scholarly journals A Markerless CRISPR-Mediated System for Genome Editing in Candida auris Reveals a Conserved Role for Cas5 in the Caspofungin Response

2021 ◽  
Author(s):  
Craig L. Ennis ◽  
Aaron D. Hernday ◽  
Clarissa J. Nobile
Keyword(s):  
Genome Editing ◽  
Fungal Pathogen ◽  
Multidrug Resistant ◽  
Candida Auris ◽  
Content Type ◽  
Life Threatening ◽  
Systemic Infections

Candida auris is a recently emerged multidrug-resistant fungal pathogen capable of causing life-threatening systemic infections in humans. Few tools are available for genome editing in C. auris .

scholarly journals SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome

mSphere ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Ben A. Evans ◽  
Douglas A. Bernstein
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Fungal Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Life Threatening ◽  
Genomic Locations

ABSTRACT Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicans ADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans. Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences. IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans. However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicans ADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.

scholarly journals Candida auris: an Emerging Fungal Pathogen

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
Emily S. Spivak ◽  
Kimberly E. Hanson
Keyword(s):  
Health Care ◽  
Fungal Pathogen ◽  
Multidrug Resistant ◽  
Health Care Facilities ◽  
Candida Auris ◽  
Environmental Persistence ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
Hospital Infection Control ◽  
Skin Colonization

ABSTRACT Candida auris has emerged globally as a multidrug-resistant health care-associated fungal pathogen. Recent reports highlight ongoing challenges due to organism misidentification, high rates of antifungal drug resistance, and significant patient mortality. The predilection for transmission within and between health care facilities possibly promoted by virulence factors that facilitate skin colonization and environmental persistence is unique among Candida species. This minireview details the global emergence of C. auris and discusses issues relevant to clinical microbiology laboratories, hospital infection control, and antimicrobial stewardship efforts.

scholarly journals Experimental Mouse Models of Disseminated Candida auris Infection

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Hong Xin ◽  
Farhan Mohiuddin ◽  
Jensen Tran ◽  
Abby Adams ◽  
Karen Eberle
Keyword(s):  
Mouse Model ◽  
Fungal Pathogen ◽  
The United States ◽  
Multidrug Resistant ◽  
Host Responses ◽  
High Dose ◽  
Invasive Infection ◽  
Global Public Health ◽  
Candida Auris ◽  
Content Type

ABSTRACT Disseminated candidiasis is a life-threatening disease and remains the most common bloodstream infection in hospitalized patients in the United States. Despite the availability of modern antifungal therapy, crude mortality in the last decade has remained unacceptably high. In particular, Candida auris is a multidrug-resistant, health care-associated fungal pathogen and has recently emerged as the first fungal pathogen to cause a global public health threat. A reliable animal model for disseminated C. auris candidiasis is therefore needed to study the unique aspects of this little-known host-pathogen interaction. In this study, we established an inbred A/J intravenous model as an appropriate model for human disseminated C. auris infection. We found that C5 deficiency in A/J mice results in a complex phenotype characterized by rapid fungal proliferation in target organs and the development of a unique and rapidly fatal response. In contrast, C57BL/6J mice and mice deficient in neutrophil elastase (NE−/−) survived high-dose C. auris intravenous challenge, even with cyclophosphamide (CY)-induced immunosuppression. Our study is the first to provide insight into the role of C5 in the host responses to C. auris invasive infection and establishes an inbred A/J mouse model of systemic C. auris infection without CY-induced immunosuppression. IMPORTANCE In the last decade, Candida auris has emerged globally as a multidrug‐resistant fungal pathogen. Although C. auris was initially isolated from the external ear canal, it can cause outbreaks of invasive infections with very high mortality and comorbidities. Recent reports highlight the ongoing challenges due to organism misidentification, high rates of multifungal drug resistance, and unacceptably high patient mortality. The assessment of C. auris virulence in a specific genetic deficiency mouse model of invasive C. auris infection in this study contributes to the little knowledge of host defense to C. auris infection, which holds promise as a model for investigating the pathogenesis of C. auris invasive infection, exploring the immune responses elicited by the fungus, evaluating the possible induction of immunity to the infection, and targeting candidates for an antifungal vaccine.

