Paracoccidioidomycosis due to Paracoccidioides lutzii complicated with adrenal injury and pulmonary arterial hypertension

Paracoccidioidomycosis caused by Paracoccidioides lutzii is endemic in the Midwest of Brazil and its clinical spectrum is still little known due to the recent identification of this fungal species. A patient resident in Southeast Brazil, but who had lived for many years in the Midwest region, presented with skin injuries, chronic cough and bilateral adrenal involvement. Paracoccidioides spp. was isolated in culture from a skin lesion biopsy. This isolate was later identified as P. lutzii using gene sequencing. A favorable initial response to treatment with itraconazole was observed, but a few weeks later, the patient developed respiratory failure and worsening of lung lesions. Evaluation by computed tomography and echocardiography were suggestive of pulmonary arterial hypertension, and a bronchoscopic biopsy showed peribronchial remodeling. The patient completed the antifungal treatment but maintained the respiratory dysfunction. The reported case shows that P. lutzii can be isolated from patients in a geographic area far from the place of infection acquisition and that, as P. brasiliensis , it can cause adrenal injury and cardio-respiratory complications as a consequence of excessive necrosis and fibrosis

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 gene encoding a protein with the signal peptide, masA, Aspergillus fumigatus

O gene MAS1/GAS2, conhecido como \"Magnaporthe Apressoria Specific\", foi identificado como altamente expresso durante a formação do apressório do fungo filamentoso fito patogênico Magnaporthe grisea. Em Aspergillus fumigatus, fungo patogênico oportunista, o gene masA, ortólogo a MAS1/GAS2 foi identificado como upregulated em uma análise transcriptômica exposto a voriconazole e anidulafungina, antifúngicos que afetam a síntese do ergosterol e a parede celular, respectivamente. Em ambos os ortólogos apresentam uma estrutura na região N-terminal da proteína denominada peptídeo sinal o qual neste trabalho foi determinado em uma análise in silico a presença de peptídeo sinal na região N-terminal da proteína. Na caracterização fenotípica de uma linhagem masA - deletada de A. fumigatus não foi identificado alteração estrutural do conidióforo e no seu aspecto macromorfológico. A linhagem masA-deletada é resistente a voriconazol e farnesol, drogas que inibem a síntese de ergosterol e a uma molécula quorum sensing, respectivamente. Ao avaliar o nível de expressão gênica do masA frente a diferentes classes de drogas, foi observado que o gene esta super expresso quando ocorre dano na parede celular, membrana citoplasmática, DNA, inibições na síntese de lipídeos e ácidos graxos e no estresse oxidativo. Na presença de dano na parede celular fúngica causados por anidulafungina, a proteína MasA está localizado na parede celular próximo a ponta da hifa. Adicionalmente, foi capaz de transferir para o meio extra-celular a proteína fusionada a ele, a GFP. Assim, possivelmente MasA participa da via secretória do fungo principalmente em momentos de estresse como o desarranjo da parede celular. A capacidade de secreção natural dos fungos filamentosos tem sido explorada no contexto industrial há décadas. Indicando para este fim, uma possível aplicabilidade para este peptídeo sinal.The MAS1/GAS2 gene, known as \"Magnaporthe Apressoria Specific\", was identified as highly expressed during the formation of the appressorium phyto pathogenic filamentous fungus Magnaporthe grisea. In Aspergillus fumigatus, opportunistic saprophytic fungus, masA gene orthologous to MAS1/GAS2 was identified as upregulated in a transcriptomic analysis exposed to voriconazole and anidulafungin, antifungals that affect the synthesis of ergosterol and the cell wall, respectively. In both orthologs have a structure at the N-terminus of the protein called signal peptide. During the phenotypic and functional characterization of masA gene in A. fumigatus, was determined on an in silico analysis the presence of signal peptide at the N-terminal region of the protein. In the phenotypic characterization of a strain masA - deleted, no structural change of conidiophores and its macromorfologic aspect was not identified. The characterization masA-deleted strain is resistant to voriconazol and farnesol drugs which inhibit ergosterol synthesis and a quorum sensing molecule, respectively. When evaluating the level of gene expression masA against different class of drugs was observed the super gene is expressed when damage occurs in the cell wall and membrane DNA, the synthesis of lipids and fatty acids, and oxidative stress. In the presence of damage in the fungal cell wall caused by anidulafungin, MasA protein is located near the cell wall and the tip of the hypha and additionally, was able to transfer the protein to the extracellular the protein fused to GFP. Thus, possibly MasA participates in the secretory pathway of the fungus especially in stress of the cell wall. The natural secretion capability of filamentous fungi has been exploited in the industrial context for decades. Indicating for this purpose, a possible applicability for this signal peptide

Molecular identification and serological study of clinical and environmental isolates of Paracoccidioides spp. from the Northeast of the State of São Paulo and the Southwest of the State of Minas Gerais

