Gene expression of Paracoccidioides virulence factors after interaction with macrophages and fibroblasts

BACKGROUND Paracoccidioidomycosis (PCM) is a systemic mycosis with high prevalence in Latin America that is caused by thermodimorphic fungal species of the Paracoccidioides genus. OBJECTIVES In this study, we used quantitative polymerase chain reaction (qPCR) to investigate the expression of genes related to the virulence of Paracoccidioides brasiliensis (Pb18) and P. lutzii (Pb01) strains in their mycelial (M) and yeast (Y) forms after contact with alveolar macrophages (AMJ2-C11 cell line) and fibroblasts (MRC-5 cell line). METHODS The selected genes were those coding for 43 kDa glycoprotein (gp43), enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 14-3-3 protein (30 kDa), phospholipase, and aspartyl protease. FINDINGS In the Pb18 M form, the aspartyl protease gene showed the highest expression among all genes tested, both before and after infection of host cells. In the Pb18 Y form after macrophage infection, the 14-3-3 gene showed the highest expression among all genes tested, followed by the phospholipase and gp43 genes, and their expression was 50-fold, 10-fold, and 6-fold higher, respectively, than that in the M form. After fibroblast infection with the Pb18 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 25-fold, 10-fold, and 10-fold higher, respectively, than that in the M form. Enolase and aspartyl protease genes were expressed upon infection of both cell lines. After macrophage infection with the Pb01 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 18-fold, 12.5-fold, and 6-fold higher, respectively, than that in the M form. MAIN CONCLUSIONS In conclusion, the data show that the expression of the genes analysed may be upregulated upon fungus-host interaction. Therefore, these genes may be involved in the pathogenesis of paracoccidioidomycosis.

Paracoccidioidomycosis in the Central Nervous System

Paracoccidioidomycosis is a systemicmycosis caused by the Paracoccidioides brasiliensis fungus, which is endemic in Latin America. Brazil is the country with the highest number of cases. The affection of the central nervous system (CNS), a potentially fatal condition, occurs in 12% of the cases. The following forms of presentation are identified:meningeal, which is unusual;meningoencephalitic; and pseudotumoral, the latter two being more frequent. Imaging tests are essential for the diagnosis, but the histological identification of the fungus is required for confirmation of the pathology. The clinical picture depends on the neuraxial location.We present a case of amale rural worker, with expansive lesions in the CNS compatible with paracoccidioidomycosis.

Fungal-host interactions: insights into microRNA in response to Paracoccidioides species

BACKGROUND Paracoccidioides spp. causes paracoccidioidomycosis (PCM), an important and frequent systemic mycosis that occurs in Latin America. The infectious process begins with contact between the fungus and lung cells, and the molecular pattern of this interaction is currently poorly understood. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the gene expression in many biological processes, including in the infections. OBJECTIVE This study aimed to analyse the expression of miRNAs in lung cells as response to infection by Paracoccidioides spp. METHODS A quantitative real-time polymerase chain reaction (RT-qPCR) based screening was employed to verify differentially expressed miRNAs in human lung cells infected with three different species; Paracoccidioides lutzii, Paracoccidioides americana, and Paracoccidioides brasiliensis. Furthermore, the in silico predictions of target genes and pathways for miRNAs were obtained. FINDINGS The results showed that miRNAs identified in the lung cells were different according to the species studied. However, based on the predicted targets, the potential signaling pathways regulated by miRNAs are common and related to adhesion, actin cytoskeleton rearrangement, apoptosis, and immune response mediated by T cells and TGF-β. MAIN CONCLUSIONS In summary, this study showed the miRNAs pattern of epithelial cells in response to infection by Paracoccidioides species and the potential role of these molecules in the regulation of key pathogenesis mechanisms of PCM.

Influência de TLR3 em modelo de Paracoccidioidomicose experimental / TLR3 influence in experimental PCM model