scholarly journals An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Namkha Nguyen ◽  
Morgan M. F. Quail ◽  
Aaron D. Hernday
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Fungal Pathogen ◽  
Gene Knockout ◽  
Strain Engineering ◽  
Molecular Genetic Analysis ◽  
Content Type ◽  
Highly Efficient ◽  
Discovery Research ◽  
Gene Knockouts

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

scholarly journals Environmental Isolation of Candida auris from the Coastal Wetlands of Andaman Islands, India

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Parth Arora ◽  
Prerna Singh ◽  
Yue Wang ◽  
Anamika Yadav ◽  
Kalpana Pawar ◽  
...  
Keyword(s):  
Salt Marsh ◽  
Coastal Wetlands ◽  
Ecological Niche ◽  
Multidrug Resistant ◽  
Sandy Beaches ◽  
Andaman Islands ◽  
Human Pathogen ◽  
Candida Auris ◽  
Content Type ◽  
The Indian Ocean

ABSTRACT Candida auris is a multidrug resistant pathogen that presents a serious global threat to human health. As C. auris is a newly emerged pathogen, several questions regarding its ecological niche remain unexplored. While species closely related to C. auris have been detected in different environmental habitats, little is known about the natural habitat(s) of C. auris. Here, we explored the virgin habitats around the very isolated Andaman Islands in the Indian Ocean for evidence of C. auris. We sampled coastal wetlands, including rocky shores, sandy beaches, tidal marshes, and mangrove swamps, around the Andaman group of the Andaman & Nicobar Islands, Union Territory, in India. Forty-eight samples of sediment soil and seawater were collected from eight sampling sites representing the heterogeneity of intertidal habitats across the east and west coast of South Andaman district. C. auris was isolated from two of the eight sampling sites, a salt marsh and a sandy beach. Interestingly, both multidrug-susceptible and multidrug-resistant C. auris isolates were found in the sample. Whole-genome sequencing analysis clustered the C. auris isolates into clade I, showing close similarity to other isolates from South Asia. Isolation of C. auris from the tropical coastal environment suggests its association with the marine ecosystem. The fact that viable C. auris was detected in the marine habitat confirms C. auris survival in harsh wetlands. However, the ecological significance of C. auris in salt marsh wetland and sandy beaches to human infections remains to be explored. IMPORTANCE Candida auris is a recently emerged multidrug-resistant fungal pathogen capable of causing severe infections in hospitalized patients. Despite its recognition as a human pathogen a decade ago, so far the natural ecological niche(s) of C. auris remains enigmatic. A previous hypothesis suggested that C. auris might be native to wetlands, that its emergence as a human pathogen might have been linked to global warming effects on wetlands, and that its enrichment in that ecological niche was favored by the ability of C. auris for thermal tolerance and salinity tolerance. To understand the mystery of environmental niches of C. auris, we explored the coastal wetland habitat around the very isolated Andaman Islands in the Indian Ocean. C. auris was isolated from the virgin habitats of salt marsh area with no human activity and from a sandy beach. C. auris isolation from the marine wetlands suggests that prior to its recognition as a human pathogen, it existed as an environmental fungus.

scholarly journals In Vitro Antifungal Combination of Flucytosine with Amphotericin B, Voriconazole, or Micafungin against Candida auris Shows No Antagonism

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
A. L. Bidaud ◽  
F. Botterel ◽  
A. Chowdhary ◽  
E. Dannaoui
Keyword(s):  
Amphotericin B ◽  
Inhibitory Concentration ◽  
Geometric Mean ◽  
Multidrug Resistant ◽  
Candida Auris ◽  
Content Type ◽  
Hospital Acquired Infections ◽  
Resistant Mutants ◽  
Hospital Acquired