A paracoccidioidomicose (PCM) é uma infecção fúngica sistêmica e endêmica em países da América Latina. A maioria dos casos de PCM ocorrem em grandes áreas no Brasil, compreendendo as regiões Sul, Sudeste e Centro-Oeste do país. O complexo Paracoccidioides brasiliensis (espécies filogenéticas S1a, S1b, PS2, PS3 e PS4) e a espécie P. lutzii são os agentes etiológicos da PCM e existem poucos estudos filogenéticos que mostram, de forma incompleta, a distribuição geográfica dessas espécies na América do Sul. Este estudo realizou a identificação molecular de isolados clínicos e ambientais obtidos no Nordeste do Estado de São Paulo e Sudoeste do Estado de Minas Gerais, associando-os com características epidemiológicas e clínicas dos respectivos pacientes com PCM. Também foi avaliada a reatividade de exoantígenos de diferentes espécies do gênero Paracoccidioides spp. produzidos a partir de células miceliais frente a soros de pacientes cujo isolado fúngico foi identificado. Cinquenta amostras de Paracoccidioides spp. foram genotipadas, 46 de origem clínica e predominantemente isoladas na área geográfica de Ribeirão Preto, SP e 4 isolados ambientais coletados na cidade Ibiá, MG, Sudeste do Brasil. Estes isolados foram avaliados por PCR - RFLP do gene tub1 e sequenciamento do gp43 exon 2 loci. A espécie P. lutzii foi confirmada pelo sequenciamento da região \"internal transcribed spacer\" (ITS) do DNA ribossomal. Entre os isolados clínicos foram identificados P. brasiliensis sensu stricto S1b (n = 42) e S1a (n = 5), P. americana (PS2) (n = 1), P. restrepiensis (PS3) (n = 1) e P. lutzii (n = 1). Todos os isolados ambientais foram caracterizados como P. brasiliensis sensu stricto S1b. Não foi encontrada associação entre o genótipo infectante e a forma da doença. O histórico de migração de dois pacientes sugeriu que as infecções por P. americana (PS2) e P. lutzii ocorreram, respectivamente, nos Estados do Paraná e Mato Grosso. A reatividade dos exoantígenos miceliais de espécies do complexo P. brasiliensis e P. lutzii, produzidas por cepas de referência e clínicas, utilizando as técnicas de imunodifusão dupla (IDD) e contraimunoeletroforese (CIE) foi avaliada frente a 15 soros de pacientes cujo isolado fúngico foi identificado genotipicamente. Observou-se que as frações antigênicas de espécies do complexo P. brasiliensis tiveram 100% de reatividade em ambas as técnicas sorológicas, mas o exoantígeno de P. lutzii mostrou baixa reatividade, 53% em IDD e 20% em CIE. Os imunoprecipitados obtidos com exoantígenos de espécies do complexo P. brasiliensis foram idênticos entre si, mas não mostraram identidade com os precipitados resultantes da reação entre os soros e o exoantígeno P. lutzii. Este estudo contribui para o conhecimento da biogeografia de Paracoccidioides spp. e mostra que a análise da distribuição territorial de espécies deste fungo deve considerar a migração humana. Fenotipicamente, enquanto exoantígenos das espécies do complexo P. brasiliensis tem antigenicidade similar em teste de imunoprecipitação, o exoantígeno de P. lutzii apresentou reatividade quantitativa e qualitativamente distinta, possivelmente pela liberação de fração antigênica peculiar a esta última espécie.Paracoccidioidomycosis (PCM) is a systemic and endemic fungal infection that occurs in Latin American countries. Most cases of PCM occur in large areas in Brazil, including the South, Southeast and Midwest regions. The Paracoccidioides brasiliensis complex (phylogenetic species S1a, S1b, PS2, PS3 and PS4) and the species P. lutzii are the etiological agents of PCM and there are few phylogenetic studies that, incompletely, show the geographic distribution of these species in South America. This study identified molecular genotypes of Paracoccidioides spp. clinical and environmental isolates obtained in the northeast of São Paulo state and the southwest of Minas Gerais state, Brazil. The genotypes were associated with epidemiological and clinical characteristics of the respective PCM patients. It was also evaluated the reactivity of exoantigens from different species of the genus Paracoccidioides spp. produced by reference and clinical strains, mycelial cells that was tested against sera from patients whose fungal isolate was identified. Fifty samples (50) of Paracoccidioides spp. were genotyped, 46 of clinical origin and predominantly isolated in the geographical area of Ribeirão Preto, SP and 4 environmental isolates collected in the Ibiá city, MG, southeastern Brazil. These isolates were evaluated by PCR - RFLP of the tub1 gene and sequencing of gp43 exon 2 loci. The species P. lutzii was confirmed by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA. Among the clinical isolates were identified P. brasiliensis sensu stricto S1b (n = 42) and S1a (n = 5), P. americana (PS2) (n = 1), P. restrepiensis (PS3) (n = 1) and P lutzii (n = 1). All environmental isolates were characterized as P. brasiliensis sensu stricto S1b. No association was found between the infecting genotype and the form of the disease. The reactivity of the mycelial exoantigens from species of the P. brasiliensis complex and P. lutzii, produced from reference and clinical strains was evaluated using the techniques of double immunodiffusion (IDD) and counterimmunoelectrophoresis (CIE) against 15 sera from patients whose fungal isolate was genotypically identified. It was observed that the antigenic fraction of the P. brasiliensis complex species had obtained 100% reactivity in both serological techniques applied but the P. lutzii exoantigen showed low serum reactivity of 53% in IDD and 20% in CIE. The immune precipitates obtained with exoantigens of the P. brasiliensis complex were all identical, but this identity was not verified with the gel precipitates formed between the sera and the P. lutzii exoantigens. This study expanded the knowledge of the biogeography of Paracoccidioides spp. and shows that the analysis of the territorial distribution of this fungus species must consider human migration. Phenotypically, exoantigens of the P. brasiliensis complex had similar antigenicity in immunoprecipitation test while P. lutzii exoantigen presented differences in the aspect and positivity of the precipitate in agar gel, possibly by liberation of an antigenic fraction peculiar to this last species

