RESUMO
Quinupristin-dalfopristin (Q-D), which is active against bacteria and Toxoplasma gondii, was examined for its activity against Pneumocystis carinii. After 72 h of incubation with rat P. carinii in an ATP cytotoxicity assay, the 50% inhibitory concentration of Q-D was 10.6 microg/ml, a level that can be achieved in serum with high-dose administration. Q-D administered intraperitoneally at doses of 50 to 200 mg per kg of body weight per day in the treatment and 100 mg/kg/day three times per week in the prophylaxis of pneumocystosis in immunosuppressed mice reduced the organism burden up to 15- and 302-fold, respectively. We conclude that Q-D has activity against P. carinii in vitro and in vivo.
Assuntos
Antibacterianos/uso terapêutico , Infecções por Pneumocystis/tratamento farmacológico , Pneumocystis/efeitos dos fármacos , Virginiamicina/análogos & derivados , Virginiamicina/uso terapêutico , Trifosfato de Adenosina/metabolismo , Animais , Antibacterianos/administração & dosagem , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos C3H , Testes de Sensibilidade Microbiana , Infecções por Pneumocystis/microbiologia , Virginiamicina/administração & dosagemRESUMO
Although the clinical aspects of Pneumocystis carinii pneumonia are well characterized, the basic biology of the causative organism is poorly understood. Most proposed life cycles of P. carinii include both asexual and sexual replicative cycles. The two most prominent morphological forms are a trophic form, thought to undergo asexual replication by binary fission, and a cystic form or ascus containing intracystic bodies or ascospores, the products of sexual replication. To facilitate the Pneumocystis genome project, a P. carinii f. sp. carinii genomic cosmid library and an additional lambda cDNA library were generated. A partial expressed sequence tag database, created as part of the genome project, revealed the transcription of meiosis-specific genes and other genes related to sexual reproduction. The ortholog of Ste3, an a-factor pheromone receptor, was cloned and genes surrounding the ste3 locus were examined. Clustered around the ste3 gene are genes encoding elements functional in the pheromone response signal transduction cascade of model fungal organisms. These include the Ste20 protein kinase, the Ste12 homoeodomain transcriptional regulator, a potential pheromone mating factor, and other DNA-binding proteins. The genomic organization of the ste3 locus bears significant similarity to that of the mating locus recently described in Cryptococcus neoformans. The P. carinii genome contains much of the genetic machinery necessary for pheromone responsiveness, and these data support the existence of a sexual replication cycle.
Assuntos
Genoma Fúngico , Família Multigênica , Feromônios/genética , Pneumocystis/genética , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Receptores de Feromônios , Reprodução/genética , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais/genética , Sequência de Aminoácidos , Southern Blotting , Clonagem Molecular , Cosmídeos/genética , DNA Complementar/metabolismo , Etiquetas de Sequências Expressas , Biblioteca Gênica , Modelos Genéticos , Dados de Sequência Molecular , Receptores de Fator de Acasalamento , Homologia de Sequência de AminoácidosRESUMO
The prevalence of Pneumocystis carinii pneumonia (PCP) in humans caused by more than a single genotype has been reported to range from 10 to 67%, depending on the method used for detection (3, 19). Most coinfections were associated with primary rather than recurrent disease. To better understand the factors influencing the development of coinfections, the time periods between inoculations and the genotype of the infecting organisms were evaluated in the chronically immunosuppressed-inoculated rat model of PCP. P. carinii f. sp. carinii infecting rats differentiated by karyotypic profiles exhibit the same low level of genetic divergence manifested by organisms infecting humans. P. carinii f. sp. carinii karyotype forms 1, 2, and 6 were inoculated into immunosuppressed rats, individually and in dual combinations, spaced 0, 10, and 20 days apart. Infections comprised of both organism forms resulted from admixtures inoculated at the same time. In contrast, coinfections did not develop in most rats, where a 10- or 20-day gap was inserted between inoculations; only the first organism form inoculated was detected by pulsed-field gel electrophoresis in the resultant infection. Organism burdens were reduced with combinations of forms 1 and 2 spaced 20 days apart but not in rats inoculated with forms 1 and 6. A role for the host response in the elimination of the second population and in reduction of the organism burden was suggested by the lack of direct killing of forms 1 and 2 in an in vitro ATP assay, by reduction of the burden by autoclaved organisms, and by the specific reactions of forms 1 and 2 but not forms 1 and 6. These studies showed that the time between inoculations was critical in establishing coinfections and P. carinii f. sp. carinii karyotype profiles were associated with differences in biological responses. This model provides a useful method for the study of P. carinii coinfections and their transmission in humans.
