Cell biology life cycle and interactions with the host

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Several aspects of the life cycle, transmission, pathogenesis, and host response in P. carinii infection remain unknown. However, in recent years we have gained much information on the complex interplay of P. carinii with host inflammatory cells, release of cytokines, generation of toxic metabolites, and involvement of both cellular and humoral immunity. Although much remains unknown about the pathogenesis and host response, studies of P. carinii in recent years have provided us with an increasing base of knowledge about this organism and its relationship to the host. These studies have led to a better understanding of mechanisms of P. carinii attachment and injury to host cells, new information about the interaction of P. carinii with pulmonary epithelial cells and the interplay of the organism with host inflammatory cells (Su and Martin 1994).

Two dominant stages of P. carinii have been identified in the mammalian lung, the mature thick walled cysts or asci containing eight intracystic bodies or ascospores and the polymorphic 'trophozoites' (Figure 12.2). The latter arise from ascospores liberated from ruptured

Figure 12.2 Pneumocystis carinii organisms with eight 'intracystic bodies' in broncho-alveolar lavage sample from a patient with PCP seen in a Giemsa-stained preparation.

Figure 12.3 Pneumocystis carinii organisms identified in histological section of infected lung tissue and in broncho-alveolar lavage fluid obtained from a patient with fulminant PCP. Immunoperoxidase staining of intra-alveolar masses of Pneumocystis carinii is seen. Using fluorescence microscopy of lavage fluid both asci (cysts) and trophozoites are seen using monoclonal anti-pneumocystis antibody 3F6 as the marker (100). (See Colour Plate II.)

Figure 12.3 Pneumocystis carinii organisms identified in histological section of infected lung tissue and in broncho-alveolar lavage fluid obtained from a patient with fulminant PCP. Immunoperoxidase staining of intra-alveolar masses of Pneumocystis carinii is seen. Using fluorescence microscopy of lavage fluid both asci (cysts) and trophozoites are seen using monoclonal anti-pneumocystis antibody 3F6 as the marker (100). (See Colour Plate II.)

asci which can be seen as empty, banana-shaped shells in electron micrographs of bronchial lavage fluid sediments from infected lungs. Alveolar spaces filled with organisms can be seen in pathology specimens and both stages identified in lavage fluids by immunocytochemistry, (Figure 12.3). The composition of the cyst wall is distinct from the surface of the trophozoites. The thick middle electron-lucent layer of the cyst wall seen in transmission electron microscopy (Figure 12.4) contains important immunogens. The cyst wall contains carbohydrates (De Stefano et al. 1989) and some appear to contain beta-1-3-glucan (Goheen et al. 1994). Degradation by glucanase and chitinase confirms that this layer contains branched glucan and chitin. The susceptibility of the polysaccharide-rich electron-lucent layer to proteolysis reveals that proteins are also relevant in building up the cyst-wall glucan skeleton (Roth et al. 1997). Interestingly beta-D-glucan can be detected in sera obtained from patients with PCP (Yasuoka et al. 1996).

The trophozoites have a plasma membrane surrounded by a glycocalyx. Upon development of trophozoites to thin walled asci and subsequently thick walled asci, a typical trilaminated wall is seen

(Figure 12.1). Numerous tubular extensions of the ascus wall are apparently involved in the attachment of the organism to type-I lung epithelial cells. Binding seems to involve penetration, but not invasion, into the host cell cytoplasm without damaging the plasma membrane. All P. carinii, irrespective of their host of origin, express an abundant mannosylated surface glycoprotein, commonly referred to as gp120, or because its size varies in different species, as surface glycoprotein A (Gigliotti 1992). The protein moiety of the major high molecular

Figure 12.4 Ultrastructural appearance of Pneumocystis ascus (cyst) in lung tissue from a patient with fatal PCP.

weight surface antigen, represented by numerous isoforms, is encoded by different genes. These proteins are post-transcriptionally modified by carbohydrates and lipids. Pneumocystis surface glycoproteins, especially the mannose-rich major surface glycoproteins, appear to mediate adherence to type-I epithelial cells. Extracellular matrix proteins are necessary for the attachment of Pneumocystis to host cells, a phenomenon well known in several other host-parasite interactions. Integrin receptors on the epithelial cells take up extracellular matrix proteins fibronectin and vitronectin produced by alveolar macrophages in the inflammatory reaction in the alveolar space (Limper et al. 1996, 1997; Limper 1997).

