Immunohistochemical and ultra-structural detection of Pneumocystis in wild boars (Sus scrofa) co-infected with porcine circovirus type 2 (PCV2) in Southern Brazil.

Pneumocystis spp. are fungi that are able to infect a variety of host species and, occasionally, lead to severe pneumonia. Porcine circovirus type 2 (PCV2) is an important viral pathogen which affects both swine and wild boar herds worldwide. Co-infection between PCV2 and other pathogens has been reported, and the secondary immunodeficiency caused by the virus may predispose to these co-infections. In the present study, postmortem tissue samples obtained from wild boar herds in Southern Brazil were analyzed by histopathology, ultra-structural observation, and immunohistochemistry. Forty-seven out of seventy-eight (60%) wild boars showed clinical signs, gross, and histopathological lesions characteristic of infection by PCV2. Pneumocystis was detected by immunohistochemistry in 39 (50%) lungs and viral antigens of PCV2 were found in 29 (37.2%) samples. Concomitant presence of Pneumocystis and PCV2 were observed in 16 (20.5%) of the wild boars. Cystic and trophic forms of Pneumocystis were similar to previously described ultra-structural observations in other mammals.

Defective nitric oxide production by alveolar macrophages during Pneumocystis pneumonia.

The effect of nitric oxide (NO) on Pneumocystis (Pc) organisms, the role of NO in the defense against infection with Pc, and the production of NO by alveolar macrophages (AMs) during Pneumocystis pneumonia (PCP) were investigated. The results indicate that NO was toxic to Pc organisms and inhibited their proliferation in culture. When the production of NO was inhibited by intraperitoneal injection of rats with the nitric oxide synthase inhibitor L-N(5)-(1-iminoethyl) ornithine, progression of Pc infection in immunocompetent rats was enhanced. Concentrations of NO in bronchoalveolar lavage fluids from immunosuppressed, Pc-infected rats and mice were greatly reduced, compared with those from uninfected animals, and AMs from these animals were defective in NO production. However, inducible nitric oxide synthase (iNOS) mRNA and protein concentrations were high in AMs from Pc-infected rats and mice. Immunoblot analysis showed that iNOS in AMs from Pc-infected rats existed primarily as a monomer, but the homo-dimerization of iNOS monomers was required for the production of NO. When iNOS dimerization cofactors, including calmodulin, were added to macrophage lysates, iNOS dimerization increased, whereas incubation of the same lysates with all cofactors except calmodulin did not rescue iNOS dimer formation. These data suggest that NO is important in the defense against Pc infection, but that the production of NO in AMs during PCP is defective because of the reduced dimerization of iNOS.

Pneumocystis: from a doubtful unique entity to a group of highly diversified fungal species.

At the end of the 20th century the unique taxonomically enigmatic entity called Pneumocystis carinii was identified as a heterogeneous group of microscopic Fungi, constituted of multiple stenoxenic biological entities largely spread across ecosystems, closely adapted to, and coevolving in parallel with, mammal species. The discoveries and reasoning that led to the current conceptions about the taxonomy of Pneumocystis at the species level are examined here. The present review also focuses on the biological, morphological and phylogenetical features of Pneumocystis jirovecii, Pneumocystis oryctolagi, Pneumocystis murina, P. carinii and Pneumocystis wakefieldiae, the five Pneumocystis species described until now, mainly on the basis of the phylogenetic species concept. Interestingly, Pneumocystis organisms exhibit a successful adaptation enabling them to dwell and replicate in the lungs of both immunocompromised and healthy mammals, which can act as infection reservoirs. The role of healthy carriers in aerial disease transmission is nowadays recognized as a major contribution to Pneumocystis circulation, and Pneumocystis infection of nonimmunosuppressed hosts has emerged as a public health issue. More studies need to be undertaken both on the clinical consequences of the presence of Pneumocystis in healthy carriers and on the intricate Pneumocystis life cycle to better define its epidemiology, to adapt existing therapies to each clinical context and to discover new drug targets.

Biofilm formation by Pneumocystis spp.

