Search

Previous work from our lab indicates that the heterotrimeric Gβ subunit 1 (Gpb1) enhances hyphal development and virulence in Mucor lusitanicus. In this study, three Gγ- and two additional Gβ-encoding genes were deleted to identify which ones might have a similar role as Gpb1. Deletion of gpg1 reduces hyphal growth, virulence, cyclic adenosine monophosphate (cAMP) levels, and protein kinase A (PKA) activity, similar to gpb1 deletion, suggesting that gpg1 participates in the same regulatory pathway as gpb1. The defects observed in Δgpg1 or Δgpb1 were suppressed by overexpression of the gene pkaR1 encoding the regulatory subunit 1 of PKA, indicating that this pathway is controlled by Gpg1 and Gpb1. Moreover, Δgpg1 and Δgpb1 show a downregulation of the transcription factors tec1 and tec2. Furthermore, tec-overexpression in Δgpg1, Δgpb1, and ΔpkaR1 restores the wild-type phenotype, indicating that both Tec are under control by the Gpb1, Gpg1, and PKA pathway. Moreover, the Δgpb1/Δgbg1 (+)(-) exhibits lower aerobic germination, hyphal growth and downregulates NAD + -glutamate dehydrogenases (gdh2a/b), whereas virulence is similar to that of the wild-type (WT) strain. These alterations in Δgpb1/Δgbg1 (+)(-) were reversed by the presence of glutamate during growth, suggesting that NAD + -Gdh2 could be under control of these subunits. Compared to the WT and Δgpb1/Δgbg1 (+)(-) strains under aerobic growth, single deletion strains showed lower rhizoferrin levels, respiration and reactive oxygen species levels. Our results suggest that Gpg1 interacts with Gpb1 to positively control the hyphal development and virulence by repressing the PKA pathway, thereby regulating the mitochondrial oxidative metabolism in M. lusitanicus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for this journal and was not involved in the editorial., (Copyright © 2025. Published by Elsevier Inc.)

Mucor circinelloides is an etiologic agent of mucormycosis, a fungal infection produced by Mucorales often associated with mortality due to unavailability of antifungal drugs. Arl proteins belong to the Arf family and are involved in vesicle trafficking and tubulin assembly. This study identified two Arl (Arf-like)-encoding genes, arl1 and arl2, in M. circinelloides and explored their function in morphogenesis, virulence, and antifungal susceptibility. Although Arl1 and Arl2 proteins shared 55% amino acid sequence identity, arl1 and arl2 genes showed distinct transcriptional expression patterns. arl1 was expressed at higher levels than arl2 and induced in mycelia, suggesting a role in morphological transitions. Disruption of the arl1 and arl2 genes led to heterokaryon (Δarl1 (+)(-) ) and homokaryon (Δarl2) genotypes, respectively. The incapacity to generate homokaryon mutants for arl1 suggested that it is essential for growth of M. circinelloides. Deletion of each gene reduced the expression of the other, suggesting the existence of a positive cross-regulation between them. Thus, deletion of arl2 resulted in a ~60% reduction of arl1 expression, whereas the Δarl1 (+)(-) showed ∼90% reduction of arl1 expression. Mutation of arl2 showed no phenotype or a mild phenotype between Δarl1 (+)(-) and wild-type (WT), suggesting that all observed phenotypes in both mutant strains corresponded to arl1 low expression. The Δarl1 (+)(-) produced a small amount of spores that showed increased sensitivity to dodecyl-sulfate and azoles, suggesting a defect in the cell wall that was further supported by decrease in saccharide content. These defects in the cell wall were possibly originated by abnormal vesicle trafficking since FM4-64 staining of both mutants Δarl1 (+)(-) and Δarl2 revealed less well-localized endosomes compared to the WT. Moreover, aberrant vesicle trafficking may be responsible for the secretion of specific virulence-related proteins since cell-free medium from Δarl1 (+)(-) were found to increase killing of Caenorhabditis elegans compared to WT., (Copyright © 2019 Elsevier Inc. All rights reserved.)