Giardia lamblia: autoradiographic analysis of nuclear replication

Giardia lamblia trophozoites, grown in axenic culture, were labeled for various periods of time with [3H]thymidine. After autoradiography, grains were counted over each of the two nuclei in each trophozoite. Analysis of the fraction of trophozoites labeled for each time period resulted in an estimate of a generation time of 15 hr. The DNA synthetic or S phase for a trophozoite in culture was calculated to be 1.8 hr. G1 and G2 periods were determined to be 8.5 and 3 hr, respectively. A comparison of the labeling density between the two nuclei indicated that replication takes place simultaneously in both nuclei for at least 70% of S period. The fraction of asymmetrically labeled trophozoites is consistent with a model in which the nuclei replicate out of phase by 15-30 min, but, due to the small diameter of the nuclei relative to the grain size, the possibility that replication takes place simultaneously in both nuclei of a trophozoite throughout the S phase cannot be ruled out.     

Eugregarine trophozoite detachment from the host epithelium via epimerite retraction: Fiction or fact?

Eugregarines represent a diverse group of Apicomplexa parasitising numerous invertebrates. Their sporozoites generally develop into epicellular trophozoites attached to the host epithelium by a specialised attachment organelle known as an epimerite. They are considered peculiar protists due to their unique cell architecture and dimensions as well as their attachment strategy which is similar to that of cryptosporidia. Using electron and fluorescence microscopy, the fine structure of the epimerite with associated structures and the mechanism of trophozoite detachment from the host epithelium were studied in Gregarina polymorpha parasitising the intestine of Tenebrio molitor larvae. The epimerite appears to be a very dynamic structure whose shape dramatically changes depending on whether or not it is embedded into the host epithelium. The trophozoite’s most fragile zone is the area below the membrane fusion site at the epimerite base. The epimerite plasma membrane forms basal radial ribs which are involved in increasing its surface and strengthening the epimerite-host cell junction. FITC-phalloidin labelling demonstrated the presence of filamentous actin in trophozoites along with its accumulation at the epimerite base and in the apical end of the protomerite, as well as a patch accumulation of filamentous actin in the protomerite of maturing and mature trophozoites. Indirect immunofluorescence revealed the presence of myosin in the cortical zone of the epimerite and in the membrane fusion site area. The data obtained strongly suggest that these structures could facilitate the detachment of a mature trophozoite from the host epithelium. Supported by data presented herein and our previous observations, we propose a new hypothesis on the mechanism of trophozoite detachment from the host epithelium based on epimerite retraction into the protomerite. This is contrary to the commonly accepted hypothesis describing gradual epimerite constriction and subsequent separation facilitated by contractility of the membrane fusion site (osmiophilic ring).     

Pathogenesis of infection byEntamoeba histolytica

Entamoeba histolytica, a protozoan parasite, is the etiologic agent of amoebiasis in humans. It exists in two forms—the trophozoite which is the active, dividing form, and the cyst which is dormant and can survive for prolonged periods outside the host. In most infected individuals the trophozoites exist as commensals. In a small percentage of infections, the trophozoites become invasive and penetrate the intestinal mucosa, causing ulcers. The trophozoites may reach other parts of the body—mainly liver, where they cause tissue necrosis, leading to lifethreatening abscesses. It is thought that pathogenesis of infection byEntamoeba histolytica is governed at several levels, chief among them are (i) adherence of trophozoite to the target cell, (ii) lysis of target cell, and (iii) phagocytosis of target cell. Several molecules which may be involved in these processes have been identified. A lectin inhibitable by galactose and N-acetyl-D-galactosamine is present on the trophozoite surface. This is implicated in adherence of trophozoite to the target cell. Various amoebic poreforming proteins are known, of which 5kDa protein (amoebapore) has been extensively studied. These can insert into the lipid bilayers of target cells, forming ion-channels. The phagocytic potential of trophozoites is directly linked to virulence as measured in animal models. Factors like association of bacteria with trophozoites also influence virulence. Thus, pathogenesis is determined by multiple factors and a unifying picture taking into account the relative contributions of each factor is sought. Recent technical advances, which includes the development of a transfection system to introduce genes into trophozoites, should help to understand the mechanism of pathogenesis in amoebiasis.     

