Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5′-CACAGCCAC-3′ and 5′-CCCTGCCCC-3′ sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -B and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The ΔpmcA and ΔpmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the ΔcalA and ΔcrzA mutant strains. However, only the A. fumigatus ΔpmcA was avirulent in the murine model of invasive pulmonary aspergillosis

Cloning, functional expression, and biochemical characterization of an alternative oxidase mitochondrial gene from A. fumigatus

O Aspergillus fumigatus é um fungo filamentoso e saprofítico, encontrado em todas as regiões do mundo, desempenhando um importante papel na reciclagem de carbono e nitrogênio do solo. A principal forma de infecção ocorre através da inalação dos conídios com predominância de infecções no trato respiratório, principalmente em pacientes imunocomprometidos. A mitocôndria de A. fumigatus foi caracterizada em nosso laboratório, que revelou a presença de uma respiração resistente a cianeto mediada pela oxidase alternativa. A clonagem e sequenciamento deste gene foram realizadas através de screening de uma biblioteca de DNA genômico. O alinhamento das sequências genômica e de cDNA mostrou a presença de dois introns, que após splicing codifica uma proteína contendo 352 aminoácidos, possuindo uma massa molecular estimada de 40,84 kDa e um pI teórico de 9,51. Além disso, foram identificados domínios altamente conservados (LET, NERMHL, LEEA e RADEH) que interagem com átomos de ferro e estão contidos em -hélices propostas como responsáveis pela organização estrutural da enzima. A fim de caracterizar bioquimicamente esta proteína, a sequência de cDNA do gene foi clonada em plasmídeo pYES2 e expressa em S. cerevisiae INVSc1 como um modelo eucarioto. Após expressão, a proteína encontrou-se de forma ativa, conferindo à levedura uma respiração resistente a cianeto. Esta característica herdada provocou uma discreta diminuição na taxa de crescimento em meio não-fermentativo e uma capacidade de sobrevivência na presença de KCN. Acredita-se que a atividade das AOXs esteja diretamente relacionada com a presença de diferentes espécies reativas de oxigênio (ERO). Neste contexto, a avaliação do efeito de diferentes agentes pró-oxidantes provocou um aumento na atividade e na expressão da enzima. Paralelamente, a caracterização funcional do gene foi realizada através da técnica de interferência por RNA, utilizando o vetor de expressão pALB1. Em meio contendo maltose, as cepas pALB1/aoxAf apresentaram coloração branca devido ao silenciamento do gene alb1. Os níveis de mRNA do gene aoxAf foram determinados por Real time RT-PCR, mostrando o eficiente silenciamento do gene alvo com a construção utilizada. Devido à relação já descrita entre ERO e a atividade das AOXs, avaliou-se a produção de espécies reativas de oxigênio nas cepas silenciadas utilizando-se a sonda CM-H2DCFDA, observando maior produção na cepa pALB1/aoxAf. Além disso, a viabilidade destas cepas foi determinada por citometria de fluxo após a exposição com agentes pró-oxidantes, a qual indicou maior letalidade na cepa pALB1/aoxAf, quando comparada com CEA e pALB1. Da mesma forma, após a incubação dos conídios das cepas silenciadas com macrófagos de camundongos foi verificada uma maior atividade microbicida dos macrófagos na cepa duplamente silenciada pALB1/aoxAf, quando comparada com as outras cepas. Com estes resultados podemos concluir que a oxidase alternativa apresenta uma importante atividade antioxidante, além de contribuir nos mecanismos de defesa durante o processo de infecção de A. fumigatus.The saprophytic species Aspergillus fumigatus is a deuteromycete fungus found worldwide, which has an essential role in recycling carbon and nitrogen. Following inhalation of conidia by the immunocompetent host, the innate cellular immune system is responsible for killing the conidia, exposing them to reactive oxygen. However, A. fumigatus is capable of surviving and replicating within the phagolysosomal compartment of immunocompromised macrophages. It was previously demonstrated that A. fumigatus mitochondria possess an alternative oxidase (aoxAf) wich is a cyanide-resistant protein. A partial genomic DNA library was screened to cloning an aoxAf gene. The alignment between the cDNA and genomic DNA sequences revealed the existence of two introns which after splicing encodes a 352 amino acid sequence with a calculated molecular mass of 40 kDa and a theoretical pI of 9.51. The deduced amino acid sequence revealed four regions completely conserved among the AOXs sequences (LET, NERMHL, LEEA and RADE-H), where six conserved amino acids residues are proposed to be a metal ligand site. To characterize the AOX protein, a cDNA of aoxAf gene was cloned into pYES2 plasmid and transformed in S. cerevisiae INVSc1. After the incubation of the cells in a nonfermentable medium in the presence of KCN, S. cerevisiae expressing AOX was able to grow, while it was lethal for the control yeast. These results suggest that the recombinant AOXAf is properly targeted to the S. cerevisiae mitochondria where it has functional activity. Studies with different species demonstrated that AOX is induced by a variety of treatments usually labeled as stresses. To verify the function of AOX in A. fumigatus under oxidative stress conditions, conidia were treated with different donors of ROS. These treatments caused an increase in aoxAf activity and transcription levels. To identify genetically attributes of virulence and oxidative defense in A. fumigatus, we construct a RNA interference plasmid. Two inverted repeated sequences of conserved region of an interest gene were amplified and cloned in pALB1 plasmid. In maltose medium pALB1 and pALB1/aoxAf transformants demonstrated white colonies, attributable to the reduction of alb1 gene expression. The aoxAf mRNA levels were analyzed by Real time RT-PCR, showing an efficient alternative oxidase gene silencing in pALB1 plasmid construction. It was previously demonstrated that ROS can stimulate the AOXs activity, so, we used the dye CM-H2DCFDA to measure ROS production in RNAi transformants, showing that the decrease in aoxAf gene expression caused an increase in ROS production. After incubation with ROS donors the viability of these strains was determined by flow cytometry analysis. The pALB1/aoxAf strain showed higher lethality, when compared with CEA and pALB1, suggesting the involvement of AOX in antioxidant defense in A. fumigatus. Besides, ROS produced by alveolar macrophages play an essential role in the killing of A. fumigatus conidia. In the same way, phagocytosis assay revealed that pALB1/aoxAf strain was more lethal than CEA and pALB1. With these results, we concluded that alternative oxidase is an efficient antioxidant system and might contribute with defense mechanism of A. fumigatus

