Fig. 1. Intrapulmonary life cycle of Pneumocystis carinii proposed by Yoshida.
Pneumocystis carinii is found in the lungs (pneumo-) of mammals where a morphologically characteristic life cycle stage (cyst) develops. It was established as a newly described organism by Delanöe and Delanöe in 1912 who named it in recognition of Antonio Carini, who provided them with the specimen slides from which they characterized it.
Prior to the AIDS epidemic, the organism was only sporadically reported (e.g., malnourished children and patients undergoing immunosuppressive therapy for cancer or solid organ transplants). In the 1980s as the HIV infection spread and AIDS became a serious public health problem in the USA and Europe, Pneumocystis carinii was found to be the most prevalent infectious agent and the most common immediate cause of death among these patients (Pneumocystosis).
Since its discovery, the taxonomic and phylogenetic status of Pneumocystis carinii has been debated. Currently classified as an atypical, exotic fungus, it may represent an early branch in fungal evolution independent of those that led to the ascomycetes and basidiomycetes. Terms used for higher fungal structures may not be appropriate to adopt at this stage of understanding the organism's natural history, hence the terms most commonly found in the literature will be used. The following may be correlates: trophozoites (trophic form); pre-cyst (sporocyte); cyst (spore case); intracystic bodies (ICB, spores), excystation (spore release).
What is currently known about its life cycle is restricted to forms found in the mammalian lung. Whether or not proliferation occurs in another cycle outside the mammalian host has not been ruled out. The proposals for the life cycle within the lung alveolus are based on morphological features and cytochemical studies, but are restricted to static light and electron micrographs of the forms identified as Pneumocystis carinii (Fig. 1). With the possible exception of visual observations of intracystic bodies (ICB, spores) released from mature cysts (spore cases), no other developmental transformations have been directly observed; how one form develops into the next has not been directly observed.
Elliptical to pleomorphic irregularly shaped trophic forms ranging in size from 1.5 to 10.0 μm are found in very great numbers in infected lungs (Fig. 2). These have been subgrouped into “small” (1–2 μm) and “large” trophozoites. It is believed that the small spherical-to-oval cells are directly derived from ICB, whereas the “large” cells may be derived from asexual vegetative mitotic division of preexisting trophozoites. The pellicle (cortex) of the trophozoites is thin (20–30 nm) compared to the cyst wall. It is comprised of two unit membranes separated by a middle electron-lucent layer which is very thin in these forms. The cell's exterior is coated with a distinct thick glycocalyx which is of uniform thickness (15 nm) in all life cycle stages found in the lung. Tubular extensions, which are thin, long, tubular evaginations of the cell surface, are distinct features of the organism, especially of large trophozoites. Tubular extensions may function in anchoring the organisms in the alveolus, or enhance nutrient uptake by increasing cell surface area.
The organism adheres to thin type I epithelial cells, but not to type II cells (thick surfactant-secreting epithelial cells). In heavy infections, three to four layers of organisms can form caps above type II cells, and organisms are found attached to thin elongated outfoldings of the type I cells.
Most organisms in the lung appear to be haploid. The organism has a single nucleus which is distinguished by its small size and represents among the smallest found in eukaryotic cells; the 1C nucleus is estimated to contain 6.6 fg DNA (8.9 Mbp nucleotides). The nucleus contains a single nucleolus and a nuclear envelope with relatively few (8 pores/μm2), large (95 nm) nuclear pores. Nuclear division with spindle microtubules radiating from electron-dense structures called nucleus-associated organelles (NAO) and condensed chromosomes have been observed in trophozoites. Asexual reproduction by binary fission is believed to produce most of the trophic forms found in infected lungs.
A single mitochondrion with lamellar cristae is probably typical of Pneumocystis carinii; when several mitochondrial profiles are seen in a section through the cell this may represent lobes of the organelle. Other cytoplasmic structures include lipid droplets, rough and smooth endoplasmic reticulum, primary lysosomes, glycogen-like granules, and a number of unidentified vacuoles and vesicles present in the cytoplasm. Golgi elements, identified cytochemically, are not elaborate structures, but appear as a number of small vesicles. A large smooth endoplasmic saccule takes up a large proportion of the cytoplasm in both trophic and cystic forms.
