Gene deletion suggests a role for Trypanosoma cruzi surface glycoprotein GP72 in the insect and mammalian stages of the life cycle

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1 Journal of Cell Science 106, (1993) Printed in Great Britain The Company of Biologists Limited Gene deletion suggests a role for Trypanosoma cruzi surface glycoprotein GP72 in the insect and mammalian stages of the life cycle Amelia Ribeiro de Jesus 1, *, Robin Cooper 1,, Martha Espinosa 2, José Eugenio P. L. Gomes 3, Eloi S. Garcia 3, Simon Paul 1 and George A. M. Cross 1, 1 Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA 2 Centro de Investigacion y de Estudios Avanzados del IPN, Apartado Postal , Mexico DF, CP Department of Biochemistry and Molecular Biology, Oswaldo Cruz Foundation, Avenida Brasil 4365, CEP , Rio de Janeiro, RJ, Brazil *Present address: Hospital University Prof. Edgard Santos, Laboratório de Imunologia, Rua João das Botas, Salvador, Bahia, Brazil Present address: Casilla de Correo 248, Montevideo, Uruguay Author for correspondence SUMMARY We have explored the biological function of a surface glycoprotein (GP72) of Trypanosoma cruzi by studying a null mutant parasite, generated by targeted gene deletion. GP72 deletion affected parasite morphology in several stages of the life cycle. Insect midgut (epimastigote) forms had a detached flagellum (apomastigote) in the null mutant. The abnormal flagellar phenotype persisted during development of the infective (metacyclic) forms but there was no impairment in the aquisition of complement resistance, sialidase expression or cell infectivity. The GP72 null mutant could efficiently infect and proliferate in mouse macrophages and non-phagocytic L6E9 cells. The mammalian stages of the life cycle also showed major morphological abnormalities. During early subcultures in L6E9 cells, few extracellular fully flagellated forms, expressing markers characteristic of trypomastigotes, were seen. The extracellular population consisted almost exclusively of rounded forms with short flagella (micromastigote), which expressed an amastigote-specific surface marker and no sialidase. The propagation of the parasite was not affected, despite the apparent lack of the trypomastigote forms, which are thought to be primarily responsible for cell invasion. After some subcultures, the extracellular population changed to about equal numbers of micromastigotes and a range of flagellated forms that still did not include true trypomastigotes. Instead, the kinetoplast remained close to the nucleus and the flagellum emerged from the middle of the cell (mesomastigote). Half of the flagellum adhered to the cell body and the remainder was free at the anterior end. In Triatoma infestans, the survival of the mutant was dramatically reduced, suggesting that either GP72 itself, or the altered properties of the flagellum, were critical for establishment in the insect vector. Key words: Trypanosoma cruzi, flagellum, surface glycoprotein, gene disruption INTRODUCTION Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle consisting of four major stages. The parasite undergoes distinct biochemical and morphological changes during the life-cycle (Andrews et al., 1987; Garcia and Azambuja, 1991; Nogueira et al., 1981; Nogueira et al., 1982; Snary et al., 1981). These changes are important for survival within the gut of the insect vector, for invasion of and survival within mammalian cells, and for evading the immune system of the mammalian host. The nomenclature of the distinct morphological forms of trypanosomes (Hoare, 1972) is based on the point of emergence of the flagellum from the cell body and whether the kinetoplast (the mitochondrial DNA) is anterior or posterior to the nucleus, in relation to the direction of locomotion (the flagellum points forwards). The emergence of the flagellum from the cell is always coupled to the position of the kinetoplast (Robinson and Gull, 1991). The parasite multiplies as an epimastigote form within the gut of its invertebrate host, the reduviid bug, where it differentiates into metacyclic trypomastigotes, non-dividing invasive forms that are transmitted to the mammalian host in the insect feces by contaminative infection. In the mammalian host, T. cruzi invades macrophages and muscle cells and multiplies as an amastigote form (lacking a flagellum), which differentiates into trypomastigote forms, subsequently released to infect other cells. Several stage-specific surface antigens of T. cruzi have been cloned and sequenced (for review see Frasch et al., 1991). Efforts to establish their functions have been hampered by the lack of sexual exchange in this species and,