Assuntos
Infecções por Pneumocystis/etiologia , Pneumocystis/genética , Trifosfato de Adenosina/análise , Animais , Sequência de Bases , Humanos , Cariotipagem , Pulmão/microbiologia , Masculino , Dados de Sequência Molecular , Infecções por Pneumocystis/microbiologia , Reação em Cadeia da Polimerase , Ratos , Fatores de TempoAssuntos
Antifúngicos/farmacologia , Criopreservação/métodos , Pentamidina/farmacologia , Pneumocystis/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Contagem de Colônia Microbiana , Criopreservação/normas , Meios de Cultura , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Pneumocystis/crescimento & desenvolvimento , Pneumocystis/isolamento & purificação , Ratos , Ratos Long-EvansRESUMO
Pneumocystis carinii synthesizes sterols with a double bond at C-7 of the sterol nucleus and an alkyl group with one or two carbons at C-24 of the side chain. Also, some human-derived Pneumocystis carinii f. sp. hominis strains contain lanosterol derivatives with an alkyl group at C-24. These unique sterols have not been found in other pathogens of mammalian lungs. Thus, P. carinii may have important differences in its susceptibility to drugs known to block reactions in ergosterol biosynthesis in other fungi. In the present study, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, squalene synthase, squalene epoxidase, squalene epoxide-lanosterol cyclase, lanosterol demethylase, Delta(8) to Delta(7) isomerase, and S-adenosylmethionine:sterol methyltransferase were tested for their effects on P. carinii viability as determined by quantitation of cellular ATP levels in a population of organisms. Compounds within each category varied in inhibitory effect; the most effective included drugs targeted at squalene synthase, squalene epoxide-lanosterol cyclase, and Delta(8) to Delta(7) isomerase. Some drugs that are potent against ergosterol-synthesizing fungi had little effect against P. carinii, suggesting that substrates and/or enzymes in P. carinii sterol biosynthetic reactions are distinct. Amphotericin B is ineffective in clearing P. carinii infections at clinical doses; however, this drug apparently binds to sterols and causes permeability changes in P. carinii membranes, since it reduced cellular ATP levels in a dose-dependent fashion.
Assuntos
Anfotericina B/antagonistas & inibidores , Anfotericina B/metabolismo , Inibidores Enzimáticos/farmacologia , Lanosterol/antagonistas & inibidores , Pneumocystis/crescimento & desenvolvimento , Pneumocystis/metabolismo , Esteróis/antagonistas & inibidores , Esteróis/biossíntese , Trifosfato de Adenosina/metabolismo , Humanos , Lanosterol/biossínteseRESUMO
Atovaquone (also called Mepron, or 566C80) is a napthoquinone used for the treatment of infections caused by pathogens such as Plasmodium spp. and Pneumocystis carinii. The mechanism of action against the malarial parasite is the inhibition of dihydroorotate dehydrogenase (DHOD), a consequence of blocking electron transport by the drug. As an analog of ubiquinone (coenzyme Q [CoQ]), atovaquone irreversibly binds to the mitochondrial cytochrome bc(1) complex; thus, electrons are not able to pass from dehydrogenase enzymes via CoQ to cytochrome c. Since DHOD is a critical enzyme in pyrimidine biosynthesis, and because the parasite cannot scavenge host pyrimidines, the drug is lethal to the organism. Oxygen consumption in P. carinii is inhibited by the drug; thus, electron transport has also been identified as the drug target in P. carinii. However, unlike Plasmodium DHOD, P. carinii DHOD is inhibited only at high atovaquone concentrations, suggesting that the organism may salvage host pyrimidines and that atovaquone exerts its primary effects on ATP biosynthesis. In the present study, the effect of atovaquone on ATP levels in P. carinii was measured directly from 1 to 6 h and then after 24, 48, and 72 h of exposure. The average 50% inhibitory concentration after 24 to 72 h of exposure was 1.5 microgram/ml (4.2 microM). The kinetics of ATP depletion were in contrast to those of another family of naphthoquinone compounds, diospyrin and two of its derivatives. Whereas atovaquone reduced ATP levels within 1 h of exposure, the diospyrins required at least 48 h. After 72 h, the diospyrins were able to decrease ATP levels of P. carinii at nanomolar concentrations. These data indicate that although naphthoquinones inhibit the electron transport chain, the molecular targets in a given organism are likely to be distinct among members of this class of compounds.