Os receptores do tipo Toll compreendem a família de receptores de reconhecimento de padrões melhor caracterizados, que podem ativar diferentes respostas imunes, dependendo de quais receptores e conjuntos de adaptadores são utilizados. Os TLRs, como TLR2, TLR4 e TLR9, e sua sinalização foram implicados no reconhecimento de P. brasiliensis e na regulação da resposta imune, no entanto, o papel do TLR3 ainda não está claro. Assim, a compreensão da função endossomal do TLR3 na PCM experimental é crucial. Utilizamos modelos in vitro e in vivo de infecção por P. brasiliensis, camundongos C57Bl/6 e TLR3-/-, para avaliar a contribuição da TLR3 no desenvolvimento da infecção. Mostramos que ausência de TLR3 leva o aumento de óxido nítrico e a capacidade fagocítica por macrófagos nas primeiras 4 horas de interação com leveduras P. brasiliensis. Mostramos ainda que os camundongos TLR3-/- desempenham papel protetor após 30 dias de infecção intratraqueal com P. brasiliensis, mostrando diminuição do aumento de CFU, perfil de resposta Th1 e Th17, bem como aumento de células citotóxicas T CD8+ produtoras de IFN-γ e IL-17. As células citotóxicas T CD8+ mostraram ser essenciais para o controle da infecção nos camundongos TLR3-/-, uma vez que a depleção dessas células levou a progressão da doença. Em estágios iniciais, 3 e 5 dias de infecção, observamos aumento do recrutamento de neutrófilos para o pulmão. Estudos recentes indicam que o TLR3 é um receptor importante para a resposta imune na micose e sua ausência favorece a infecção por fungos. Em contraste, nossos resultados mostram que, no caso do PCM, o TLR3 é prejudicial ao hospedeiro, sugerindo que a ativação do TLR3 pode ser um possível mecanismo de escape de P. brasiliensis

Toll-like receptors comprise the best-characterized pattern-recognition receptor family that can activate different immune responses, depending on which receptor and adaptor set are utilized. TLRs, such as TLR2, TLR4 and TLR9, and their signaling have been implicated in the recognition of P. brasiliensis and regulation of the immune response, however, the role of TLR3 remains unclear. Thus, understanding the endosomal function of TLR3 in experimental PCM is crucial. We used in vitro and in vivo models of infection by P. brasiliensis, C57Bl/6 and TLR3-/- mice, to assess the contribution of TLR3 on development of infection. We show that absence of TLR3 leads to increased nitric oxide and phagocytic capacity by macrophages in the first 4 hours of interaction with yeasts P. brasiliensis. We also showed that TLR3-/- mice play a protective role after 30 days of intratracheal infection with P. brasiliensis, showing a decrease in the CFU increase, Th1 and Th17 response profile, as well as an increase in cytotoxic CD8+ cells producing IFN-γ and IL-17. The cytotoxic T CD8+ cells were shown to be essential for the control of infection in TLR3-/- mice, since the depletion of these cells led to the progression of the disease. In the initial stages, 3 and 5 days of infection, we observed increased recruitment of neutrophils to the lung. Recent studies indicate that TLR3 is an important receptor for the immune response in mycosis and its absence favors fungal infection. In contrast, our results show that in the case of PCM, TLR3 is detrimental to the host, suggesting that TLR3 activation may be a possible escape mechanism of P. brasiliensis

THE POWER OF THE SMALL: THE EXAMPLE OF Paracoccidioides brasiliensis CONIDIA / O poder do pequeno: o exemplo dos conídios do Paracoccidioides brasiliensis

SUMMARYResearch on Paracoccidioides brasiliensis has centered in the yeast cell probably because of the lack of distinctive features in the mycelium. In 1942 and for the first time, lateral conidia were noticed in the fungus' hyphae. Later on, Brazilian, Venezuelan and Argentinean researchers described "aleurias" when the fungus was grown in natural substrates. In 1970 authors became interested in the conidia and were able to obtain them in large numbers and treat them as individual units. Their shape and size were defined and the presence of all the elements of a competent eukaryotic cell were demonstrated. Conidia exhibited thermal dimorphism and, additionally, when given intranasally to BALB/c male mice, they converted into yeasts in the lungs and produce progressive pulmonary lesions with further dissemination to other organs. Studies on the phagocyte-conidia interaction were revealing and showed that these versatile structures allow a better understanding of the host- P. brasiliensisinteractions.

RESUMOA investigação sobre Paracoccidioides brasiliensis tem-se centrado na célula de levedura, provavelmente devido à falta de características distintas no micélio. Em 1942 e, pela primeira vez, conídios laterais foram notados nos hifas dos fungos. Mais tarde, pesquisadores brasileiros, venezuelanos e argentinos descreveram "aleurias" quando o fungo foi cultivado em substratos naturais. Em 1970, os autores se interessaram pelos conídios e foram capazes de obtê-los em grande número e tratá-los como unidades individuais. A sua forma e tamanho foram definidos, e a presença de todos os elementos de uma célula eucariótica competente foram demonstrados. Conídios apresentam dimorfismo térmico e, além disso, quando administrados por via intranasal a camundongos BALB/c machos, são convertidos em leveduras nos pulmões e produzem lesões pulmonares progressivas com posterior disseminação para outros órgãos. Estudos sobre a interação de fagócitos-conídios foram reveladores e mostraram que estas estruturas versáteis permitem melhor compreensão das interacções entre hospedeiro e P. brasiliensis.

