Florent Morio1,2,3, Lisa Lombardi3, Ulrike Binder4, Cédric Loge5, Estelle Robert2, Denise Graessle4, Marine Bodin2, Cornelia Lass-Flörl4, Geraldine Butler3, Patrice Le Pape1,2. 1. Laboratoire de Parasitologie-Mycologie, CHU de Nantes, Nantes, France. 2. Département de Parasitologie et Mycologie Médicale, Université de Nantes, Nantes Atlantique Universités, EA1155 - IICiMed, Faculté de Pharmacie, Nantes, France. 3. School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin, Ireland. 4. Department of Hygiene, Microbiology and Public Health, Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Tirol, Austria. 5. Département de Chimie Thérapeutique, Université de Nantes, Nantes Atlantique Universités, EA1155 - IICiMed, Faculté de Pharmacie, Nantes, France.
Abstract
BACKGROUND: Azoles are one of the main antifungal classes for the treatment of candidiasis. In the current context of emerging drug resistance, most studies have focused on Candida albicans, Candida glabrata or Candida auris but, so far, less is known about the underlying mechanisms of resistance in other species, including Candida orthopsilosis. OBJECTIVES: We investigated azole resistance in a C. orthopsilosis clinical isolate recovered from a patient with haematological malignancy receiving fluconazole prophylaxis. METHODS: Antifungal susceptibility to fluconazole was determined in vitro (CLSI M27-A3) and in vivo (in a Galleria mellonella model of invasive candidiasis). The CoERG11 gene was then sequenced and amino acid substitutions identified were mapped on the predicted 3D structure of CoErg11p. A clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) genome-editing strategy was used to introduce relevant mutations into a fluconazole-susceptible C. orthopsilosis isolate. RESULTS: Compared with unrelated C. orthopsilosis isolates, the clinical isolate exhibited both in vitro and in vivo fluconazole resistance. Sequencing of the CoERG11 gene identified several amino acid substitutions, including two possibly involved in fluconazole resistance (L376I and G458S). Both mutations mapped close to the active site of CoErg11p. Engineering these mutations in a different genetic background using CRISPR-Cas9 demonstrated that G458S, but not L376I, confers resistance to fluconazole and voriconazole. CONCLUSIONS: Our data show that the G458S amino acid substitution in CoERG11p, but not L376I, contributes to azole resistance in C. orthopsilosis. In addition to highlighting the potential of CRISPR-Cas9 technology for precise genome editing in the field of antifungal resistance, we discuss some points that are critical to improving its efficiency.
BACKGROUND:Azoles are one of the main antifungal classes for the treatment of candidiasis. In the current context of emerging drug resistance, most studies have focused on Candida albicans, Candida glabrata or Candida auris but, so far, less is known about the underlying mechanisms of resistance in other species, including Candida orthopsilosis. OBJECTIVES: We investigated azole resistance in a C. orthopsilosis clinical isolate recovered from a patient with haematological malignancy receiving fluconazole prophylaxis. METHODS: Antifungal susceptibility to fluconazole was determined in vitro (CLSI M27-A3) and in vivo (in a Galleria mellonella model of invasive candidiasis). The CoERG11 gene was then sequenced and amino acid substitutions identified were mapped on the predicted 3D structure of CoErg11p. A clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) genome-editing strategy was used to introduce relevant mutations into a fluconazole-susceptible C. orthopsilosis isolate. RESULTS: Compared with unrelated C. orthopsilosis isolates, the clinical isolate exhibited both in vitro and in vivo fluconazole resistance. Sequencing of the CoERG11 gene identified several amino acid substitutions, including two possibly involved in fluconazole resistance (L376I and G458S). Both mutations mapped close to the active site of CoErg11p. Engineering these mutations in a different genetic background using CRISPR-Cas9 demonstrated that G458S, but not L376I, confers resistance to fluconazole and voriconazole. CONCLUSIONS: Our data show that the G458S amino acid substitution in CoERG11p, but not L376I, contributes to azole resistance in C. orthopsilosis. In addition to highlighting the potential of CRISPR-Cas9 technology for precise genome editing in the field of antifungal resistance, we discuss some points that are critical to improving its efficiency.
Authors: Farnaz Daneshnia; João N de Almeida Júnior; Amir Arastehfar; Lisa Lombardi; Erika Shor; Lis Moreno; Ana Verena Mendes; Maria Goreth Barberino; Danilo Thomaz Yamamoto; Geraldine Butler; David S Perlin; Arnaldo Lopes Colombo Journal: Emerg Microbes Infect Date: 2022-12 Impact factor: 19.568