抗菌肽AMP-17抗白念珠菌作用靶点鉴定

Identification of the target site of antimicrobial peptide AMP-17 against Candida albicans

摘要:

白念珠菌(Candida albicans)是侵袭性真菌感染的主要致病性病原体。抗菌肽AMP-17有较强的抗念珠菌活性，经AMP-17作用白念珠菌后的蛋白组学结果显示，细胞壁(XOG1)和氧化应激(SRR1)蛋白基因表达差异显著，提示AMP-17可能通过影响XOG1和SRR1基因发挥其抗白念珠菌作用。为进一步探究XOG1和SRR1基因是否为AMP-17的作用靶点，本研究利用规律成簇间隔短回文重复序列相关蛋白9 (clustered regulatory interspaced short palindromic repeats-associated protein 9, CRISPR/Cas9)系统构建了白念珠菌xog1Δ/Δ和srr1Δ/Δ缺失菌；表型观察结果发现除XOG1基因缺失可影响白念珠菌的体外应激和菌丝形成，2个基因缺失对白念珠菌生长繁殖和生物膜形成无明显影响；药敏实验分析显示AMP-17对xog1Δ/Δ和srr1Δ/Δ缺失菌的MIC₈₀值从野生菌的8 μg/mL增至16 μg/mL，而对srr1Δ/Δ::srr1回补菌的MIC₈₀值降至野生菌水平。此外，AMP-17抑制2株缺失菌生物膜形成的能力均较野生菌明显降低，表明缺失菌在酵母态及生物膜形成过程中对AMP-17的敏感性均降低，提示XOG1和SRR1基因可能是AMP-17发挥抗白念珠菌的潜在靶点。本研究结果为进一步探讨新型肽类抗真菌药物的抗菌机制提供了一定的实验依据和基础。

关键词:抗菌肽;AMP-17;白念珠菌;XOG1;SRR1;规律成簇间隔短回文重复序列相关蛋白9(CRISPR/Cas9)

Abstract:

Candida albicans is one of the major causes of invasive fungal infections and a serious opportunistic pathogen in immunocompromised individuals. The antimicrobial peptide AMP-17 has prominent anti-Candida activity, and proteomic analysis revealed significant differences in the expression of cell wall (XOG1) and oxidative stress (SRR1) genes upon the action of AMP-17 on C. albicans, suggesting that AMP-17 may exert anti-C. albicans effects by affecting the expression of XOG1 and SRR1 genes. To further investigate whether XOG1 and SRR1 genes were the targets of AMP-17, C. albicans xog1Δ/Δ and srr1Δ/Δ mutants were constructed using the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system. Phenotypic observations revealed that deletion of two genes had no significant effect on C. albicans growth and biofilm formation, whereas XOG1 gene deletion affected in vitro stress response and mycelium formation of C. albicans. Drug sensitivity assay showed that the MIC₈₀ values of AMP-17 against xog1Δ/Δ and srr1Δ/Δ mutants increased from 8 μg/mL (for the wild type C. albicans SC5314) to 16 μg/mL, while the MIC₈₀ values against srr1Δ/Δ::srr1 revertants decreased to the level of the wild type SC5314. In addition, the ability of AMP-17 to inhibit biofilm formation of both deletion strains was significantly reduced compared to that of wild type SC5314, indicating that the susceptibility of the deletion mutants to AMP-17 was reduced in both the yeast state and during biofilm formation. These results suggest that XOG1 and SRR1 genes are likely two of the potential targets for AMP-17 to exert anti-C. albicans effects, which may facilitate further exploration of the antibacterial mechanism of novel peptide antifungal drugs.

Key words:antimicrobial peptide;AMP-17;Candida albicans;XOG1;SRR1;clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9)