2 1024 A. Ribeiro de Jesus and others until recently, the unavailability of methods for direct genetic manipulation. We previously cloned the gene encoding what was thought to be an insect stage-specific 72 kda glycoprotein, GP72 (Cooper et al., 1991), expressed primarily in the insect epimastigote and, to a lesser extent, in metacyclic trypomastigote stages. Prior efforts to establish the function of GP72 had suggested that it might control the differentiation of the non-infective epimastigote into the infective metacyclic trypomastigote (Sher and Snary, 1982). Knowledge of the amino acid sequence of GP72 provided no additional clues to its function (Cooper et al., 1991). Using targeted gene replacement by homologous recombination, we created a GP72 null mutant in T. cruzi epimastigotes (Cooper et al., 1993). The epimastigote from the null mutant (YNIH D5C3) had an unexpected morphology: the flagellum, instead of adhering to the cell body after emerging from the flagellar pocket, was completely detached, and the overall shape of the parasite was altered. We have now investigated the behavior of the null mutant throughout the life-cycle, using morphological, biochemical and functional parameters. MATERIALS AND METHODS Parasites T. cruzi YNIH and Dm28c (obtained from Dr Samuel Goldenberg, Instituto Oswaldo Cruz) strain epimastigotes were grown in liver infusion tryptose (LIT) medium (Nogueira et al., 1981) at C without agitation. Tissue culture trypomastigotes (TCT) and amastigotes were grown with monolayers of gamma-irradiated (3,000 rads) L6E9 cells, a rat skeletal muscle cell line, in Dulbecco s Minimal Essential Medium supplemented with 10% heat-inactivated fetal bovine serum (Nogueira et al., 1982). Complement-mediated lysis Metacyclogenesis was induced in vitro in YNIH wild-type and GP72 null mutants by incubating late log-phase epimastigotes in TAU3AAG medium plus 0.035% sodium carbonate for hours (Goldenberg et al., 1987). Transformation of the parasite was assessed by resistance to complement lysis. Serum obtained from a healthy human volunteer was used as a source of complement. The parasites were washed with Eagle s Minimal Essential Medium (MEM) and resuspended to m l 1 i n MEM. Then 50 µl samples of parasites were mixed with equal volumes of 60% (v/v) fresh or heat-inactivated (56 C for 1 hour) serum (final concentration of 30% (v/v)) and incubated for 1 hour at 37 C. Surviving parasites were counted in a hemocytometer. Indirect immunofluorescence studies mab WIC 29.26, which recognizes a glycan epitope on GP72 (Ferguson et al., 1983), was purified from ascitic fluid by ammonium sulfate precipitation and used at a 1:50 or 1:100 dilution in PBS containing 1% BSA. Trypomastigote and amastigote-specific mabs used in these studies were a generous gift from Dr Norma Andrews, Yale University School of Medicine. They were purified by ion-exchange chromatography. Antibodies 2H11 and 2A1 recognize trypomastigote-specific antigens Ssp1 and Ssp2, and were used separately or together. Antibody 2C2 recognizes the amastigote specific antigen Ssp4 (Andrews et al., 1987). These antibodies were used at a concentration of 50 µg ml 1 in PBS plus 1% BSA. Trypanosomes, at a concentration of ml 1, were fixed overnight at 4 C in 2% paraformaldehyde, spread onto slides and air dried. The slides were rehydrated in PBS containing 0.2% BSA and incubated with mab for 1 hour. All incubations were carried out at room temperature in a humid chamber. The slides were then washed 3 times in PBS, 0.2% BSA and incubated with a FITCconjugated goat anti-mouse IgG/IgM antibody (Jackson ImmunoResearch, West Grove, Pennsylvania) diluted 1:100 in PBS, 1% BSA. After 3 washes in the same buffer, the slides were stained with % Hoechst stain in PBS, washed in water and air dried. The slides were mounted with 25 mm Tris- HCl, ph 8.0, 50% glycerol and visualized using a