RESUMO
The Pneumocystis genus is an opportunistic fungal pathogen that infects patients with AIDS and immunocompromised individuals. The study of this fungus has been hampered due to the inability to grow it in a (defined media/pure) culture. However, the use of modern molecular techniques and genomic analysis has helped researchers to understand its complex cell biology. The transcriptional process in the Pneumocystis genus has not been studied yet, although it is assumed that it has conventional transcriptional machinery. In this work, we have characterized the function of the RNA polymerase II (RNAPII) general transcription factor TFIIB from Pneumocystis carinii using the phylogenetically related biological model Schizosaccharomyces pombe. The results of this work show that Pneumocystis carinii TFIIB is able to replace the essential function of S. pombe TFIIB both in in vivo and in vitro assays. The S. pombe strain harboring the P carinii TFIIB grew slower than the parental wild-type S. pombe strain in complete media and in minimal media. The S. pombe cells carrying out the P. carinii TFIIB are larger than the wild-type cells, indicating that the TFIIB gene replacement confers a phenotype, most likely due to defects in transcription. P. carinii TFIIB forms very weak complexes with S. pombe TATA-binding protein on a TATA box promoter but it is able to form stable complexes in vitro when S. pombe TFIIF/RNAPII are added. P. carinii TFIIB can also replace the transcriptional function of S. pombe TFIIB in an in vitro assay. The transcription start sites (TSS) of the endogenous adh gene do not change when P. carinii TFIIB replaces S. pombe TFIIB, and neither does the TSS of the nmt1 gene, although this last gene is poorly transcribed in vivo in the presence of P. carinii TFIIB. Since transcription by RNA polymerase II in Pneumocystis is poorly understood, the results described in this study are promising and indicate that TFIIB from P. carinii can replace the transcriptional functions of S. pombe TFIIB, although the cells expressing the P. carini TFIIB show an altered phenotype. However, performing studies using a heterologous approach, like this one, could be relevant to understanding the basic molecular processes of Pneumocystis such as transcription and replication.
Assuntos
Pneumocystis carinii , Pneumocystis , Pneumonia por Pneumocystis , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Humanos , Pneumocystis/genética , Pneumocystis/metabolismo , Pneumocystis carinii/genética , Pneumocystis carinii/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Fator de Transcrição TFIIB , Transcrição GênicaRESUMO
Pneumocystis jirovecii can cause severe potentially life-threatening pneumonia (PCP) in kidney transplant patients. Prophylaxis of patients against PCP in this setting is usually performed during 6 months after transplantation. The aim of this study is to describe the molecular epidemiology of a cluster of PCP in renal transplant recipients in Brazil. Renal transplant patients who developed PCP between May and December 2011 had their formalin-fixed paraffin-embedded (FFPE) lung biopsy samples analysed. Pneumocystis jirovecii 23S mitochondrial large subunit of ribosomal RNA (23S mtLSU-rRNA), 26S rRNA, and dihydropteroate synthase (DHPS) genes were amplified by polymerase chain reaction (PCR), sequenced, and analysed for genetic variation. During the study period, 17 patients developed PCP (only four infections were documented within the first year after transplantation) and six (35.3%) died. Thirty FFPE samples from 11 patients, including one external control HIV-infected patient, had fungal DNA successfully extracted for further amplification and sequencing for all three genes. A total of five genotypes were identified among the 10 infected patients. Of note, four patients were infected by more than one genotype and seven patients were infected by the same genotype. DNA extracted from FFPE samples can be used for genotyping; this approach allowed us to demonstrate that multiple P. jirovecii strains were responsible for this cluster, and one genotype was found infecting seven patients. The knowledge of the causative agents of PCP may help to develop new initiatives for control and prevention of PCP among patients undergoing renal transplant and improve routine PCP prophylaxis.
