S phase checkpoint and, interestingly, is Tup1 independent in C. auris. Intriguingly, the morphogenetic switching capability is strain specific in C. auris, highlighting the heterogenous nature of the species as a whole. IMPORTANCE Candida auris is a newly emerged fungal pathogen of humans. This species was first reported in 2009 when it was identified in an ear infection of a patient in Japan. However, despite intense interest in this organism as an often multidrug-resistant fungus, there is little knowledge about its cellular biology. During infection of human patients, fungi are able to change cell shape from ellipsoidal yeast cells to elongated filaments to adapt to various conditions within the host organism. There are different types of filaments, which are triggered by reactions to different cues. Candida auris fails to form filaments when exposed to triggers that stimulate yeast filament morphogenesis in other fungi. Here, we show that it does form filaments when its DNA is damaged. These conditions might arise when Candida auris cells interact with host immune cells or during growth in certain host tissues (kidney or bladder) or during treatment with antifungal drugs.

scholarly journals 1579. Multidrug-Resistant Candida auris Isolates From New York Hospitals and Healthcare Facilities Are Susceptible to Antifungal Combinations

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S576-S577
Author(s):  
Brittany O’Brien ◽  
Sudha Chaturvedi ◽  
Vishnu Chaturvedi
Keyword(s):  
New York ◽  
Diagnostic System ◽  
Multidrug Resistant ◽  
Drug Combinations ◽  
Antifungal Drugs ◽  
Drug Resistant ◽  
Candida Auris ◽  
Current Case ◽  
Healthcare Facilities ◽  
Broth Microdilution Method

Abstract Background Candida auris outbreak continues unabated in New York with the current case counts exceeding 300 patients. We used a modification of standard CLSI broth microdilution method (BMD) if two-drug combinations are efficacious against C. auris isolates with high-resistance to fluconazole (FZ, MIC50 >256 mg/L), and variable resistance to other broad-spectrum antifungal drugs. Methods BMD plates were custom-designed and quality controlled by TREK Diagnostic System. The combination tests of 15 drug-resistant C. auris involved microtiter wells with the initial 144 two-drug combinations and their two-fold dilutions (1/2–1/32) to get 864 two-drug combinations finally. We utilized MIC100 endpoints for the drug combination readings as reported earlier for the intra- and inter-laboratory agreements obtained against Candida species and Aspergillus fumigatus (Antimicrob Agents Chemother. 2015. 59:1759–1766). We also tested minimum fungicidal concentrations (MFC). Results We tested all possible 864 two-drug antifungal combinations for nine antifungal drugs in use to yield 12,960 MIC100 readings, and MFC readings for 15 C. auris isolates. Flucytosine (FLC) at 2.0 mg/L potentiated most successful combinations with other drugs. Micafungin (MFG), Anidulafungin (AFG), Caspofungin (CAS) at individual concentrations of 0.25 mg/L combined well with FLC (2.0 mg/L) to yield MIC100 for 14, 13, and 12 of 15 C. auris isolates tested, respectively. MFG/FLC combination was also fungicidal for 4 of 15 isolates. AMB / FLC (0.25/1.0 mg/L) yielded MIC100 for 13 isolates and MFC for three test isolates. Posaconazole (POS), and Isavuconazole (ISA) and Voriconazole (VRC) also combined well with FLC (0.25/2.0 mg/L) to yield MIC100 for 12, 13, and 13 isolates, respectively. POS/FLC combination was fungicidal for three isolates. Conclusion We identified seven two drug-combinations of antifungals efficacious against drug-resistant C. auris strains. The modified BMD combination susceptibility testing could be used by the clinical laboratories to assist providers with the selection of optimal treatment for C. auris candidemia. Disclosures All authors: No reported disclosures.

scholarly journals A Role for Saccharomyces cerevisiae Chk1p in the Response to Replication Blocks

