Does the phenotypic adaptability of Candida auris enable its success as a multi-drug resistant human pathogen?

耳念珠菌最近已成为一种主要的医院获得性病原体，并引起全球卫生关注。耳念珠菌具有高度耐药性，对皮肤和医疗器械具有粘性，难以从重症监护病房根除。侵袭性疾病的死亡率接近50%。因此，主要的卫生当局，包括布里斯托尔的疾病控制中心（CDC）和英国公共卫生部（PHE）都发布了关于金黄色葡萄球菌的警报。抗逆性和适应性促进真菌的致病性和免疫逃避。因此，我们迫切需要表征金黄色葡萄球菌的应激适应如何影响宿主-病原体相互作用。我们假设，在压力下，金黄色葡萄球菌表现出强烈的基因组和表型适应倾向，这驱动了它对抗真菌药物的耐药性和耐受性，以及它逃避宿主免疫的能力。(1)逆境适应对金黄色葡萄球菌细胞壁的影响我们最近研究了覆盖4个地理分支的金黄色葡萄球菌临床分离株的细胞壁超微结构和生物膜形成。我们观察到，与白色念珠菌SC5314菌株相比，在YPD上生长的金黄色念珠菌往往具有更厚的细胞壁，但甘露聚糖原纤维更短，并且金黄色念珠菌不是特别有效的生物膜生产者。重要的是，临床auris分离株在进化支内部和进化支之间显示出这些特征的实质性差异。这强烈表明金黄色葡萄球菌在这些细胞特性上表现出可塑性，这可能有助于真菌适应宿主的生态位。这当然是白色念珠菌的情况，我们已经表明，念珠菌细胞壁在响应宿主体内生态位所带来的生理相关环境压力时，实质上进行了重塑。为了阐明金黄色葡萄球菌对宿主相关的渗透/阳离子和氧化应激的反应，我们将选择金黄色葡萄球菌菌株暴露于sorbose、H2O2和NaCl中，并使用一系列方法监测它们的超微结构和生化细胞壁特征。此外，我们将通过qRT-PCR监测关键细胞壁蛋白和调节因子编码基因的表达，以确定它们是否在胁迫适应过程中被诱导。(2)应激适应对金黄色葡萄球菌抗真菌药物耐受性和耐药性的影响为了评估应激适应是否能保护金黄色葡萄球菌抗真菌治疗，我们将金黄色葡萄球菌分离物置于渗透、氧化/阳离子胁迫下，然后用一系列临床相关抗真菌药物对这些应激适应细胞进行mic测试。此外，我们将利用已建立的策略对金黄色葡萄球菌进行微进化，以探索金黄色葡萄球菌表观遗传应激适应与遗传应激抗性之间的机制联系以及由此产生的抗真菌药物保护作用。为了描述遗传机制，我们将测试应激和抗真菌药物耐药性是否依赖于应激调节因子和ABC外排转运蛋白。(3)在宿主-病原体相互作用中，应激适应是否使金黄色葡萄球菌占上风？为了测试压力适应和抗压力的金黄色葡萄球菌细胞是否在与宿主免疫系统的相互作用中获得优势，我们将采用离体巨噬细胞测定和体内斑马鱼感染模型。体内模型将通过测试特定菌株通过后脑注射杀死斑马鱼幼虫的速度和效率，来确定应力适应菌株和应力抗性菌株是否比未处理的对照菌株更具毒性。我们还将利用斑马鱼巨噬细胞和中性粒细胞上的荧光报告团来观察酵母-巨噬细胞和酵母-中性粒细胞在体内的相互作用。对宿主-病原体相互作用的详细了解将通过对金黄色葡萄球菌细胞和小鼠骨髓源性巨噬细胞（bmdm）在体外相互作用的成像提供。我们将测定真菌识别率、吞噬率和杀伤率。

Candida auris has recently emerged as a major hospital-acquired pathogen and poses a global health concern. C. auris is highly drug resistant, adhesive to skin and medical devices, and difficult to eradicate from intensive care wards. Mortality rates for invasive disease approach 50%. Consequently, major health authorities, including the CDC (Centre for Disease Control) and PHE (Public Health England) at Bristol have posted alerts about C. auris.Stress resistance and adaptation promote fungal pathogenicity and immune evasion. Therefore, we urgently need to characterise how stress adaptation in C. auris impacts host-pathogen interactions. We hypothesize that, under stress, C. auris shows a strong propensity for genomic and phenotypic adaptations, which drive its resistance and tolerance to antifungal drugs, and its ability to evade host immunity.(1) Impact of stress adaptation upon the C. auris cell wall We have recently surveyed cell wall ultrastructure and biofilm formation in multiple clinical isolates of C. auris covering all four geographical clades. We observed, that C. auris tends to have thicker cell walls but shorter mannan fibrils than the C. albicans SC5314 strain, when grown on YPD, and that C. auris is not a particularly efficient biofilm producer. Importantly, the clinical C. auris isolates display substantial variability in these features both within and between clades. This strongly indicates that C. auris shows plasticity in these cellular properties, which probably contributes to fungal adapation to host niches. This certainly is the case for C. albicans, where we have shown that the Candida cell wall is substantially remodelled in response to physiologically relevant environmental stresses posed by niches in the host body. To elucidate how C. auris reacts to host-relevant osmotic/cationic and oxidative stresses, we will expose a selection of C. auris strains to sorbose, H2O2, and NaCl, and monitor their ultrastructural and biochemical cell wall features using an array o0f methods. In addition, we will monitor the expression of genes encoding key cell wall proteins and regulators by qRT-PCR to ascertain whether they are induced during stress adaptation.(2) Impact of stress adaptation upon antifungal drug tolerance and resistance in C. auris To assess whether stress adaptation protects C. auris against antifungal treatment, we will subject the C. auris isolates to osmotic, oxidative/cationic stresses, and then expose these stress-adapted cells to MIC-testing with an array of clinically relevant antifungals. Furthermore, using an established strategy we will micro-evolve stress-resistant C. auris strains to explore the mechanistic links between epigenetic stress adaptation and genetic stress resistance in C. auris and the resultant protection against antifungal drugs. To characterise genetic mechanisms, we will test whether stress and antifungal drug resistances depend on stress regulators and ABC efflux transporters.(3) Does stress adaptation give C. auris the upper hand during host-pathogen interactions?To test whether stress-adapted and stress-resistant C. auris cells gain an advantage during interaction with the host immune system, we will employ ex vivo macrophages assays, and in vivo zebrafish infection models. The in vivo model will establish whether stress-adapted and stress-resistant strains are more virulent than the untreated control strain, by testing how quickly and efficiently particular strains kill zebrafish larvae via hindbrain-injection. We will also utilise fluorophore reporters on macrophages and on neutrophils in zebrafish to visualise yeast-macrophage and yeast-neutrophil interactions in vivo. Detailed insight into the host-pathogen interactions will be provided by imaging interactions between C. auris cells and murine bone marrow-derived macrophages (BMDMs) ex vivo. We will assay rates of fungal recognition, phagocytosis and killing.