Giardia lamblia: Evaluation of roller bottle cultivation

A modified “outside-in” roller bottle with a high ratio of surface area to volume was used to cultivate Giardia lamblia. Yields were high, more so when bottles were rotated at 6 rph (9.3 ± 4.0 × 10⁸ trophozoites/bottle) than at 12 rph (4.2 ± 1.9 × 10⁸ trophozoites/bottle). The method was more efficient than stationary tube culture with respect to utilization of culture medium; trophozoite concentration after roller bottle culture (1.7 ± 0.8 × 10⁶ trophozoites/ml) was significantly higher (by a factor of 2.8) than concentrations obtained from stationary tube culture (0.6 ± 0.4 × 10⁶ trophozoites/ml, P < 0.002). Increased yields from roller bottle culture were not accounted for by a reduction in mean trophozoite generation time (roller culture, 10.7 ± 1.2 hr; stationary tube culture, 10.3 ± 0.6 hr) but may be related to prolongation of the period of log phase growth or increased trophozoite survival. Trophozoite yields expressed per unit surface area were significantly higher from roller bottle culture (7.2 ± 3.1 × 10⁵ trophozoites/cm²) than from stationary tubes (1.9 ± 1.0 × 10⁵ trophozoites/cm², P < 0.002). Attempts to cultivate G. lamblia in spin culture using polystyrene beads (Biosilon) as a microcarrier were unsuccessful, trophozoite growth being inhibited rather than promoted. Roller bottle culture of G. lamblia, however, is efficient, economical, and less laborious than stationary tube culture, particularly when more than 10⁸ trophozoites are required.     

Plasmodium falciparum: isolation and purification of spontaneously released merozoites by nylon membrane sieves

A new procedure for isolating spontaneously released merozoites from in vitro cultures of Plasmodium falciparum (FVO and FCB strains) is described. The mature forms of relatively synchronous cultures containing predominantly trophozoites and few schizonts were concentrated with Plasmagel and then incubated at 37 C, without adding fresh red blood cells, until trophozoites matured into schizonts. Merozoites which were subsequently released were harvested and freed from host red blood cell material by low-speed centrifugations and nylon membrane sieves (3- and 1.2-μm pore size). From a culture containing about 5.2 × 10⁹ mature-form parasites, a total of about 10.7 × 10⁹ merozoites were released during three consecutive harvests and about 69% of these merozoites were recovered after the isolation and purification procedures. As demonstrated by both light and electron microscopy, most merozoites were morphologically intact and the merozoite preparations were free of host cell constituents. SDS-acrylamide gel electrophoresis confirmed the absence of host cell material and also showed that merozoites had a complex protein pattern of apparent molecular weights between 225 and 15 kdaltons. Such purified merozoite preparations will be invaluable for malaria immunization studies, for identification of protective antigens of P. falciparum, and for other immunological and biochemical studies.     

Inhibition of Giardia intestinalis by Extracellular Factors from Lactobacilli: an In Vitro Study

The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent. Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G₁ phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation of Giardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.     