2004 ◽  
Vol 15 (9) ◽  
pp. 4051-4063 ◽  
Author(s):  
Kaila L. Schollaert ◽  
Julie M. Poisson ◽  
Jennifer S. Searle ◽  
Jennifer A. Schwanekamp ◽  
Craig R. Tomlinson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Transcriptional Response ◽  
S Phase ◽  
Yeast Cells ◽  
Dna Damage Checkpoint ◽  
Checkpoint Kinases ◽  
Checkpoint Pathway ◽  
Dna Replication And Repair ◽  
S Phase Checkpoint

Replication blocks and DNA damage incurred during S phase activate the S-phase and intra-S-phase checkpoint responses, respectively, regulated by the Atrp and Chk1p checkpoint kinases in metazoans. In Saccharomyces cerevisiae, these checkpoints are regulated by the Atrp homologue Mec1p and the kinase Rad53p. A conserved role of these checkpoints is to block mitotic progression until DNA replication and repair are completed. In S. cerevisiae, these checkpoints include a transcriptional response regulated by the kinase Dun1p; however, dun1Δ cells are proficient for the S-phase-checkpoint-induced anaphase block. Yeast Chk1p kinase regulates the metaphase-to-anaphase transition in the DNA-damage checkpoint pathway via securin (Pds1p) phosphorylation. However, like Dun1p, yeast Chk1p is not required for the S-phase-checkpoint-induced anaphase block. Here we report that Chk1p has a role in the intra-S-phase checkpoint activated when yeast cells replicate their DNA in the presence of low concentrations of hydroxyurea (HU). Chk1p was modified and Pds1p was transiently phosphorylated in this response. Cells lacking Dun1p were dependent on Chk1p for survival in HU, and chk1Δ dun1Δ cells were defective in the recovery from replication interference caused by transient HU exposure. These studies establish a relationship between the S-phase and DNA-damage checkpoint pathways in S. cerevisiae and suggest that at least in some genetic backgrounds, the Chk1p/securin pathway is required for the recovery from stalled or collapsed replication forks.

scholarly journals A marine microbiome antifungal targets urgent-threat drug-resistant fungi

Science ◽  
2020 ◽  
Vol 370 (6519) ◽  
pp. 974-978 ◽  
Author(s):  
Fan Zhang ◽  
Miao Zhao ◽  
Doug R. Braun ◽  
Spencer S. Ericksen ◽  
Jeff S. Piotrowski ◽  
...  
Keyword(s):  
Mode Of Action ◽  
Fungal Pathogens ◽  
Antifungal Drugs ◽  
Multiple Drug ◽  
Drug Resistant ◽  
Candida Auris ◽  
Marine Animals ◽  
Multiple Drug Resistant

New antifungal drugs are urgently needed to address the emergence and transcontinental spread of fungal infectious diseases, such as pandrug-resistant Candida auris. Leveraging the microbiomes of marine animals and cutting-edge metabolomics and genomic tools, we identified encouraging lead antifungal molecules with in vivo efficacy. The most promising lead, turbinmicin, displays potent in vitro and mouse-model efficacy toward multiple-drug–resistant fungal pathogens, exhibits a wide safety index, and functions through a fungal-specific mode of action, targeting Sec14 of the vesicular trafficking pathway. The efficacy, safety, and mode of action distinct from other antifungal drugs make turbinmicin a highly promising antifungal drug lead to help address devastating global fungal pathogens such as C. auris.

scholarly journals In Vitro Evaluation of Antifungal Drug Combinations against Multidrug-Resistant Candida auris Isolates from New York Outbreak

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Brittany O’Brien ◽  
Sudha Chaturvedi ◽  
Vishnu Chaturvedi
Keyword(s):  
New York ◽  
Drug Combination ◽  
Multidrug Resistant ◽  
Drug Combinations ◽  
Health Care Facilities ◽  
Antifungal Drugs ◽  
Candida Auris ◽  
Pathogenic Yeast ◽  
Content Type

