scholarly journals Essential Gene Discovery in the Basidiomycete Cryptococcus neoformans for Antifungal Drug Target Prioritization

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Giuseppe Ianiri ◽  
Alexander Idnurm
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Fungal Infections ◽  
Essential Gene ◽  
Essential Genes ◽  
Fungal Diseases ◽  
Evolutionary Divergence ◽  
Diploid Strain ◽  
Content Type ◽  
Ascomycete Fungi

ABSTRACTFungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly.Cryptococcus neoformansis a basidiomycete fungus that causes cryptococcosis worldwide in both immunocompromised and healthy individuals. As a basidiomycete, it diverged from other common pathogenic or model ascomycete fungi more than 500 million years ago. Here, we reportC. neoformansgenes that are essential for viability as identified through forward and reverse genetic approaches, using an engineered diploid strain and genetic segregation after meiosis. The forward genetic approach generated random insertional mutants in the diploid strain, the induction of meiosis and sporulation, and selection for haploid cells with counterselection of the insertion event. More than 2,500 mutants were analyzed, and transfer DNA (T-DNA) insertions in several genes required for viability were identified. The genes include those encoding the thioredoxin reductase (Trr1), a ribosome assembly factor (Rsa4), an mRNA-capping component (Cet1), and others. For targeted gene replacement, theC. neoformanshomologs of 35 genes required for viability in ascomycete fungi were disrupted, meiosis and sporulation were induced, and haploid progeny were evaluated for their ability to grow on selective media. Twenty-one (60%) were found to be required for viability inC. neoformans. These genes are involved in mitochondrial translation, ergosterol biosynthesis, and RNA-related functions. The heterozygous diploid mutants were evaluated for haploinsufficiency on a number of perturbing agents and drugs, revealing phenotypes due to the loss of one copy of an essential gene inC. neoformans. This study expands the knowledge of the essential genes in fungi using a basidiomycete as a model organism. Genes that have no mammalian homologs and are essential in bothCryptococcusand ascomycete human pathogens would be ideal for the development of antifungal drugs with broad-spectrum activity.IMPORTANCEFungal infections are very common in humans but may be neglected due to misdiagnosis and inattention.Cryptococcus neoformansis a yeast that infects mainly immunocompromised people, causing high mortality rates in developing countries. The fungus infects the lungs, crosses the blood-brain barrier, and invades the cerebrospinal fluid, causing fatal meningitis.C. neoformansinfections are treated with amphotericin B, flucytosine, and azoles, all developed decades ago. However, problems with antifungal agents highlight the urgent need for more-effective drugs to treatC. neoformansand other invasive fungal infections. These issues include the negative side effects of amphotericin B, the spontaneous resistance ofC. neoformansto azoles, and the inefficacy of the echinocandin antifungals. In this study, we report the identification ofC. neoformansessential genes as targets for the development of novel antifungals. Because of the level of evolutionary divergence betweenC. neoformansand the ascomycetes, a subset of these genes is likely essential in all fungi. Genes identified in this study represent an excellent starting point for the future development of new antifungals by pharmaceutical companies.

scholarly journals Trends in Antifungal Drug Susceptibility of Cryptococcus neoformans Isolates Obtained through Population-Based Surveillance in South Africa in 2002-2003 and 2007-2008

2011 ◽  
Vol 55 (6) ◽  
pp. 2606-2611 ◽  
Author(s):  
Nelesh P. Govender ◽  
Jaymati Patel ◽  
Marelize van Wyk ◽  
Tom M. Chiller ◽  
Shawn R. Lockhart ◽  
...  
Keyword(s):  
South Africa ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Fungal Infections ◽  
Antifungal Susceptibility ◽  
Drug Susceptibility ◽  
Population Based ◽  
Content Type ◽  
Microdilution Method ◽  
Broth Microdilution Method

