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A subunit of protein kinase a regulates growth and differentiation in the fungus Mucor circinelloides.

ABSTRACT: The cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling pathway plays a role in regulating development, growth, and virulence in a number of fungi. To determine whether PKA plays a similar function in zygomycete fungi, a mutant of Mucor circinelloides was generated that lacks pkaR1, one of the regulatory subunits of PKA. The mutant showed a reduction in growth and alterations in germination rates, cell volume, germ tube length, and asexual sporulation. The lack of pkaR1 gene resulted in a highly decreased, but not null, cAMP binding activity and in a protein kinase activity that was still dependent on cAMP, although with a higher -/+ cAMP activity ratio, suggesting the existence of other cAMP binding activities. Consequently, three proteins analogous to pkaR1 were predicted from the recently sequenced genome of M. circinelloides and were named pkaR2, pkaR3, and pkaR4. Two of the proteins, pkaR2 and pkaR3, with cAMP binding activity were isolated from the wild-type strain and identified by mass spectrometry. The expression of all genes was detected at the mRNA level by semiquantitative reverse transcription-PCR, and they showed a differential expression at different developmental stages. This is the first time that a fungus is reported to have more than one gene encoding the regulatory subunit of PKA.

SUBMITTER: Ocampo J

PROVIDER: S-EPMC2708453 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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A subunit of protein kinase a regulates growth and differentiation in the fungus Mucor circinelloides.

Ocampo J J Fernandez Nuñez L L Silva F F Pereyra E E Moreno S S Garre V V Rossi S S

Eukaryotic cell 20090501 7

The cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling pathway plays a role in regulating development, growth, and virulence in a number of fungi. To determine whether PKA plays a similar function in zygomycete fungi, a mutant of Mucor circinelloides was generated that lacks pkaR1, one of the regulatory subunits of PKA. The mutant showed a reduction in growth and alterations in germination rates, cell volume, germ tube length, and asexual sporulation. The lack of pkaR1 gene resulted in ...[more]

PMID: 19411621

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Project description:Mucormycosis is an emerging lethal invasive fungal infection. The infection caused by fungi belonging to the order Mucorales has been reported recently as one of the most common fungal infections among COVID-19 patients. The lack of understanding of pathogens, particularly at the molecular level, is one of the reasons for the difficulties in the management of the infection. Myosin is a diverse superfamily of actin-based motor proteins that have various cellular roles. Four families of myosin motors have been found in filamentous fungi, including myosin I, II, V, and fungus-specific chitin synthase with myosin motor domains. Our previous study on Mucor circinelloides, a common pathogen of mucormycosis, showed that the Myo5 protein (ID 51513) belonging to the myosin type V family had a critical impact on the growth and virulence of this fungus. In this study, to investigate the roles of myosin II proteins in M. circinelloides, silencing phenotypes and null mutants corresponding to myosin II encoding genes, designated mcmyo2A (ID 149958) and mcmyo2B (ID 136314), respectively, were generated. Those mutant strains featured a significantly reduced growth rate and impaired sporulation in comparison with the wild-type strain. Notably, the disruption of mcmyo2A led to an almost complete lack of sporulation. Both mutant strains displayed abnormally short, septate, and inflated hyphae with the presence of yeast-like cells and an unusual accumulation of pigment-filled vesicles. In vivo virulence assays of myosin-II mutant strains performed in the invertebrate model Galleria mellonella indicated that the mcmyo2A-knockout strain was avirulent, while the pathogenesis of the mcmyo2B null mutant was unaltered despite the low growth rate and impaired sporulation. The findings provide suggestions for critical contributions of the myosin II proteins to the polarity growth, septation, morphology, pigment transportation, and pathogenesis of M. circinelloides. The findings also implicate the myosin family as a potential target for future therapy to treat mucormycosis.

Project description:BackgroundRNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants.ResultsDeletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway.ConclusionThis work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model.