ABSTRACT Since 2016, New York hospitals and health care facilities have faced an unprecedented outbreak of the pathogenic yeast Candida auris. We tested over 1,000 C. auris isolates from affected facilities and found high resistance to fluconazole (MIC > 256 mg/liter) and variable resistance to other antifungal drugs. Therefore, we tested if two-drug combinations are effective in vitro against multidrug-resistant C. auris. Broth microdilution antifungal combination plates were custom manufactured by TREK Diagnostic System. We used 100% inhibition endpoints for the drug combination as reported earlier for the intra- and interlaboratory agreements against Candida species. The results were derived from 12,960 readings, for 15 C. auris isolates tested against 864 two-drug antifungal combinations for nine antifungal drugs. Flucytosine (5FC) at 1.0 mg/liter potentiated the most combinations. For nine C. auris isolates resistant to amphotericin B (AMB; MIC ≥ 2.0 mg/liter), AMB-5FC (0.25/1.0 mg/liter) yielded 100% inhibition. Six C. auris isolates resistant to three echinocandins (anidulafungin [AFG], MIC ≥ 4.0 mg/liter; caspofungin [CAS], MIC ≥ 2.0 mg/liter; and micafungin [MFG], MIC ≥ 4.0 mg/liter) were 100% inhibited by AFG-5FC and CAS-5FC (0.0078/1 mg/liter) and MFG-5FC (0.12/1 mg/liter). None of the combinations were effective for C. auris 18-1 and 18-13 (fluconazole [FLC] > 256 mg/liter, 5FC > 32 mg/liter) except MFG-5FC (0.1/0.06 mg/liter). Thirteen isolates with a high voriconazole (VRC) MIC (>2 mg/liter) were 100% inhibited by the VRC-5FC (0.015/1 mg/liter). The simplified two-drug combination susceptibility test format would permit laboratories to provide clinicians and public health experts with additional data to manage multidrug-resistant C. auris.

scholarly journals Prevalence and Therapeutic Challenges of Fungal Drug Resistance: Role for Plants in Drug Discovery

Antibiotics ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 150 ◽  
Author(s):  
Lewis Marquez ◽  
Cassandra L. Quave
Keyword(s):  
United States ◽  
Drug Resistance ◽  
Fungal Pathogens ◽  
Fungal Infections ◽  
The United States ◽  
Multidrug Resistant ◽  
Modern Medicine ◽  
Candida Auris ◽  
Medical Ethnobotany ◽  
Global Issue

Antimicrobial resistance is a global issue that threatens the effective practice of modern medicine and global health. The emergence of multidrug-resistant (MDR) fungal strains of Candida auris and azole-resistant Aspergillus fumigatus were highlighted in the Centers for Disease Control and Prevention’s (CDC) 2019 report, Antibiotic Resistance Threats in the United States. Conventional antifungals used to treat fungal infections are no longer as effective, leading to increased mortality. Compounding this issue, there are very few new antifungals currently in development. Plants from traditional medicine represent one possible research path to addressing the issue of MDR fungal pathogens. In this commentary piece, we discuss how medical ethnobotany—the study of how people use plants in medicine—can be used as a guide to identify plant species for the discovery and development of novel antifungal therapies.

scholarly journals In Vitro Activity of Manogepix against Multidrug-Resistant and Panresistant Candida auris from the New York Outbreak

2020 ◽  
Vol 64 (11) ◽  
Author(s):  
YanChun Zhu ◽  
Shannon Kilburn ◽  
Mili Kapoor ◽  
Sudha Chaturvedi ◽  
Karen Joy Shaw ◽  
...  
Keyword(s):  
New York ◽  
Fungal Infections ◽  
Geometric Mean ◽  
Multidrug Resistant ◽  
Antifungal Drugs ◽  
New Drugs ◽  
Wild Type ◽  
Candida Auris ◽  
Content Type