Assuntos
Trifosfato de Adenosina/metabolismo , Antifúngicos/farmacologia , Naftoquinonas/farmacologia , Pneumocystis/efeitos dos fármacos , Animais , Atovaquona , Relação Dose-Resposta a Droga , Masculino , Pentamidina/farmacologia , Pneumocystis/metabolismo , Infecções por Pneumocystis/microbiologia , Ratos , Ratos Endogâmicos BN , Ratos Long-EvansAssuntos
Trifosfato de Adenosina/metabolismo , Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , Ergosterol/biossíntese , Pneumocystis/efeitos dos fármacos , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Oxigenases/antagonistas & inibidores , Pneumocystis/crescimento & desenvolvimento , Pneumocystis/metabolismo , Esqualeno Mono-OxigenaseRESUMO
Histopathologic evaluation combined with a period of immunosuppression has been the standard procedure for detection of Pneumocystis carinii in commercial rat colonies. Variation in induction regimens and in the sensitivity of detection methods may result in underreporting of the presence of P. carinii in breeding colonies or delay its detection. In the present study, methylprednisolone and cyclophosphamide were evaluated for the ability to induce P. carinii infection in rats from an enzootically infected commercial barrier colony. The presence of P. carinii was detected by histopathologic methods and by amplification of a targeted region of the P. carinii thymidylate synthase gene by PCR over the 8-week study period. Sera taken from rats prior to either induction regimen were evaluated for the presence of P. carinii-specific antibodies by the immunoblotting technique. Few significant differences in ability to induce organism burden or in histopathology were observed between the two immunosuppressive regimens. However, a dramatic loss of weight over the study period was observed in rats treated with methylprednisolone but not in rats treated with cyclophosphamide. Although histopathologic changes attributable to P. carinii did not appear before 2 weeks with either immunosuppressant, the presence of the organism in these animals was detected by immunoblotting and PCR. Cyst scores and the intensities of the histopathologic lesions increased during the study period, but the number of rats exhibiting evidence of P. carinii infection did not change after week 3. These results suggest that use of the PCR method on postmortem lung tissue of rats without prior induction regimens or identification of anti-P. carinii antibodies in antemortem serum samples is a sufficiently sensitive method for detection of the presence of a P. carinii carrier state in rodent breeding colonies.
Assuntos
Imunossupressores/farmacologia , Infecções por Pneumocystis/diagnóstico , Animais , Pulmão/patologia , Masculino , Metilprednisolona/farmacologia , Infecções por Pneumocystis/patologia , Reação em Cadeia da Polimerase , Ratos , Ratos Endogâmicos F344 , Testes SorológicosRESUMO
A series of over 60 agents representing several different classes of compounds were evaluated for their effects on the ATP pools of Pneumocystis carinii populations derived from immunosuppressed rats. A cytotoxicity assay based on an ATP-driven bioluminescent reaction was used to determine the concentration of agent which decreased the P. carinii ATP pools by 50% versus untreated controls (IC50). A ranking system based on the IC50 value was devised for comparison of relative responses among the compounds evaluated in the cytotoxic assay and for comparison to in vivo efficacy. With few exceptions, there was a strong correlation between results from the ATP assay and the performance of the compound in vivo. Antibiotics, with the exception of trimethoprim-sulfamethoxazole (TMP-SMX), were ineffective at reducing the ATP pools and were not active clinically or in the rat model of P. carinii pneumonia. Likewise, other agents not expected to be effective, e.g., antiviral compounds, did not show activity. Standard anti-P. carinii compounds, e.g., TMP-SMX, pentamidine, and dapsone, dramatically reduced ATP levels. Analogs of the quinone and topoisomerase inhibitor groups were shown to reduce ATP concentrations and hold promise for further in vivo investigation. The cytotoxicity assay provides a rapid assessment of response, does not rely on replicating organisms, and should be useful for assessment of structure-function relationships.