A Detailed, Hierarchical Study of Giardia lamblia's Ventral Disc Reveals Novel Microtubule-Associated Protein Complexes

Giardia lamblia is a flagellated, unicellular parasite of mammals infecting over one billion people worldwide. Giardia''s two-stage life cycle includes a motile trophozoite stage that colonizes the host small intestine and an infectious cyst form that can persist in the environment. Similar to many eukaryotic cells, Giardia contains several complex microtubule arrays that are involved in motility, chromosome segregation, organelle transport, maintenance of cell shape and transformation between the two life cycle stages. Giardia trophozoites also possess a unique spiral microtubule array, the ventral disc, made of approximately 50 parallel microtubules and associated microribbons, as well as a variety of associated proteins. The ventral disc maintains trophozoite attachment to the host intestinal epithelium. With the help of a combined SEM/microtome based slice and view method called 3View® (Gatan Inc., Pleasanton, CA), we present an entire trophozoite cell reconstruction and describe the arrangement of the major cytoskeletal elements. To aid in future analyses of disc-mediated attachment, we used electron-tomography of freeze-substituted, plastic-embedded trophozoites to explore the detailed architecture of ventral disc microtubules and their associated components. Lastly, we examined the disc microtubule array in three dimensions in unprecedented detail using cryo-electron tomography combined with internal sub-tomogram volume averaging of repetitive domains. We discovered details of protein complexes stabilizing microtubules by attachment to their inner and outer wall. A unique tri-laminar microribbon structure is attached vertically to the disc microtubules and is connected to neighboring microribbons via crossbridges. This work provides novel insight into the structure of the ventral disc microtubules, microribbons and associated proteins. Knowledge of the components comprising these structures and their three-dimensional organization is crucial toward understanding how attachment via the ventral disc occurs in vivo.     

Culture and filtration methods for obtaining Pneumocystis carinii trophozoites and cysts.

Two methods for acquisition of Pneumocystis carinii (Pc) trophozoites and cysts are reported. One method, the isolation of Pc from infected rat lung, provides large numbers of trophozoites and cysts but retains rat proteins. Ground lung is filtered through a series of Nucleopore filters from 10 to 3 microns; 1 g of rat lung yields an average of 1.1 x 10(9) Pc trophozoites and 1 x 10(7) cysts. The second method, propagation of Pc in culture with human embryonic lung cells on microcarrier beads, provides Pc trophozoites which are relatively free of host lung material. Cultured organisms may be filtered to remove rare culture monolayer cells. Organisms harvested from filtered lung are free from intact host cells and cell nuclei, however, host cell proteins and host DNA remain. Organisms from culture have minimal host contamination.     

Ultrastructural observations on the attachment of Pneumocystis carinii in vitro.

Pneumocystis carinii trophozoites grow in vivo in close contact with host cells. The attachment of Pneumocystis to the lung cells seems to be a critical step in the parasite's development. Up to now, the contact of Pneumocystis with mammalian tissue culture cells was shown using light and scanning electron microscopy. The methods are not sufficient to observed in detail the parasite-feeder cell area of contact. In this work, the attachment of Pneumocystis trophozoites to feeder cells was examined in serial sections using transmission electron microscopy. When the contact of a trophozoite with a feeder cell took place, the development of filopodia penetrating deeply into invaginations of the feeder cell plasma membrane was observed. Then, the apical tips of filopodia become bulged anchoring the parasite to the feeder cell. The behaviour of Pneumocystis in feeder cell cultures is compared to that of the parasite in other in vitro or in vivo experimental models.     

Directed migration of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) in its natural host Aedes albopictus (Diptera: Culicidae)

Directed migration of trophozoites from the midgut toward the Malpighian tubules is essential for Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) to complete its developmental cycle within the natural host Aedes albopictus. We have obtained a 275-bp actin cDNA fragment amplified from extracted mRNAs of migrating trophozoites, suggesting the involvement of actin in trophozoite motility. Down-regulation on the migration of the trophozoite was seen in mosquito larvae fed with cytochalasin D, ML-7, and BDM, indicating that myosin, in the form of an actomyosin system, may also be involved in driving motility of the trophozoite. The "protruding apparatus" (PA) formed at the anterior end of trophozoites during the migrating stage had significant deposits of actin by immunofluorescent microscopy. Moreover, PA formation was enhanced in response to elevated levels of 20-hydroxyecdysone (20-HE) in cultures of alimentary canals in which the trophozite was contained. Thus, 20-HE may also promote expression of actin and perhaps myosin simultaneously.     

