Degani Genny, Ragni Enrico, Botias Pedro, Ravasio Davide, Calderon Julia, Pianezzola Elena, Rodriguez-Peña Jose Manuel, Vanoni Maria Antonietta, Arroyo Javier, Fonzi William A, Popolo Laura
Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133, Milano, Italy.
Present address: Unit of Cell therapy and Cryobiology, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, Milano, Italy.
BMC Genomics. 2016 Jul 2;17:482. doi: 10.1186/s12864-016-2853-5.
The cell wall is essential for the yeast to hypha (Y-H) transition that enables Candida albicans to invade human tissues and evade the immune system. The main constituent, β(1,3)-glucan, is remodeled by glucanosyltransferases of the GH72 family. Phr1p is responsible of glucan remodeling at neutral-alkaline pH and is essential for morphogenesis and virulence. Due to the pH-regulated expression of PHR1, the phr1Δ phenotype is manifested at pH > 6 and its severity increases with the rise in pH. We exploited the pH-conditional nature of a PHR1 null mutant to analyze the impact of glucan remodeling on the hyphal transcriptional program and the role of chitin synthases in the hyphal wall stress (HWS) response.
In hyphal growth inducing conditions, phr1Δ germ tubes are defective in elongation, accumulate chitin, and constitutively activate the signaling pathways mediated by the MAP kinases Mkc1p, Cek1p and Hog1p. The transcriptional profiles revealed an increase of transcript levels for genes involved in cell wall formation (CHS2 and CHS8, CRH11, PGA23, orf19.750, RBR1, RBT4, ECM331, PGA6, PGA13), protein N-glycosylation and sorting in the ER (CWH8 and CHS7), signaling (CPP1, SSK2), ion transport (FLC2, YVC1), stress response and metabolism and a reduced expression of adhesins. A transient up-regulation of DNA replication genes associated with entry into S-phase occurred whereas cell-cycle regulating genes (PCL1, PCL2, CCN1, GIN4, DUN1, CDC28) were persistently up-regulated. To test the physiological relevance of altered CHS gene expression, phr1Δ chsxΔ (x = 2,3,8) mutant phenotypes were analyzed during the Y-H transition. PHR1 deletion was synthetic lethal with CHS3 loss on solid M199 medium-pH 7.5 and with CHS8 deletion on solid M199-pH 8. On Spider medium, PHR1 was synthetic lethal with CHS3 or CHS8 at pH 8.
The absence of Phr1p triggers an adaptive response aimed to reinforce the hyphal cell wall and restore homeostasis. Chs3p is essential in preserving phr1Δ cell integrity during the Y-H transition. Our findings also unveiled an unanticipated essential role of Chs8p during filamentation on solid media. These results highlight the flexibility of fungal cells in maintaining cell wall integrity and contribute to assessments of glucan remodeling as a target for therapy.
细胞壁对于白色念珠菌从酵母形态转变为菌丝形态（Y-H转变）至关重要，这一转变使白色念珠菌能够侵入人体组织并逃避免疫系统。其主要成分β(1,3)-葡聚糖由GH72家族的葡糖基转移酶进行重塑。Phr1p负责在中性至碱性pH条件下的葡聚糖重塑，对形态发生和毒力至关重要。由于PHR1的表达受pH调节，phr1Δ突变体表型在pH > 6时表现出来，且其严重程度随pH升高而增加。我们利用PHR1缺失突变体的pH条件依赖性来分析葡聚糖重塑对菌丝转录程序的影响以及几丁质合成酶在菌丝壁应激（HWS）反应中的作用。
在诱导菌丝生长的条件下，phr1Δ芽管伸长存在缺陷，几丁质积累，并组成性激活由丝裂原活化蛋白激酶Mkc1p、Cek1p和Hog1p介导的信号通路。转录谱显示，参与细胞壁形成的基因（CHS2和CHS8、CRH11、PGA23、orf19.750、RBR1、RBT4、ECM331、PGA6、PGA13）、内质网中的蛋白质N-糖基化和分选（CWH8和CHS7）、信号传导（CPP1、SSK2）、离子转运（FLC2、YVC1）、应激反应和代谢相关基因的转录水平增加，而黏附素的表达降低。与进入S期相关的DNA复制基因出现短暂上调，而细胞周期调节基因（PCL1、PCL2、CCN1、GIN4、DUN1、CDC28）持续上调。为了测试CHS基因表达改变的生理相关性，在Y-H转变过程中分析了phr1ΔchsxΔ（x = 2、3、8）突变体的表型。在固体M199培养基-pH 7.5上，PHR1缺失与CHS3缺失具有合成致死性，在固体M199-pH 8上与CHS8缺失具有合成致死性。在蜘蛛培养基上，在pH 8时PHR1与CHS3或CHS8具有合成致死性。
Phr1p的缺失触发了一种适应性反应，旨在加强菌丝细胞壁并恢复体内平衡。Chs3p对于在Y-H转变过程中维持phr1Δ细胞完整性至关重要。我们的研究结果还揭示了Chs8p在固体培养基上丝状化过程中出人意料的重要作用。这些结果突出了真菌细胞在维持细胞壁完整性方面的灵活性，并有助于评估将葡聚糖重塑作为治疗靶点的可能性。
