Modifications of the composition of the hyphal outerlayer of Aspergillus fumigatus affects HUVEC inflammatory and immune response

Aspergillus fumigatus is an angioinvasive fungal pathogen and the main etiologic agent causing invasive aspergillosis. Upon contact with human umbilical vein endothelial cells (HUVECs), this fungus induces cellular injury, inflammatory response and a pro-thrombotic phenotype. The pathogen molecules involved in this processes are however still unknown. A. fumigatus hyphae have been shown to produce, both in vivo and in vitro, an extracellular matrix composed of galactomannan, galactosaminogalactan and α-(1,3)-glucan. The deletion of the UDP-Galp mutase results in a mutant with a very sticky phenotype, due to the increase in the galactosaminogalactan content in the cell surface. The overexpression of galactosaminogalactan in this mutant was confirmed by immunofluorescence microscopy with a monoclonal antibody. Interestingly, the adhesion of ∆ugm1 germlings to HUVECs was significantly higher in comparison to the wild type strain. The ∆ugm1 adhesion capacity was followed by an increment in the cytokine secretion and tissue factor expression by infected endothelial cells. Using a shotgun proteomic approach with label-free quantification, we were able to search and compare pathways regulated in endothelial cells challenged with A. fumigatus, wild type and ∆ugm1. The deletion of the UGM1 gene in A. fumigatus induced changes in the endothelial cell response, maily related to immune and stress responses. In addition, the purified urea soluble fraction of galactosaminogalactan was able to induce TNF-α secretion by HUVECs and also regulate coincident pathways identified in the mutant interaction condition. This work describes for the first time the impact of the modifications of the hyphal surface galactosaminogalactan on the endothelial cell responses to A. fumigatus.

烟曲霉（Aspergillus fumigatus）是一种血管侵袭性真菌病原体，也是引发侵袭性曲霉病的主要致病原。当该真菌与人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）接触后，可诱导细胞损伤、炎症反应及促血栓表型。然而，参与该过程的病原体相关分子仍未被阐明。已有研究证实，烟曲霉菌丝可在体内及体外产生由半乳甘露聚糖、半乳聚糖胺半乳糖（galactosaminogalactan）及α-(1,3)-葡聚糖组成的细胞外基质。UDP-Galp变位酶（UDP-Galp mutase）的缺失会导致突变株产生极强的黏附表型，究其原因，该突变株的细胞表面半乳聚糖胺半乳糖含量显著升高。利用针对半乳聚糖胺半乳糖的单克隆抗体进行免疫荧光显微镜检测，已证实该突变株中半乳聚糖胺半乳糖呈现过表达状态。值得注意的是，Δugm1芽管对HUVECs的黏附能力显著高于野生型菌株。该黏附能力的提升，同时伴随着被感染内皮细胞的细胞因子分泌量增加与组织因子表达上调。本研究采用无标记定量鸟枪蛋白质组学（shotgun proteomic approach with label-free quantification）方法，对野生型烟曲霉与Δugm1菌株分别侵染的内皮细胞的调控通路进行了检索与比对。烟曲霉UGM1基因的缺失可改变内皮细胞的应答模式，且该改变主要与免疫应答及应激反应相关。此外，纯化得到的尿素可溶性半乳聚糖胺半乳糖组分，可诱导HUVECs分泌肿瘤坏死因子α（tumor necrosis factor-α, TNF-α），同时能够调控在Δugm1菌株与内皮细胞互作实验中鉴定到的重合通路。本研究首次阐明了菌丝表面半乳聚糖胺半乳糖的修饰对内皮细胞应答烟曲霉过程的影响。