The AGC kinase YpkA regulates sphingolipids biosynthesis and physically interacts with SakA MAP kinase in Aspergillus fumigatus

Sphingolipids (SL) are complex lipids and components of the plasma membrane which are involved in numerous cellular processes, as well as important for virulence of different fungal pathogens. In yeast, SL biosynthesis is regulated by the "AGC kinases" Ypk1 and Ypk2, which also seem to connect the SL biosynthesis with the cell wall integrity (CWI) and the High Osmolarity Glycerol (HOG) pathways. Here, we investigate the role of ypkA(YPK1) in SL biosynthesis and its relationship with the CWI and the HOG pathways in the opportunistic human pathogen Aspergillus fumigatus. We found that ypkA is important for fungal viability, since the 1 ypkA strain presented a drastically sick phenotype and complete absence of conidiation. We observed that under repressive condition, the conditional mutant niiA::ypkA exhibited vegetative growth defects, impaired germination and thermosensitivity. In addition, the ypkA loss of function caused a decrease in glycosphingolipid (GSL) levels, especially the metabolic intermediates belonging to the neutral GSL branch including dihydroceramide (DHC), ceramide (Cer), and glucosylceramide (GlcCer), but interestingly a small increase in ergosterol content. Genetic analyzes showed that ypkA genetically interacts with the MAP kinases of CWI and HOG pathways, mpkA and sakA, respectively, while only SakA physically interacts with YpkA. Our results suggest that YpkA is important for fungal survival through the regulation of GSL biosynthesis and cross talks with A. fumigatus MAP kinase pathways9CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP462383/2014-82009/53546-5; 2015/17541-0; 2017/19694-3This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP Grant Numbers: 2009/53546-5, 2015/17541-0, and 2017/19694-3) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Grant Number 462383/2014-8), Brazil to IM. This work was also supported by NIH grants (AI116420 and AI125770) and by the VA Merit Award grant (I01BX002624) to MDP. The funding body had no role in designing the study or in collecting, analyzing and interpreting the data, or in writing the manuscrip

The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus

Sphingolipids (SL) are complex lipids and components of the plasma membrane which are involved in numerous cellular processes, as well as important for virulence of different fungal pathogens. In yeast, SL biosynthesis is regulated by the “AGC kinases” Ypk1 and Ypk2, which also seem to connect the SL biosynthesis with the cell wall integrity (CWI) and the High Osmolarity Glycerol (HOG) pathways. Here, we investigate the role of ypkAY PK1 in SL biosynthesis and its relationship with the CWI and the HOG pathways in the opportunistic human pathogen Aspergillus fumigatus. We found that ypkA is important for fungal viability, since the ΔypkA strain presented a drastically sick phenotype and complete absence of conidiation. We observed that under repressive condition, the conditional mutant niiA::ypkA exhibited vegetative growth defects, impaired germination and thermosensitivity. In addition, the ypkA loss of function caused a decrease in glycosphingolipid (GSL) levels, especially the metabolic intermediates belonging to the neutral GSL branch including dihydroceramide (DHC), ceramide (Cer), and glucosylceramide (GlcCer), but interestingly a small increase in ergosterol content. Genetic analyzes showed that ypkA genetically interacts with the MAP kinases of CWI and HOG pathways, mpkA and sakA, respectively, while only SakA physically interacts with YpkA. Our results suggest that YpkA is important for fungal survival through the regulation of GSL biosynthesis and cross talks with A. fumigatus MAP kinase pathways