Significance of Aspergillus spp. isolation in defining cases of COVID-19 Associated Pulmonary Aspergillosis – CAPA

COVID-19-Associated Pulmonary Aspergillosis (CAPA) is a relatively common complication in patients with severe forms of the disease caused by the SARS-CoV-2 virus. Diagnosing and confirming CAPA is challenging. In this study, Aspergillus spp. isolation in respiratory specimens from patients with COVID-19 was evaluated for identifying cases of CAPA. In 2020‒2021, 17 Aspergillus spp. were isolated from 15 COVID-19 patients admitted to a university hospital in Brazil. Patient records were retrospectively reviewed to obtain clinical-epidemiological data and other markers of Aspergillus spp. infection and then compared with the ECMM/ISHAM criteria for defining CAPA. Probable CAPA was defined in 5/10 patients, who had Aspergillus spp. isolated from Bronchoalveolar Lavage (BAL) or a positive galactomannan blood test. Additionally, anti-Aspergillus antibodies were detected in two of these patients, during active or follow-up phases of CAPA. In another seven patients with Aspergillus spp. isolated from tracheobronchial aspirate or sputum, CAPA was presumed, mainly due to deterioration of clinical conditions and new lung imaging suggestive of fungal infection. Antifungal agents to control CAPA, particularly voriconazole, were used in 9/15 cases. In cases of probable CAPA and remaining patients, clinical conditions and comorbidities were similar, with lethality being high, at 60% and 71%, respectively. The number of CAPA cases defined by scientific criteria was lower than that assumed in the clinical context. This was largely due to the lack of BAL collection for fungal culture and the non-intensive use of other markers of invasive aspergillosis. The isolation of Aspergillus spp. in different respiratory specimens should alert clinicians to the diagnosis of CAPA

An update on the occurrence of Paracoccidioides species in the Midwest region, Brazil: Molecular epidemiology, clinical aspects and serological profile of patients from Mato Grosso do Sul State.

BackgroundParacoccidioidomycosis (PCM) is a systemic and endemic fungal infection in Latin American, mainly in Brazil. The majority of PCM cases occur in large areas in Brazil, comprising the South, Southeast and Midwest regions, with the latter demonstrating a higher incidence of the species Paracoccidioides lutzii.Methodology and main findingsThis study presents clinical, molecular and serological data of thirteen new PCM cases during 2016 to 2019 from the state of Mato Grosso do Sul, located in the Midwest region, Brazil. From these thirteen cases, sixteen clinical isolates were obtained and their genomic DNAs were subjected to genotyping by tub1 -PCR-RFLP. Results showed Paracoccidioides brasiliensis sensu stricto (S1) (11/16; 68.8%), Paracoccidioides restrepiensis (PS3) (4/16; 25.0%) and P. lutzii (1/16; 6.2%) as Paracoccidiodes species. Therefore, in order to understand whether the type of phylogenetic species that are circulating in the state influence the reactivity profile of serological tests, we performed double agar gel immunodiffusion (DID), using exoantigens from genotyped strains found in this series of PCM cases. Overall, our DID tests have been false negative in about 30% of confirmed PCM cases. All patients were male, most with current or previous rural activity, with ages ranging from 17 to 59 years, with 11 patients (84.6%) over 40 years of age. No clinical or epidemiological differences were found between Paracoccidioides species. However, it is important to note that the only case of P. lutzii died as an outcome.ConclusionsThis study suggests P. brasiliensis sensu stricto (S1) as the predominant species, showing its wide geographic distribution in Brazil. Furthermore, our findings revealed, for the first time, the occurrence of P. restrepiensis (PS3) in the state of Mato Grosso do Sul, Brazil. Despite our setbacks, it would be interesting to provide the complete sequencing of these clinical isolates to complement the molecular information presented