Aspergillus fumigatus mitochondrial electron transport chain mediates oxidative stress homeostasis, hypoxia responses and fungal pathogenesis

We previously observed that hypoxia is an important component of host microenvironments during pulmonary fungal infections. However, mechanisms of fungal growth in these in vivo hypoxic conditions are poorly understood. Here, we report that mitochondrial respiration is active in hypoxia (1% oxygen) and critical for fungal pathogenesis. We generated Aspergillus fumigatus alternative oxidase (aoxA) and cytochrome C (cycA) null mutants and assessed their ability to tolerate hypoxia, macrophage killing and virulence. In contrast to ?aoxA, ?cycA was found to be significantly impaired in conidia germination, growth in normoxia and hypoxia, and displayed attenuated virulence. Intriguingly, loss of cycA results in increased levels of AoxA activity, which results in increased resistance to oxidative stress, macrophage killing and long-term persistence in murine lungs. Thus, our results demonstrate a previously unidentified role for fungal mitochondrial respiration in the pathogenesis of aspergillosis, and lay the foundation for future research into its role in hypoxia signalling and adaptation.National Institutes of Health, COBRE [RR020185]National Institutes of Health, COBRENIH/NIAID [R01AI81838]NIH/NIAIDM. J. Murdock Charitable TrustM. J. Murdock Charitable TrustMontana State University Agricultural Experiment StationMontana State University Agricultural Experiment StationFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) from BrazilConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) from Brazi

The roles played by Aspergillus nidulans apoptosis-inducing factor (AIF)-like mitochondrial oxidoreductase (AifA) and NADH-ubiquinone oxidoreductases (NdeA-B and NdiA) in farnesol resistance