ABSTRACTCryptococcus neoformansis the most common cause of meningitis among adult South Africans with HIV infection/AIDS. Widespread use of fluconazole for treatment of cryptococcal meningitis and other HIV-associated opportunistic fungal infections in South Africa may lead to the emergence of isolates with reduced fluconazole susceptibility. MIC testing using a reference broth microdilution method was used to determine if isolates with reduced susceptibility to fluconazole or amphotericin B had emerged among cases of incident disease. Incident isolates were tested from two surveillance periods (2002-2003 and 2007-2008) when population-based surveillance was conducted in Gauteng Province, South Africa. These isolates were also tested for susceptibility to flucytosine, itraconazole, voriconazole, and posaconazole. Serially collected isolate pairs from cases at several large South African hospitals were also tested for susceptibility to fluconazole. Of the 487 incident isolates tested, only 3 (0.6%) demonstrated a fluconazole MIC of ≥16 μg/ml; all of these isolates were from 2002-2003. All incident isolates were inhibited by very low concentrations of amphotericin B and exhibited very low MICs to voriconazole and posaconazole. Of 67 cases with serially collected isolate pairs, only 1 case was detected where the isolate collected more than 30 days later had a fluconazole MIC value significantly higher than the MIC of the corresponding incident isolate. Although routine antifungal susceptibility testing of incident isolates is not currently recommended in clinical settings, it is still clearly important for public health to periodically monitor for the emergence of resistance.

scholarly journals Identification of Off-Patent Drugs That Show Synergism with Amphotericin B or That Present Antifungal Action against Cryptococcus neoformans and Candida spp.

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Suélen Andreia Rossi ◽  
Haroldo Cesar de Oliveira ◽  
Daniel Agreda-Mellon ◽  
José Lucio ◽  
Maria José Soares Mendes-Giannini ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Mammalian Cells ◽  
Fungal Infections ◽  
New Drugs ◽  
Minor Effect ◽  
Candida Spp ◽  
Content Type ◽  
A Minor

ABSTRACT Amphotericin B (AmB) is the antifungal with the strongest fungicidal activity, but its use has several limitations, mainly associated with its toxicity. Although some lipidic and liposomal formulations that present reduced toxicity are available, their price limits their application in developing countries. Flucytosine (5FC) has shown synergistic effect with AmB for treatment of some fungal infections, such as cryptococcosis, but again, its price is a limitation for its use in many regions. In the present work, we aimed to identify new drugs that have a minor effect on Cryptococcus neoformans, reducing its growth in the presence of subinhibitory concentrations of AmB. In the initial screening, we found fourteen drugs that had this pattern. Later, checkerboard assays of selected compounds, such as erythromycin, riluzole, nortriptyline, chenodiol, nisoldipine, promazine, chlorcyclizine, cloperastine, and glimepiride, were performed and all of them confirmed for their synergistic effect (fractional inhibitory concentration index [FICI] < 0.5). Additionally, toxicity of these drugs in combination with AmB was tested in mammalian cells and in zebrafish embryos. Harmless compounds, such as the antibiotic erythromycin, were found to have synergic activity with AmB, not only against C. neoformans but also against some Candida spp., in particular against Candida albicans. In parallel, we identified drugs that had antifungal activity against C. neoformans and found 43 drugs that completely inhibited the growth of this fungus, such as ciclopirox and auranofin. Our results expand our knowledge about antifungal compounds and open new perspectives in the treatment of invasive mycosis based on repurposing off-patent drugs.

scholarly journals Dectin-2-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Suresh Ambati ◽  
Emma C. Ellis ◽  
Jianfeng Lin ◽  
Xiaorong Lin ◽  
Zachary A. Lewis ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Aspergillus Fumigatus ◽  
Effective Dose ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Antifungal Drugs ◽  
Extracellular Matrices ◽  
Content Type ◽  
Order Of Magnitude ◽  
Life Threatening

ABSTRACT Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2’s mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases. IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

scholarly journals In Vitro Activity of a Novel Antifungal Compound, MYC-053, against Clinically Significant Antifungal-Resistant Strains of Candida glabrata, Candida auris, Cryptococcus neoformans, and Pneumocystis spp.

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
G. Tetz ◽  
M. Collins ◽  
D. Vikina ◽  
V. Tetz
Keyword(s):  
Cryptococcus Neoformans ◽  
Candida Glabrata ◽  
Fungal Infections ◽  
Antifungal Compound ◽  
Candida Auris ◽  
Content Type ◽  
Treatment And Prevention ◽  
Resistant Strains ◽  
Clinically Significant

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.

scholarly journals New Triazole NT-a9 Has Potent Antifungal Efficacy against Cryptococcus neoformans In Vitro and In Vivo

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Ren-Yi Lu ◽  
Ting-Jun-Hong Ni ◽  
Jing Wu ◽  
Lan Yan ◽  
Quan-Zhen Lv ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Pathogenic Fungi ◽  
Control Group ◽  
Survival Times ◽  
Content Type ◽  
Fungal Burden ◽  
Wide Range