Pneumocystis spp. can cause a lethal pneumonia in hosts with debilitated immune systems. The manner in which these fungal infections spread throughout the lung, the life cycles of the organisms, and their strategies used for survival within the mammalian host are largely unknown, due in part to the lack of a continuous cultivation method. Biofilm formation is one strategy used by microbes for protection against environmental assaults, for communication and differentiation, and as foci for dissemination. We posited that the attachment and growth of Pneumocystis within the lung alveoli is akin to biofilm formation. An in vitro system comprised of insert wells suspended in multiwell plates containing supplemented RPMI 1640 medium supported biofilm formation by P. carinii (from rat) and P. murina (from mouse). Dramatic morphological changes accompanied the transition to a biofilm. Cyst and trophic forms became highly refractile and produced branching formations that anastomosed into large macroscopic clusters that spread across the insert. Confocal microscopy revealed stacking of viable organisms enmeshed in concanavalin A-staining extracellular matrix. Biofilms matured over a 3-week time period and could be passaged. These passaged organisms were able to cause infection in immunosuppressed rodents. Biofilm formation was inhibited by farnesol, a quorum-sensing molecule in Candida spp., suggesting that a similar communication system may be operational in the Pneumocystis biofilms. Intense staining with a monoclonal antibody to the major surface glycoproteins and an increase in (1,3)-beta-D-glucan content suggest that these components contributed to the refractile properties. Identification of this biofilm process provides a tractable in vitro system that should fundamentally advance the study of Pneumocystis.

Current insights into the biology and pathogenesis of Pneumocystis pneumonia.

The fungal infection Pneumocystis pneumonia is the most prevalent opportunistic infection in patients with AIDS. Although the analysis of this opportunistic fungal pathogen has been hindered by the inability to isolate it in pure culture, the use of molecular techniques and genomic analysis have brought insights into its complex cell biology. Analysis of the intricate relationship between Pneumocystis and the host lung during infection has revealed that the attachment of Pneumocystis to the alveolar epithelium promotes the transition of the organism from the trophic to the cyst form. It also revealed that Pneumocystis infection elicits the production of inflammatory mediators, culminating in lung injury and impaired gas exchange. Here we discuss these and other recent findings relating to the biology and pathogenesis of this intractable fungus.

Localization of cytoskeletal proteins in Pneumocystis carinii by immuno-electron microscopy.

Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms.

Immunosuppressed surfactant protein A-deficient mice have increased susceptibility to Pneumocystis carinii infection.

Immunosuppressed Swiss Black mice deficient in surfactant protein A (SP-A(-/-)) and wild-type control mice (SP-A(+/+)) were exposed to Pneumocystis carinii by environmental exposure, intratracheal inoculation, and direct exposure to other infected animals. The frequency and intensity of P. carinii infection were significantly greater in the SP-A(-/-) mice by all 3 methods of exposure. P. carinii free of SP-A and alveolar macrophages were isolated from SP-A(-/-) mice and were tested in an in vitro attachment assay. Pretreatment of P. carinii with human SP-A resulted in a significant dose-dependent increase of the adherence of P. carinii to the macrophages. Thus, SP-A plays a role in host defense against P. carinii in vivo, perhaps by functioning as a nonimmune opsonin.

Differential regulation of growth and checkpoint control mediated by a Cdc25 mitotic phosphatase from Pneumocystis carinii.

Pneumocystis carinii is an opportunistic fungal pathogen phylogenetically related to the fission yeast Schizosaccharomyces pombe. P. carinii causes severe pneumonia in immunocompromised patients with AIDS and malignancies. Although the life cycle of P. carinii remains poorly characterized, morphologic studies of infected lung tissue indicate that P. carinii alternates between numerous small trophic forms and fewer large cystic forms. To understand further the molecular mechanisms that regulate progression of the cell cycle of P. carinii, we have sought to identify and characterize genes in P. carinii that are important regulators of eukaryotic cell cycle progression. In this study, we have isolated a cDNA from P. carinii that exhibits significant homology, but unique functional characteristics, to the mitotic phosphatase Cdc25 found in S. pombe. P. carinii Cdc25 was shown to rescue growth of the temperature-sensitive S. pombe cdc25-22 strain and thus provides an additional tool to investigate the unique P. carinii life cycle. Although P. carinii Cdc25 could also restore the DNA damage checkpoint in cdc25-22 cells, it was unable to restore fully the DNA replication checkpoint. The dissociation of checkpoint control at the level of Cdc25 indicates that Cdc25 may be under distinct regulatory control in mediating checkpoint signaling.

Pneumocystis carinii uses a functional cdc13 B-type cyclin complex during its life cycle.

Pneumocystis carinii causes severe pneumonia in immunocompromised patients. Recent studies indicate that P. carinii uses a Cdc2 cyclin-dependent kinase to control its proliferation. To further study the regulation of the life cycle of P. carinii, we characterized the P. carinii B-type cyclin termed Cdc13, whose binding to Cdc2 is necessary for kinase activity. Antibodies to B-type cyclins (Cdc13) specifically immunoprecipitated Cdc2/ Cdc13 complexes with associated kinase activity from P. carinii extracts. To clone P. carinii cdc13, degenerate polymerase chain reaction was undertaken using primers generated from amino-acid motifs conserved in fungal Cdc13 proteins. This amplicon was used to obtain full-length genomic and complementary DNA (cDNA) clones. A specific synthetic peptide antibody generated to P. carinii Cdc13 further demonstrated differential Cdc2/Cdc13 activity over the life cycle of P. carinii, with greater activity in cysts compared with trophic forms of the organism. Finally, P. carinii cdc13 cDNA was used to rescue mutant Schizosaccharomyces pombe strains containing temperature-sensitive deficiencies of endogenous Cdc13 activity, thus verifying function of the P. carinii Cdc13 protein. Therefore, P. carinii contains a Cdc13 cyclin, which is variably active over its life cycle and which promotes fungal proliferation.