Farnesol (FOH) is a nonsterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. Here, we show that Aspergillus nidulans AifA encoding the apoptosis-inducing factor (AIF)-like mitochondrial oxidoreductase plays a role in the function of the mitochondrial Complex I. Additionally, we demonstrated that ndeA-B and ndiA encode external and internal alternative NADH dehydrogenases, respectively, that have a function in FOH resistance. When exposed to FOH, the ΔaifA and ΔndeA strains have increased ROS production while ΔndeB, ΔndeA ΔndeB, and ΔndiA mutant strains showed the same ROS accumulation than in the absence of FOH. We observed several compensatory mechanisms affecting the differential survival of these mutants to FOH.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, John Simon Guggenheim Memorial Foundation, USA

Molecular Characterization of the Putative Transcription Factor SebA Involved in Virulence in Aspergillus fumigatus

Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for A. fumigatus virulence and survival in the mammalian host. This study characterized the A. fumigatus SebA transcription factor, which is the putative homologue of the factor encoded by Trichoderma atroviride seb1. The Delta sebA mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl2, and poor nutritional conditions, while viability associated with sebA was also affected by heat shock exposure. Accordingly, SebA:GFP (SebA:green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the Delta sebA mutant. The A. fumigatus Delta sebA strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the Delta sebA mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain. A. fumigatus SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvi-mento Cientifico e Tecnologico (CNPq), Brazi

Number of genes and the associated functions of the proteins they encode that had restored or elevated expression in the <i>alcA::pkcA ΔcnaA</i> strain when compared to the Δ<i>cnaA</i> strain.

<p>sP is number of genes in each category.</p

Overexpression of <i>pkcA</i> in a <i>ΔcnaA</i> background rescues aberrant septa formation in a Δ<i>cnaA</i> background.

<p>Wild-type, Δ<i>cnaA</i> and <i>alcA</i>::<i>pkcA</i> Δ<i>cnaA</i> strains were grown in minimal medium supplemented with 2% (w/v) glycerol plus 100 mM threonine for 16 hours at 30°C before being fixed for 30 minutes at room temperature (RT) and stained with calcofluor white (CFW) for 5 minutes at RT. Mycelial fluorescence was then assessed under the microscope (scale bars indicate 5 µm).</p

Protein Kinase C Overexpression Suppresses Calcineurin-Associated Defects in <i>Aspergillus nidulans</i> and Is Involved in Mitochondrial Function

<div><p>In filamentous fungi, intracellular signaling pathways which are mediated by changing calcium levels and/or by activated protein kinase C (Pkc), control fungal adaptation to external stimuli. A rise in intracellular Ca²⁺ levels activates calcineurin subunit A (CnaA), which regulates cellular calcium homeostasis among other processes. Pkc is primarily involved in maintaining cell wall integrity (CWI) in response to different environmental stresses. Cross-talk between the Ca²⁺ and Pkc-mediated pathways has mainly been described in <i>Saccharomyces cerevisiae</i> and in a few other filamentous fungi. The presented study describes a genetic interaction between CnaA and PkcA in the filamentous fungus <i>Aspergillus nidulans</i>. Overexpression of <i>pkcA</i> partially rescues the phenotypes caused by a <i>cnaA</i> deletion. Furthermore, CnaA appears to affect the regulation of a mitogen-activated kinase, MpkA, involved in the CWI pathway. Reversely, PkcA is involved in controlling intracellular calcium homeostasis, as was confirmed by microarray analysis. Furthermore, overexpression of <i>pkcA</i> in a <i>cnaA</i> deletion background restores mitochondrial number and function. In conclusion, PkcA and CnaA-mediated signaling appear to share common targets, one of which appears to be MpkA of the CWI pathway. Both pathways also regulate components involved in mitochondrial biogenesis and function. This study describes targets for PkcA and CnaA-signaling pathways in an <i>A. nidulans</i> and identifies a novel interaction of both pathways in the regulation of cellular respiration.</p></div