Permselectivity changes in malaria (Plasmodium falciparum) infected human red blood cell membranes

The development of the malaria parasite Plasmodium falciparum in human red blood cells induces parasite-dependent perturbations in the permselectivity properties of the host cell membrane. The changes appear as parasites develop from ring to the trophozoite stage and persist during schizogony. In the present work we assessed the permeability changes of the infected cells to anionic substances by the use of radioactive and fluorescent probes. Our data show that i) covalent binding probes, such as diisothiocyano ditritiostilbene disulfonic acid [3H]H2DIDS, which are virtually impermeant to normal red blood cells, became markedly permeant to trophozoites and schizonts, as evidenced by high labeling of intracellular hemoglobin; ii) permeation of the fluorescent anion transport substrate NBD-taurine, measured in the efflux mode, was very rapid and substantially enhanced in parasitized erythrocytes, as compared with noninfected cells; iii) this efflux could not be blocked by H2DIDS, which is a specific inhibitor of anion transport in normal red blood cells; iv) permeation of anionic probes was temperature dependent (Ea:11 +/- 1 kcal/mole); and v) could be blocked by nonspecific transport inhibitors that are known to interact with membrane lipids. The appearance of a new permeation pathway in the host cell membrane of trophozoites is associated with structural modification of the host cell membrane matrix.     

New advances in the in-vitro culture of Dientamoeba fragilis

SUMMARYDientamoeba fragilis is an intestinal protozoan in humans that is commonly associated with diarrhoea and other gastrointestinal complaints. Studies conducted to investigate the biology of this parasite are limited by methods for in vitro cultivation. The objective of this study was to improve a biphasic culture medium, based on the Loeffler's slope, by further supplementation in order to increase the yield of trophozoites in culture. The current in vitro culture of D. fragilis is a xenic culture with a mix of bacteria. Three different liquid overlays were evaluated including Earle's balanced salt solution (EBSS), PBS and Dulbecco's modified PBS (DPBS), for their ability to support the in vitro growth of D. fragilis trophozoites. Out of these 3 overlays EBSS gave the highest increase in the trophozoite numbers. The effect of supplementation was analysed by supplementing EBSS with ascorbic acid, ferric ammonium citrate, L-cysteine, cholesterol and alpha-lipoic acid and quantification of in vitro growth by cell counts. A new liquid overlay is here described based upon EBSS supplemented with cholesterol and ferric ammonium citrate that, in conjunction with the Loeffler's slope, supports the growth of D. fragilis trophozoites in vitro. This modified overlay supported a 2-fold increase in the numbers of trophozoite in culture from all 4 D. fragilis isolates tested, when compared to a PBS overlay. These advances enable the harvest of a larger number of trophozoites needed for further studies on this parasite.     

Functional characterization of EhADH112: an Entamoeba histolytica Bro1 domain-containing protein

EhADH112 is part of the EhCPADH complex, a protein involved in key events of the Entamoeba histolytica host invasion. EhADH112 participates in trophozoite adherence to target cells and in phagocytosis. We report here the finding of two EhADH112 homologues in the E. histolytica genome (EhADH112-like proteins). EhADH112 and its relatives have a Bro1 domain at their amino-terminus and a consensus context for phosphorylation by Src-tyrosine kinases, both involved in signal transduction processes in other organisms. Our findings associate EhADH112 to supplementary functions related to those reported for the Alix/AIP1 family. To elucidate the precise function of EhADH112, we studied the phenotypes displayed by trophozoites transfected with the Ehadh112 full gene. Transfected trophozoites overexpressed a 78 kDa protein, which was mainly targeted to the EhCPADH complex. Moreover, these trophozoites exhibited enhanced phagocytic rates, providing further evidence of EhADH112 contribution to adhesion and phagocytosis activities.     

FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells

Background

During its intraerythrocytic asexual reproduction cycle Plasmodium falciparum consumes up to 80% of the host cell hemoglobin, in large excess over its metabolic needs. A model of the homeostasis of falciparum-infected red blood cells suggested an explanation based on the need to reduce the colloid-osmotic pressure within the host cell to prevent its premature lysis. Critical for this hypothesis was that the hemoglobin concentration within the host cell be progressively reduced from the trophozoite stage onwards.

Methodology/Principal Findings

The experiments reported here were designed to test this hypothesis by direct measurements of the hemoglobin concentration in live, infected red cells. We developed a novel, non-invasive method to quantify the hemoglobin concentration in single cells, based on Förster resonance energy transfer between hemoglobin molecules and the fluorophore calcein. Fluorescence lifetime imaging allowed the quantitative mapping of the hemoglobin concentration within the cells. The average fluorescence lifetimes of uninfected cohorts was 270±30 ps (mean±SD; N = 45). In the cytoplasm of infected cells the fluorescence lifetime of calcein ranged from 290±20 ps for cells with ring stage parasites to 590±13 ps and 1050±60 ps for cells with young trophozoites and late stage trophozoite/ early schizonts, respectively. This was equivalent to reductions in hemoglobin concentration spanning the range from 7.3 to 2.3 mM, in line with the model predictions. An unexpected ancillary finding was the existence of a microdomain under the host cell membrane with reduced calcein quenching by hemoglobin in cells with mature trophozoite stage parasites.

Conclusions/Significance

The results support the predictions of the colloid-osmotic hypothesis and provide a better understanding of the homeostasis of malaria-infected red cells. In addition, they revealed the existence of a distinct peripheral microdomain in the host cell with limited access to hemoglobin molecules indicating the concentration of substantial amounts of parasite-exported material.     

Pathogenic bacteria prime the induction of Toll-like receptor signalling in human colonic cells by the Gal/GalNAc lectin Carbohydrate Recognition Domain of Entamoeba histolytica

In mixed intestinal infections with Entamoeba histolytica trophozoites and enteropathogenic bacteria, which are wide-spread in areas of endemic amoebiasis, interaction between the pathogens could be an important factor in the occurrence of invasive disease. It has been reported that exposure of human colonic cells to enteropathogenic bacteria increased trophozoite adherence to the cells and their subsequent damage. We report here that the Carbohydrate Recognition Domain (CRD) of the amoebic Gal/GalNAc lectin binds to Toll-like receptors TLR-2 and TLR-4 in human colonic cells, activating the “classic” signalling pathway of these receptors. Activation induced expression of TLR-2 and TLR-4 mRNAs and the mRNAs of pro-inflammatory cytokines, as well as an increase in the corresponding proteins. Direct correlation was observed between the increased expression of TLRs and pro-inflammatory cytokines, the enhanced adhesion of trophozoites to the cells and the inflicted cell damage. When cells were exposed to pathogenic bacteria Staphylococcus aureus (Gram+) or Shigella dysenteriae (Gram−), elements of an innate immune response were induced. CRD by itself elicited a similar cell response, while exposure to a commensal Escherichia coli had a null effect. Pre-exposure of the cells to pathogenic bacteria and then to CRD rendered an inflammatory-like microenvironment that after addition of trophozoites facilitated greater cell destruction. Our results suggest that CRD is recognised by human colonic cells as a pathogen-associated-molecular-pattern-like molecule and as such can induce the expression of elements of an innate immune response. In the human host, an exacerbated inflammatory environment, derived from pathogen interplay, may be an important factor for development of invasive disease.     