Assuntos
Variação Genética , Transplante de Rim/efeitos adversos , Pneumocystis/isolamento & purificação , Pneumonia por Pneumocystis/microbiologia , Complicações Pós-Operatórias/microbiologia , Adulto , Brasil , Estudos Transversais , DNA Fúngico/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Pneumocystis/classificação , Pneumocystis/genética , Pneumonia por Pneumocystis/diagnóstico , Complicações Pós-Operatórias/diagnóstico , Estudos Retrospectivos , Subunidades Ribossômicas Maiores/genética , Adulto JovemRESUMO
BACKGROUND: Despite antiretroviral therapy and trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis, Pneumocystis pneumonia remains an important serious opportunistic infection in HIV-infected persons. Pneumocystis (Pc) colonization in HIV-infected individuals and in HIV-uninfected smokers is associated with chronic obstructive pulmonary disease (COPD). We previously developed a nonhuman primate model of HIV infection and Pc colonization and demonstrated that Pc colonization correlated with COPD development. In the present study, we examined kinetics of COPD development in non-human primate and tested the effect of Pc burden reduction on pulmonary function by TMP-SMX treatment. METHODS: Cynomolgus macaques (n = 16) were infected with simian/human immunodeficiency virus (SHIV89.6P), and natural Pc colonization was examined by nested polymerase chain reaction of serial bronchoalveolar lavage fluid and anti-Pc serology. RESULTS: Eleven of 16 monkeys became Pc colonized by 16 weeks post simian-human immunodeficiency virus (SHIV) infection. Pc colonization of SHIV-infected monkeys led to progressive declines in pulmonary function as early as 4 weeks after Pc detection. SHIV-infected and Pc-negative monkeys maintained normal lung function. At 25 weeks post-SHIV infection, TMP-SMX treatment was initiated in 7 Pc-positive (Pc+) (TMP: 20 mg/kg and SMX: 100 mg/kg, daily for 48 weeks) and 5 Pc-negative (Pc-) monkeys. Four SHIV+/Pc+ remained untreated for the duration of the experiment. Detection frequency of Pc in serial bronchoalveolar lavage fluid (P < 0.001), as well as plasma Pc antibody titers (P = 0.02) were significantly reduced in TMP-SMX-treated macaques compared with untreated. CONCLUSIONS: Reduction of Pc colonization by TMP-SMX treatment did not improve pulmonary function, supporting the concept that Pc colonization results in early, permanent obstructive changes in the lungs of immunosuppressed macaques.
Assuntos
Anti-Infecciosos/uso terapêutico , Pneumopatias Obstrutivas/tratamento farmacológico , Infecções por Pneumocystis/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/complicações , Combinação Trimetoprima e Sulfametoxazol/uso terapêutico , Animais , Anticorpos Antifúngicos/sangue , Líquido da Lavagem Broncoalveolar/microbiologia , Pneumopatias Obstrutivas/complicações , Pneumopatias Obstrutivas/patologia , Macaca , Pneumocystis/genética , Pneumocystis/isolamento & purificação , Infecções por Pneumocystis/complicações , Infecções por Pneumocystis/patologia , Reação em Cadeia da Polimerase , Resultado do TratamentoRESUMO
Molecular techniques have revealed a high prevalence of Pneumocystis colonization in wild mammals. Accurate quantification of Pneumocystis sp. is essential for the correct interpretation of many research experiments investigating this organism. The objectives of this study were to detect the presence of Pneumocystis sp. in bats by qPCR, and to distinguish colonization from infection. Probes and primers for real time PCR (qPCR) were designed based on the gene of major surface glycoprotein (MSG) of Pneumocystis sp., in order to analyze 195 lung tissue samples from bats captured (2007-2009). All samples were also analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit rRNA (mtSSU rRNA) to confirm the results. The qPCR assay was standardized using a standard curve made with the DNA extracted from bronchoalveolar lavage positive for Pneumocystis jirovecii. The average Ct was found to be between 13 and 14 (calibration curve) for the detection of infection with Pneumocystis sp. and above these values for colonization. It was considered as negative samples the ones that had Ct values equal to 50. Out of the total 195 samples, 47 (24.1%) bat lung DNA samples were positive for Pneumocystis sp. by qPCR. The most common bat species found were: Tadarida brasiliensis (23.4%), Histiotus velatus (17.0%), Desmodus rotundus (14.9%) and Molossus molossus (8.5%). The average cycle threshold of the positive samples (bats) was 25.8 and standard deviation was 1.7. The DNA samples with Ct values greater than 14 suggest that these animals might be colonized by Pneumocystis sp. Results obtained in this study demonstrated the usefulness of the qPCR procedure for identification of Pneumocystis sp. and for distinction between its colonizing or infectious status in bats.