DOSE RESPONSE EFFECT OF Paracoccidioides brasiliensis IN AN EXPERIMENTAL MODEL OF ARTHRITIS / Efeito da dose resposta de Paracoccidioides brasiliensis no modelo experimental de artrite

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb) and corresponds to prevalent systemic mycosis in Latin America. The aim of the present work was to evaluate the dose response effect of the fungal yeast phase for the standardization of an experimental model of septic arthritis. The experiments were performed with groups of 14 rats that received doses of 103, 104 or 105 P. brasiliensis (Pb18) cells. The fungi were injected in 50 µL of phosphate-buffered saline (PBS) directly into the knee joints of the animals. The following parameters were analyzed in this work: the formation of swelling in knees infused with yeast cells and the radiological and anatomopathological alterations, besides antibody titer by ELISA. After 15 days of infection, signs of inflammation were evident. At 45 days, some features of damage and necrosis were observed in the articular cartilage. The systemic dissemination of the fungus was observed in 11% of the inoculated animals, and it was concluded that the experimental model is able to mimic articular PCM in humans and that the dose of 105 yeast cells can be used as standard in this model.

A paracoccidioidomicose (PCM) é causada pelo fungo dimórfico Paracoccidioides brasiliensis (Pb) e corresponde à micose sistêmica de maior prevalência na América Latina. O objetivo do presente trabalho foi avaliar a dose resposta de leveduras do fungo para padronização do modelo experimental de artrite séptica. Os experimentos foram realizados com grupos de 14 ratos que receberam doses de 103, 104 ou 105 células de P. brasiliensis (Pb18). Os fungos foram injetados em 50 µL de solução salina em tampão fosfatado (PBS) diretamente na articulação do joelho dos animais. Os seguintes parâmetros foram analisados neste trabalho: a formação de edema nos joelhos infundidos com as células das leveduras e alterações radiológicas, anatopalógicas além de titulação de anticorpos por Elisa. Após 15 dias de infecção, os sinais de inflamação foram evidentes. Aos 45 dias, algumas características de dano e necrose foram observadas na cartilagem articular. A disseminação sistêmica do fungo foi observada em 11% dos animais inoculados, concluiu-se que o modelo experimental é capaz de mimetizar a PCM articular em humanos e que a dose de 105 leveduras representa a dose padrão para o desenvolvimento do modelo.

Paracoccidioidomicosis: clínica, epidemiología y tratamiento / Paracoccidioidomicosis: clinical considerations, epidemiology and treatment

Objetivo: caracterizar desde el punto de vista clínico, epidemiológico y de tratamiento, los pacientes con diagnóstico de paracoccidioidomicosis evaluados en la Consulta Externa de la Sección de Micología Médica del IMT-UCV y procedentes de otros hospitales del área de la Gran Caracas, cuyas muestras fueron procesadas en dicha Sección, entre los años 2001-2011. Métodos: estudio descriptivo de registros, donde se tomaron en cuenta las historias de los pacientes con diagnóstico de paracoccidioidomicosis. Resultados: la relación hombre:mujer obtenida fue de 6,5:1, sólo el 29,16% de los pacientes tenían ocupaciones agrícolas. Los síntomas más frecuentes fueron tos (50%) y pérdida de peso (45,06%) y los signos más comunes fueron lesiones en orofaringe (53,6%) y adenopatías (27,2%). 7,65% cursaron concomitantemente con infección por VIH/SIDA, 5,61% con tuberculosis y 6,63% con histoplasmosis. En cuanto al diagnóstico, el examen directo fue positivo en 87,5% de los casos, el cultivo en el 62,90% y la serología en el 88,46%. La mortalidad general fue de 4,11%. Discusión: La paracoccidioidomicosis es una enfermedad que puede ocurrir en pacientes inmunocompetentes e inmunocomprometidos; tiende a observarse ahora con mayor frecuencia en los pacientes con SIDA. El número de casos entre los pacientes del sexo femenino ha aumentado. La investigación acerca del lugar de origen, de la procedencia y la ocupación del paciente es importante a la hora de plantearse el diagnóstico. Esto debe ser tomado en consideración en un país endémico como el nuestro. Su diagnóstico es relativamente fácil en manos de personal entrenado.

