Mancuso Ryan, Chinnici Jennifer, Tsou Charlene, Busarajan Sujay, Munnangi Raveena, Maddi Abhiram
Periodontics & Endodontics, State University of New York at Buffalo, Buffalo, NY, United States of America.
Periodontics & Endodontics, Oral Biology, State University of New York at Buffalo, Buffalo, NY, United States of America.
PeerJ. 2018 Sep 28;6:e5685. doi: 10.7717/peerj.5685. eCollection 2018.
is a commensal fungus that inhabits the oral mucosal surface and causes oral and systemic candidiasis. Oral candidiasis most commonly occurs in patients with AIDS, denture wearers and newborn children. Systemic candidiasis occurs mainly in immunocompromised patients and patients admitted to hospitals for prolonged periods. homologous genes, and , encode for two closely related cell wall proteins with putative glycosyltransferase enzyme activity and C-terminal GPI-anchors. Past studies have shown that individual and mutations are viable but simultaneous deletion of and in results in lethality. However, the exact functions of these cell wall based enzymes, which represent potential drug targets, are not understood.
heterologous and conditional double mutant strains were assessed for growth and biofilm formation in comparison to wild type and parental strains. Cell wall and heat stress susceptibility of the mutant and control strains were assessed using agar spotting assays. Growth was assessed under normal and osmotic stress conditions along with light microscopy imaging. Biofilm dry weight and microscopic imaging analysis of biofilms was performed. Hypha formation in response to serum was analyzed using light microscopy imaging. Western blot analysis of mutant strains and control strains was performed to assess Hog1 basal levels and phosphorylation status.
Analysis of the heterologous mutants indicated that Dfg5p is more important for growth while Dcw1p appeared to play a role in cell wall integrity response. The conditional double mutant was observed to be less resistant to cell wall stress. However, growth of the mutants was similar under control and osmotic stress conditions. The mutants were also able to grow similar to wild type under heat stress. Biofilm formation was reduced in the mutants where was deleted or suppressed. Hyphal morphogenesis was reduced although germ tube formation was observed in the biofilms of the mutant strains. Basal Hog1 protein levels were reduced or absent in the and mutants. However, osmotic stress was able to induce Hog1 protein levels comparable to wild type. Hog1 phosphorylation appeared to be slightly reduced although not significantly. In addition to biofilm assays, serum dose response imaging analysis indicated that hyphae formation in and mutants was defective.
These data indicate that and are required for hyphal morphogenesis and biofilm formation in . These functions may be regulated via basal Hog1 MAPK which is required for transcriptional regulation of chitin synthesis.
是一种共生真菌，栖息于口腔黏膜表面，可引起口腔和系统性念珠菌病。口腔念珠菌病最常见于艾滋病患者、佩戴假牙者和新生儿。系统性念珠菌病主要发生在免疫功能低下的患者以及长期住院的患者中。同源基因和编码两种密切相关的细胞壁蛋白，具有假定的糖基转移酶活性和C末端糖基磷脂酰肌醇（GPI）锚定。过去的研究表明，单个和突变是可行的，但在中同时缺失和会导致致死性。然而，这些基于细胞壁的酶的确切功能尚不清楚，而它们是潜在的药物靶点。
与野生型和亲本菌株相比，评估异源和条件性双突变菌株的生长和生物膜形成。使用琼脂点样试验评估突变株和对照株对细胞壁和热应激的敏感性。在正常和渗透应激条件下评估生长情况，并进行光学显微镜成像。进行生物膜干重和生物膜的显微镜成像分析。使用光学显微镜成像分析血清刺激下的菌丝形成。对突变株和对照株进行蛋白质免疫印迹分析，以评估Hog1的基础水平和磷酸化状态。
对异源突变体的分析表明，Dfg5p对生长更为重要，而Dcw1p似乎在细胞壁完整性反应中起作用。观察到条件性双突变体对细胞壁应激的耐受性较低。然而，在对照和渗透应激条件下，突变体的生长情况相似。在热应激下，突变体也能够与野生型相似地生长。在缺失或抑制的突变体中，生物膜形成减少。尽管在突变株的生物膜中观察到芽管形成，但菌丝形态发生减少。在和突变体中，基础Hog1蛋白水平降低或缺失。然而，渗透应激能够诱导Hog1蛋白水平与野生型相当。Hog1磷酸化似乎略有降低，但不显著。除了生物膜试验外，血清剂量反应成像分析表明，和突变体中的菌丝形成存在缺陷。
这些数据表明，和是菌丝形态发生和生物膜形成所必需的。这些功能可能通过基础Hog1丝裂原活化蛋白激酶（MAPK）进行调节，而Hog1 MAPK是几丁质合成转录调控所必需的。
