Characterization of the Cellular and Immunological Functions of an Atypical PDR-Type Transporter in the Human Fungal Pathogen Cryptococcus neoformans

Fungal infections kill close to 3.8 million people yearly, with the biggest burden in resource-limited regions. One of the leading causes of fungal infections is the environmental, opportunistic pathogen, Cryptococcus neoformans, which is also one of the leading causes of death in the HIV+ population. Moreover, with recent medical advances, the population at risk of developing disease from this fungus is increasing, hence C. neoformans represents a serious neglected health concern. Given that cryptococcal infection is becoming more widespread and that current therapies are inappropriate, C. neoformans is on the top priority group of the WHO’s list of fungal pathogens threatening human health, with a mortality rate as high as 81%. Hence, there is a clear need for a better understanding of the cryptococcal-host interactions that can lead to novel and more effective therapeutics. Previous clinical studies have correlated fungal uptake by macrophages with mortality, hence we performed a screen of 1,200 C. neoformans single-gene deletions for fungal regulators of phagocytosis. One of the mutants identified with increased uptake was missing the gene CNAG_06909. We have called this uncharacterized gene PDR6 due to similarities to Pleiotropic Drug Resistance genes in other fungi and hypothesized that it may play a role in the virulence of C. neoformans. Sequence analysis showed that PDR6 encodes a unique half-size ATP-binding cassette (ABC) transporter protein. Microbial ABC transporters often play a role in the efflux of substrates across a membrane and have specifically been implicated in the development of antimicrobial resistance. Consistently, we found that the pdr6? strain was hypersensitive to fluconazole (FLC), the most common antifungal drug. In fact, antifungal ETEST strips and broth dilution assays with other antifungals demonstrated that the pdr6? strain had altered antifungal profiles relative to WT control, particularly against the azole-class antifungals. Additionally, we found that the pdr6? strain has significantly less ergosterol in its plasma membrane when compared to the WT, which could explain the hypersensitivity to azoles, defects in biofilm formation, capsule shedding, and host recognition exhibited by this mutant. Cellularly, these results are consistent with a model where Pdr6 regulates ergosterol transport into the plasma membrane, affecting both azole resistance as well as host-fungal interactions.
Given that PDR6 regulates the interaction with macrophages, which subsequently influences the outcome of the disease, we next characterized the immunological functions of PDR6 in vivo. We found that animals infected with the pdr6? strain lived significantly longer than those infected with the WT fungus. Although analysis of the colony forming units over the course of infection revealed reduced growth and dissemination of the pdr6? strain, histological analysis of pulmonary tissue identified increased damage and inflammation in the lungs of the pdr6?-infected mice at late time points when compared to the WT control. These findings were further supported by quantification of immune cells and pro-inflammatory cytokines. Additionally, survival was significantly extended in the pdr6?-infected mice when the immune response was dampened using the corticosteroid dexamethasone. Together, these data support our hypothesis that the pdr6?-infected mice succumb to infection because of pneumonia and an uncontrolled pro-inflammatory immune response in the lungs rather than the typical meningitis associated with the WT fungus. This highlights the importance of the biological function of Pdr6 in the progression and development of cryptococcal meningitis. Clinically, given its indirect involvement in antifungal resistance, PDR6 represents a promising and novel therapeutic target in treating cryptococcal meningitis. Furthermore, determining the cellular and immunological functions of PDR6 will not only open new avenues of investigation in the cryptococcal field, but also affect the PDR transporter field since these half-size, atypical transporters are conserved in evolution but their function has not been characterized.

真菌感染每年导致近380万人死亡，其中资源匮乏地区负担最重。真菌感染的主要诱因之一是环境机会性病原体新型隐球菌，它也是HIV阳性人群中死亡的主要原因之一。此外，随着近年来医学的进步，易受这种真菌感染风险的人群正在增加，因此新型隐球菌构成了一个严重的被忽视的健康问题。鉴于隐球菌感染正在日益蔓延，而现有的治疗方法又不尽人意，新型隐球菌已成为世界卫生组织真菌病原体威胁人类健康优先级最高的病原体之一，死亡率高达81%。因此，对隐球菌与宿主相互作用有更深入的理解，以引领新型且更有效的治疗策略，显得尤为迫切。既往的临床研究表明，巨噬细胞对真菌的摄取与死亡率相关，因此我们对1200个新型隐球菌单基因缺失体进行了筛选，以识别真菌吞噬作用的调节因子。其中，一个摄取能力增强的突变体缺失了基因CNAG_06909。我们根据该基因与其他真菌中多效性药物耐药基因的相似性，将其命名为PDR6，并假设它在新型隐球菌的致病性中可能发挥作用。序列分析显示，PDR6编码一种独特的半大小ATP结合 cassette（ABC）转运蛋白。微生物ABC转运蛋白通常在底物跨膜外排中发挥作用，并且已被特别指出与抗菌耐药性的发展有关。一致地，我们发现pdr6?菌株对氟康唑（FLC）——最常用的抗真菌药物——表现出超敏感性。实际上，使用抗真菌ETEST条带和 broth 稀释法对其他抗真菌药物进行测试也表明，与野生型（WT）对照相比，pdr6?菌株的抗真菌谱发生了改变，尤其是在针对唑类药物的抗真菌药物方面。此外，我们发现与WT相比，pdr6?菌株的质膜中麦角甾醇含量显著降低，这可能解释了其对唑类药物的超敏感性、生物膜形成缺陷、荚膜脱落和宿主识别等突变体表型。从细胞层面来看，这些结果与一种模型一致，其中Pdr6调节麦角甾醇进入质膜的过程，从而影响唑类药物耐药性以及宿主-真菌相互作用。

鉴于PDR6调节与巨噬细胞的相互作用，进而影响疾病的结局，我们进一步在体内表征了PDR6的免疫学功能。我们发现，感染pdr6?菌株的动物比感染野生型真菌的动物存活时间显著更长。尽管感染过程中的菌群形成单位分析显示pdr6?菌株的生长和传播减少，但肺组织学分析显示，与野生型对照相比，在晚期时间点感染pdr6?菌株的小鼠肺部损伤和炎症增加。这些发现进一步得到了免疫细胞和促炎细胞因子的量化支持。此外，当使用皮质类固醇地塞米松抑制免疫反应时，感染pdr6?菌株的小鼠的存活时间也显著延长。总之，这些数据支持我们的假设，即pdr6?感染的小鼠死于肺炎和肺部无法控制的促炎免疫反应，而不是与野生型真菌相关的典型脑膜炎。这突出了Pdr6在隐球菌性脑膜炎的进展和发展中的生物学功能的重要性。在临床上，鉴于PDR6在抗真菌耐药性中的间接作用，它代表了治疗隐球菌性脑膜炎的一个有希望的、新颖的治疗靶点。此外，确定PDR6的细胞和免疫学功能不仅将为隐球菌领域的研究开辟新的途径，也将影响PDR转运蛋白领域，因为这些半大小、非典型的转运蛋白在进化中得以保守，但其功能尚未得到表征。