1. Precyst. It is generally assumed that cystic forms develop from trophic forms; an intermediate stage called the pre-cyst has been proposed and described. This spherical-to-oval intermediate form is larger (approximately 4–5 μm) and has a rigid pellicle (40–120 nm) which is thicker than that of trophozoites and thinner than that of mature cysts. The thicker wall results from the thickening of the middle electron-lucent layer, which is believed to contain β-glucan, α-glucan polymers, and chitin.
Synaptonemal complexes (pairing of homologous chromosomes) have been reported to occur at this stage, but these structures apparently are rare since most workers have not observed them. Nonetheless, since the synaptomenal complex is a hallmark of sexual reproduction, it is generally accepted that meiosis occurs during this stage, and that ICB are meiotic products (sporogenesis). It is presumed that mating of forms that appear to be trophic give rises to the zygotic nucleus. Mitochondrial elements appear to surround the nucleus in pre-cysts.
2. Thick-walled cysts containing round ICB. It is assumed that the single nucleus in the pre-cyst stage divides to give rise to a cyst containing two ICB, which then gives rise to a four-ICB stage. After division of these four ICB, the mature spores, containing eight ICB, are formed.
At this stage, most cysts are spherical and the middle layer of the cyst wall becomes very thick, giving a total pellicle thickness of 100–160 nm. By digesting the polysaccharides in the outermost layer with zymolyase, a bilayer membrane became evident by transmission electron microscopy. In mature cysts, a specialized region in the middle layer of the cyst wall becomes thicker than the rest of this layer. This thickened region may be the site where a pore develops through which ICB are released during excystation. After excystation, the collapsed crescent-shaped cyst (spore case), containing remnants of cytoplasm and organelles, is found with a rupture in its wall.
The ICB is formed by delineation of its nucleus and cytoplasm by membranes derived from the cyst inner surface membrane. These forms resemble the small trophozoites with a thin pellicle containing two membranes and a barely visible middle electron-lucent layer; no tubular extensions are present on their surfaces at this stage.
3. Thick-walled cysts containing banana-shaped ICB. Distinct from cysts with spherical-to-ovoid ICB are thick-walled cysts containing banana-shaped ICB (Fig. 2). Banana-shaped ICB in some cysts clearly exhibit cell motility (wiggling, flexing) and the cytoplasm of these forms is more electron-dense than that of round ICB. The ICB appear to be attached to the inner membrane of the cyst wall by a stalk-like structure. It is not known whether this form represents a stage in a cycle independent from the cycle which gives rise to spherical cysts; or whether banana-shaped ICB develop from spherical ICB.
4. Thin-walled cysts containing pleomorphic ICB. Another form of which even less is understood with respect to its position in a proposed life cycle scheme is described as a thin-walled cyst. These generally do not have walls as thick as those surrounding the more abundant forms with round or banana-shaped ICB. The characteristic feature that distinguishes this form from the others is that the ICB are irregularly shaped and resemble trophozoites. The irregularly shaped ICB are closely packed and appear attached to each other by their glycocalyxes, resembling adhesions between organisms and between the organisms and type I pneumocytes. Based strictly on morphology, it has been proposed that these forms arise from mitotic divisions within a parent cell analogous to a process known among some protozoans as endogeny.
5. Dormant form. There appears to be a life cycle stage which remains dormant and infective outside the mammalian host. Its morphological features may differ from most other forms described in the lung.
Evidence for sexual reproduction in the intrapulmonary cycle comes from one electron microscopic study in which a synaptonemal complex was observed in a precyst found in a rat lung. This observation has yet to be independently confirmed.
Most experimental studies are done on organisms obtained from infected lungs or bronchoalveolar lavage fluid of laboratory animals. Infections are provoked by immunosuppression (mainly rats, mice, ferrets) using corticosteroids. Animals with prior exposure to Pneumocystis carinii, or housed in facilities with PcP animals, develop PcP under corticosteroid treatment (given in drinking water or by injections). Alternatively, corticosteroid-treated animals reared under strict barrier (filtered air) conditions are inoculated (intratracheally or intranasally) with freshly isolated or cryopreserved organisms. Other animal models include helper T-cell-depleted animals treated with anti-CD4 monoclonal antibodies; neonatal rabbits and genetically immunodeficient animals (e.g., nude and SCID mice).
