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ABSTRACT Mitogen-activated protein kinase (MAPK) pathways are fundamental to the regulation of biological processes in eukaryotic organisms. The basidiomycete Cryptococcus neoformans, known for causing fungal meningitis worldwide, possesses five MAPKs. Among these, Cpk1, Hog1, and Mpk1 have established roles in sexual reproduction, stress responses, and cell wall integrity. However, the roles of Cpk2 and Mpk2 are less understood. Our study elucidates the functional interplay between the Cpk1/Cpk2 and Mpk1/Mpk2 MAPK pathways in C. neoformans. We discovered that CPK2 overexpression compensates for cpk1Δ mating deficiencies via the Mat2 transcription factor, revealing functional redundancy between Cpk1 and Cpk2. We also found that Mpk2 is phosphorylated in response to cell wall stress, a process regulated by the MAPK kinase (MAP2K) Mkk2 and MAP2K kinases (MAP3Ks) Ssk2 and Ste11. Overexpression of MPK2 partially restores cell wall integrity in mpk1Δ by influencing key cell wall components, such as chitin and the polysaccharide capsule. Contrarily, MPK2 overexpression cannot restore thermotolerance and cell membrane integrity in mpk1Δ. These results suggest that Mpk1 and Mpk2 have redundant and opposing roles in the cellular response to cell wall and membrane stresses. Most notably, the dual deletion of MPK1 and MPK2 restores wild-type mating efficiency in cpk1Δ mutants via upregulation of the mating-regulating transcription factors MAT2 and ZNF2, suggesting that the Mpk1 and Mpk2 cooperate to negatively regulate the pheromone-responsive Cpk1 MAPK pathway. Our research collectively underscores a sophisticated regulatory network of cryptococcal MAPK signaling pathways that intricately govern sexual reproduction and cell wall integrity, thereby controlling fungal development and pathogenicity.IMPORTANCEIn the realm of fungal biology, our study on Cryptococcus neoformans offers pivotal insights into the roles of specific proteins called mitogen-activated protein kinases (MAPKs). Here, we discovered the cryptic functions of Cpk2 and Mpk2, two MAPKs previously overshadowed by their dominant counterparts Cpk1 and Mpk1, respectively. Our findings reveal that these “underdog” proteins are not just backup players; they play crucial roles in vital processes like mating and cell wall maintenance in C. neoformans. Their ability to step in and compensate when their dominant counterparts are absent showcases the adaptability of C. neoformans. This newfound understanding not only enriches our knowledge of fungal MAPK mechanisms but also underscores the intricate balance and interplay of proteins in ensuring the organism’s survival and adaptability.

ABSTRACT The human-pathogenic yeast Cryptococcus neoformans assembles two types of O-linked glycans on its proteins. In this study, we identified and functionally characterized the C. neoformans CAP6 gene, encoding an α1,3-mannosyltransferase responsible for the second mannose addition to minor O-glycans containing xylose in the Golgi apparatus. Two cell surface sensor proteins, Wml1 (WSC/Mid2-like) and Wml2, were found to be independent substrates of Cap6-mediated minor or Ktr3-mediated major O-mannosylation, respectively. The double deletion of KTR3 and CAP6 (ktr3Δ cap6Δ) completely blocked the mannose addition at the second position of O-glycans, resulting in the accumulation of proteins with O-glycans carrying only a single mannose. Tunicamycin (TM)-induced phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) was greatly decreased in both ktr3Δ cap6Δ and wml1Δ wml2Δ strains. Transcriptome profiling of the ktr3Δ cap6Δ strain upon TM treatment revealed decreased expression of genes involved in the Mpk1-dependent cell wall integrity (CWI) pathway. Consistent with its defective growth under several stress conditions, the ktr3Δ cap6Δ strain was avirulent in a mouse model of cryptococcosis. Associated with this virulence defect, the ktr3Δ cap6Δ strain showed decreased adhesion to lung epithelial cells, decreased proliferation within macrophages, and reduced transcytosis of the blood-brain barrier (BBB). Notably, the ktr3Δ cap6Δ strain showed reduced induction of the host immune response and defective trafficking of ergosterol, an immunoreactive fungal molecule. In conclusion, O-glycan extension in the Golgi apparatus plays critical roles in various pathobiological processes, such as CWI signaling and stress resistance and interaction with host cells in C. neoformans. IMPORTANCE Cryptococcus neoformans assembles two types of O-linked glycans on its surface proteins, the more abundant major O-glycans that do not contain xylose residues and minor O-glycans containing xylose. Here, we demonstrate the role of the Cap6 α1,3-mannosyltransferase in the synthesis of minor O-glycans. Previously proposed to be involved in capsule biosynthesis, Cap6 works with the related Ktr3 α1,2-mannosyltransferase to synthesize O-glycans on their target proteins. We also identified two novel C. neoformans stress sensors that require Ktr3- and Cap6-mediated posttranslational modification for full function. Accordingly, the ktr3Δ cap6Δ double O-glycan mutant strain displays defects in stress signaling pathways, CWI, and ergosterol trafficking. Furthermore, the ktr3Δ cap6Δ strain is completely avirulent in a mouse infection model. Together, these results demonstrate critical roles for O-glycosylation in fungal pathogenesis. As there are no human homologs for Cap6 or Ktr3, these fungus-specific mannosyltransferases are novel targets for antifungal therapy.