Objective: to characterize from the clinical, epidemiological and treatment viewpoints, patients diagnosed with paracoccidioidomycosis and evaluated in the outpatient clinic of the Medical Mycology Section, at the IMT-UCV and from other hospitals in the greater Caracas area, whose samples were processed in this section between 2001-2011. Methods: descriptive study of records, taking into account the histories of patients diagnosed with paracoccidioidomicosis. Results: the male:female ratio obtained was 6.5:1; only 29.16% of the patients had agricultural occupations. The most common symptoms were cough (50%) and weight loss (45.06%), and the most common signs were oropharyngeal lesions (53.6%) and adenopathy (27.2%). Concomitantly, 7.65% had HIV/AIDS infection, 5.61% tuberculosis and 6.63% histoplasmosis. In terms of diagnosis, the direct exam was positive in 87.5% of cases, the culture in 62.90% and serology in 88.46%. Overall mortality was 4.11%. Discussion: paracoccidioidomycosis is a disease that can occur in immunocompetent and immunocompromised patients; currently, it tends to appear more frequently in patients with AIDS. The number of cases among female patients has increased. Research about the place of birth, the origin and the occupation of the patient is important when considering the diagnosis; this should be taken into consideration in an endemic country like Venezuela. Its diagnosis is relatively easy in the hands of trained personnel.

Humoral response of paracoccidioidomycosis sera in hamsters with different Venezuelan isolates

Humoral response of paracoccidioidomycosis sera in hamsters with different Venezuelan isolates. Paracoccidioidomycosis (PCM) is a progressive systemic mycosis caused by the fungus Paraccocidioides brasiliensis (Pb), endemic to Venezuela and Latin America. In this study, eight different Venezuelan isolates obtained from patients with PCM, were inoculated intraperitoneally in Syrian hamsters (Cricetus auratus) and studied by immune-serum. Each strain was collected by gently scraping the surface of the culture medium (Sabouraud Dextrose Agar) and suspended in 3ml of 0.15 M phosphate-buffered saline. The antigen obtained was called Paraccocidioides brasiliensis Crude Antigen (CAP). Immunoblotting results showed that the immune-sera from hamsters recognized at least 3 bands: one over 200 kDa, and two of 80 and 15-20 kDa. This study suggests that IgG anti-CAP can reveal a significant variability in the eight Venezuelan isolates. Sera from 88 infected hamsters were evaluated by ELISA with eight different CAPs and Western blot with CAP 37383. ELISA results showed that, the antigen of the virulent isolate 37383 had the highest percentage (38%) of positivity, while the non-virulent isolate 1458 had the lowest one (13.6%). Furthermore, scanning densitometry revealed that the isolate 37383 had less bands than the non-virulent isolates. These results suggest that the ELISA test with CAP 37383 can detect circulating antibodies, and that this virulent isolate may be useful for the diagnosis of PCM, and to monitor disease responses to treatments. Rev. Biol. Trop. 57 (3): 505-513. Epub 2009 September 30.

La Paracoccidioidomicosis (PCM), es una micosis sistémica causada por el hongo Paraccocidioides brasiliensis (Pb), endémica en Venezuela y Latino América. En este estudio ocho diferentes aislados venezolanos, obtenidos de pacientes con PCM, fueron inoculados intraperitonealmente en hámsteres y fueron estudiados por ELISA e inmunoblotting. Los antígenos obtenidos de P. brasiliensis fueron llamados, Antígeno Crudo (CAP). Los resultados del immunoblotting mostraron que los sueros inmunes de hámsteres reconocieron al menos tres bandas: una sobre 200, y otras de 80, y 15-20 kDa. Este estudio sugiere que la IgG anti-CAP muestra una variabilidad en los ocho aislados Venezolanos. Sueros de 88 hámsteres infectados fueron evaluados usando ELISA, el antígeno del aislado virulento 37383 mostró el más alto porcentaje de positividad (38%) en los sueros de los hámsteres estudiados. El aislado novirulento 1458 mostró un porcentaje bajo de positividad (13.6%). Además, un escaneo densitométrico reveló que el aislado 37383 tiene menos bandas que el otro aislado no-virulento. Por lo tanto, estos resultados sugieren que el ensayo de ELISA con CAP 37383 puede detectar anticuerpos circulantes y este aislado virulento puede ser útil para el diagnostico de PCM, y para el monitoreo de la respuesta al tratamiento de la enfermedad.