Continuous axenic cultivation of Pneumocystis carinii.

Continuous axenic culture of Pneumocystis carinii has been achieved. A culture vessel is used that allows for frequent medium exchange without disturbance of organisms that grow attached to a collagen-coated porous membrane. The growth medium is based on Minimal Essential Medium with Earle's salt supplemented with S-adenosyl-L-methionine, putrescine, ferric pyrophosphate, N-acetyl glucosamine, putrescine, p-aminobenzoic acid, L-cysteine and L-glutamine, and horse serum. Incubation is in room air at 31 degrees C. The pH of the medium begins at 8.8 and rises to approximately 9 as the cells grow. Doubling times calculated from growth curves obtained from cultures inoculated at moderate densities ranged from 35 to 65 hours. With a low-density inoculum, the doubling time is reduced to 19 hours. The morphology of cultured organisms in stained smears and in transmission electron micrographs is that of P. carinii, and P. carinii-specific mAbs label the cultured material. Cultured organisms are infective for immunosuppressed rats and can be stored frozen and used to reinitiate culture.

Pneumocystis carinii contains a functional cell-division-cycle Cdc2 homologue.

Pneumocystis carinii causes life-threatening pneumonia in immunocompromised patients. The inability to culture P. carinii has hampered basic investigations of the organism's life cycle, limiting the development of new therapies directed against it. Recent investigations indicate that P. carinii is a fungus phylogenetically related to other ascomycetes such as Schizosaccharomyces pombe. The cell cycles of S. pombe and homologous fungi are carefully regulated by cell-division-cycle molecules (cdc), particularly cell-division-cycle 2 (Cdc2), a serine-threonine kinase with essential activity at the G1 restriction point and for entry into mitosis. Antibodies to the proline-serine-threonine-alanine-isoleucine-arginine (PSTAIR) amino-acid sequence conserved in Cdc2 proteins specifically precipitated, from P. carinii extracts, a molecule with kinase activity consistent with a Cdc2-like protein. Cdc2 molecules exhibit differential activity throughout the life cycle of the organisms in which they occur. In accord with this, the P. carinii Cdc2 showed greater specific activity in P. carinii trophic forms (trophozoites) than in spore-case forms (cysts). In addition, complete genomic and complementary DNA (cDNA) sequences of P. carinii Cdc2 were cloned and found to be most closely homologus to the corresponding sequences of other pathogenic fungi. The function of P. carinii cdc2 cDNA was further documented through its ability to complement the DNA of mutant strains of S. pombe with temperature-sensitive deficiencies in Cdc2 activity. The P. carinii cdc2 cDNA restored normal Cdc2 function in these mutant strains of S. pombe, and promoted fungal proliferation. These studies represent the first molecular analysis of the cell-cycle-regulatory machinery in P. carinii. Further understanding of P. carinii's life cycle promises novel insights for preventing and treating the intractable infection it causes in immunocompromised patients.

Alveolar damage in AIDS-related Pneumocystis carinii pneumonia.

OBJECTIVE: Pneumocystis carinii pneumonia is the most common and serious of the pulmonary complications of AIDS. Despite this, many basic aspects in the pathogenesis of HIV-associated P carinii pneumonia are unknown. We therefore undertook a light and electron microscopic study of transbronchial biopsy specimens to compare pathologic features of P carinii pneumonia and other HIV-related lung diseases. DESIGN AND PATIENTS: Thirty-seven consecutive HIV-infected patients undergoing a diagnostic bronchoscopy. RESULTS: P carinii pneumonia was characterized by an increase in inflammation, edema, exudate, fibrosts, type II pneumocyte proliferation, and cellular infiltration of the alveolar wall when compared with other lung diseases (all p < 0.05). Electron microscopy showed apposition of the trophozoite to the type I pneumocyte. Erosion of type I pneumocytes was observed in 13 of 15 patients with P carinii pneumonia, whereas none without P carinii pneumonia had this finding (p < 0.05). Erosion of the type II pneumocyte was not observed. CONCLUSION: Inflammation, interstitial fibrosis, and alveolar epithelial erosion are characteristic features of P carinii pneumonia. The changes may form the pathologic basis for the respiratory failure seen in patients with P carinii pneumonia. Electron microscopy did not show any diagnostie advantage over conventional light microscopy using routine stains.

