Garfoot Andrew L, Shen Qian, Wüthrich Marcel, Klein Bruce S, Rappleye Chad A
Departments of Microbiology and Microbial Infection and Immunity, Ohio State University, Columbus, Ohio, USA.
Department of Pediatrics, University of Wisconsin-Madison, Madison, Wisconsin, USA.
mBio. 2016 Apr 19;7(2):e01388-15. doi: 10.1128/mBio.01388-15.
The fungal pathogen Histoplasma capsulatum parasitizes host phagocytes. To avoid antimicrobial immune responses, Histoplasma yeasts must minimize their detection by host receptors while simultaneously interacting with the phagocyte. Pathogenic Histoplasma yeast cells, but not avirulent mycelial cells, secrete the Eng1 protein, which is a member of the glycosylhydrolase 81 (GH81) family. We show that Histoplasma Eng1 is a glucanase that hydrolyzes β-(1,3)-glycosyl linkages but is not required for Histoplasma growth in vitro or for cell separation. However, Histoplasma yeasts lacking Eng1 function have attenuated virulence in vivo, particularly during the cell-mediated immunity stage. Histoplasma yeasts deficient for Eng1 show increased exposure of cell wall β-glucans, which results in enhanced binding to the Dectin-1 β-glucan receptor. Consistent with this, Eng1-deficient yeasts trigger increased tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) cytokine production from macrophages and dendritic cells. While not responsible for large-scale cell wall structure and function, the secreted Eng1 reduces levels of exposed β-glucans at the yeast cell wall, thereby diminishing potential recognition by Dectin-1 and proinflammatory cytokine production by phagocytes. In α-glucan-producing Histoplasma strains, Eng1 acts in concert with α-glucan to minimize β-glucan exposure: α-glucan provides a masking function by covering the β-glucan-rich cell wall, while Eng1 removes any remaining exposed β-glucans. Thus, Histoplasma Eng1 has evolved a specialized pathogenesis function to remove exposed β-glucans, thereby enhancing the ability of yeasts to escape detection by host phagocytes.
The success of Histoplasma capsulatum as an intracellular pathogen results, in part, from an ability to minimize its detection by receptors on phagocytic cells of the immune system. In this study, we showed that Histoplasma pathogenic yeast cells, but not avirulent mycelia, secrete a β-glucanase, Eng1, which reduces recognition of fungal cell wall β-glucans. We demonstrated that the Eng1 β-glucanase promotes Histoplasma virulence by reducing levels of surface-exposed β-glucans on yeast cells, thereby enabling Histoplasma yeasts to escape detection by the host β-glucan receptor, Dectin-1. As a consequence, phagocyte recognition of Histoplasma yeasts is reduced, leading to less proinflammatory cytokine production by phagocytes and less control of Histoplasma infection in vivo Thus, Histoplasma yeasts express two mechanisms to avoid phagocyte detection: masking of cell wall β-glucans by α-glucan and enzymatic removal of exposed β-glucans by the Eng1 β-glucanase.
真菌病原体荚膜组织胞浆菌寄生于宿主吞噬细胞。为避免抗菌免疫反应，荚膜组织胞浆菌酵母必须在与吞噬细胞相互作用的同时，尽量减少被宿主受体的检测。致病性荚膜组织胞浆菌酵母细胞而非无毒力的菌丝体细胞分泌Eng1蛋白，它是糖基水解酶81（GH81）家族的成员。我们发现荚膜组织胞浆菌Eng1是一种葡聚糖酶，可水解β-(1,3)-糖苷键，但对荚膜组织胞浆菌的体外生长或细胞分离并非必需。然而，缺乏Eng1功能的荚膜组织胞浆菌酵母在体内的毒力减弱，尤其是在细胞介导的免疫阶段。Eng1缺陷的荚膜组织胞浆菌酵母显示细胞壁β-葡聚糖的暴露增加，这导致与Dectin-1β-葡聚糖受体的结合增强。与此一致，Eng1缺陷的酵母引发巨噬细胞和树突状细胞产生更多的肿瘤坏死因子α（TNF-α）和白细胞介素-6（IL-6）细胞因子。虽然不负责大规模的细胞壁结构和功能，但分泌的Eng1降低了酵母细胞壁上暴露的β-葡聚糖水平，从而减少了被Dectin-1识别的可能性以及吞噬细胞产生促炎细胞因子的可能性。在产生α-葡聚糖的荚膜组织胞浆菌菌株中，Eng1与α-葡聚糖协同作用以尽量减少β-葡聚糖的暴露：α-葡聚糖通过覆盖富含β-葡聚糖的细胞壁提供一种掩盖功能，而Eng1去除任何剩余的暴露β-葡聚糖。因此，荚膜组织胞浆菌Eng1进化出一种特殊的致病功能来去除暴露的β-葡聚糖，从而增强酵母逃避宿主吞噬细胞检测的能力。
荚膜组织胞浆菌作为一种细胞内病原体取得成功，部分原因在于其有能力尽量减少被免疫系统吞噬细胞上的受体检测到。在本研究中，我们表明致病性荚膜组织胞浆菌酵母细胞而非无毒力的菌丝体分泌一种β-葡聚糖酶Eng1，它可减少对真菌细胞壁β-葡聚糖的识别。我们证明Eng1β-葡聚糖酶通过降低酵母细胞表面暴露的β-葡聚糖水平来促进荚膜组织胞浆菌的毒力，从而使荚膜组织胞浆菌酵母能够逃避宿主β-葡聚糖受体Dectin-1的检测。结果，吞噬细胞对荚膜组织胞浆菌酵母的识别减少，导致吞噬细胞产生的促炎细胞因子减少，以及体内对荚膜组织胞浆菌感染的控制减弱。因此，荚膜组织胞浆菌酵母表达两种避免被吞噬细胞检测的机制：通过α-葡聚糖掩盖细胞壁β-葡聚糖以及通过Eng1β-葡聚糖酶酶解去除暴露的β-葡聚糖。