Induction of apoptosis in A549 pulmonary cells by two Paracoccidioides brasiliensis samples

Paracoccidioidomycosis presents a variety of clinical manifestations and Paracoccidioides brasiliensis can reach many tissues, most importantly the lungs. The ability of the pathogen to interact with host surface structures is essential to its virulence. The interaction between P. brasiliensis and epithelial cells has been studied, with particular emphasis on the induction of apoptosis. To investigate the expression of different apoptosis-inducing pathways in human A549 cells, we infected these cells with P. brasiliensis Pb18SP (subcultured) and 18R (recently isolated from cell culture and showing a high adhesion pattern) samples in vitro. The expressions of Bcl-2, Bak and caspase 3 were analysed by flow cytometry and DNA fragmentation using the TUNEL technique. Apoptosis of human A549 cells was induced by P. brasiliensis in a sample and time-dependent manner. Using an in vitro model, our data demonstrates that caspase 3, Bak, Bcl-2 and DNA fragmentation mediate P. brasiliensis-induced apoptosis in A549 cells. The overall mechanism is a complex process, which may involve several signal transduction pathways. These findings could partially explain the efficient behaviour of this fungus in promoting tissue infection and/or blood dissemination.

Resistance of melanized yeast cells of Paracoccidioides brasiliensis to antimicrobial oxidants and inhibition of phagocytosis using carbohydrates and monoclonal antibody to CD18

Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.

Epidemiological study of paracoccidioidomycosis and histoplasmosis in a suburb of San Félix city Bolívar state Venezuela

Epidemiologic studies of deep mycosis have been scarce in Bolívar state, where paracoccidioidomycosis and histoplasmosis are considered as endemic diseases. The aim of this study was to determine paracoccidioidomycosis and histoplasmosis prevalences in people from a suburb of San Félix, Bolívar state, Venezuela. Three-hundred volunteers agreed to participate in this study and they were inoculated with paracoccidioidine and histoplasmine. Identification and epidemiologic data were registered. Reading of skin tests after 24 hours was performed in 275 persons. Paracoccidioidine test was positive in 10.2% (n=28). A higher percentage of positive reactions in the age group of 40-50 years old (n=10; 35.7%) was observed. Bricklayers, farmers and miners were positive in 27.3% (3 out of 11), a higher percentage than in people with other occupations. Histoplasmine test was positive in 7.6 percent of cases (n= 21). The higher percentage of reactivity was observed in the age group of 40-50 years old (n=9; 42.9%). There was a direct proportional relationship between staying time in the locality and H. capsulatum infection mainly in persons staying in the area for more than 30 years (p<0.05). These results showed low prevalences of P. brasiliensis and H. capsulatum infection in this area.

El estudio epidemiológico de las micosis profundas ha sido escaso en el estado Bolívar donde la paracoccidioidomicosis e histoplasmosis son endémicas. El objetivo de este estudio fue determinar la prevalencia de infecciones por Paracoccidioides brasiliensis e Histoplasma capsulatum en individuos residenciados en una población suburbana de San Félix, estado Bolívar, Venezuela. Se administró paracoccidioidina e histoplasmina a 300 personas voluntarias. Se realizó lectura de las pruebas a las 24 horas. La paracoccidioidina fue positiva en el 10,2% (n=28). Se observó mayor porcentaje de positividad en el grupo de 40-50 años (n=10; 35,7%). Las ocupaciones de riesgo: albañiles, agricultores y mineros, presentaron un porcentaje de positividad de 27,3% (3 de 11), mayor que el de los individuos sin riesgo aparente: mecánicos, oficios del hogar y estudiantes (25 de 264; 9,5 %) (p=0,04). La histoplasmina fue positiva en el 7,6% (n=21). El mayor porcentaje de intradermorreacción positiva se observó entre los 40-50 años (n=9; 42,9%). Hubo relación significativa entre el tiempo de residencia en la localidad y la infección por H. capsulatum, demostrándose en los individuos con más de 30 años en esa localidad (p<0,05). Estos resultados muestran una prevalencia relativamente baja de infecciones por P. brasiliensis y de H. capsulatum en el área estudiada.