ABSTRACT An ongoing Candida auris outbreak in the New York metropolitan area is the largest recorded to date in North America. Laboratory surveillance revealed NY C. auris isolates are resistant to fluconazole, with variable resistance to other currently used broad-spectrum antifungal drugs, and that several isolates are panresistant. Thus, there is an urgent need for new drugs with a novel mechanism of action to combat the resistance challenge. Manogepix (MGX) is a first-in-class agent that targets the fungal Gwt1 enzyme. The prodrug fosmanogepix is currently in phase 2 clinical development for the treatment of fungal infections. We evaluated the susceptibility of 200 New York C. auris isolates to MGX and 10 comparator drugs using CLSI methodology. MGX demonstrated lower MICs than comparators (MIC50 and MIC90, 0.03 mg/liter; range, 0.004 to 0.06 mg/liter). The local epidemiological cutoff value (ECV) for MGX indicated all C. auris isolates were within the population of wild-type (WT) strains; 0.06 mg/liter defines the upper limit of wild type (UL-WT). MGX was 8- to 32-fold more active than the echinocandins, 16- to 64-fold more active than the azoles, and 64-fold more active than amphotericin B. No differences were found in the MGX or comparators’ MIC50, MIC90, or geometric mean (GM) values when subsets of clinical, surveillance, and environmental isolates were evaluated. The range of MGX MIC values for six C. auris panresistant isolates was 0.008 to 0.015 mg/liter, and the median and mode MIC values were 0.015 mg/liter, demonstrating that MGX retains activity against these isolates. These data support further clinical evaluation of fosmanogepix for the treatment of C. auris infections, including highly resistant isolates.

scholarly journals Evaluation of in vitro activity of manogepix against multidrug-resistant and pan-resistant Candida auris from the New York Outbreak

2020 ◽  
Author(s):  
YanChun Zhu ◽  
Shannon Kilburn ◽  
Mili Kapoor ◽  
Sudha Chaturvedi ◽  
Karen Joy Shaw ◽  
...  
Keyword(s):  
New York ◽  
Fungal Infections ◽  
Multidrug Resistant ◽  
Antifungal Drugs ◽  
New Drugs ◽  
Candida Auris ◽  
York Metropolitan Area ◽  
Clinical Surveillance ◽  
Laboratory Surveillance

ABSTRACTAn ongoing Candida auris outbreak in the New York metropolitan area is the largest recorded to date in North America. Laboratory surveillance revealed NY C. auris isolates are resistant to fluconazole, with variable resistance to other currently used broad-spectrum antifungal drugs, and that several isolates are pan-resistant. Thus, there is an urgent need for new drugs with a novel mechanism of action to combat the resistance challenge. Manogepix (MGX) is a first-in-class agent that targets the fungal Gwt1 enzyme. The prodrug, fosmanogepix, is currently in Phase 2 clinical development for the treatment of fungal infections. We evaluated the susceptibility of 200 New York C. auris isolates to MGX and 10 comparator drugs using CLSI methodology. MGX demonstrated lower MICs than comparators (MIC50 and MIC90 0.03 mg/L; range 0.004-0.06 mg/L). The MGX epidemiological cutoff value (ECV, 99% cutoff) for the tested C. auris isolates was 0.06 mg/L. MGX was 8-32-fold more active than the echinocandins, 16-64-fold more active than the azoles, and 64-fold more active than amphotericin B. No differences were found in the MGX or comparators’ MIC50, MIC90, or GEOMEAN values when subsets of clinical, surveillance, and environmental isolates were evaluated. The range of MGX MIC values for six C. auris pan-resistant isolates was 0.008-0.015 mg/L, and the median and mode MIC values were 0.015 mg/L, demonstrating that MGX retains activity against these isolates. These data support further clinical evaluation of fosmanogepix for the treatment of C. auris infections, including highly resistant isolates.

scholarly journals Fungicidal Potency and Mechanisms of θ-Defensins against Multidrug-Resistant Candida Species

