Binding of Serum Mannan Binding Lectin to a Cell Integrity-DefectiveCryptococcus neoformans ccr4Δ Mutant

Abstract

Mannan binding lectin (MBL) is an innate immune mediator belonging to the collectin family known to bind to the surfaces of many viruses, bacteria, and fungi. However, pathogenic strains of the fungus Cryptococcus neoformans are resistant to MBL binding. To dissect the mechanism of cryptococcal resistance to MBL, we compared MBL binding to an encapsulated wild-type strain, an encapsulated ccr4⌬ mutant defective in cell integrity, and an acapsular cap60⌬ strain. No MBL binding was detected on wild-type C. neoformans. In contrast, the ccr4⌬ mutant bound MBL to the cell wall, predominantly at the ends of enlarged buds, whereas the acapsular strain bound MBL only at the bud neck and bud scars. In addition, the ccr4⌬ mutant was sensitive to the cell wall-active antifungal caspofungin and other cell wall stress inducers, and its virulence was reduced in a mouse model of cryptococcosis. Interestingly, treatment of wild-type cells with caspofungin also increased MBL binding to C. neoformans. These results suggest that both the presence of capsule and wild-type cell wall architecture preclude MBL binding to C. neoformans.

Ccr4 Promotes Resolution of the Endoplasmic Reticulum Stress Response during Host Temperature Adaptation in Cryptococcus neoformans

“…Our previous studies of a C. neoformans ccr4⌬ mutant demonstrate a role for the mRNA degradation machinery in host temperature adaptation, cell integrity, and pathogenesis (24). Our data, coupled with data from S. cerevisiae and higher eukaryotes, highlight the importance of posttranscriptional gene regulation in the ability of cells to respond to temperature stress.…”

“…Our previous studies of the ccr4⌬ mutant demonstrate a cell integrity phenotype, as the ccr4⌬ mutant is sensitive to a range of cell integrity stress-inducing agents (24). Constitutive activation of the UPR is known to cause cell integrity defects in S. cerevisiae, as induction of the UPR by overexpression of a mutant form of carboxypeptidase Y, CPY*, results in cell integrity defects, as well as enhanced release of cell wall proteins with mild DTT extraction and increased secretion of proteins into the medium (26).…”

“…We previously reported that a C. neoformans ccr4⌬ mutant exhibited a defect in growth at 37°C, suggesting that Ccr4-dependent posttranscriptional gene regulation is important for host temperature adaptation in C. neoformans (24). To determine if there were defects in gene regulation during the initial phase of 37°C adaptation in a ccr4⌬ mutant, we performed microarray analysis on RNA pools from the wild-type and ccr4⌬ strains shifted from 30°C to 37°C for 10 min.…”

“…Cryptococcus neoformans var. grubii strain H99 and a ccr4⌬ mutant described previously were propagated on yeast extract-peptone-dextrose (YPD) agar at 30°C (24). Liquid cultures were cultivated in 250-ml baffled cotton-plugged Erlenmeyer flasks with a culture volume not exceeding 30 ml.…”

“…This modulation of gene expression likely requires alterations in mRNA synthesis rates through activation of transcriptional transactivators and repressors, as well as alterations in chromatin structure. In addition to mRNA synthesis, our previous studies of a C. neoformans ccr4⌬ mutant lacking the major mRNA deadenylase involved in regulated mRNA turnover suggest a role for posttranscriptional regulation of gene expression in C. neoformans host temperature adaptation (24).…”