Prevalencia de la infección por Paracoccidioides brasiliensis e Histoplasma capsulatum en agricultores del “Caserío La Entrada”. Estado Aragua-Venezuela / Prevalence of Paracoccidioides brasiliensis and Histoplasma capsulatum infection in agricultural workers from the "Caserío La Entrada". State of Aragua. Venezuela

La paracoccidioidomicosis es una de las micosis sistémicas más frecuente en Venezuela. Su agente etiológico es Paracoccidioides brasiliensis. Investigaciones previas, sugieren que en el municipio Tovar del estado Aragua (Venezuela) existen zonas endémicas para Paracoccidioides brasiliensis en las que los agricultores están ocupacionalmente expuestos. Además, han sido descritas algunas áreas endémicas comunes para Paracoccidioides brasiliensis y Histoplasma capsulatum. En consecuencia, se determinó la prevalencia de la infección por ambos hongos en 131 agricultores del “Caserío La Entrada” (municipio Tovar), mediante la intradermorreacción a la paracoccidioidina e histoplasmina. La prevalencia fue 90,1% para Paracoccidioides brasiliensis y 72,5% para Histoplasma capsulatum, encontrándose copositividades en 88 agricultores (67%). No se encontró asociación estadísticamente significativa (p=0,186) entre las intradermopositividades. La frecuencia de infección por Paracoccidioides brasiliensis y/o Histoplasma capsulatum resultó independiente de la edad, sexo y del tiempo laborando en la agricultura. Altos porcentajes de intradermopositividades fueron encontrados en menores de 10 años de edad (paracoccidiodina: 89,3%; histoplasmina: 75%) y en aquellos que tenían menos de un año como agricultores (paracoccidiodina: 85,4%; histoplasmina: 65,9%). Los hallazgos obtenidos sugieren que en el área geográfica estudiada coexisten Paracoccidioides brasiliensis y Histoplasma capsulatum. Además, que la infección ocurre rápida y tempranamente en la vida de estos individuos, permitiendo inferir que la endemicidad de estos microorganismos y el riesgo de contraer la infección son elevados en los trabajadores agrícolas

The paracoccidioidomycosis is one of the more frequent systemic mycosis in Venezuela. His etiologic agent is Paracoccidioides brasiliensis. Previous investigations, suggest the existence of endemic zones for Paracoccidioides brasiliensis in which the farmers are occupationally exposed in the Tovar Municipality of the Aragua state (Venezuela). In addition, some common endemic areas for Paracoccidioides brasiliensis and Histoplasma capsulatum have been described. Consequently, the prevalence of the infection by both fungi was determined in 131 farmers from the “Caserío La Entrada” (Tovar Municipality), by means of the intradermoreaction to paracoccidioidin and histoplasmin. The prevalence was 90.1% for Paracoccidioides brasiliensis and 72.5% for Histoplasma capsulatum, being the skin reactivity positive for both antigens in 88 farmers (67%). Association was not statistically significant (p=0,186) among the skin reactivities. The frequency of infection by Paracoccidioides brasiliensis and/or Histoplasma capsulatum was independent from age, sex and time working in farms. High percentages of skin reactivity were found in individuals under 10 years of age (paracoccidiodin: 89.3%; histoplasmin: 75%) and in people working as farmers less than one year (paracoccidiodin: 85.4%; histoplasmin: 65.9%). Findings suggest that there is a coexistance of Paracoccidioides brasiliensis and Histoplasma capsulatum in the studied geographic area. In addition, the infection occurs fast and early in the life of these individuals, allowing to infer that those microorganisms are endemic and the risk of contracting the infection are elevated in the farmers

Preferential transcription of Paracoccidioides brasiliensis genes: host niche and time-dependent expression

Paracoccidioides brasiliensis causes infection through inhalation by the host of airborne propagules from the mycelium phase of the fungus. This fungus reaches the lungs, differentiates into the yeast form and is then disseminated to virtually all parts of the body. Here we review the identification of differentially-expressed genes in host-interaction conditions. These genes were identified by analyzing expressed sequence tags (ESTs) from P. brasiliensis cDNA libraries. The P. brasiliensis was recovered from infected mouse liver as well as from fungal yeast cells incubated in human blood and plasma, mimicking fungal dissemination to organs and tissues and sites of infection with inflammation, respectively. In addition, ESTs from a cDNA library of P. brasiliensis mycelium undergoing the transition to yeast were previously analyzed. Together, these studies reveal significant changes in the expression of a number of genes of potential importance in the host-fungus interaction. In addition, the unique and divergent representation of transcripts when the cDNA libraries are compared suggests differential gene expression in response to specific niches in the host. This analysis of gene expression patterns provides details about host-pathogen interactions and peculiarities of sites within the host.

