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  • 1
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    678. Galactomannan Is a Biomarker of APX001 (Fosmanogepix) Efficacy in Treating Experimental Invasive Pulmonary Aspergillosis
    Oxford University Press (OUP) ; 2019
    In:  Open Forum Infectious Diseases Vol. 6, No. Supplement_2 ( 2019-10-23), p. S309-S309
    Details
    In: Open Forum Infectious Diseases, Oxford University Press (OUP), Vol. 6, No. Supplement_2 ( 2019-10-23), p. S309-S309
    Abstract: Invasive pulmonary aspergillosis (IPA) is a serious fungal infection afflicting immunocompromised patients. Galactomannan (GM) detection in biological samples using the Platelia ELISA has been shown to predict therapy response by azoles, and polyenes. We previously reported on the activity of APX001 (fosmanogepix) in treating murine IPA. Here, we investigated the potential use of GM as a biomarker of APX001 efficacy in an immunosuppressed murine model of IPA. Methods ICR mice (n = 8/group) were immunosuppressed with cyclophosphamide and cortisone acetate on days −2, and +3, relative to infection with Aspergillus fumigatus via inhalation. Treatment with placebo (diluent control), APX001 (104 mg/kg, PO, a human equivalent dose), or posaconazole (POSA, 30 mg/kg, BID [equivalent to 6× the humanized dose]) began 16-hour post-infection and continued daily. To extend the half-life of APX001, mice were administered 50 mg/kg of the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) 2 hours prior to APX001 administration. Mice were sacrificed 48-, 72-, or 96-hour post-infection and their lungs, bronchoalveolar lavage (BAL) and sera were collected. Lung fungal burden was determined by conidial equivalent (CE) using qPCR, while GM was determined using the Platelia ELISA. Results Compared with placebo, APX001 or POSA treatment resulted in a gradual decrease in tissue fungal burden over time with APX001 or POSA showing significant reduction as early as 96 and 72 hours, respectively (P & lt; 0.005). Although the super-therapeutic dose of POSA resulted in faster reduction in lung fungal burden after 72 hours, both drugs resulted in similar reduction (~6–7 log) in lung CE vs. placebo after 96 hours. Changes in GM levels in BAL or serum samples mirrored reductions in lung CFU with significant decrease seen after 96 hours or 72 hours for APX001 or POSA, respectively, vs. placebo (P & lt; 0.02) (figure). Conclusion A human equivalent dose of APX001 and a super humanized dose of POSA resulted in a time-dependent reduction of lung fungal burden and GM levels when compared with placebo. These results show that GM can be used as a biomarker of APX001 efficacy in immunosuppressed mice. Disclosures All authors: No reported disclosures.
    Type of Medium: Online Resource
    ISSN: 2328-8957
    URL: Article
    DOI: 10.1093/ofid/ofz360.746
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2019
    detail.hit.zdb_id: 2757767-3
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  • 2
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    An evolutionarily diverged mitochondrial protein controls biofilm growth and virulence in Candida albicans
    Public Library of Science (PLoS) ; 2021
    In:  PLOS Biology Vol. 19, No. 3 ( 2021-3-15), p. e3000957-
    Details
    In: PLOS Biology, Public Library of Science (PLoS), Vol. 19, No. 3 ( 2021-3-15), p. e3000957-
    Abstract: A forward genetic screening approach identified orf19.2500 as a gene controlling Candida albicans biofilm dispersal and biofilm detachment. Three-dimensional (3D) protein modeling and bioinformatics revealed that orf19.2500 is a conserved mitochondrial protein, structurally similar to, but functionally diverged from, the squalene/phytoene synthases family. The C . albicans orf19.2500 is distinguished by 3 evolutionarily acquired stretches of amino acid inserts, absent from all other eukaryotes except a small number of ascomycete fungi. Biochemical assays showed that orf19.2500 is required for the assembly and activity of the N A D H u biquinone oxidoreductase Complex I (CI) of the respiratory electron transport chain (ETC) and was thereby named NDU1 . NDU1 is essential for respiration and growth on alternative carbon sources, important for immune evasion, required for virulence in a mouse model of hematogenously disseminated candidiasis, and for potentiating resistance to antifungal drugs. Our study is the first report on a protein that sets the Candida -like fungi phylogenetically apart from all other eukaryotes, based solely on evolutionary “gain” of new amino acid inserts that are also the functional hub of the protein.