Assuntos
Antifúngicos/farmacologia , Pneumocystis/efeitos dos fármacos , Trifosfato de Adenosina/análise , Animais , Bactérias , Avaliação Pré-Clínica de Medicamentos , Técnicas In Vitro , Medições Luminescentes , Pneumocystis/metabolismo , RatosRESUMO
Several pathogenic fungi and protozoa are known to have sterols distinct from those of their mammalian hosts. Of particular interest as targets for drug development are the biosyntheses of the sterols of important parasites such as the kinetoplastid flagellates and the AIDS-associated opportunistic protist Pneumocystis carinii. These pathogens synthesize sterols with an alkyl group at C-24, and some have a double bond at C-22 of the side chain. Humans and other mammalian hosts are incapable of C-24 alkylation and C-22 desaturation. In the present study, three steroidal compounds with side chains substituted by phosphonyl-linked groups were synthesized and tested for their effects on Leishmania donovani and L. mexicana mexicana culture growth. The compounds inhibited organism proliferation at concentrations in micrograms per milliliter. The most potent inhibitors of this group of compounds were characterized by two ethyl groups at the phosphate function. Leishmania organisms treated with 17-[2-(diethylphosphonato) ethylidienyl]3-methoxy-19-norpregna-1,3,5-triene exhibited reduced growth after transfer into inhibitor-free medium. Because there are currently no axenic methods available for the continuous subcultivation of P. carinii, the effects of these drugs on this organism were evaluated by two alternative screening methods. The same two diethyl phosphonosteroid compounds that inhibited Leishmania proliferation were also the most active against P. carinii as determined by the potent effect they had on reducing cellular ATP content. Cystic as well as trophic forms responded to the drug treatments, as evaluated by a dual fluorescent staining live-dead assay. Other modifications of steroidal phosphonates may lead to the development of related drugs with increased activity and specificity for the pathogens.
Assuntos
Leishmania donovani/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Organofosfonatos/farmacologia , Pneumocystis/efeitos dos fármacos , Esteroides/farmacologia , Trifosfato de Adenosina/análise , Animais , Contagem de Colônia Microbiana , Ésteres/farmacologiaRESUMO
Pneumocystis carinii is a family of organisms found in a wide variety of mammalian lungs. In immunocompromised hosts, the organisms are able to produce an oftentimes fatal pneumonia. The existence of distinct types of Pneumocystis populations is strongly supported by antigenic and genetic evidence. In the present study, we assessed the antigenic profiles of two genetically distinct Pneumocystis carinii populations, P. carinii f. sp. carinii and P. carinii f. sp. ratti, as well as two types of P. carinii f. sp. carinii defined by electrophoretic karyotyping (forms 1 and 2). The separated and blotted proteins of the organism preparations were probed with four monoclonal antibodies (MAbs) generated to the major surface glycoproteins of rat-derived P. carinii, one anti-human P. carinii MAb, and two polyclonal antisera made with rat-derived P. carinii as the immunogen. Differences in reactivities between the P. carinii f. sp. carinii and P. carinii f. sp. ratti preparations were detected with two of the MAbs, and both of the rat P. carinii polyclonal antisera in the 45- to 55-kDa molecular mass range, but not with the human P. carinii MAb. The reactivities of the 16 P. carinii f. sp. carinii preparations were the same with two exceptions. Two preparations of form 1 showed strong reactivity with the anti-MSG MAb RA-C11. The ratios of cyst forms to trophic forms evaluated by microscopy were not associated with any of the differences observed in the antigenic profiles. The antigenic differences between P. carinii f. sp. carinii and P. carinii f. sp. ratti are consistent with the distinction of these two populations made by molecular genetic techniques, while the two differences detected among the P. carinii f. sp. carinii preparations suggest the organism may be able to modulate antigenic epitopes. The use of immunoblotting to differentiate infecting organism populations and assess antigenic modulation holds promise for future epidemiologic studies.