Experimental infections with Paracoccidioides brasiliensis obtained from armadillos: comparison to clinical isolates

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM) that is one of the most prevalent systemic human mycoses in Latin America. Armadillos show a high incidence of PCM infection and could, therefore, be a natural reservoir for this fungus. In this study were compared the virulence profiles of isolates obtained from nine-banded armadillos (Dasypus novemcinctus) (PbT1 and PbT4) and isolates from PCM patients (Pb265 and Bt83). Pathogenicity was evaluated by fungal load and analysis of colony morphology. Immunity against the fungus was tested by delayed type hypersensitivity test (DTH) and antibody quantification by ELISA. The higher virulence of PbT1 and PbT4 was suggested by higher fungal load in spleen and lungs. Armadillo isolates and Bt83 presented a cotton-like surface contrasting with the cerebriform appearance of Pb265. All isolates induced cellular and humoral immune responses in infected BALB/c mice. DTH reactions were similarly induced by the four isolates, however, a great variability was observed in specific antibody levels, being the highest ones induced by Bt83 and PbT4. The present work confirms that armadillos harbor P. brasiliensis, whose multiplication and induced immunity in experimentally infected mice are heterogeneous, resembling the behavior of isolates from human PCM. This study reinforces the possibility that armadillos play an important role in the biological cycle of this pathogen.

Virulence attenuation and phenotypic variation of Paracoccidioides brasiliensis isolates obtained from armadillos and patients

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, the most important systemic mycosis in Latin America. The virulence profiles of five isolates of P. brasiliensis were studied in two different moments and correlated with some colonial phenotypic aspects. We observed a significant decrease in the virulence and an intense phenotypic variation in the mycelial colony. The recognition of all ranges of phenotypic and virulence variation of P. brasiliensis, as well as its physiological and genetic basis, will be important for a better comprehension of its pathogenic and epidemiological features.

Virulence profile of ten Paracoccidioides brasiliensis isolates: Association with morphologic and genetic patterns

Dez isolados de P. brasiliensis foram avaliados em relação à patogenicidade por inoculação intravenosa em camundongos e associação com morfologia miceliana e padrão genético por amplificação genônica do DNA polimórfico (RAPD). A patogenicidade, avaliada por recuperação de fungos viáveis a partir de tecido pulmonar e por lesões histopatológicas em diferentes órgãos, mostrou que os isolados apresentaram quatro graus de virulência: alta virulência, virulência intermediária, baixa virulência e não virulência. A técnica de RAPD agrupou os isolados em dois grupos com 56% de similaridade genética. Amostras com baixa virulência Pb265 ou não virulência Pb192 apresentaram morfologia glabra/cerebriforme e alta similaridade genética (98,7%) quando comparadas com os outros isolados estudados. O mesmo foi observado com os isolados Bt79 e Bt83, que compartilharam 96% de semelhança genética, colônias cotonosas e alta virulência. Essa técnica pode discriminar apenas isolados com morfologia glabra da cotonosa e com alta e baixa virulência. Isolados com virulência intermediária como Pb18, Pb18B6, Bt32 e Bt54 mostraram variabilidade no coeficiente de similaridade, sugerindo que a técnica de RAPD permite mostrar variabilidade genética nessa espécie fúngica. O estudo do perfil de virulência das amostras de P. brasiliensis demonstrou que os dois fenótipos extremos de morfologia miceliana podem ser associados com a virulência do fungo e com o tempo de subcultivo in vitro. Assim, a análise de RAPD, utilizada em conjunto com aspectos de virulência, morfológicos e imunológicos pode ser considerada adequada para detectar diferenças entre isolados de P. brasiliensis.

Transcriptome overview of Paracoccidioides brasiliensis proteases

Proteases perform a wide variety of functions inside and outside cells, regulating many biological processes. Infectious microorganisms use proteases, either secreted or attached to their cell surface to weaken and invade their hosts. Therefore, proteases are targets for drugs against a diverse set of diseases. Paracoccidioides brasiliensis is the most prevalent fungal pathogen causing systemic mycosis in Latin America. The development of paracoccidioidomycosis depends on interactions between fungal and host components and proteases have been described as important factors implicated in the mechanism of host colonization by fungi. The primary goal for this study is to present an overview of the transcriptome sequences--identified cDNAs that encode proteases. We obtained a total of 53 cDNAs encoding proteases; 15 were classified as ATP-independent, 12 as ATP-dependent, 22 as proteasome subunits, and 4 as deubiquitinating proteases. The mechanisms and biological activity of these proteases differ in substrate specificity and in catalytic mechanisms.