    Type of Medium: Online Resource
    ISSN: 1545-7885
    URL: Article
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    DOI: 10.1371/journal.pbio.3000957
    DOI: 10.1371/journal.pbio.3000957.g001
    DOI: 10.1371/journal.pbio.3000957.g002
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    DOI: 10.1371/journal.pbio.3000957.s001
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    DOI: 10.1371/journal.pbio.3000957.s008
    Language: English
    Publisher: Public Library of Science (PLoS)
    Publication Date: 2021
    detail.hit.zdb_id: 2126773-X
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  • 3
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    1724. Plasmid-free CRISPR-Cas9 System for Genetic Engineering of Rhizopus delemar
    Oxford University Press (OUP) ; 2019
    In:  Open Forum Infectious Diseases Vol. 6, No. Supplement_2 ( 2019-10-23), p. S632-S632
    Details
    In: Open Forum Infectious Diseases, Oxford University Press (OUP), Vol. 6, No. Supplement_2 ( 2019-10-23), p. S632-S632
    Abstract: Mucormycosis is a serious infection caused by fungi of the order Mucorales. Rhizopus delemar is the most common etiologic agent of mucormycosis. Pathogenesis studies of mucormycosis have been hampered by poor genetic trackability of the organism, owing to rare chromosomal integration events and multinucleated nature of the cells. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system has been widely used in genetic manipulation through efficient homologous and non-homologous break points in a variety of organisms including R. delemar. However, plasmid-free CRISPR/Cas9 system has not been previously described in the fungus. Here, we introduce a rapid plasmid-free system for inducing orotidine 5’-phosphate decarboxylase (pyrF) gene mutation in R. delemar. Methods Protoplasts of R. delemar 99–880 strain were transformed with 20 nucleotide gRNA targeting the N-terminus of pyrF gene and the Cas9 enzyme. Screening for pyrF auxotrophy was carried out by plating transformed protoplasts on potato dextrose agar (PDA) plates containing 1 mg/mL 5-fluoroorotic acid (5-FOA) and 100 µg/mL uracil. Putative mutant strains were selected for uracil auxotrophy by plating simultaneously on media with or without uracil. pyrF disruption was verified by using PCR and qRT–PCR. Results Approximately100 transformants were generated through plating on 5-FOA plates. Only three transformants did not grow on minimal medium lacking uracil, indicating that they were true pyrF null mutants. PCR analysis showed that these three transformants have undergone nucleotide deletion events within the pyrF gene. The lack of pyrF gene expression was further verified by using qRT–PCR relative to wild-type R. delemar 99–880. Conclusion Similar to the plasmid-based genome manipulation strategy, the plasmid-free CRISPR/Cas9 system can induce gene editing in R. delemar. This rapid and simple approach adds an additional tool in our conquest to understand pathogenesis of mucormycosis. Disclosures All authors: No reported disclosures.
    Type of Medium: Online Resource
    ISSN: 2328-8957
    URL: Article
    DOI: 10.1093/ofid/ofz360.1587
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2019
    detail.hit.zdb_id: 2757767-3
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  • 4
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    726. APX001 (Fosmanogepix) Is Effective in an Immunosuppressed Mouse Model of Rhizopus oryzae Infection
    Oxford University Press (OUP) ; 2019
    In:  Open Forum Infectious Diseases Vol. 6, No. Supplement_2 ( 2019-10-23), p. S325-S326
    Details
    In: Open Forum Infectious Diseases, Oxford University Press (OUP), Vol. 6, No. Supplement_2 ( 2019-10-23), p. S325-S326
    Abstract: Mucormycosis is a life-threatening infection that predominantly occurs in immunocompromised hosts. The antifungal APX001A (manogepix) inhibits Gwt1, an enzyme required for the conserved glycosylphosphatidyl inositol (GPI) post-translational modification in eukaryotes. We previously reported the activity of APX001 (fosmanogepix, the prodrug of APX001A) against Rhizopus delemar (minimum effective concentration [MEC] = 0.25 µg/mL). Here we assessed the activity against R. oryzae, which has an elevated MEC value. Methods R. oryzae 99–892 MIC and MEC values were 0.125 µg/mL and 4.0 µg/mL for isavuconazole (ISAV) and APX001A, respectively. ICR mice were immunosuppressed