An expanded toolkit of drug resistance cassettes for Candida glabrata, Candida auris, and Candida albicans leads to new insights into the ergosterol pathway.
An expanded toolkit of drug resistance cassettes for Candida glabrata, Candida auris, and Candida albicans leads to new insights into the ergosterol pathway.
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DOI：
10.1128/msphere.00311-23
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发表时间：
2023-12-20
期刊：
影响因子：
4.8
通讯作者：
Briggs, Scott D.
中科院分区：
文献类型：
--
作者：
Gregor, Justin B.;Gutierrez-Schultz, Victor A.;Hoda, Smriti;Baker, Kortany M.;Saha, Debasmita;Burghaze, Madeline G.;Vazquez, Cynthia;Burgei, Kendra E.;Briggs, Scott D.
关键词：
世界卫生组织最近公布了第一份重点真菌病原体清单，强调了多种念珠菌，包括光秃念珠菌、白色念珠菌和耳念珠菌。然而，先前对这些病原体的研究主要局限于使用NatMX和HphMX两种耐药磁带，限制了对原生营养实验室菌株和临床分离株的遗传操作能力。在这项研究中，我们扩展了裸毛念珠菌、耳念珠菌和白色念珠菌的工具包，将KanMX和BleMX与体外组装的CRISPR-Cas9核糖核蛋白（RNP）系统结合。将这些耐药磁带重新用于念珠菌，我们能够进行单基因缺失，顺序和同时双基因缺失，表位标签和拯救构建。我们使用这些耐药磁带来询问麦角甾醇途径，这是唑类和多烯类抗真菌药物的关键途径。利用我们的方法，我们首次确定了C. glabrata， C. auris和C. albicans原生营养菌株中ERG3的缺失导致了唑耐药性，这进一步支持了ERG3依赖性毒性甾醇模型的保守性。此外，我们还发现，在亚抑制浓度下，光棘草中的ERG5缺失对唑敏感，这表明ERG5可以作为Erg11的唑缓冲剂。最后，我们在C. glabrata中发现了一个合成的生长缺陷，当ERG3和ERG5都被删除时，这表明可能有另一种有毒甾醇影响生长。总的来说，我们已经扩展了可用的遗传工具来询问原生营养菌株和临床分离株的复杂途径。日益严重的耐药性和新出现的病原体问题是一个紧迫的全球卫生问题，需要开发和扩大研究真菌耐药性和发病机制的工具。先前对光秃念珠菌、耳念珠菌和白色念珠菌的研究主要局限于使用NatMX/SAT1和HphMX/CaHyg对原生营养菌株和临床分离株进行遗传操作。在这项研究中，我们证明了NatMX/SAT1、HphMX、KanMX和/或BleMX耐药盒与基于crispr -核糖核蛋白（RNP）的系统结合后，可以有效地用于删除或修饰C. glabrata、C. auris和C. albicans麦角甾醇通路中的基因。此外，这些工具的使用为ERG基因及其与念珠菌抗唑的关系提供了新的见解。总的来说，我们已经扩展了念珠菌病原体的工具包，以增加参与耐药性和发病机制的基因修饰复杂途径的多功能性。
The World Health Organization recently published the first list of priority fungal pathogens highlighting multiple Candida species, including Candida glabrata, Candida albicans, and Candida auris. However, prior studies in these pathogens have been mainly limited to the use of two drug resistance cassettes, NatMX and HphMX, limiting genetic manipulation capabilities in prototrophic laboratory strains and clinical isolates. In this study, we expanded the toolkit for C. glabrata, C. auris, and C. albicans to include KanMX and BleMX when coupled with an in vitro assembled CRISPR-Cas9 ribonucleoprotein (RNP)-based system. Repurposing these drug resistance cassettes for Candida, we were able to make single gene deletions, sequential and simultaneous double gene deletions, epitope tags, and rescue constructs. We applied these drug resistance cassettes to interrogate the ergosterol pathway, a critical pathway for both the azole and polyene antifungal drug classes. Using our approach, we determined for the first time that the deletion of ERG3 in C. glabrata, C. auris, and C. albicans prototrophic strains results in azole drug resistance, which further supports the conservation of the Erg3-dependent toxic sterol model. Furthermore, we show that an ERG5 deletion in C. glabrata is azole susceptible at subinhibitory concentrations, suggesting that Erg5 could act as an azole buffer for Erg11. Finally, we identified a synthetic growth defect when both ERG3 and ERG5 are deleted in C. glabrata, which suggests the possibility of another toxic sterol impacting growth. Overall, we have expanded the genetic tools available to interrogate complex pathways in prototrophic strains and clinical isolates. The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies in Candida glabrata, Candida auris, and Candida albicans have been mainly limited to the use of NatMX/SAT1 and HphMX/CaHyg for genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated that NatMX/SAT1, HphMX, KanMX, and/or BleMX drug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway of C. glabrata, C. auris, and C. albicans. Moreover, the utility of these tools has provided new insights into ERG genes and their relationship to azole resistance in Candida. Overall, we have expanded the toolkit for Candida pathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis.
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DOI：
10.1016/j.cub.2021.09.084
发表时间：
2021-12-06
期刊：
Current biology : CB
影响因子：
--
作者：
Ksiezopolska E;Schikora-Tamarit MÀ;Beyer R;Nunez-Rodriguez JC;Schüller C;Gabaldón T
通讯作者：
Gabaldón T
影响因子：
4.8
作者：
Grahl N;Demers EG;Crocker AW;Hogan DA
通讯作者：
Hogan DA
影响因子：
3.8
作者：
Boroujeni, Zeinab Borjian;Shamsaei, Sina;Raiesi, Omid
通讯作者：
Raiesi, Omid
影响因子：
4.1
作者：
HITCHCOCK, CA;DICKINSON, K;ADAMS, DJ
通讯作者：
ADAMS, DJ
影响因子：
5.3
作者：
Chandrasekharan, Mahesh B.;Huang, Fu;Sun, Zu-Wen
通讯作者：
Sun, Zu-Wen