Nikon Optiphot microscope, with an excitation wavelength of nm and a 520 nm emission filter. In general, 1 to 4 minute exposures were taken using Kodak Ektachrome P800/1600 film pushed to 1600 ASA. Invasion assays Mouse macrophages were obtained by peritoneal lavage of CD1 mice with PBS plus heparin. The cells were adjusted to 10 6 m l 1 in RPMI 1640 (Gibco) supplemented with glutamine, HEPES, sodium bicarbonate and 10% heat-inactivated fetal bovine serum and plated in 24-well plates (1 ml per well) containing sterile glass coverslips. Macrophages were allowed to attach to the coverslips for 48 hours at 37 C. Each coverslip was incubated with a mixture of epimastigotes and metacyclic trypomastigotes obtained from TAU3AAG medium. After 2 hours, unattached parasites were washed off. After 48 to 120 hours, the coverslips were fixed in 2.5% glutaraldehyde in PBS, stained in % Hoechst stain, and visualized using a Nikon Optiphot microscope with an excitation wavelength of nm and an emission filter of 420 nm. L6E9 cells were infected with complement-resistant metacyclic trypomastigotes produced in vitro. A mixture of amastigotes and trypomastigotes isolated from the supernatants of these cultures were used to infect other cultures of L6E9 cells. After three subcultures in L 6 E 9 cells, these parasites were used to infect irradiated L6E9 cells attached to round glass coverslips, for quantitative analysis of invasion. At 24 hours after infection, unattached parasites were washed off and, at intervals of 24, 48, 72 and 120 hours, the coverslips were fixed and stained with Hoechst 33258, as described. Infection rates were determined by averaging the counts from triplicate samples, measured by two observers who did not know the identities of the samples. Infection of mice Female C3H mice (6- to 8-weeks-old) were injected intraperitoneally with mixtures of 10 5, 10 6, or 10 7 trypomastigotes and amastigotes derived from L6E9 cells. At intervals of 4 to 7 days, blood was collected from the tail veins and parasitemias determined by viewing 100 fields at 400 magnification of Giemsastained thin smears. Scanning electron microscopy Log phase epimastigotes, grown in LIT medium, and a mixture of amastigotes and trypomastigotes from L6E9 infected cells, were harvested and fixed in 2% glutaraldehyde in PBS and processed as described (Andrews et al., 1987). The samples were criticalpoint dried (maximum temperature 34 C; maximum pressure 110 pounds per square inch (750 kpa)) using a Samdri apparatus (Tousimis Research Corporation, Rockville, Maryland) and lightly coated with gold (approximately 30 nm thick) with a Jeol-110 ion sputtering device and examined in a Jeol 35C scanning electron microscope set at 10 kv, to reduce charging. Sialidase activity assay Cells, at a concentration of 10 9 ml 1, were washed once in PBS

3 Trypanosoma cruzi developmental mutant 1025 and lysed in PBS, 0.5% NP40, 1mM PMSF, 0.5 µg ml 1 leupeptin. Then 10 µl of each lysate was tested for sialidase activity in duplicate, by measuring the hydrolysis of the fluorogenic substrate 2 -(4-methylumbelliferyl) α-d-n-acetylneuraminic acid (4MU-NANA) as described (Schenkman et al., 1992). The reaction was carried out with 0.1 mm MU-NANA (Sigma) in 0.17 M sodium acetate, ph 6.0, 0.5 mg ml 1 BSA, in a total volume of 50 µl, for 8 hours at 37 C. The reaction was stopped by adding 150 µl of 0.1 M glycine, ph Fluorescence was measured using a Titertek Multiskan fluorometer, excitation 355 nm, emission 480 nm. Enzyme activity was calculated by comparison with a Clostridium perfringens sialidase (Type V, Sigma) of known activity (1 unit = 1 µmole min 1 ). Surviving parasites (%) Insect infections Fifth-instar larvae of Triatoma infestans were reared synchronously from eggs (Garcia and Azambuja, 1991). The experimental insects, weighing 34.7±2.2 mg (n=40), which had been starved for days, were allowed to feed on citrated and de-complemented rabbit blood containing epimastigotes ml 1 of the wild-type or null mutants. Only fully gorged insects were used for these experiments; partially fed ones were discarded. After 0, 10, 20 and 30 days, the intestinal tract (crop, midgut and rectum) was removed and gently homogenized in 1 ml PBS. The total number of parasites, and the percentage of