Sodium New Houttuyfonate Inhibits Candida albicans Biofilm Formation by Inhibiting the Ras1-cAMP-Efg1 Pathway Revealed by RNA-seq.

Jiadi Wu,Yuanqing Si,Yeye Zhao,Jing Shao,Changzhong Wang,Daqiang Wu,Tianming Wang,Longfei Mei,Guiming Yan

doi:10.3389/fmicb.2020.02075

Abstract

Here, we aim to investigate the antifungal effect and mechanism of action of sodium new houttuyfonate (SNH) against Candida albicans. Microdilution analysis results showed that SNH possesses potent inhibitory activity against C. albicans SC5314, with a MIC80 of 256 μg/mL. Furthermore, we found that SNH can effectively inhibit the initial adhesion of C. albicans. Inverted microscopy, crystal violet staining, scanning electron microscopy and confocal laser scanning microscopy results showed that morphological changes during the transition from yeast to hypha and the biofilm formation of C. albicans are repressed by SNH treatment. We also found that SNH can effectively inhibit the biofilm formation of clinical C. albicans strains (Z103, Z3044, Z1402, and Z1407) and SNH in combination with fluconazole, berberine chloride, caspofungin and itraconazole antifungal agents can synergistically inhibit the biofilm formation of C. albicans. Eukaryotic transcriptome sequencing and qRT-PCR results showed that SNH treatment resulted in significantly down-regulated expression in several biofilm formation related genes in the Ras1-cAMP-Efg1 pathway (ALS1, ALA1, ALS3, EAP1, RAS1, EFG1, HWP1, and TEC1) and significantly up-regulated expression in yeast form-associated genes (YWP1 and RHD1). We also found that SNH can effectively reduce the production of key messenger cAMP in the Ras1-cAMP-Efg1 pathway. Furthermore, using Galleria mellonella as an in vivo model we found that SNH can effectively treat C. albicans infection in vivo. Our presented results suggest that SNH exhibits potential antibiofilm effects related to inhibiting the Ras1-cAMP-Efg1 pathway in the biofilm formation of C. albicans.

Highlights

Fungal infections caused by Candida albicans, an opportunistic fungal pathogen (Naglik et al, 2006), are a growing threat in the immune deficient population (Wang et al, 2018), and C. albicans is the species most frequently implicated in different forms of invasive candidiasis (Rodriguezcerdeira et al, 2019)
To verify the transcriptome results described above, we examined several genes related to adhesion and biofilms, especially those related to the Ras1-cyclic adenosine monophosphate (cAMP)-Efg1 pathway through the semiquantitative polymerase chain reaction (PCR) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods
To prevent the formation of biofilms, the transformation of yeast cells to hyphae should be prevented (Kavanaugh et al, 2014), and we found that sodium new houttuyfonate (SNH) can significantly repress the yeast-to-hypha morphological transition, which is key to the biofilm maturation of C. albicans

Summary

Introduction

Fungal infections caused by Candida albicans, an opportunistic fungal pathogen (Naglik et al, 2006), are a growing threat in the immune deficient population (Wang et al, 2018), and C. albicans is the species most frequently implicated in different forms of invasive candidiasis (Rodriguezcerdeira et al, 2019). Adherence of C. albicans to host cells, a prerequisite for infection, plays a significant role in pathogenesis, as it allows the establishment of a strong link to host cell surfaces and provides a focal point for infection by enabling persistence in harsh conditions (Pizarrocerda and Cossart, 2006; Martin et al, 2018). This high adherence is the main cause of fungal infection in human hosts, indicating that the highly infectious nature of this fungus may be related to its strong adherence capacity (Albrecht et al, 2006). New anti-adherence and anti-biofilm agents and strategies for C. albicans are urgently needed

Objectives

Methods

Results

Discussion

Conclusion