Assuntos
Portador Sadio/veterinária , Quirópteros/microbiologia , Reservatórios de Doenças/microbiologia , Infecções por Pneumocystis/transmissão , Pneumocystis/isolamento & purificação , Animais , Brasil , Líquido da Lavagem Broncoalveolar/microbiologia , Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Quirópteros/classificação , DNA Fúngico/análise , DNA Fúngico/genética , Proteínas Fúngicas/genética , Especificidade de Hospedeiro , Pulmão/microbiologia , Glicoproteínas de Membrana/genética , Pneumocystis/genética , Pneumocystis carinii/genética , Pneumocystis carinii/isolamento & purificação , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Ribotipagem , Especificidade da EspécieRESUMO
BACKGROUND: Pneumocystis without obvious accompanying pathology is occasionally reported in autopsied infant lungs. Its prevalence and significance are unknown. Interestingly, this mild infection induces a strong activation of mucus secretion-related genes in young immunocompetent rodents that has not been explored in infants. Excess mucus is induced by multiple airway offenders through nonspecific pathways and would explain a cofactor role of Pneumocystis in respiratory disease. We undertook characterization of the prevalence of Pneumocystis and associated mucus in infant lungs. METHODS: Samples from 128 infants (mean age, 101 days) who died suddenly and unexpectedly in Santiago during 1999-2004 were examined for Pneumocystis using nested polymerase chain reaction (nPCR) amplification of the P. jirovecii mtLSU ribosomal RNA gene and immunofluorescence microscopy (IF). Pneumocystis-negative infants 28 days and older and their age-closest positives were studied for MUC5AC expression and Pneumocystis burden by Western blot and quantitative PCR, respectively. RESULTS: Pneumocystis DNA was detected by nPCR in 105 of the 128 infants (82.0%) and Pneumocystis organisms were visualized by IF in 99 (94.3%) of the DNA-positive infants. The infection was commonest at 3-4 months with 40 of 41 (97.6%) infants of that age testing positive. MUC5AC was significantly increased in Pneumocystis-positive tissue specimens (P = .013). Death was unexplained in 113 (88.3%) infants; Pneumocystis was detected in 95 (84.0%) of them vs 10 of 15 (66.7%) with explained death (P = .28). CONCLUSIONS: A highly focal Pneumocystis infection associated to increased mucus expression is almost universally present in the lungs of infants dying unexpectedly in the community regardless of autopsy diagnosis.
Assuntos
Pulmão/metabolismo , Muco/metabolismo , Pneumocystis/isolamento & purificação , Pneumonia por Pneumocystis/epidemiologia , Morte Súbita do Lactente/epidemiologia , Autopsia , Contagem de Colônia Microbiana , DNA Fúngico/genética , Feminino , Humanos , Lactente , Recém-Nascido , Pulmão/microbiologia , Pulmão/patologia , Masculino , Microscopia , Mucina-5AC/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Pneumocystis/genética , Pneumonia por Pneumocystis/diagnóstico , Pneumonia por Pneumocystis/microbiologia , Prevalência , Sensibilidade e Especificidade , Morte Súbita do Lactente/diagnósticoRESUMO
First recognised as 'schizonts' of Trypanosoma cruzi, Pneumocystis organisms are now considered as part of an early-diverging lineage of Ascomycetes. As no robust long-term culture model is available, most data on the Pneumocystis cell cycle have stemmed from ultrastructural images of infected mammalian lungs. Although most fungi developing in animals do not complete a sexual cycle in vivo, Pneumocystis species constitute one of a few exceptions. Recently, the molecular identification of several key players in the fungal mating pathway has provided further evidence for the existence of conjugation and meiosis in Pneumocystisorganisms. Dynamic follow-up of stage-to-stage transition as well as studies of stage-specific proteins and/or genes would provide a better understanding of the still hypothetical Pneumocystislife cycle. Although difficult to achieve, stage purification seems a reasonable way forward in the absence of efficient culture systems. This mini-review provides a comprehensive overview of the historical milestones leading to the current knowledge available on the Pneumocystis life cycle.
Assuntos
Ciclo Celular/fisiologia , Genes Fúngicos Tipo Acasalamento/fisiologia , Estágios do Ciclo de Vida/fisiologia , Pneumocystis/crescimento & desenvolvimento , Animais , Ciclo Celular/genética , Genes Fúngicos Tipo Acasalamento/genética , Microscopia Eletrônica de Transmissão , Pneumocystis/genética , Pneumocystis/ultraestruturaRESUMO
First recognised as "schizonts" of Trypanosoma cruzi, Pneumocystis organisms are now considered as part of an early-diverging lineage of Ascomycetes. As no robust long-term culture model is available, most data on the Pneumocystis cell cycle have stemmed from ultrastructural images of infected mammalian lungs. Although most fungi developing in animals do not complete a sexual cycle in vivo, Pneumocystis species constitute one of a few exceptions. Recently, the molecular identification of several key players in the fungal mating pathway has provided further evidence for the existence of conjugation and meiosis in Pneumocystisorganisms. Dynamic follow-up of stage-to-stage transition as well as studies of stage-specific proteins and/or genes would provide a better understanding of the still hypothetical Pneumocystislife cycle. Although difficult to achieve, stage purification seems a reasonable way forward in the absence of efficient culture systems. This mini-review provides a comprehensive overview of the historical milestones leading to the current knowledge available on the Pneumocystis life cycle.
