Heterotrimeric G-alpha subunits Gpa11 and Gpa12 define a transduction pathway that control spore size and virulence in Mucor circinelloides

Abstract

Mucor circinelloides is one of the causal agents of mucormycosis, an emerging and high mortality rate fungal infection produced by asexual spores (sporangiospores) of fungi that belong to the order Mucorales. M. circinelloides has served as a model genetic system to understand the virulence mechanism of this infection. Although the G-protein signaling cascade plays crucial roles in virulence in many pathogenic fungi, its roles in Mucorales are yet to be elucidated. Previous study found that sporangiospore size and calcineurin are related to the virulence in Mucor, in which larger spores are more virulent in an animal mucormycosis model and loss of a calcineurin A catalytic subunit CnaA results in larger spore production and virulent phenotype. The M. circinelloides genome is known to harbor twelve gpa (gpa1 to gpa12) encoding G-protein alpha subunits and the transcripts of the gpa11 and gpa12 comprise nearly 72% of all twelve gpa genes transcript in spores. In this study we demonstrated that loss of function of Gpa11 and Gpa12 led to larger spore size associated with reduced activation of the calcineurin pathway. Interestingly, we found lower levels of the cnaA mRNAs in sporangiospores from the Δgpa12 and double Δgpa11/Δgpa12 mutant strains compared to wild-type and the ΔcnaA mutant had significantly lower gpa11 and gpa12 mRNA levels compared to wild-type. However, in contrast to the high virulence showed by the large spores of ΔcnaA, the spores from Δgpa11/Δgpa12 were avirulent and produced lower tissue invasion and cellular damage, suggesting that the gpa11 and gpa12 define a signal pathway with two branches. One of the branches controls spore size through regulation of calcineurin pathway, whereas virulences is controlled by an independent pathway. This virulence-related regulatory pathway could control the expression of genes involved in cellular responses important for virulence, since sporangiospores of Δgpa11/Δgpa12 were less resistant to oxidative stress and phagocytosis by macrophages than the ΔcnaA and wild-type strains. The characterization of this pathway could contribute to decipher the signals and mechanism used by Mucorales to produce mucormycosis.

Mucorales Species and Macrophages

“…The signals and the transduction pathways that regulate the NCRIP are unknown, but typical components of transduction signal pathways as heterotrimeric G proteins are required for full virulence in M. circinelloides. Genes gpa11 and gpa12 encoding α subunits of heterotrimeric G proteins show high transcript levels in resting spores [68] and are required to survive oxidative stress, phagocytosis and virulence in DKA mice [69]. Moreover, spores produced from cultures supplemented with native human blood serum show increased germination, resistance to oxidative stress, and survival to phagocytosis by macrophages, suggesting thermolabile components in the blood serum triggering a M. circinelloides response involved in macrophage interaction [70].…”

Genetic Manipulation in Mucorales and New Developments to Study Mucormycosis

“…Different genomic and transcriptomic approaches identified gene profiles related to virulence, and many genes from these profiles were mutated and functionally validated in survival assays [ 25 , 28 ]. In addition, the study of the transduction pathways in M. lusitanicus led to identifying new genes and pathways related to virulence [ 79 , 105 ]. Thus, M. lusitanicus has been an invaluable genetic model in studying genes and pathways associated with the virulence of Mucorales.…”

Recent Advances and Future Directions in the Understanding of Mucormycosis

“…Interestingly, most of the genes responding to phagocytosis, including atf1 and atf2 , are repressed by a non-canonical RNAi pathway (NCRIP) during saprophytic growth, suggesting that interaction of spores with macrophages inhibits NCRIP leading to the activation of the genetic program to overcome host defense mechanisms ( Pérez-Arques et al., 2020 ). The transduction pathways that regulate the NCRIP and other pathogenic processes are unknown, but the identification of heterotrimeric G proteins involved in M. lusitanicus virulence suggests that we may be approaching to their identification ( Patiño-Medina et al., 2019a ; Valle‐Maldonado et al., 2020 ).…”