Assuntos
Antígenos de Fungos/imunologia , Pneumocystis/genética , Pneumocystis/imunologia , Ratos Sprague-Dawley/microbiologia , Síndrome da Imunodeficiência Adquirida/microbiologia , Animais , Anticorpos Antifúngicos , Anticorpos Monoclonais , Southern Blotting , Diferenciação Celular , Contagem de Colônia Microbiana , Proteínas Fúngicas/imunologia , Humanos , Immunoblotting , Hospedeiro Imunocomprometido , Glicoproteínas de Membrana/imunologia , Pneumocystis/classificação , Ratos , Especificidade da EspécieRESUMO
The use of semiquantitative PCR (SQPCR) to assess Pneumocystis carinii pneumonia (PCP) infection and its response to treatment was studied with rats. Groups of eight rats were immunosuppressed with steroids for 3 to 12 weeks. Untreated controls were maintained for the same periods. Three groups of rats were treated with pentamidine, three groups were treated with trimethoprim-sulfamethoxazole, and three groups of rats were tapered from steroids. At various times during suppression, rats from the different groups were sacrificed. At necropsy, lungs were lavaged to obtain bronchoalveolar fluids and then homogenized. Bronchoalveolar fluids and homogenates were assayed by cyst counting and SQPCR. An increase in the SQPCR signal was seen throughout immunosuppression, with a slow decrease upon the withdrawal of steroids and a faster decrease with drug treatment. SQPCR results with lung homogenates and bronchoalveolar fluids strongly correlated with each other and with cyst counts. These results warrant investigation of SQPCR for assessing treatment results of human P. carinii pneumonia infection.
Assuntos
Infecções por Pneumocystis/tratamento farmacológico , Infecções por Pneumocystis/microbiologia , Pneumocystis/isolamento & purificação , Reação em Cadeia da Polimerase , Animais , Sequência de Bases , Líquido da Lavagem Broncoalveolar/microbiologia , DNA Fúngico/análise , Terapia de Imunossupressão , Dados de Sequência Molecular , Pentamidina/uso terapêutico , Pneumocystis/efeitos dos fármacos , Pneumocystis/genética , Infecções por Pneumocystis/diagnóstico , Ratos , Ratos Sprague-Dawley , Combinação Trimetoprima e Sulfametoxazol/uso terapêuticoRESUMO
A bioluminescent assay which employs the luciferin-luciferase ATP-dependent reaction was used to evaluate the viability of populations of Pneumocystis carinii derived from infected rat lungs. Contamination with host cells was reduced by a purification method which involved a combination of low- and high-speed centrifugations resulting in a 1,000-fold reduction of the rat cells while enriching for the trophic form of P. carinii. A linear correlation for the number of P. carinii nuclei versus the amount of ATP was observed. The ATP content of the organism populations could be maintained at inoculum levels for one week, although the number of organisms did not increase. Addition of respiratory chain inhibitors dramatically decreased the ATP content of the P. carinii after 24 h of incubation, with the exception of the antibiotic oligomycin B. Low concentrations of trimethoprim-sulfamethoxazole and pentamidine isethionate reduced the organism ATP content by over 50% after 24 h of exposure, while no effect was observed with 100-fold greater concentrations of ampicillin. The bioluminescent assay was found to be a more sensitive indicator of viability than a dual fluorescent staining technique. This assay does not require replication of P. carinii and should be a useful method for in vitro drug screening and viability assessment of P. carinii populations.