metacyclic trypomastigotes (identified by morphology), was established using a Neubauer hemocytometer. Six insects were used for each time point with each of the parasite strains. RESULTS In vitro metacyclogenesis of the GP72 null mutant We evaluated the ability of the GP72 null mutant, YNIH D5C3, to differentiate from epimastigotes to metacyclic trypomastigotes in an in vitro system that mimics insect urine. Late log-phase epimastigotes from the parental wild-type strain, YNIH, the null mutant, and a third strain, Dm28c, which differentiates with high efficiency, were incubated in TAU3AAG medium for hours. Samples of these parasites and mid-log-phase parasites obtained directly from LIT medium, were incubated with 30% of fresh or heatinactivated human serum and surviving parasites counted. We found little difference in the metacyclogenesis rate among the three strains (Fig. 1). The mean differentiation rate for parasites incubated in TAU3AAG medium was 18%, 30% and 32% for YNIH, YNIH D5C3 and Dm28c, respectively. Parasites that were growing in LIT medium taken at mid-log-phase had very low differentiation rates: 0.56%, 0% and 1.5% for YNIH, YNIH D5C3 and Dm28c, respectively. Expression of GP72 in epimastigotes and metacyclic trypomastigotes The mab WIC29.26 recognizes a glycan epitope on GP72 (Ferguson et al., 1983). Western blot analysis demonstrated that the GP72 null mutant lacks a 72 kda glycoprotein, but other bands that have the same glycan epitope remain (Cooper et al., 1993). Immunofluorescence was performed to clarify the location of GP72. In YNIH wild-type epimastigotes, WIC reacted with the entire surface, but a concentration of reaction was seen in the flagellar region, and at the posterior end of the parasite (Fig. 2A-C). In wildtype metacyclic trypomastigotes, mab WIC reaction 0 TAU LIT TAU LIT TAU LIT YNIH YNIH D5C3 Dm28c Fig. 1. Metacyclogenesis induced in vitro by TAU3AAG medium and analyzed by parasite resistance to complement lysis. The results shown are the means ± s.d. of 5 experiments from pairwise comparisons of the percentage survival, in fresh versus heatinactivated serum, of parasites from TAU or LIT media. was much weaker and more distinctly restricted to the flagellum and an area at the posterior end (Fig. 2A-C). In the null mutant epimastigote, mab WIC reacted with the surface but less strongly with the flagellum, especially in the third of the flagellum close to the cell body (Fig. 2D- F). In the GP72 null mutant we observed two morphologically distinct metacyclic trypomastigotes. Both forms exhibited a detached flagellum, a thinner cell body than epimastigotes, and an elongated nucleus (Fig. 2G-M). The first type had the kinetoplast at one end of the nucleus (due to the flagellum abnormality one cannot determine the anterior and posterior ends of the parasite). This form of complement-resistant parasite was positive for the mab WIC epitope at the end of the flagellum and part of the cell body (Fig. 2G,H). The second form had a similar aspect but the nucleus was thinner and the kinetoplast was at the middle of the parasite superimposed on the nucleus, as if it was migrating, as is observed in the process of differentiation from epimastigote to trypomastigote forms. In this form we observed two patterns of mab WIC reaction: very weak internal fluorescence at the cell body only (Fig. 2I,J) or negative (Fig. 2K-M). The first type is possibly starting to differentiate and, although it is complement resistant, the kinetoplast is still in the anterior end. In the second form the kinetoplast is migrating to the posterior end. No parasites were found where the kinetoplast had migrated completely to the posterior end, as in normal trypomastigotes. These data suggest that kinetoplast migration in metacyclics produced in vitro was affected by GP72 deletion. The absence of fluorescence in the differentiated null mutant suggests that, in metacyclic trypomastigotes, the epitope recognized by mab WIC is expressed only on GP72. Because the flagellum emerges close to the middle of the parasite cell body of the metacyclic forms and the tissue culture-derived flagellated forms (data shown later), we have adopted the term mesomastigote for these forms (meso, middle), following standard nomenclature (Hoare, 1972).