Development of culture methods for studying the organism in the laboratory has progressed slowly. Most current in vitro experimental studies rely on primary cultures initiated by organisms isolated from infected animal models. Some investigators performed experiments on axenic short-term cultures whereas others incubate the organisms with one of several mammalian cell lines.
Broad genetic diversity exists among populations of organisms currently called Pneumocystis carinii. The organisms are host species-specific and different species names may be eventually assigned to those in different mammalian species. Distinct genetic populations have also been found among organisms infecting the same host species (e.g., humans, rats, ferrets); therefore there may be several different species that can inhabit a single mammalian species. An interim trinomial nomenclature is currently used to designate organisms from different hosts: e.g., P. carinii carinii and P. carinii ratti (rat); P. carinii hominis (human); P. carinii mustelae (ferret), P. carinii muris (mouse), P. carinii equi (horse), P. carinii suis (pig), P. carinii oryctolagi (rabbit).
Genetic diversity has been identified by nucleotide sequences, karyotype (chromosomal) patterns and antigens. The number of chromosomes resolved by pulsed field gel electrophoretic techniques varies even within a population in a single animal. They range from 12 to 16 in number; individual chromosomes are 850–300 kb in size. Analyses of samples from a single infected host in which 22–24 chromosomes are resolved very likely represent a mixed infection of two distinct populations.
The fungal assignment of Pneumocystis carinii was initially based on the small subunit 16S-like RNA gene and complementary DNA (cDNA). Subsequently, nucleotide sequence analyses of genes encoding for dihydrofolate reductase, thymidylate synthetase, P-type cation-translocating ATPase pre-chorismate shikimic acid pathway pentafunctional arom gene β-tubulin, the presence of elongation factor 3 (needed for fungal protein synthesis), and several mitochondrial genes (NADH and cytochrome oxidase) show close homologies to their counterparts in fungi. However, these data also indicate that Pneumocystis carinii is significantly divergent from other extant fungi.
The environment in which Pneumocystis carinii rapidly proliferates in the mammalian host is the lung alveolus. The alveolus is lined with fluid rich in lung surfactant secreted by alveolar type II epithelial cells. Lung surfactant is comprised mainly of lipids dominated by dipalmitoylphosphatidylcholine (disaturated PC), but also contains substantial amounts of other molecular species of PC, phosphatidylglycerol, phosphatidylinositol, and cholesterol. Surfactant proteins are minor components, but they potently influence the surface tension of the alveolar lining fluid. Surfactant proteins avidly bind to the surfaces of Pneumocystis carinii.
The type I epithelial cells are thin and they are the site for most of the gas exchange between the alveolar lumen and the capillaries of the circulatory system. The pathogen adheres to these cells, causes damage to these epithelial cells, and the physical presence of numerous organisms covering the epithelium invokes a serious barrier to gas exchange.
Unlike most common fungal pathogens of animals, Pneumocystis carinii does not contain ergosterol (C28). Cholesterol (C27), believed to be scavenged from the host, constitutes 75% of sterols in freshly isolated organisms. The absence of ergosterol explains the lack of sensitivity of Pneumocystis carinii to polyene antimycotics (e.g. amphotericin B, nystatin), drugs that target ergosterol in fungal membranes. Unlike most fungi, and more in common with some plants and rust fungi, Pneumocystis carinii sterols can undergo one or two methyl transfer reactions at C-24 of the sterol side chain to form C28 and C29 sterols. The major sterol components have a double bond at Δ7 (Fig. 4). The identification in P. carinii hominis of 24-methylenelanost-8,24(28)-diene-3 β-ol (C31) and pneumocysterol (C32), which are C-24-alkylated lanosterol molecules, can explain why Pneumocystis carinii is insensitive to azole drugs. These drugs inhibit sterol nucleus demethylation and are effective against fungi in which demethylation of lanosterol is required before alkylation of C-24 of the sterol side chain can take place; zymosterol is the preferred substrate for the C-24 transmethylase reaction in those pathogens that are sensitive to azole drugs.
The major ubiquinone homolog in Pneumocystis carinii is CoQ10, and the organism synthesizes de novo both the benzoquinone ring and the polyprenyl chain moieties of this compound, which plays a pivotal role in mitochondrial electron transport. Hydroxynaphthoquinones have good anti-Pneumocystis carinii activity. As ubiquinone analogs, these drugs disrupt electron transport in the organism.