The more rapid turnover of plasma membrane constituents of type-II cells may inhibit attachment of Pneumocystis. Also, type-II cells may have a direct toxic effect on Pneumocystis (Pesanti and Shanley 1988). There is evidence for Pneumocystis-induced reduced alveolar type-II epithelial cell function upon secretagogue stimulation (Rice et al. 1993).

Pulmonary surfactants appear to play a role in the disease process, containing mainly phospholipids and four surfactant-specific proteins (SP-A, SP-B, SP-C, and SP-D). Pulmonary surfactant protein A (SP-A), an alveolar glycoprotein containing collagen-like and carbohydrate-recognition domains, binds P. carinii and enhances adherence to alveolar macrophages (McCormack et al. 1997). Also, SP-D seems to facilitate binding of the parasite to alveolar macrophages. SP-A and SP-D may also mediate attachment to the type-I pneumocyte. In rodent models of PCP a reduction in phospholipid levels as well as a decrease in SP-B have been suggested to play an important role in the hypoxemic respiratory insufficiency associated with PCP (Beers et al. 1997).

Several aspects of Pneumocystis metabolism have been clarified recently, which have led to the recognition of novel therapeutic possibilities. Lipid transfer from human alveolar epithelial cells to P. carinii has been demonstrated (Furlong et al. 1997), and uncommon lipids have been identified in P. carinii. (Kaneshiro et al. 1989). The organism has the shikimic acid pathway that leads to the formation of compounds which mammals cannot synthesize (e.g. folic acid); hence drugs that inhibit these pathways are effective against the pathogen. The high concentration of free fatty acids and the relatively low level of triglycerides in P. carinii suggest that fatty acids may represent major carbon sources for ATP production by the organism (Ellis et al. 1996). Pneumocystis carinii also possesses the biochemical pathway for de novo synthesis of the CoQ benzoquinone ring (Sul and Kaneshiro 1997). Instead of ergosterol (the major sterol of higher fungi), P. carinii synthesizes distinct delta(7), C-24-alkylated sterols. An unusual C-32 sterol, pneumocysterol, has been identified in human-derived P. carinii. Another signature lipid discovered is cis-9,10-epoxy stearic acid. CoQ(10), identified as the major ubiquinone homologue, is synthesized de novo by P. carinii (Kaneshiro 1998). Pneumocystis carinii glycoprotein A stimulates interleukin-8 production and inflammatory cell activation in alveolar macrophages and cultured monocytes (Lipschik et al. 1996). The role of cytokines in PCP has been discussed by Perenboom et al. (1996). Vitronectin, fibronectin, and gp120 antibody enhance macrophage release of TNF-alpha in response to P. carinii (Neese et al. 1994) and the intercellular adhesion molecule-1 which is important in leukocyte accumulation is enhanced in lung epithelium during P. carinii infection, in part, through TNF-alpha-mediated mechanisms (Yu and Limper 1997).

During recovery from PCP in a dexamethasone rat model, the trophozoite-to-cyst ratio significantly changed. The number of cysts decreased from week 0 to week 4 whereas there was a much lesser decrease in the number of trophozoites suggesting that in particular the cyst form of P. carinii is sensitive to the host response mounted during recovery. The results support the possibility that trophozoites may multiply by extracystic asexual fission (Sukura 1995).

Immune response to clear the infection is manifested by phagocytosis by alveolar macrophages added by CD4+ lymphocytes and serum antibodies.

Pneumocystis carinii adheres to alveolar macrophages and is engulfed and digested in the presence of an opsonizing antibody. The adherence of P. carinii occurs via multiple pathways and stimulates oxidative burst, cytokine production, phagocytosis, and killing of the organism. A number of cytokines are involved in pneumonia. TNF-a and IL-1b are considered to be important mediators of host resistence against P. carinii (Chen et al. 1992a,b) while IL-6 is involved in the inflammatory and antibody responses during resolution of the infection (Chen et al. 1993). Pneumocystis carinii induces production of TNF-a by alveolar macrophages in vivo and in vitro. (Krishnan et al. 1990; Pesanti et al. 1991; Kandil et al. 1994). TNF-a release is also augmented by opsonization of P. carinii with not only antibodies but also adhesive glycoproteins (Neese et al. 1994)

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