ABSTRACT In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo. NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 μg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 μg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 106 CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

scholarly journals Efficacy of Oral Encochleated Amphotericin B in a Mouse Model of Cryptococcal Meningoencephalitis

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
R. Lu ◽  
C. Hollingsworth ◽  
J. Qiu ◽  
A. Wang ◽  
E. Hughes ◽  
...  
Keyword(s):  
Mouse Model ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Oral Absorption ◽  
Oral Formulation ◽  
Significant Saving ◽  
Content Type ◽  
Cryptococcal Meningoencephalitis ◽  
Cryptococcal Disease ◽  
The Impact

ABSTRACT Cryptococcus neoformans is an encapsulated yeast responsible for approximately a quarter of a million deaths worldwide annually despite therapy, and upwards of 11% of HIV/AIDS-related deaths, rivaling the impact of tuberculosis and malaria. However, the most effective antifungal agent, amphotericin B, requires intravenous delivery and has significant renal and hematopoietic toxicity, making it difficult to utilize, especially in resource-limited settings. The present studies describe a new nanoparticle crystal encapsulated formulation of amphotericin B known as encochleated amphotericin B (CAmB) that seeks to provide an oral formulation that is low in toxicity and cost. Using a 3-day delayed model of murine cryptococcal meningoencephalitis and a large inoculum of a highly virulent strain of serotype A C. neoformans, CAmB, in combination with flucytosine, was found to have efficacy equivalent to parental amphotericin B deoxycholate with flucytosine and superior to oral fluconazole without untoward toxicity. Transport of fluorescent CAmB particles to brain as well as significant brain levels of amphotericin drug was demonstrated in treated mice, and immunological profiles were similar to those of mice treated with conventional amphotericin B. Additional toxicity studies using a standardized rat model showed negligible toxicity after a 28-day treatment schedule. These studies thus offer the potential for an efficacious oral formulation of a known fungicidal drug against intrathecal cryptococcal disease. IMPORTANCE Cryptococcus neoformans is a significant global fungal pathogen that kills an estimated quarter of a million HIV-infected individuals yearly and has poor outcomes despite therapy. The most effective therapy, amphotericin B, is highly effective in killing the fungus but is available only in highly toxic, intravenous formulations that are unavailable in most of the developing world, where cryptococcal disease in most prevalent. For example, in Ethiopia, reliance on the orally available antifungal fluconazole results in high mortality, even when initiated as preemptive therapy at the time of HIV diagnosis. Thus, alternative agents could result in significant saving of lives. Toward this end, the present work describes the development of a new formulation of amphotericin B (CAmB) that encapsulates the drug as a crystal lipid nanoparticle that facilitates oral absorption and prevents toxicity. Successful oral absorption of the drug was demonstrated in a mouse model that, in combination with the antifungal flucytosine, provided efficacy equal to a parental preparation of amphotericin B plus flucytosine. These studies demonstrate the potential for CAmB in combination with flucytosine to provide an effective oral formulation of a well-known, potent fungicidal drug combination.

scholarly journals Interactions between Triazoles and Amphotericin B against Cryptococcus neoformans

2000 ◽  
Vol 44 (9) ◽  
pp. 2435-2441 ◽  
Author(s):  
Francesco Barchiesi ◽  
Anna M. Schimizzi ◽  
Francesca Caselli ◽  
Andrea Novelli ◽  
Stefania Fallani ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Clinical Laboratory ◽  
Fungal Infections ◽  
National Committee ◽  
Positive Interaction ◽  
Pronounced Increase