In vitro effects of multi-purpose contact lens disinfecting solutions towards survivability of Acanthamoeba genotype T4 in Malaysia

The incidence of Acanthamoeba keratitis has been increasing since the previous decades, especially among contact lens users. This infection is majorly caused by the use of ineffective contact lens disinfecting solution. Thus, this study was conducted to evaluate the in vitro effects of multi-purpose disinfecting solutions (MPDS) against Acanthamoeba trophozoites and cysts. Acanthamoeba genotype T4 isolated from contact lens paraphernalia and an environmental strains were propagated for trophozoite or cyst-containing culture and adjusted in final concentration of 1 × 10⁵ cells/ml. Amoebicidal and cysticidal assays were conducted by incubating trophozoites and cysts with OPTI-FREE® Express®, ReNu® Fresh™, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive according to the manufacturer’s minimum recommended disinfectant time (MMRDT) for up to 12 h at 30 ⁰C. Trypan blue hemocytometer-based microscopic counts determined amoebicidal and cysticidal effects. The viability of Acanthamoeba trophozoites and cysts was confirmed by re-inoculated them in the 1.5% non-nutrient agar plates. It was found that none of the MPDS showed amoebicidal and cysticidal effects during the MMRDT. However, OPTI-FREE® Express® demonstrated a significant differences in average cell reduction for both stages within MMRDT. When subjected to 12 h exposure, both OPTI-FREE® Express® and ReNu® Fresh™ led to significant reduction in the number of trophozoite and cyst cells. Notably, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive did appreciably improve the solution effectiveness towards trophozoite cells when incubated for 12 h. All MPDS were largely ineffective, with 100% survival of all isolates at MMRDT, while OPTI-FREE® Express® showed limited amoebicidal activity against the contact lens paraphernalia isolate, however, it was more against the environmental strains after 12 h incubation time. The commercially available MPDS employed in this research offered minimal effectiveness against the protozoa despite the contact time. Improvement or development of new solution should consider the adjustment of the appropriate disinfectant concentration, adequate exposure time or the incorporation of novel chemical elements, which are effective against Acanthamoeba for accelerated disinfecting and more reduction of potential exposure of contact lens users to Acanthamoeba keratitis.     

Fine structure of trophozoites of the gregarine Leidyana ephestiae (Apicomplexa: Eugregarinida) parasitic in Ephestia kuehniella larvae (Lepidoptera)

The ultrastructure of the eugregarine Leidyana ephestiae, parasitic in the larval gut of the flour moth, Ephestia kuehniella, is described. Guts of experimentally infected larvae of E. kuehniella were fixed and sectioned for electron microscope studies of young and mature trophozoites. Young unsegmented trophozoites were small, oval to ovoid, and possessed a simple, globular epimerite. The plasma membrane covering the epimerite region was continuous with the plasma membrane of the protodeutomerite and was in close contact with that of the host cell. Three cortical membranes covered the protodeutomerite region. Folding of the protodeutomeritic epicyte occurred after about 2 days of development of the gregarine. After 3–4 days the body of the trophozoite became differentiated into three segments. A septum was visible between protomerite and deutomerite, but there was nothing similar to this structure between epimerite and protomerite. Fully developed trophozoites showed a large ovoid epimerite containing many mitochondria and vesicles. The epimerite was situated on a short neck filled with fibrils. The cytoplasm of protomerite and deutomerite was rich in amylopectin granules and electron-dense bodies.     

Cell Wall Antigens of Pneumocystis carinii Trophozoites and Cysts: Purification and Carbohydrate Analysis of these Glycoproteins

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat-derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life-cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose. and N-acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species-specificity of glycoprotein glycosylation, preparations of human-derived P. carinii (comprised of mixed life-cycle stages) were also examined and found to contain the same sugars as those found in rat-derived organisms. Most of the purified rat-derived glycoproteins bound Concanavalin A, which was abolished by treatment with N-glycanase. This suggested that the majority of the oligosaccharides were N-linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host. Supplementary key words. Biochemical analysis, developmental stages, opportunistic pathogen, structure.