ABSTRACT
This study analyzed the role of blood serum in enhancing the mitochondrial metabolism and virulence of Mucorales through rhizoferrin secretion. We observed that the spores of clinically relevant Mucorales produced in the presence of serum exhibited higher virulence in a heterologous infection model of Galleria mellonella. Cell-free supernatants of the culture broth obtained from spores produced in serum showed increased toxicity against Caenorhabditis elegans, which was linked with the enhanced secretion of rhizoferrin. Spores from Mucoralean species produced or germinated in serum showed increased respiration rates and reactive oxygen species levels. The addition of non-lethal concentrations of potassium cyanide and N-acetylcysteine during the aerobic or anaerobic growth of Mucorales decreased the toxicity of the cell-free supernatants of the culture broth, suggesting that mitochondrial metabolism is important for serum-induced virulence. In support of this hypothesis, a mutant strain of Mucor lusitanicus that lacks fermentation and solely relies on oxidative metabolism exhibited virulence levels comparable to those of the wild-type strain under serum-induced conditions. Contrary to the lower virulence observed, even in the serum, the ADP-ribosylation factor-like 2 deletion strain exhibited decreased mitochondrial activity. Moreover, spores produced in the serum of M. lusitanicus and Rhizopus arrhizus that grew in the presence of a mitophagy inducer showed low virulence. These results suggest that serum-induced mitochondrial activity increases rhizoferrin levels, making Mucorales more virulent.
ABSTRACT
Beauveria bassiana is a dimorphic and entomopathogenic fungus with different ecological roles in nature. In pathogenic fungi, yeast-to-mycelial conversion, which is controlled by environmental factors, is required for virulence. Here, we studied the effects of different stimuli on the morphology of two B. bassiana strains and compared the toxicities of culture filtrates. In addition, we explored the role of volatiles as quorum sensing-like signals during dimorphic transition. The killing assays in Caenorhabditis elegans (Nematoda: Rhabditidae) showed that strain AI2 isolated from a mycosed insect cadaver had higher toxicity than strain AS5 isolated from soil. Furthermore, AI2 showed earlier yeast-to-mycelial switching than AS5. However, an increase in inoculum size induced faster yeast-to-mycelium conversion in AS5 cells, suggesting a cell-density-dependent phenomenon. Gas chromatography-mass spectrometry (GC-MS) analyses showed that the fingerprint of the volatiles was strain-specific; however, during the morphological switching, an inverse relationship between the abundance of total terpenes and 3-methylbutanol was observed in both strains. Fungal exposure to 3-methylbutanol retarded the yeast-to-mycelium transition. Hence, this study provides evidence that volatile compounds are associated with critical events in the life cycle of B. bassiana.
ABSTRACT
RNA interference (RNAi) was discovered at the end of last millennium, changing the way scientists understood regulation of gene expression. Within the following two decades, a variety of different RNAi mechanisms were found in eukaryotes, reflecting the evolutive diversity that RNAi entails. The essential silencing mechanism consists of an RNase III enzyme called Dicer that cleaves double-stranded RNA (dsRNA) generating small interfering RNAs (siRNAs), a hallmark of RNAi. These siRNAs are loaded into the RNA-induced silencing complex (RISC) triggering the cleavage of complementary messenger RNAs by the Argonaute protein, the main component of the complex. Consequently, the expression of target genes is silenced. This mechanism has been thoroughly studied in fungi due to their proximity to the animal phylum and the conservation of the RNAi mechanism from lower to higher eukaryotes. However, the role and even the presence of RNAi differ across the fungal kingdom, as it has evolved adapting to the particularities and needs of each species. Fungi have exploited RNAi to regulate a variety of cell activities as different as defense against exogenous and potentially harmful DNA, genome integrity, development, drug tolerance, or virulence. This pathway has offered versatility to fungi through evolution, favoring the enormous diversity this kingdom comprises.
Subject(s)
Evolution, Molecular , Fungi/genetics , RNA Interference , Gene Expression Regulation, FungalABSTRACT
Mucor circinelloides, a dimorphic opportunistic pathogen, expresses three heterotrimeric G-protein beta subunits (Gpb1, Gpb2 and Gpb3). The Gpb1-encoding gene is up-regulated during mycelial growth compared with that in the spore or yeast stage. gpb1 deletion mutation analysis revealed its relevance for an adequate development during the dimorphic transition and for hyphal growth under low oxygen concentrations. Infection assays in mice indicated a phenotype with considerably reduced virulence and tissue invasiveness in the deletion mutants (Δgpb1) and decreased host inflammatory response. This finding could be attributed to the reduced filamentous growth in animal tissues compared with that of the wild-type strain. Mutation in a regulatory subunit of cAMP-dependent protein kinase A (PKA) subunit (PkaR1) resulted in similar phenotypes to Δgpb1. The defects exhibited by the Δgpb1 strain were genetically suppressed by pkaR1 overexpression, indicating that the PKA pathway is controlled by Gpb1 in M. circinelloides. Moreover, during growth under low oxygen levels, cAMP levels were much higher in the Δgpb1 than in the wild-type strain, but similar to those in the ΔpkaR1 strain. These findings reveal that M. circinelloides possesses a signal transduction pathway through which the Gpb1 heterotrimeric G subunit and PkaR1 control mycelial growth in response to low oxygen levels.