ABSTRACT The interaction of amphotericin B (AmB) and azole antifungal agents in the treatment of fungal infections is still a controversial issue. A checkerboard titration broth microdilution-based method that adhered to the recommendations of the National Committee for Clinical Laboratory Standards was applied to study the in vitro interactions of AmB with fluconazole (FLC), itraconazole (ITC), and the new investigational triazole SCH 56592 (SCH) against 15 clinical isolates ofCryptococcus neoformans. Synergy, defined as a fractional inhibitory concentration (FIC) index of ≤0.50, was observed for 7% of the isolates in studies of the interactions of both FLC-AmB and ITC-AmB and for 33% of the isolates in studies of the SCH-AmB interactions; additivism (FICs, >0.50 to 1.0) was observed for 67, 73, and 53% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively; indifference (FICs, >1.0 to ≤2.0) was observed for 26, 20, and 14% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively. Antagonism (FIC >2.0) was not observed. When synergy was not achieved, there was still a decrease, although not as dramatic, in the MIC of one or both drugs when they were used in combination. To investigate the effects of FLC-AmB combination therapy in vivo, we established an experimental model of systemic cryptococcosis in BALB/c mice by intravenous injection of cells of C. neoformans 2337, a clinical isolate belonging to serotype D against which the combination of FLC and AmB yielded an additive interaction in vitro. Both survival and tissue burden studies showed that combination therapy was more effective than FLC alone and that combination therapy was at least as effective as AmB given as a single drug. On the other hand, when cells of C. neoformans 2337 were grown in FLC-containing medium, a pronounced increase in resistance to subsequent exposures to AmB was observed. In particular, killing experiments conducted with nonreplicating cells showed that preexposure to FLC abolished the fungicidal activity of the polyene. However, this apparent antagonism was not observed in vivo. Rather, when the two drugs were used sequentially for the treatment of systemic murine cryptococcosis, a reciprocal potentiation was often observed. Our study shows that (i) the combination of triazoles and AmB is significantly more active than either drug alone against C. neoformans in vitro and (ii) the concomitant or sequential use of FLC and AmB for the treatment of systemic murine cryptococcosis results in a positive interaction.

scholarly journals Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli

2015 ◽  
Vol 81 (16) ◽  
pp. 5650-5659 ◽  
Author(s):  
Shan Goh ◽  
Angela Hohmeier ◽  
Timothy C. Stone ◽  
Victoria Offord ◽  
Francisco Sarabia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Target Gene ◽  
Essential Gene ◽  
Transcript Level ◽  
Essential Genes ◽  
Content Type ◽  
Reading Frame ◽  
Knockout Mutants ◽  
Bacterial Genes ◽  
Relationship Of

ABSTRACTEssential bacterial genes located within operons are particularly challenging to study independently because of coordinated gene expression and the nonviability of knockout mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene disruption of genes may help establish essentiality but can lead to polar effects on genes downstream or upstream of the target gene. Here, theEscherichia coliribF-ileS-lspA-fkpB-ispHoperon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. The gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition as the minimum transcript level required for 50% growth (MTL50). Mupirocin and globomycin, the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and open reading frame-expressing plasmids to examine deregulation of the overlappingileS-lspAgenes. We found upstream and downstream polar silencing effects when eitherileSorlspAwas silenced, indicating coupled expression. Weighted MTL50values (means and standard deviations) ofribF,ileS, andlspAwere 0.65 ± 0.18, 0.64 ± 0.06, and 0.76 ± 0.10, respectively. However, they were not significantly different (P= 0.71 by weighted one-way analysis of variance). The gene requirement forispHcould not be determined due to insufficient growth reduction. Mupirocin and globomycin sensitization experiments indicated thatileS-lspAexpression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA-silencing antimicrobials.

scholarly journals Adaptation to Fluconazole via Aneuploidy Enables Cross-Adaptation to Amphotericin B and Flucytosine in Cryptococcus neoformans

2021 ◽  
Author(s):  
Feng Yang ◽  
Vladimir Gritsenko ◽  
Hui Lu ◽  
Cheng Zhen ◽  
Lu Gao ◽  
...  
Keyword(s):  
Fungal Infection ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Invasive Fungal Infection ◽  
Cryptococcus Gattii ◽  
Content Type ◽  
Therapeutic Drugs ◽  
Cross Adaptation ◽  
Pyrimidine Analogues ◽  
Globally Distributed

Cryptococcosis is a globally distributed invasive fungal infection caused by infections with Cryptococcus neoformans or Cryptococcus gattii . Only three classes of therapeutic drugs are clinically available for treating cryptococcosis: polyenes (amphotericin B), azoles (fluconazole), and pyrimidine analogues (flucytosine).

scholarly journals A Peptide from Budding Yeast GAPDH Serves as a Promising Antifungal against Cryptococcus neoformans

2022 ◽  
Author(s):  
Yang Zhang ◽  
Liyan Zhou ◽  
Yan Liu ◽  
Xi Zhao ◽  
Xianqiang Lian ◽  
...  
Keyword(s):  
Mortality Rate ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
High Mortality ◽  
Budding Yeast ◽  
Cryptococcus Gattii ◽  
Content Type

Cryptococcus neoformans and Cryptococcus gattii can cause cryptococcosis, which has a high mortality rate. To treat the disease, amphotericin B and fluconazole are often used in clinic.

Sign in / Sign up

Export Citation Format

Share Document