Assuntos
Trifosfato de Adenosina/análise , Bactérias , Medições Luminescentes , Pneumocystis/fisiologia , Animais , Antimicina A/farmacologia , Meios de Cultura , Masculino , Pneumocystis/isolamento & purificação , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVES: We examined 96 bronchoalveolar lavage fluid (BALF) specimens from AIDS patients with proven Pneumocystis carinii pneumonia (PCP) in order to compare the relationship of organism burden, viability and antigen expression with disease severity at the time of clinical presentation. METHODS: Tinctorial analysis of BALF specimens with proven PCP using Diff-Quik, cresyl echt violet and erythrosin B stains to evaluate organism burden and viability. P. carinii antigen examination was performed by Western blot analysis. RESULTS: P. carinii cluster ratios were more sensitive than cyst counts as an indicator of organism burden, and correlated well with the alveolar-arterial oxygen gradient as a measure of disease severity. Erythrosin B, the vital stain used to measure P. carinii viability, displayed a wide range of values and provided little useful information. Antigens of 35-45 and 95kD, which were specific for P. carinii, were found by immunoblot analysis in BALF cellular fraction of most patients with pneumocystosis. By contrast, antigens of 52 and 66 kD, which were found in both BALF supernatant and cellular fractions of P. carinii patients and controls, most likely represented albumin and immunoglobulin G heavy chain, respectively, of host origin. The 35-45 kD antigen was found in 88% of the BALF specimens and appeared to represent an important marker of P. carinii infection. The 95 kD antigen was detected in 49% of the specimens. CONCLUSIONS: We conclude that analysis of P. carinii characteristics in BALF specimens of patients with pneumocystis may provide additional information. These data will also be helpful in developing more sensitive assays and in targeting specific P. carinii factors for future investigation.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/fisiopatologia , Antígenos de Fungos/análise , Líquido da Lavagem Broncoalveolar/microbiologia , Infecções por HIV/fisiopatologia , Pneumocystis/isolamento & purificação , Pneumonia por Pneumocystis/fisiopatologia , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Western Blotting , Eritrosina , Infecções por HIV/microbiologia , Humanos , Imunoglobulina G/análise , Cadeias Pesadas de Imunoglobulinas/análise , Peso Molecular , Pneumonia por Pneumocystis/microbiologia , Valores de Referência , Coloração e RotulagemRESUMO
One hundred eighty-three toluidine blue O-stained necropsy lung imprint smears from different avian species were examined microscopically for Pneumocystis carinii. No cyst forms of the organism could be identified. Seventy-eight serum samples from a total of 155 chickens were examined by a competition enzyme-linked immunosorbent assay (ELISA) for antibodies to P. carinii; 53 serum samples were from individual chickens, and 25 samples were pools of sera from two to five chickens. Diluted 1:50, the 78 serum samples showed a specific ELISA-inhibition of 4% to 56% (the 95% confidence limit being 25% to 30% inhibition). Diluted 1:50, nine serum pools representing 34 chickens and 17 of the 53 individual serum samples (32.1%) showed an inhibition greater than 30%. No specific pneumocyst DNA could be detected in serum from 13 of the 53 chickens using polymerase chain reaction and dihydrofolate reductase gene as a specific probe. Specific antibodies to a 116,000-molecular-weight antigen of rat pneumocysts were shown in two (13.3%) of 15 individual chicken serum samples. The results indicate that P. carinii organisms do not commonly reside in the lungs of birds, although some birds may be exposed to external sources of organisms.
Assuntos
Aves/microbiologia , Pulmão/microbiologia , Pneumocystis/isolamento & purificação , Aves Domésticas/microbiologia , Animais , Sequência de Bases , Southern Blotting , Galinhas/microbiologia , Columbidae/microbiologia , DNA Fúngico/análise , Dinamarca , Patos/microbiologia , Gansos/microbiologia , Amplificação de Genes , Genes Fúngicos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Pneumocystis/enzimologia , Pneumocystis/genética , Codorniz/microbiologia , Ratos , Ratos Sprague-Dawley , Tetra-Hidrofolato Desidrogenase/genética , Perus/microbiologiaRESUMO
Immunomagnetic sorting, sequential filtrations, and counterflow centrifugal elutriation were compared for their ability to obtain enriched populations of Pneumocystis carinii developmental stages from infected rat-lung homogenates. Elutriation combined with sequential filtrations resulted in highly (> 95%) enriched populations of P. carinii cysts and trophozoites with excellent viability. This approach offers advantages over previously described methods of obtaining enriched P. carinii cell populations and should have important applications to research on this organism.
Assuntos
Pneumocystis/crescimento & desenvolvimento , Pneumocystis/isolamento & purificação , Pneumonia por Pneumocystis/microbiologia , Animais , Centrifugação , Filtração , Pulmão/microbiologia , Magnetismo , Masculino , Micologia/métodos , Ratos , Ratos Sprague-DawleyRESUMO
It has long been thought that the cyst form of Pneumocystis carinii, which can resist host defenses and antimicrobial drugs, is responsible for relapses of P. carinii pneumonia. The thick wall of the cyst is immunogenic and rich in glucosyl/mannosyl and N-acetyl-D-glucosamine residues. In this study we have demonstrated the presence of a hitherto unreported outer membrane in the cyst wall of P. carinii. This membrane was detected by a combination of techniques, including transmission electron microscopy, freeze-fracture electron microscopy, and membrane labeling with fluorescent lipid analogs following treatment of P. carinii cysts from infected rats for 30 min with Zymolyase, a beta-1-3 glucanase. As in gram-negative bacteria and blue-green algae, this 2nd membrane may have an important role in osmoregulation and nutrient utilization; it may also mediate the interaction of P. carinii with its host and serve as a target for drug therapy.
