Phosphomannosylation and the Functional Analysis of the Extended -Like Gene Family.

Phosphomannosylation is a modification of cell wall proteins that occurs in some species of yeast-like organisms, including the human pathogen . 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 , the synthesis of phosphomannan relies on two enzymes, the phosphomannosyltransferase Ktr6 and its positive regulator Mnn4. However, in , at least three phosphomannosyltransferases, Mnn4, Mnt3 and Mnt5, participate in the addition of phosphomannan. In addition to has a -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 family; and demonstrate that, although Mnn4 has a major impact on the phosphomannan content, was also required for full protein phosphomannosylation. The reintroduction of , or in a genetic background lacking partially restored the phenotype associated with the Δ null mutant, suggesting that there is partial redundancy of function between some family members and that the dominant effect of 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 , restored the phosphomannan content in cells lacking both and . 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 -like genes, with exception of , and [iii] activation of Mnt3 and Mnt5 by Mnn4 and Mnn46. Therefore, although the -like genes have the potential to functionally redundant with Mnn4, they apparently do not play a major role in cell wall mannosylation under most growth conditions. In addition, our phenotypic analyses indicate that several members of this gene family influence the ability of macrophages to phagocytose cells.