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Virginia Basso ◽  
Angie Garcia ◽  
Dat Q. Tran ◽  
Justin B. Schaal ◽  
Patti Tran ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Atp Release ◽  
Multidrug Resistant ◽  
Human Saliva ◽  
Drug Resistant ◽  
Candida Auris ◽  
Cell Permeabilization ◽  
Content Type ◽  
Atp Production ◽  
New Therapeutic Approaches

ABSTRACT Systemic candidiasis is a growing health care concern that is becoming even more challenging due to the growing frequency of infections caused by multidrug-resistant (MDR) Candida species. Thus, there is an urgent need for new therapeutic approaches to candidiasis, including strategies bioinspired by insights into natural host defense against fungal pathogens. The antifungal properties of θ-defensins, macrocyclic peptides expressed in tissues of Old World monkeys, were investigated against a panel of drug-sensitive and drug-resistant clinical isolates of Candida albicans and non- albicans Candida species. Rhesus θ-defensin 1 (RTD-1), the prototype θ-defensin, was rapidly and potently fungicidal against drug-sensitive and MDR C. albicans strains. Fungal killing occurred by cell permeabilization that was temporally correlated with ATP release and intracellular accumulation of reactive oxygen species (ROS). Killing by RTD-1 was compared with that by histatin 5 (Hst 5), an extensively characterized anticandidal peptide expressed in human saliva. RTD-1 killed C. albicans much more rapidly and at a >200-fold lower concentration than that of Hst 5. Unlike Hst 5, the anticandidal activity of RTD-1 was independent of mitochondrial ATP production. Moreover, RTD-1 was completely resistant to Candida proteases for 2 h under conditions that rapidly and completely degraded Hst 5. MICs and minimum fungicidal concentrations (MFCs) of 14 natural θ-defensins isoforms against drug-resistant C. albicans isolates identified peptides that are more active than amphotericin B and/or caspofungin against fluconazole-resistant organisms, including MDR Candida auris. These results point to the potential of macrocyclic θ-defensins as structural templates for the design of antifungal therapeutics.

Quantification and assessment of viability of Cryptococcus neoformans by LightCycler amplification of capsule gene mRNA

2004 ◽  
Vol 53 (12) ◽  
pp. 1201-1206 ◽  
Author(s):  
Muhammad Amjad ◽  
Najla Kfoury ◽  
Raymond Cha ◽  
Reem Mobarak
Keyword(s):  
Cryptococcus Neoformans ◽  
Internal Control ◽  
Fungal Pathogens ◽  
Pcr Amplification ◽  
Plasmid Vector ◽  
Antifungal Drugs ◽  
Yeast Cells ◽  
Rt Pcr ◽  
Gene Mrna

Cryptococcus neoformans is an opportunistic fungal pathogen. It infects the central nervous system causing meningitis, which is fatal if untreated, especially in AIDS and immunosuppressed patients. In this study a method of quantification and assessment of viability of C. neoformans by LightCycler RT-PCR amplification of the capsule gene mRNA is established. The sequence of primers and probes were derived from C. neoformans capsular CAP10 gene mRNA (GenBank accession number AF144574), and were species specific. Agarose gel electrophoresis analysis of LightCycler RT-PCR product showed a single band of 223 bp in length. In order to develop an internal control a 223 bp exon fragment of capsule mRNA was cloned in the pCR2.1 plasmid vector and RNA was generated by in vitro transcription. To determine the sensitivity of the assay, serial dilutions of in vitro-transcribed RNA with known concentrations and copy numbers, and serially diluted cultures of viable and nonviable C. neoformans were used. Under optimal conditions as little as 0.472 fg of capsule mRNA could be detected, corresponding to 1–10 c.f.u. ml−1 of the sample. No amplification was observed from up to105 heat/UV radiation-killed yeast cells and RNA of other bacterial and fungal pathogens and human genomic DNA or RNA. The amplification of capsule mRNA represents a sensitive, specific and quantitative means of detection of viable C. neoformans in clinical specimens and can be useful in the evaluation of the therapeutic efficacy of antifungal drugs in the treatment of C. neoformans meningitis.

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