ABSTRACT Candida auris is an emerging, multidrug-resistant pathogen responsible for invasive hospital-acquired infections. Flucytosine is an effective anti-Candida species drug, but which cannot be used as a monotherapy because of the risk of development of resistant mutants during treatment. It is, therefore, noteworthy to test possible combinations with flucytosine that may have a synergistic interaction. In this study, we determined the in vitro interaction between flucytosine and amphotericin B, micafungin, or voriconazole. These combinations have been tested against 15 C. auris isolates. The MIC ranges (geometric mean [Gmean]) of flucytosine, amphotericin B, micafungin, and voriconazole were 0.125 to 1 μg/ml (0.42 μg/ml), 0.25 to 1 μg/ml (0.66 μg/ml), 0.125 to 0.5 μg/ml (0.3 μg/ml), and 0.03 to 4 μg/ml (1.05 μg/ml), respectively. When tested in combination, indifferent interactions were mostly observed with fractional inhibitory concentration index values from 0.5 to 1, 0.31 to 1.01, and 0.5 to 1.06 for the combinations of flucytosine with amphotericin B, micafungin, and voriconazole, respectively. A synergy was observed for the strain CBS 10913 from Japan. No antagonism was observed for any combination. The combination of flucytosine with amphotericin B or micafungin may be relevant for the treatment of C. auris infections.

scholarly journals Simultaneous Infection with Enterobacteriaceae and Pseudomonas aeruginosa Harboring Multiple Carbapenemases in a Returning Traveler Colonized with Candida auris

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Ayesha Khan ◽  
William C. Shropshire ◽  
Blake Hanson ◽  
An Q. Dinh ◽  
Audrey Wanger ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Susceptibility Testing ◽  
Multidrug Resistant ◽  
Critically Ill Patient ◽  
Whole Genome ◽  
Candida Auris ◽  
Content Type ◽  
Simultaneous Infection ◽  
Polymicrobial Infections ◽  
Selection Of

ABSTRACT We report our clinical experience treating a critically ill patient with polymicrobial infections due to multidrug-resistant Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa in a 56-year-old woman who received health care in India and was also colonized by Candida auris. A precision medicine approach using whole-genome sequencing revealed a multiplicity of mobile elements associated with NDM-1, NDM-5, and OXA-181 and, supplemented with susceptibility testing, guided the selection of rational antimicrobial therapy.

scholarly journals Inactivation of the Pseudomonas -Derived Cephalosporinase-3 (PDC-3) by Relebactam

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Melissa D. Barnes ◽  
Christopher R. Bethel ◽  
Jim Alsop ◽  
Scott A. Becka ◽  
Joseph D. Rutter ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Gram Negative ◽  
Content Type ◽  
Life Threatening ◽  
Lactamase Inhibitor

ABSTRACT Pseudomonas aeruginosa is a prevalent and life-threatening Gram-negative pathogen. Pseudomonas -derived cephlosporinase (PDC) is the major inducible cephalosporinase in P. aeruginosa . In this investigation, we show that relebactam, a diazabicyclooctane β-lactamase inhibitor, potently inactivates PDC-3, with a k 2 / K of 41,400 M −1 s −1 and a k off of 0.00095 s −1 . Relebactam restored susceptibility to imipenem in 62% of multidrug-resistant P. aeruginosa clinical isolates, while only 21% of isolates were susceptible to imipenem-cilastatin alone. Relebactam promises to increase the efficacy of imipenem-cilastatin against P. aeruginosa .

scholarly journals Engineering of Long-Circulating Peptidoglycan Hydrolases Enables Efficient Treatment of Systemic Staphylococcus aureus Infection

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Anna M. Sobieraj ◽  
Markus Huemer ◽  
Léa V. Zinsli ◽  
Susanne Meile ◽  
Anja P. Keller ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Human Blood ◽  
Half Life ◽  
Multidrug Resistant ◽  
Life Extension ◽  
Drug Resistant ◽  
Content Type ◽  
Peptidoglycan Hydrolases ◽  
Life Threatening

ABSTRACT Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections. IMPORTANCE Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells.

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