Assuntos
Animais , Ciclo Celular/fisiologia , Genes Fúngicos Tipo Acasalamento/fisiologia , Estágios do Ciclo de Vida/fisiologia , Pneumocystis/crescimento & desenvolvimento , Ciclo Celular/genética , Genes Fúngicos Tipo Acasalamento/genética , Microscopia Eletrônica de Transmissão , Pneumocystis/genética , Pneumocystis/ultraestruturaRESUMO
Parasites are increasingly used to complement the evolutionary and ecological adaptation history of their hosts. Pneumocystis pathogenic fungi, which are transmitted from host-to-host via an airborne route, have been shown to constitute genuine host markers of evolution. These parasites can also provide valuable information about their host ecology. Here, we suggest that parasites can be used as phylogeographic markers to understand the geographical distribution of intra-specific host genetic variants. To test our hypothesis, we characterised Pneumocystis isolates from wild bats living in different areas. Bats comprise a wide variety of species; some of them are able to migrate. Thus, bat chorology and migration behaviour can be approached using Pneumocystis as phylogeographic markers. In the present work, we find that the genetic polymorphisms of bat-derived Pneumocystis are structured by host chorology. Therefore, Pneumocystis intra-specific genetic diversity may constitute a useful and relevant phylogeographic tool.
Assuntos
Quirópteros/microbiologia , Variação Genética , Geografia , Pneumocystis/genética , Animais , Argentina , Quirópteros/classificação , França , Guiana Francesa , México , Filogenia , Pneumocystis/classificação , Pneumocystis/isolamento & purificação , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Parasites are increasingly used to complement the evolutionary and ecological adaptation history of their hosts. Pneumocystis pathogenic fungi, which are transmitted from host-to-host via an airborne route, have been shown to constitute genuine host markers of evolution. These parasites can also provide valuable information about their host ecology. Here, we suggest that parasites can be used as phylogeographic markers to understand the geographical distribution of intra-specific host genetic variants. To test our hypothesis, we characterised Pneumocystis isolates from wild bats living in different areas. Bats comprise a wide variety of species; some of them are able to migrate. Thus, bat chorology and migration behaviour can be approached using Pneumocystis as phylogeographic markers. In the present work, we find that the genetic polymorphisms of bat-derived Pneumocystis are structured by host chorology. Therefore, Pneumocystis intra-specific genetic diversity may constitute a useful and relevant phylogeographic tool.
Assuntos
Animais , Quirópteros/microbiologia , Variação Genética , Geografia , Pneumocystis/genética , Argentina , Quirópteros/classificação , França , Guiana Francesa , México , Filogenia , Pneumocystis/classificação , Pneumocystis/isolamento & purificação , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Pneumocystis sp. is transmitted through the airborne route and presents a high host-species-specificity. Occasional reports of Pneumocystis pneumonia in still births and newborn infants suggest that other routes of transmission, e.g. transplacental might occur. The latter has been reported in rabbits but available data indicate that transplacental transmission of Pneumocystis seems not to occur in corticosteroid-treated rats and in SCID mice. The present study was undertaken to evaluate transplacental transmission of Pneumocystis oryctolagi. The spontaneously-acquired pneumocystosis rabbit model using hybrid California/New Zealand white female rabbits was selected because of similarities among rabbit and human placentas. Three different experiments were conducted in France and Chile. Pneumocystis organisms were detected by microscopy in the lungs of pregnant does and Pneumocystis DNA was found in the lungs of fetuses from the multiparous does from the second week to the end of gestation. Pneumocystis DNA was not detected in fetuses from primiparous does. Detection of Pneumocystis oryctolagi--DNA in fetuses of multiparous does and not in those of primiparous ones, suggests that transplacental transmission may be favored by multiple gestations. Whether Pneumocystis-DNA in fetal tissues from multiparous does resulted from transplacental passage of viable transmissible forms requires further investigation.