Project description:BackgroundMucor circinelloides WJ11 is a high-lipid producing strain and an excellent producer of γ-linolenic acid (GLA) which is crucial for human health. We have previously identified genes that encode for AMP-activated protein kinase (AMPK) complex in M. circinelloides which is an important regulator for lipid accumulation. Comparative transcriptional analysis between the high and low lipid-producing strains of M. circinelloides showed a direct correlation in the transcriptional level of AMPK genes with lipid metabolism. Thus, the role of Snf-β, which encodes for β subunit of AMPK complex, in lipid accumulation of the WJ11 strain was evaluated in the present study.ResultsThe results showed that lipid content of cell dry weight in Snf-β knockout strain was increased by 32 % (from 19 to 25 %). However, in Snf-β overexpressing strain, lipid content of cell dry weight was decreased about 25 % (from 19 to 14.2 %) compared to the control strain. Total fatty acid analysis revealed that the expression of the Snf-β gene did not significantly affect the fatty acid composition of the strains. However, GLA content in biomass was increased from 2.5 % in control strain to 3.3 % in Snf-β knockout strain due to increased lipid accumulation and decreased to 1.83 % in Snf-β overexpressing strain. AMPK is known to inactivate acetyl-CoA carboxylase (ACC) which catalyzes the rate-limiting step in lipid synthesis. Snf-β manipulation also altered the expression level of the ACC1 gene which may indicate that Snf-β control lipid metabolism by regulating ACC1 gene.ConclusionsOur results suggested that Snf-β gene plays an important role in regulating lipid accumulation in M. circinelloides WJ11. Moreover, it will be interesting to evaluate the potential of other key subunits of AMPK related to lipid metabolism. Better insight can show us the way to manipulate these subunits effectively for upscaling the lipid production. Up to our knowledge, it is the first study to investigate the role of Snf-β in lipid accumulation in M. circinelloides.

Project description:Procedures such as solid-organ transplants and cancer treatments can leave many patients in an immunocompromised state. This leads to their increased susceptibility to opportunistic diseases such as fungal infections. Mucormycosis infections are continually emerging and pose a serious threat to immunocompromised patients. Recently there has been a sharp increase in mucormycosis cases as a secondary infection in patients battling severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Mucorales fungi are notorious for presenting resistance to most antifungal drugs. The absence of effective means to treat these infections results in mortality rates approaching 100% in cases of disseminated infection. One of the most effective antifungal drug classes currently available is the echinocandins. Echinocandins seem to be efficacious in the treatment of many other fungal infections. Unfortunately, susceptibility testing has found that echinocandins have little to no effect on Mucorales fungi. In this study, we found that the model Mucorales Mucor circinelloides genome carries three copies of the genes encoding the echinocandin target protein β-(1,3)-d-glucan synthase (fksA, fksB, and fksC). Interestingly, we found that exposing M. circinelloides to micafungin significantly increased the expression of the fksA and fksB genes, resulting in an increased accumulation of β-(1,3)-d-glucan on the cell walls. However, this overexpression of the fks genes is not directly connected to the intrinsic resistance. Subsequent investigation discovered that the serine/threonine phosphatase calcineurin regulates the expression of fksA and fksB, and the deletion of calcineurin results in a decrease in expression of all three fks genes. Deletion of calcineurin also results in a lower minimum effective concentration (MEC) of micafungin. In addition, we found that duplication of the fks gene is also responsible for the intrinsic resistance, in which lack of either fksA or fksB led a lower MEC of micafungin. Together, these findings demonstrate that calcineurin and fks gene duplication contribute to the intrinsic resistance to micafungin we observe in M. circinelloides.

Dataset Information

A subunit of protein kinase a regulates growth and differentiation in the fungus Mucor circinelloides.

Publications

A subunit of protein kinase a regulates growth and differentiation in the fungus Mucor circinelloides.

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