Virulence insights from the Paracoccidioides brasiliensis transcriptome

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus, which is found as mycelia at 22-26 degrees C and as yeasts at 37 degrees C. A remarkable feature common to several pathogenic fungi is their ability to differentiate from mycelium to yeast morphologies, or vice-versa. Although P. brasiliensis is a recognized pathogen for humans, little is known about its virulence genes. In this sense, we performed a search for putative virulence genes in the P. brasiliensis transcriptome. BLAST comparative analyses were done among P. brasilienses assembled expressed sequence tags (PbAESTs) and the sequences deposited in GenBank. As a result, the putative virulence PbAESTs were grouped into five classes, metabolism-, cell wall-, detoxification-related, secreted factors, and other determinants. Among these, we have identified orthologs of the glyoxylate cycle enzymes, a metabolic pathway involved in the virulence of bacteria and fungi. Besides the previously described alpha- and beta-glucan synthases, orthologs to chitin synthase and mannosyl transferases, also important in cell wall synthesis and stabilization, were identified. With respect to the enzymes involved in the intracellular survival of P. brasiliensis, orthologs to superoxide dismutase, thiol peroxidase and an alternative oxidase were also found. Among the secreted factors, we were able to find phospholipase and urease orthologs in P. brasiliensis transcriptome. Collectively, our results suggest that this organism may possess a vast arsenal of putative virulence genes, allowing the survival in the different host environments.

Cell signaling pathways in Paracoccidioides brasiliensis: inferred from comparisons with other fungi

The human fungal pathogen Paracoccidioides brasiliensis is an ascomycete that displays a temperature-dependent dimorphic transition, appearing as a mycelium at 22 degrees C and as a yeast at 37 degrees C, this latter being the virulent form. We report on the in silico search made of the P. brasiliensis transcriptome-expressed sequence tag database for components of signaling pathways previously known to be involved in morphogenesis and virulence in other species of fungi, including Saccharomyces cerevisiae, Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Using this approach, it was possible to identify several protein cascades in P. brasiliensis, such as i) mitogen-activated protein kinase signaling for cell integrity, cell wall construction, pheromone/mating, and osmo-regulation, ii) the cAMP/PKA system, which regulates fungal development and virulence, iii) the Ras protein, which allows cross-talking between cascades, iv) calcium-calmodulin-calcineurin, which controls cell survival under oxidative stress, high temperature, and membrane/cell wall perturbation, and v) the target of rapamycin pathway, controlling cell growth and proliferation. The ways in which P. brasiliensis responds to the environment and modulates the expression of genes required for its survival and virulence can be inferred through comparison with other fungi for which this type of data is already available.

Enfermedad de Lutz-Splendore-De Almeida sin manifestaciones mucocutáneas: a propósito de un caso / Disease Lutz-Splendore-De Almeida without mucocutaneous manifestations: a purpose of a case

La enfermedad de LUTZ-SPLENDORE-DE ALMEIDA se debe al Paracoccidiodes brasiliensis, presente en Latinoamérica. Al principio la infección pulmonar produce pocos síntomas y la diseminación hasta mucosas, ganglios linfáticos y otros; hace que el paciente acuda al médico. Los signos son: úlceras induradas en mucosas de orofaringe, y nariz, adenomegalias, lesiones cutáneas y genitales, tos productiva, pérdida de peso, disnea y fiebre. Radiológicamente muestra una neumonía con focos dispersos bilaterales, el cultivo de secreciones, las pruebas serológicas confirman el diagnóstico; se trata con Itraconazol o Trimetopín-Sulfametoxazol. Se describe caso de un masculino de 49 años, agricultor, quien consultó por tos y pérdida de peso, confirmado mediante estudios imagenológicos, serológicos, histológicos y fibrobroncospia, durante su evolución no cursó con manifestaciones cutáneo-mucosas, siendo divergentes al común de los casos, ya que dichas lesiones preceden o acompañan la forma pulmonar. Es importante integrar al pensamiento médico la existencia del Paracoccidioides brasiliensis, como agente etiológico de infecciones respiratorias aun sin manifestaciones cutáneas.