4 1026 A. Ribeiro de Jesus and others Fig. 2. Expression of GP72 in epimastigotes and metacyclic trypomastigotes from YNIH wild-type and GP72 null mutants, determined by indirect immunofluorescence with mab WIC (A) FITC staining and (B) Hoechst staining of YNIH wild-type epimastigote (upper) and metacyclic (lower); (C) FITC + Hoechst staining of another wild-type epimastigote (right) and metacyclic (left); (D) FITC and (E) Hoechst of two GP72 null mutant epimastigotes; (F) FITC + Hoechst of another GP72 null mutant epimastigote. (G-M) Metacyclic trypomastigotes from GP72 null mutant: (G) FITC and (H) Hoechst show a metacyclic with a kinetoplast at one end of the nucleus; (I) FITC and (J) Hoechst show a metacyclic in which the kinetoplast is superimposed on the nucleus. This form shows a weakly positive FITC fluorescence; (K) Phase, (L) FITC and (M) Hoechst of a FITC positive (left) and FITC negative (right) metacyclic, the latter showing a more elongated nucleus with a superimposed kinetoplast. The positions of the nucleus (n) and kinetoplast (k) are indicated by letters on representative panels. The arrows in (K) and (M) indicate the emergence of the flagellum from the cell body. Bars, 5 µm. In vitro infectivity of the GP72 null mutant Mouse peritoneal macrophages were infected with a mixture of epimastigotes and metacyclic trypomastigotes obtained from TAU3AAG medium and incubated for 48 to 120 hours. We observed many macrophages with large numbers of amastigotes in their cytoplasm. The null mutant motility was somewhat affected by the abnormal flagellum morphology. The parasite sank to the bottom of the culture, making it difficult to perform a meaningful quantitative analysis of the infection rate. Qualitative assessment suggested no significant difference in infectivity between the parental parasite and the null mutant.

5 Trypanosoma cruzi developmental mutant 1027 To ensure that the macrophage infectivity of the null mutant was not dependent on phagocytosis, L6E9 cells, a non-phagocytic rat skeletal muscle cell line, were also infected with the null mutant. The null mutant was able to infect and replicate within L6E9 cells. However, until the fourth subculture, null mutants, unlike the wild-type, did not transform to trypomastigotes. Instead, most of the L6E9 cells released micromastigote forms, resembling amastigotes but with a short flagellum (see later). All cultures were always infected with a 5:1 ratio of parasites to L6E9. In these experiments, the number of flagellated forms (mesomastigotes, as these forms had the kinetoplast and flagellar pocket in the middle of the cell body) in the null mutant was <1% of the total number of cells collected from the culture supernatant. In contrast, in the wild-type, >30% of the released cells were trypomastigotes. After 5 generations and changing the infection ratio to 10:1, the number of mesomastigotes in the null mutant-infected cultures increased considerably, and up to 10% mesomastigotes were detected in these cultures. Quantitative analysis of the trypomastigotes or mesomastigotes in L6E9 culture supernatants was performed on different days. We observed a variation in the percentage of trypomastigotes or mesomastigotes, versus amastigotes or micromastigotes in these cultures on different days but no significant differences were found between the YNIH wild-type (6.8-34%) and GP72 null mutant ( %) (P=0.047). The infectivity and growth kinetics of the parental and null mutant strains were compared using an in vitro assay with L6E9 cells attached to glass coverslips. L6E9 cell cultures were infected with equal numbers of YNIH and null mutant parasites taken from the supernatant of third subcultures of L6E9 cells. At intervals, the ratio of parasites to L6E9 cells determined. No difference in infectivity was found between the two strains (Fig. 3). Equal numbers of internalized parasites were found 24 hours after infection. As the supernatant from the third subculture of L6E9 cells infected with GP72 null mutant contained almost entirely micromastigotes (<1% trypomastigotes), we concluded that these forms were able to infect L6E9 cells with about the same efficiency as wild-type cell populations, containing 30% trypomastigotes. In addition, the growth rate of the amastigotes within L6E9 cells was also similar as indicated by the number of parasites per cell at 72 and 120 hours post-infection. Attempts were made to compare the infectivity of wildtype and GP72 null mutant strains in vivo. A highly susceptible strain of mouse, C3H, was inoculated with mixtures of trypomastigotes and amastigotes obtained from L6E9 cultures. However, despite several attempts with different inocula, we were not able to establish significant parasitemias with either parasite. Fig. 3. Kinetics of infection of YNIH wild-type and GP72 null mutant in L6E9 