白色念珠菌是最普遍的机会性人类真菌病原体之一。通过甘露糖基化蛋白层从免疫检测中掩蔽免疫原性多糖β(1,3)-葡聚糖的能力是白色念珠菌的关键毒力因子。我们先前报道了Cek1丝裂原活化蛋白(MAP)激酶途径的过度活化促进β(1,3)-葡聚糖暴露。在这份通讯中,我们报告了一种新型的Cek1活化上游调节因子,并表征了Cek1活性对真菌毒力的影响。Lrg1编码GTP酶激活蛋白(GAP),该蛋白已被建议抑制GTP酶Rho1。我们发现LRG1的破坏会导致Cek1过度激活和β(1,3)-葡聚糖掩蔽。然而,当测量一组GTP酶的GTP酶激活时,lrg1ΔΔ突变体显示Cdc42和Ras1的激活增加,但Rho1或Rac1没有激活。可以通过抑制Ste11MAP激酶激酶激酶(MAPKKK)来阻断lrg1ΔΔΔ突变体中的解掩蔽和Cek1激活,表明lrg1ΔΔΔ突变体通过典型的Cek1MAP激酶级联起作用。为了确定Cek1过度激活如何具体影响毒力,在没有多西环素的情况下,白色念珠菌中表达了一个多西环素抑制的高活性STE11ΔN467等位基因,该等位基因过表达STE11ΔN467,诱导小鼠巨噬细胞产生促炎性肿瘤坏死因子α(TNF-α)。这种体外表型与全身性感染小鼠模型中降低的定殖和毒力相关。用RNA测序(RNA-Seq)分析探索了Ste11ΔN467引起解掩蔽的机制。Ste11ΔN467的过表达导致Cph1转录因子和一组细胞壁修饰蛋白的上调,这些蛋白预计会影响细胞壁结构。重要的白色念珠菌是人类全身感染的重要来源。从宿主免疫监视中掩蔽免疫原性细胞壁聚合物β(1,3)-葡聚糖的能力有助于真菌毒力。我们以前报道过Cek1MAP激酶级联的过度激活促进细胞壁的揭开,从而增加菌株的免疫原性。在这项研究中,我们发现了一种名为Lrg1的Cek1通路的新调节因子.Lrg1是一种预测的GTPase激活蛋白(GAP),通过下调GTPaseCdc42及其下游MAPKKK来抑制Cek1活性,Ste11.Cek1活性上调降低了感染小鼠模型中的真菌毒力,这与巨噬细胞的细胞因子反应增加有关。我们还分析了在由Cek1超激活驱动的β(1,3)-葡聚糖暴露期间确定的转录谱。我们的报告提供了一个模型,其中Cek1过度激活通过上调细胞壁蛋白导致β(1,3)-葡聚糖暴露,并导致更强大的体内免疫检测。促进更有效的清除。
Candida albicans is among the most prevalent opportunistic human fungal pathogens. The ability to mask the immunogenic polysaccharide β (1,3)-glucan from immune detection via a layer of mannosylated proteins is a key virulence factor of C. albicans We previously reported that hyperactivation of the Cek1 mitogen-activated protein (MAP) kinase pathway promotes β (1,3)-glucan exposure. In this communication, we report a novel upstream regulator of Cek1 activation and characterize the impact of Cek1 activity on fungal virulence. Lrg1 encodes a GTPase-activating protein (GAP) that has been suggested to inhibit the GTPase
Rho1. We found that disruption of LRG1 causes Cek1 hyperactivation and β (1,3)-glucan unmasking. However, when GTPase activation was measured for a panel of GTPases, the lrg1ΔΔ mutant exhibited increased activation of Cdc42 and Ras1 but not
Rho1 or Rac1. Unmasking and Cek1 activation in the lrg1ΔΔ mutant can be blocked by inhibition of the Ste11 MAP kinase kinase kinase (MAPKKK), indicating that the lrg1ΔΔ mutant acts through the canonical Cek1 MAP kinase cascade. In order to determine how Cek1 hyperactivation specifically impacts virulence, a doxycycline-repressible hyperactive STE11ΔN467 allele was expressed in C. albicans In the absence of doxycycline, this allele overexpressed STE11ΔN467 , which induced production of proinflammatory tumor necrosis factor alpha (TNF-α) from murine macrophages. This in vitro phenotype correlates with decreased colonization and virulence in a mouse model of systemic infection. The mechanism by which Ste11ΔN467 causes unmasking was explored with RNA sequencing (RNA-Seq) analysis. Overexpression of Ste11ΔN467 caused upregulation of the Cph1 transcription factor and of a group of cell wall-modifying proteins which are predicted to impact cell wall architecture.IMPORTANCECandida albicans is an important source of systemic infections in humans. The ability to mask the immunogenic cell wall polymer β (1,3)-glucan from host immune surveillance contributes to fungal virulence. We previously reported that the hyperactivation of the Cek1 MAP kinase cascade promotes cell wall unmasking, thus increasing strain immunogenicity. In this study, we identified a novel regulator of the Cek1 pathway called Lrg1. Lrg1 is a predicted GTPase-activating protein (GAP) that represses Cek1 activity by downregulating the GTPase Cdc42 and its downstream MAPKKK, Ste11. Upregulation of Cek1 activity diminished fungal virulence in the mouse model of infection, and this correlates with increased cytokine responses from macrophages. We also analyzed the transcriptional profile determined during β (1,3)-glucan exposure driven by Cek1 hyperactivation. Our report provides a model where Cek1 hyperactivation causes β (1,3)-glucan exposure by upregulation of cell wall proteins and leads to more robust immune detection in vivo, promoting more effective clearance.