Objectives：
The calcineurin signaling pathway plays critical roles in antifungal resistance and virulence in various pathogenic fungi, including Candida albicans, Candida glabrata, Cryptococcus neoformans, and Aspergillus fumigatus, and has attracted attention as a novel target for antifungal therapy. The serine/threonine protein kinase Elm1 is regulated by calcineurin and involved in multiple cellular functions, including mitosis, cytokinesis, and morphogenesis in Saccharomyces cerevisiae. In this study, we performed functional analysis of Elm1 in the clinically important fungal pathogen Candida glabrata.

Methods： 
An ELM1 deletion construct was generated using the one-step PCR-based technique and a Δelm1 deletion mutant was constructed by homologous recombination in C. glabrata. Cell morphology was examined under fluorescence microscope and transmission electron microscope. The major cell wall components were measured using alkali-insoluble cell wall fraction. A spot dilution assay was performed to evaluate sensitivity to cell wall damaging agents and high temperature. Antifungal susceptibility was examined using a commercially prepared colorimetric panel, Sensititre YeastOne microtiter panel. Virulence of the Δelm1 strain was examined using Galleria mellonella infection model. In this model, C. glabrata cells（2.0 × 106 cells / larva）were injected into the hemocoel through the last left pro-leg of larvae and survival was monitored daily for 7 days.

Results：
The Δelm1 strain displayed impaired cell division and elongated morphology. In addition, the Δelm1 strain had significantly thicker cell wall and increased chitin content compared with the wild-type and ELM1-reconstituted strains, although there was no difference in the amount of β-glucan. The loss of ELM1 also resulted in a decreased growth capacity and increased sensitivity to high temperature and cell wall damaging agents including echinocandins, Congo red and calcofluor white. However, the Δelm1 strain was more virulent than the wild-type strain in Galleria mellonella infection model. 

Conclusion：
This study characterized various phenotypes of the C. glabrata Δelm1 mutant for the first time, demonstrating that Elm1 is related to cell wall integrity and virulence in C. glabrata. To understand molecular basis of these phenomena, further analyses using an elm1 strain controlled by the tetracycline-regulatable promoter and an elm1 kinase-dead mutant are now in progress.

Role of Elm1 in cell wall integrity and virulence of Candida glabrata