cells. Characterization of tissue culture-derived trypomastigotes (TCT) and amastigotes The morphology of the parental and null mutant parasites from all stages of their life cycle is shown in Fig. 4. The detached flagellum of the epimastigote form of the GP72 null mutant, which we will refer to as the apomastigote form (apo, detached), can be seen clearly in Fig. 4B,C (compare with wild-type parasite in (A)). Dividing parasites also showed a detached flagellum (Fig. 4E), as compared with the wild-type parasite (D). The amastigote forms of the wild-type and null mutant are shown in Fig. 4F-H. Although a micro flagellum was observed in a few wildtype parasites, micromastigotes were dominant (>90%) in the null mutant. The null mutant flagellated forms (mesomastigotes) did not have the normal morphology of a true trypomastigote (Fig. 4J-L): there was no undulating membrane and the flagellum emerged from the middle of the cell, having a much longer free end (compare with a typical wild-type trypomastigote, Fig. 4I). In the majority of the null mutant mesomastigotes, the flagellum was attached to the cell body (Fig. 4J,K), while others had a detached flagellum (Fig. 4L). mabs that recognize either amastigote or trypomastigote-specific surface antigens were used in indirect immunofluorescence assays to assess whether the few flagellated parasites observed were biochemically differentiated. A trypomastigote-specific (Andrews et al., 1987) anti-ssp1 and anti-ssp2 mab mixture stained both wild-type and null mutant flagellated forms with weak but equivalent intensity and did not stain amastigote forms (Fig. 5A,B and G-L, respectively). Wild-type (Fig. 5E,F) and null mutant (Fig. 5M,N) amastigotes/micromastigotes fluoresced brightly with mab 2C2, specific for the Ssp-4 amastigote-specific antigen (Andrews et al., 1987), but wild-type trypomastigotes (Fig. 5E,F) or mutant mesomastigotes (Fig. 5M,N) did not. These experiments demonstrated that the null mutant transforms into flagellated forms that are antigenically similar to wild-type trypomastigotes. The morphology of the null mutant, however, is quite distinct. In the wild-type trypomastigotes the kinetoplast and flagellar pocket are at the posterior end, and the flagellum follows the body of the parasite, forming the undulating membrane, until the anterior end (Fig. 5A,B). Some intermediate forms, much smaller and with weaker fluorescence, were also observed, but these intermediate forms already have the kinetoplast at the posterior end (Fig. 5C,D). Notice that, in the wildtype trypomastigote, it is always hard to discern the flagellum, due to its attachment to the parasite cell body (Fig. 5A,B,E). In the null mutants, several morphological vari-

6 1028 A. Ribeiro de Jesus and others Fig. 4. Scanning electromicrographs of YNIH wild-type and GP72 null mutants produced in vitro. The wild-type parasite is shown in (A) epimastigote, (D) dividing epimastigote, (F) amastigotes, and (I) tissue culture trypomastigote. The GP72 null mutant is represented in (B,C) epimastigotes, (E) dividing epimastigote, (G,H) amastigotes, and (J-L) tissue culture trypomastigotes. Bars, 1 µm. ants that were positive for the trypomastigote stage-specific markers were observed (Fig. 5G-N). Some were similar to amastigotes, with flagella of different sizes (Fig. 5I); the majority of them showed an elongated body shape but had the kinetoplast next to the center of the cell, superimposed on the nucleus, and the flagellum also emerged from the

7 Trypanosoma cruzi developmental mutant 1029 middle of the cell (Fig. 5K). Forms that seemed to be intermediate between the flagellated amastigotes and the elongated forms were also seen (Fig. 4J), and a few where the kinetoplast has migrated to a more posterior position (Fig. 5H,L,N), but still has not migrated completely to the posterior end, as in the wild-type parasite. In this last form, the overall shape of the parasite is still different from the wild-type parasite (compare (A) and (G)). There were no differences in the parasite reactivity to the anti-ssp1 and anti-ssp2 antibodies when the immunofluorescence was performed with each antibody independently (data not shown). Neither the wild-type nor the null mutant trypomastigotes/mesomastigotes and amastigotes/micromastigotes expressed the epitope recognized by mab WIC (data not shown). Sialidase activity Sialidase/trans-sialidase is a surface enzyme of T. cruzi, present in the invasive stages of its life cycle (Cross and Takle, 1993; Pereira, 1983; Schenkman et al., 1992). Sialidase activity was tested in parental and GP72 null mutant parasites, as an additional marker for differentiation and as a comparison between the strains (Fig. 6). Epimastigotes or apomastigotes showed no measurable activity, while in vitro-induced metacyclic trypomastigotes or mesomastigotes showed