Assuntos
Pneumocystis/ultraestrutura , Animais , Parede Celular/ultraestrutura , Técnica de Fratura por Congelamento , Glucana Endo-1,3-beta-D-Glucosidase , Masculino , Membranas/ultraestrutura , Fosfatidiletanolaminas , Ratos , Ratos Endogâmicos Lew , Esporos Fúngicos/ultraestruturaRESUMO
Pneumocystis carinii cysts are capable of resisting host defenses and antimicrobial drugs and are therefore thought to be responsible for relapses of P. carinii pneumonia in AIDS and other immunocompromised patients. The interaction of P. carinii with its host, and other P. carinii, might be mediated by molecules which form the outer surfaces of this organism. Carbohydrates are known to play many roles in cell-cell adhesion, and have been detected on the surface of P. carinii by lectin labeling experiments. In this study P. carinii cyst wall material was obtained from Zymolyase treatment. Alditol acetate derivatives of neutral and amino sugars or trimethylsilyl derivatives of methyl glycosides were prepared from the monosaccharides released from the sample by acid hydrolysis. Analyses were done by a combination of gas chromatography and mass spectrometry. Glucose was found to be the major sugar constituent. Mannose and galactose were present in equal ratios. A lesser amount of N-acetyl-D-glucosamine, and trace amounts of ribose and sialic acid were present in the cyst wall samples analyzed. These sugars may mediate P. carinii-host interaction and play an important protective role by creating a permeability barrier around the cyst.
Assuntos
Carboidratos/análise , Pneumocystis/análise , Acetatos/química , Carboidratos/química , Parede Celular/química , Cromatografia Gasosa , Cromatografia Gasosa-Espectrometria de Massas , Polissacarídeos/química , Esporos Fúngicos/análise , Álcoois Açúcares/química , Compostos de Trimetilsilil/químicaRESUMO
An ultrastructural study of life cycle stages of Pneumocystis carinii in infected rat lungs in situ was undertaken utilizing 8 different modes of fixation. Three of the fixatives employed gave good fixation of cysts and intracystic bodies, but for the trophic forms fixation was only fair. Both the trophic forms and intracystic bodies have nuclear pores. The mitochondria of the organism have cristae that appear lamellar. One of the fixation modes revealed a thin, electron-dense layer on the outer surface of the cell wall, a "fuzzy coat" that had not been described previously. This material appears to mediate tight adhesion of trophic forms with other trophic forms, cysts, and with pneumocytes of the lung alveolus.
Assuntos
Pneumocystis/crescimento & desenvolvimento , Animais , Fixadores , Pneumocystis/ultraestrutura , Pneumonia por Pneumocystis/parasitologia , Pneumonia por Pneumocystis/patologia , RatosRESUMO
Pneumocystis carinii obtained from rat lungs (RLH) and in vitro culture (RTC) were reacted with a panel of 14 fluorescein isothiocyanate conjugated lectins. Percentage fluorescence and fluorescent intensity were determined for both trophic and cyst forms. All RLH and RTC derived organisms bound strongly concanavalin A (Con A), and wheat germ agglutinin (WGA). However, differences in soybean agglutinin (SBA) binding between RLH and RTC organisms was observed. Different subsets of the organism bound lectins from Griffonia simplicifolia I, Maclura pomifera, and Bauhinia purpurea, indicating heterogeneity in the surface carbohydrates within each of the RLH and RTC populations. Eight lectins reacted very weakly or not at all: Dolichos biflorus, Arachis hypogaea, Griffonia simplicifolia I-beta 4, Solanum tuberosum, Ulex europeus, Griffonia simplicifolia II, Viscum album, and Limax flavus. The results indicate that P. carinii trophic and cyst forms have surface constituents containing mannose, N-acetylglucosamine and N-acetylgalactosamine as the predominant carbohydrates. Molecules resembling sialic acid and beta-galactose are absent or inaccessible. The surface glycoconjugates identified in these studies may play a role in the adherent properties of P. carinii.