Phosphomannosylation and the Functional Analysis of the Extended Candida albicans MNN4-Like Gene Family

Abstract

Phosphomannosylation is a modification of cell wall proteins that occurs in some species of yeast-like organisms, including the human pathogen Candida albicans. These modified mannans confer a negative charge to the wall, which is important for the interactions with phagocytic cells of the immune systems and cationic antimicrobial peptides. In Saccharomyces cerevisiae, the synthesis of phosphomannan relies on two enzymes, the phosphomannosyltransferase Ktr6 and its positive regulator Mnn4. However, in C. albicans, at least three phosphomannosyltransferases, Mnn4, Mnt3 and Mnt5, participate in the addition of phosphomannan. In addition to MNN4, C. albicans has a MNN4-like gene family composed of seven other homologous members that have no known function. Here, using the classical mini-Ura-blaster approach and the new gene knockout CRISPR-Cas9 system for gene disruption, we generated mutants lacking single and multiple genes of the MNN4 family; and demonstrate that, although Mnn4 has a major impact on the phosphomannan content, MNN42 was also required for full protein phosphomannosylation. The reintroduction of MNN41, MNN42, MNN46, or MNN47 in a genetic background lacking MNN4 partially restored the phenotype associated with the mnn4Δ null mutant, suggesting that there is partial redundancy of function between some family members and that the dominant effect of MNN4 over other genes could be due to its relative abundance within the cell. We observed that additional copies of alleles number of any of the other family members, with the exception of MNN46, restored the phosphomannan content in cells lacking both MNT3 and MNT5. We, therefore, suggest that phosphomannosylation is achieved by three groups of proteins: [i] enzymes solely activated by Mnn4, [ii] enzymes activated by the dual action of Mnn4 and any of the products of other MNN4-like genes, with exception of MNN46, and [iii] activation of Mnt3 and Mnt5 by Mnn4 and Mnn46. Therefore, although the MNN4-like genes have the potential to functionally redundant with Mnn4, they apparently do not play a major role in cell wall mannosylation under most in vitro growth conditions. In addition, our phenotypic analyses indicate that several members of this gene family influence the ability of macrophages to phagocytose C. albicans cells.

Loss of Kex2 Affects the Candida albicans Cell Wall and Interaction with Innate Immune Cells

“…Freshly grown yeast cells adjusted to a concentration of 2 × 10 7 cell/mL were washed twice with PBS and labeled with 1 mg/mL Acridine orange (Sigma) as described [47]. The excess of dye was removed by washing cells twice with PBS and resuspended at a cell concentration of 3 × 10 7 yeast cells/mL.…”

“…Plates were incubated for 2 h at 37 • C and 5% (v/v) CO 2 . Then, the macrophages were washed twice with cold PBS and suspended in 1.25 mg/mL Trypan blue as an external fluorescence quencher [47]. Samples were analyzed by flow cytometry in a MoFlo XDP system (Beckman Coulter) collecting 50,000 events gated for macrophage cells.…”

Antifungal Activity of Anionic Defense Peptides: Insight into the Action of Galleria mellonella Anionic Peptide 2

“…It is generally accepted that electrostatic attraction between AMPs and the negatively charged microbial cell surface is an important factor for their antimicrobial activity. The modification of C. albicans surface proteins by phosphomannosylation determines the generally negative charge of the C. albicans cell surface [38][39][40], which seems to exclude the electrostatic interaction with the anionic AP2. However, according to the bioinformatics analysis, the peptide molecule contains five cationic lysine residues with side chains exposed on the surface of the putative amphipathic α-helices and also three lysine residues on the C-terminus (Figure 8).…”

Advances in Molecular Tools and In Vivo Models for the Study of Human Fungal Pathogenesis

“…Additionally, Cas13 has been developed as an RNA editing nuclease, which allows RNA knockdown and precise editing in mammalian cells [62]. While these more recent advancements have been demonstrated in mammalian cells, they can potentially be exploited to study fungal biology and pathogenesis [63].…”