Light and electron microscopy study of carbohydrate antigens found in the electron-lucent layer of Pneumocystis carinii cysts.

The localization and biochemical nature of antigens found in the electron-lucent layer (ELL) of Pneumocystis carinii cysts using polyclonal rabbit antibodies are described. These antigens, specific for the cystic stages of the parasite, were shared by organisms from different hosts, suggesting that they represent functionally important components of the cyst cell wall. The binding sites were situated on an interwoven net of fibrils in the ELL produced by mild to strong proteolysis. Degradation of this residue by glucanase and chitinase confirms that this layer contains branched glucan and chitin. In contrast, the prompt susceptibility of the polysaccharide-rich ELL to proteolysis reveals that proteins are also relevant in building up the cyst-wall glucan skeleton. It is therefore concluded that the formation of the Pneumocystis cyst wall shows differences to the typical fungal cell-wall architecture. The taxonomical debate regarding this unique protist is ongoing, and consideration of these immunological and morphological findings may be useful for the study of the biology and phylogeny of Pneumocystis.

Attachment of Pneumocystis carinii to primary cultures of rat alveolar epithelial cells.

Pneumocystis carinii (PC) is an exclusively extracellular pathogen which causes pneumonia in immunocompromised individuals. Histologic studies have demonstrated that PC organisms attach preferentially to type I alveolar epithelial cells and rarely bind to type II cells. Previous reports have demonstrated that cultured type II cells develop a type I cell-like phenotype and express type I cell surface antigens. The current study examines the attachment of PC organisms to isolated rat type II alveolar epithelial cells as a function of time in culture. PC attachment to isolated type II cells increased as the type II cells differentiated in culture from 2.3 +/- 1.2% on Day 2 to 18.4 +/- 2.7% by Day 8. Previous studies have indicated a role for fibronectin (Fn) and Fn receptors as mediators of PC attachment. Addition of anti-Fn antibodies decreased attachment of PC to Day 8 type II cells from 19.4 +/- 2.5% to 9.4 +/- 1.9% (P < 0.01). Addition of antibodies to the alpha v and alpha 5 integrin subunits resulted in significant decreases in PC attachment to Day 8 type II cells. Examination of expression of alpha v and alpha 5 integrins on Day 2 and Day 8 type II cells demonstrated increased expression of both alpha v and alpha 5 integrin subunits on Day 8 type II cells. Overall these data indicate that attachment of PC to isolated rat type II cells increases as the cells differentiate into a type I cell-like phenotype in vitro and correlates with increased expression of Fn-binding integrins on the cell surface of the cultured type II cells.

Relation of cyst counts with numbers of total nuclei of Pneumocystis carinii in rats.

Wistar rats were induced of Pneumocystis carinii infection by injection with methyl-prednisolone to correlate the cyst counts and numbers of nuclei. Seven sections of the lungs were examined by impression smears and also whole lung homogenates were screened for nucleus counting for each rat. At the first week of the experiment, all of the impression smears except one were cyst negative but trophic forms were counted around 10(6). At the third week, the cysts appeared one per 20 immersion oil lens fields. The nuclei were on the order of 10(7) at this period, and this amount of Pc is regarded as the limit of cyst detection on impression smears. When the nuclei were over 10(9) in the lungs, the cysts were counted about 50 in 20 microscopic fields. The organisms were distributed in the lungs without any predilection focus. The present data suggest that the trophic forms, proliferate first and the cysts appear later in the lungs.

A novel in situ model to study Pneumocystis carinii adhesion to lung alveolar epithelial cells.

Pneumocystis carinii, an extracellular parasite thriving in the lungs of immunosuppressed mammals, is a major cause of death in AIDS patients in the USA. As a prelude to growth, the parasite adheres mostly to type I pneumocytes lining the alveolar spaces. The mechanism of adherence remains unknown, largely because of difficulties in isolating type I pneumocytes and maintaining them in vitro. As a first step to understand P. carinii adherence to its natural substrate, we developed an in situ method to directly study parasite binding to lung alveolar cells. We used formaldehyde-fixed paraffin-embedded sections of normal rat lung as substrate for adhesion. As in its binding to the lungs in vivo, P. carinii adhered preferentially to type I pneumocytes. Adherence was saturable, time and dose dependent, and selectively blocked by glycoconjugates, in particular bovine submaxillary mucin, fetuin, and asialofetuin, suggesting that it may be mediated by a lectin type of interaction. Further, IgG of rats with P. carinii pneumonia inhibited adherence, suggesting that it may react with parasite ligands involved in the recognition of type I cell receptors. Our results demonstrate the usefulness of the in situ model for studying the mechanisms of P. carinii adherence to alveolar cells. In addition, this method may be valuable for identifying neutralizing antibodies and drugs potentially useful for controlling the infection in vivo.