Supporting data for "Role of Candida auris Extracellular Vesicles in Pathogenesis and Treatment"

“The role of Candida auris extracellular vesicles on pathogenesis and treatment”
Submitted by
Walton Man To Chan
for the degree of Master of Philosophy
at the University of Hong Kong
in August 2022
Candida auris is an emergent pathogenic fungus prominently known for its unusual transmissibility, environmental persistence, and multidrug resistance. It is the only fungi to be listed in the highest priority for the American Centers for Disease Control’s Antimicrobial Resistance Threats List. Until now, few of its known antifungal resistance mechanisms could adequately explain differences in survival in vitro and in vivo when challenged with antifungal drugs. Therefore, research into phenotypic survival mechanisms for C. auris was undertaken.
Here, naturally produced nanometer-sized secreted particles known as extracellular vesicles (EVs) are shown to play a key role in attenuating the potency of the essential frontline drug, amphotericin B. In in vitro assays, these EVs acted in a dose-dependent manner, with nanogram levels of them capable of doubling the minimum inhibitory concentration needed to kill the yeast. The results were reproducible in drastically different clinical formulations of the drug as well, suggesting a possible clinical translation to the research. Furthermore, this effect proved reproducible and transferable among isolates of C. auris, though could not be replicated in other pathogenic fungi such as Candida albicans. Chemical ablation of EV endocytosis restored wild-type susceptibility towards the drug in the face of EV treatment. Proteomic analysis of C. auris EVs revealed the abundance of an uncharacterized protein suspected to have glucanase activity. Its nearest homologue in C. albicans revealed potential functions in both dampening immune responses and biofilm catalysing ability. Quantification of C. auris EVs incubated with amphotericin B suggested that EVs likely were not inducing resistance through reducing bioavailability. 
Together, these results reveal extracellular vesicles to likely be a novel and unique mechanism for the pathogenic yeast C. auris to evade amphotericin B treatment. Successful inhibition of this process restored wild-type susceptibility and provide justification for research into synergies between EV-inhibiting and existing antifungal drugs. Further research will be required to detail the exact mechanism in which C. auris EVs confer resistance against amphotericin B, and whether these results can be clinically translated. 
- An abstract of 328 words

《曲霉菌auris细胞外囊泡在致病机制及治疗中的作用》

本研究由沃尔顿·曼·托·陈提交，旨在香港大学攻读哲学硕士学位，并于2022年8月完成。

曲霉菌auris作为一种新兴的致病真菌，以其独特的传播性、环境持久性和多重耐药性而著称。它是唯一被列入美国疾病控制与预防中心抗微生物耐药性威胁清单最高优先级的真菌。迄今为止，关于其已知的抗真菌耐药机制，尚不能充分解释在抗真菌药物挑战下，其在体外和体内生存差异的原因。因此，本研究开展了针对C. auris表型生存机制的研究。

在本研究中，天然产生的纳米级分泌颗粒，即细胞外囊泡（EVs），被证明在降低关键一线药物两性霉素B的效力方面起着关键作用。在体外实验中，这些EVs以剂量依赖性方式发挥作用，其中纳克级的EVs足以将杀灭酵母的最低抑菌浓度加倍。这些结果在药物的不同临床配方中也得到了重复，这表明了研究可能具有临床转化潜力。此外，这种效应在C. auris的分离株之间也得到了重复和转移，尽管在其他致病真菌如白色念珠菌中未能复制。化学消除EV内吞作用在EV治疗面前恢复了野生型对药物的敏感性。对C. auris EVs的蛋白质组学分析揭示了大量未表征的蛋白质，该蛋白质被认为具有葡萄糖苷酶活性。其在白色念珠菌中的最近同源物揭示了在降低免疫应答和生物膜催化能力方面的潜在功能。与两性霉素B孵育的C. auris EVs的定量分析表明，EVs可能不是通过降低生物利用度来诱导耐药性的。

综上所述，这些结果揭示了细胞外囊泡可能是致病酵母C. auris逃避两性霉素B治疗的全新且独特的机制。成功抑制这一过程恢复了野生型敏感性，并为研究EV抑制剂与现有抗真菌药物之间的协同作用提供了依据。进一步的研究将需要详细阐述C. auris EVs赋予对两性霉素B耐药性的确切机制，以及这些结果是否可以转化为临床应用。

——摘要，328字