with cyclophosphamide (200 mg/kg) and cortisone acetate (500 mg/kg) on Days -2, +3, and +8 relative to intratracheal infection with 2.5 × 105 cells of R. oryzae 99–892. For survival studies, treatment with 104 mg/kg APX001 was compared with ISAV (110 mg/kg TID). Oral treatment started on Day +1 through Day +7, relative to infection for survival studies, and through Day +4 for tissue fungal burden studies (assessed by conidial equivalent [CE] using qPCR). Placebo mice received vehicle control. To extend the half-life of APX001, mice were administered 50 mg/kg of the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) 2 h prior to APX001 administration. Results APX001 and ISAV equally prolonged median survival time of mice (n = 20) vs. placebo (12 and 14 days for APX001 and ISAV, respectively, vs. 8 days for placebo). Furthermore, APX001 and ISAV treatment both resulted in 30% 21-day survival vs. 0% survival of placebo mice (P 〈 0.05 by log-rank test). Both drug treatments resulted in ~1.5 log10 reduction in lung and brain CE vs. placebo-treated mice (n = 10, P 〈 0.005 by Wilcoxon rank-sum test). Conclusion Despite a higher MEC value, APX001 showed significant efficacy against R. oryzae that was as protective as ISAV in immunosuppressed mice. Given the previously reported activity of APX001 against a strain of R. delemar with a lower MEC value,APX001 has now been shown to be efficacious against both species of Rhizopus, which together are responsible for ~60–70% of isolates causing lethal mucormycosis. Thus, continued investigation of APX001 against mucormycosis is warranted. Disclosures All authors: No reported disclosures.
    Type of Medium: Online Resource
    ISSN: 2328-8957
    URL: Article
    DOI: 10.1093/ofid/ofz360.794
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2019
    detail.hit.zdb_id: 2757767-3
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  • 5
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    Online Resource
    Fosmanogepix (APX001) Is Effective in the Treatment of Immunocompromised Mice Infected with Invasive Pulmonary Scedosporiosis or Disseminated Fusariosis
    American Society for Microbiology ; 2020
    In:  Antimicrobial Agents and Chemotherapy Vol. 64, No. 3 ( 2020-02-21)
    Details
    In: Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Vol. 64, No. 3 ( 2020-02-21)
    Abstract: There are limited treatment options for immunosuppressed patients with lethal invasive fungal infections due to Fusarium and Scedosporium . Manogepix (MGX; APX001A) is a novel antifungal that targets the conserved Gwt1 enzyme required for localization of glycosylphosphatidylinositol-anchored mannoproteins in fungi. We evaluated the in vitro activity of MGX and the efficacy of the prodrug fosmanogepix (APX001) in immunosuppressed murine models of hematogenously disseminated fusariosis and pulmonary scedosporiosis. The MGX minimum effective concentration (MEC) for Scedosporium isolates was 0.03 μg/ml and ranged from 0.015 to 0.03 μg/ml for Fusarium isolates. In the scedosporiosis model, treatment of mice with 78 mg/kg and 104 mg/kg of body weight fosmanogepix, along with 1-aminobenzotriazole (ABT) to enhance the serum half-life of MGX, significantly increased median survival time versus placebo from 7 days to 13 and 11 days, respectively. Furthermore, administration of 104 mg/kg fosmanogepix resulted in an ∼2-log 10 reduction in lung, kidney, or brain conidial equivalents/gram tissue (CE). Similarly, in the fusariosis model, 78 mg/kg and 104 mg/kg fosmanogepix plus ABT enhanced median survival time from 7 days to 12 and 10 days, respectively. A 2- to 3-log 10 reduction in kidney and brain CE was observed. In both models, reduction in tissue fungal burden was corroborated with histopathological data, with target organs showing reduced or no abscesses in fosmanogepix-treated mice. Survival and tissue clearance were comparable to a clinically relevant high dose of liposomal amphotericin B (10 to 15 mg/kg). Our data support the continued development of fosmanogepix as a first-in-class treatment for infections caused by these rare molds.
    Type of Medium: Online Resource
    ISSN: 0066-4804 , 1098-6596
    URL: Article
    DOI: 10.1128/AAC.01735-19
    RVK:
    XA 24552
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2020
    detail.hit.zdb_id: 1496156-8
    SSG: 12
    SSG: 15,3
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