Functional characterization of the ypkA gene, an ypk1 homologous in yeast, on the human opportunist pathogenous fungus Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic pathogenic filamentous fungus, and the causative agent of aspergillosis, including the invasive pulmonary aspergillosis in immunocompromised individuals, most lethal form of the disease. As in all eukaryotes, the plasma membrane of A. fumigatus is composed of sterols, glycolipids and sphingolipids. The sphingolipids molecules are structural elements that participate in the regulation of the actin cytoskeleton endocytosis, and cell growth. In Saccharomyces cerevisiae, the sphingolipid synthesis is regulated by proteins such as the "AGC kinases" known as Ypk1p and Ypk2p that inactivate Orm1p and Orm2p, which are inhibitory proteins of the serine palmitoyltransferase (SPT) enzyme, the first enzyme that participates in biosynthetic pathway of sphingolipids. Thus, Ypk1/2p are responsible for stimulating the production of sphingolipids in response to several cellular stress factors, such as cell wall stress and heat stress. Here, we aimed to expand the study of sphingolipid biosynthesis in A. fumigatus by functional characterization of the ypkAYPK1 gene by establishing its role in the regulation of sphingolipid synthesis and in the maintenance of cell integrity. To accomplish this goal, one null mutant strain ΔypkAypk1 and a conditional mutant strain niiA::ypkA of A. fumigatus were obtained. The mutant strains were characterized by phenotypic tests aimed at understanding the role of this gene in sphingolipid synthesis, virulence and pathogenicity of this fungus. Subsequently real time RT-PCR experiments were performed to quantify the expression of this gene in conditions of heat stress. The results indicated that deletion of the gene ypkA promotes changes in the development of the fungus, which presents severe defects in the vegetative growth and absence of conidia. In addition, the conditional mutant shows increased sensitivity to lipid synthesis inhibitory drugs. The results also indicate that there is no interaction between genetic ypkA and pathway genes of the cell wall. Thus, we propose that protein kinase YpkA in A. fumigatus is related to vegetative growth and participates in the sphingolipid biosynthesis in A. fumigatus, contributing to the development and integrity of the fungal cell.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Aspergillus fumigatus é um fungo filamentoso patogênico oportunista, e o agente etiológico da aspergilose, que em imunocomprometidos pode vir a ocasionar a forma mais letal da doença, a aspergilose pulmonar invasiva. Como em todos os eucariotos, a membrana plasmática de A. fumigatus é composta por esteróis, glicolipídios e esfingolipídios. As moléculas de esfingolipídios são elementos estruturais que participam da regulação da endocitose, do citoesqueleto de actina e do crescimento celular. Em Saccharomyces cerevisiae, a síntese de esfingolipídios é regulada pelas proteínas do tipo “AGC quinases” conhecidas como Ypk1p e Ypk2p, que inativam Orm1p e Orm2p, as quais são proteínas inibidoras da enzima serina palmitoil-transferase (SPT), a primeira enzima que participa da via de biossíntese dos esfingolipídios. Dessa forma Ypk1/2p são responsáveis por estimular a produção de esfingolipídios na membrana plasmática em resposta a diversos fatores de estresse celular, como por exemplo o estresse de parede celular, estresse térmico entre outros. Diante disso, este trabalho teve como objetivo ampliar o estudo da biossíntese de esfingolipídios em A. fumigatus através da caracterização funcional do gene ypkAYPK1, de forma a estabelecer sua função na regulação da síntese de esfingolipídios e na manutenção da integridade celular. Para cumprir esse objetivo, uma linhagem mutante nulo ΔypkAypk1 e uma linhagem mutante condicional niiA::ypkA em A. fumigatus foram obtidas. As linhagens mutantes foram caracterizadas através de ensaios fenotípicos visando a compreensão do papel desse gene na síntese de esfingolipídios, na virulência e patogenicidade desse fungo. Posteriormente, foram realizados experimentos de RT-PCR em tempo real para a quantificação da expressão desse gene em condições de estresse térmico. Os resultados indicaram que a deleção do gene ypkA promove alterações no desenvolvimento vegetativo do fungo, o qual cresce na forma de colônias pequenas as quais não formam conídios. Ainda, o mutante condicional, quando reprimido, apresenta sensibilidade a drogas inibidoras da síntese de lipídeos e esfingolipídios. Os resultados indicam também que não há interação genética entre ypkA e os genes da via da integridade da parede celular. Assim, podemos propor que a proteína quinase YpkA de A. fumigatus está relacionada com o crescimento vegetativo e participa da biossíntese de esfingolipídios em A. fumigatus, contribuindo com o desenvolvimento e integridade da célula fúngica