Genotypic, proteomic, and phenotypic approaches to decipher the response to caspofungin and calcineurin inhibitors in clinical isolates of echinocandin-resistantCandida glabrata

Abstract

Background Echinocandin resistance represents a great concern, as these drugs are recommended as first-line therapy for invasive candidiasis. Echinocandin resistance is conferred by mutations in FKS genes. Nevertheless, pathways are crucial for enabling tolerance, evolution, and maintenance of resistance. Therefore, understanding the biological processes and proteins involved in the response to caspofungin may provide clues indicating new therapeutic targets. Objectives We determined the resistance mechanism and assessed the proteome response to caspofungin exposure. We then evaluated the phenotypic impact of calcineurin inhibition by FK506 and cephalosporine A (CsA) on caspofungin-resistant Candida glabrata isolates. Methods Twenty-five genes associated with caspofungin resistance were analysed by NGS, followed by studies of the quantitative proteomic response to caspofungin exposure. Then, susceptibility testing of caspofungin in presence of FK506 and CsA was performed. The effects of calcineurin inhibitor/caspofungin combinations on heat stress (40°C), oxidative stress (0.2 and 0.4 mM menadione) and on biofilm formation (polyurethane catheter) were analysed. Finally, a Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of the combinations on larval survival. Results F659-del was found in the FKS2 gene of resistant strains. Proteomics data showed some up-regulated proteins are involved in cell-wall biosynthesis, response to stress and pathogenesis, some of them being members of calmodulin–calcineurin pathway. Therefore, the impact of calmodulin inhibition was explored. Calmodulin inhibition restored caspofungin susceptibility, decreased capacity to respond to stress conditions, and reduced biofilm formation and in vivo pathogenicity. Conclusions Our findings confirm that calmodulin-calcineurin-Crz1 could provide a relevant target in life-threatening invasive candidiasis.

Evaluation of Anti-Candida Potential of Piper nigrum Extract in Inhibiting Growth, Yeast-Hyphal Transition, Virulent Enzymes, and Biofilm Formation

“…IFIs are responsible for killing more than 1.5 million people yearly, similar to mortality rates due to tuberculosis and about three times more than malaria [ 68 ]. The limited number of antifungal drugs available (i.e., polyenes, azoles, and echinocandins) and the spread of antifungal resistance amplifies the need to identify new fungal targets for developing novel therapeutic alternatives [ 58 ].…”

“…Furthermore, antifungal resistance involves the regulation of several signaling pathways associated with drug response (e.g., Ras/cAMP/PKA pathway, calmodulin/calcineurin pathway (CaM/CaL), and mitogen-activated protein kinase [MAPK] signaling pathway). These pathways may favor not only resistance but also pathogenicity, so it is possible to observe different phenotypes across isolates [ 58 ]. However, these complex and multifactorial processes need to be studied in depth.…”

“…The effect of P. nigrum ethanolic extract on the biofilm formation of C. albicans strains was tested in 96-well microplates [ 57 , 58 ]. One hundred µL of Candida cell suspensions (1 × 10 6 cells/mL) in RPMI-1640 with MOPS were dispensed in 96 microdilution wells with or without P. nigrum extract (8 to 2048 μg/mL).…”

Molecular investigations on Candida glabrata clinical isolates for pharmacological targeting

“…In recent years, researchers have extensively investigated the clinical versions of pathogenic fungi at gene level and highlighted the variations in growth pattern, virulence, antifungal susceptibility, morphology and physiology of clinical isolates in comparison to wild-type counterpart. [48][49][50][51][52] The phenomenon of natural selection plays key role in helping microbes to evolve themselves in order to survive and tolerate stress present inside the host. 53 Therefore, time to time updation in our understanding about the fungal clinical strains is the most logical way of rationalizing the antifungal treatment practice.…”

In vitro synergistic antifungal activities of caspofungin in combination with fluconazole or voriconazole against Candida species determined by the Etest method

“…Amphotericin B (AmB) and echinocandins could be used to treat patients with Candida infection. However, with the use of echinocandins, C.glabrata began to show drug resistance (Ceballos-Garzon et al, 2022;Pfaller, 2012). In recent years, the incidence of C.glabrata infection has been increasing, and became the most common type of infection in candidiasis after C.albicans (Schmiedel, 2016).…”