activities of 0.27±0.005, and 0.48±0.085 µu per 10 7 cells, respectively, for wild-type and null mutant in two independent experiments. A null mutant micromastigote population, which contained <1% mesomastigotes, showed very low activity (0.024±0.001 µu per 10 7 cells). Mixtures of tissue culture amastigotes/trypomastigotes and micromastigotes/mesomastigotes from different days of culture of parental and null mutant parasites were tested. Over a 4-day period, the activity ranged from µu per 10 7 cells in the wild-type parasite, and from µu per 10 7 cells in the null mutant. The percentages of flagellated forms in these cultures (Fig. 6) did not correlate with enzyme activity. The null mutant appeared to have higher activity per mesomastigote. One possible explanation for this finding is the problem of the aberrant morphology of mutant parasites, which resulted in an underestimation of the numbers of mutant cells that were biochemically rather than morphologically equivalent to trypomastigotes. Nevertheless, it is clear that the null mutant parasites can differentiate to forms that express the infectious stage-specific sialidase in amounts comparable to parental cells. In vivo viability and metacyclogenesis in the insect vector Triatoma infestans Groups of 40 fifth-instar larvae of Triatoma infestans were allowed to feed on citrated rabbit blood to which was added either wild-type YNIH, single mutant YNIH D5, or double mutant YNIH D5C3 parasites taken from LIT medium. Immediately after feeding, six insects from each group were killed and the numbers of parasites in the crop determined. No significant difference was found in the number of parasites of each strain ingested by each set of insects (Fig. 7). Six insects from each set were killed at 10, 20 and 30 days post-infection and the number of parasites in the intestine and rectum determined. No significant difference in number was found between YNIH and YNIH D5 parasites. From an initial infection of or YNIH and YNIH D5 parasites, respectively, the number of surviving parasites declined to an average value of and , an insignificant difference in the ability of these parasites to establish in the vector. However, in insects infected with the null mutant, the parasites declined much faster and were not detectable by day 10 (in 2 out of 6 insects), day 20 (4 out of 6), and day 30 (5 out of 6). Therefore, the viability of the parasite in the vector is seriously affected by the absence of GP72. The percentages of metacyclic trypomastigotes at days 10, 20 and 30 were also determined. As expected, the percentage of trypomastigotes in insects infected with YNIH parasites increased from 47% at day 10 to 91% at day 30. YNIH D5 parasites underwent metacyclogenesis to approximately the same extent, from 34% at day 10 to 82% at day 30. However, we observed a marked difference in insects infected with YNIH D5C3. In these insects, the metacyclogenesis percentage remained low. In the four insects in which parasites were found at day 10, the mean percentage was 15%. In the single insect with parasites at day 30 the percentage was 20%. Of the few parasites that remained in the insect environment, some were apparently able to undergo metacyclogenesis, but this was assessed only by morphology and not by complement resistance, sialidase activity or cell invasion assays. Analysis of GP72 expression in trypomastigotes As defined by mab WIC 29.26, GP72 was apparently only expressed in the insect stages of the parasite, but the results described above suggested that GP72 was necessary in the tissue culture trypomastigotes. Attempts were made to confirm this in wild-type parasites by RNA analysis. Northern analysis of total RNA indicated the presence of GP72 transcripts in trypomastigotes (data not shown), but confirmatory experiments will be necessary to show whether these lower RNA levels are translated into protein. DISCUSSION These results extend our previous observations on the morphological and physiological effects of deleting the genes encoding GP72 (Cooper et al., 1993). Initially it was demonstrated that the GP72 null mutant strain had an abnormal flagellar morphology in the epimastigote stage. Here we show that effects of the mutation are evident throughout the life cycle. Morphological changes were seen in the infective metacyclic mutant (mesomastigote), where the flagellum remains detached from the cell body and the kinetoplast does not migrate completely to the posterior end. Complement-resistant metacyclics have a thinner body and elongated nucleus, but the kinetoplast either remains in the anterior end or in the middle of the cell superimposed on the nucleus. The present data show that, in spite of its morphological abnormalities, the GP72 null mutant metacyclics are complement resistant, express sialidase activity and are able to infect L6E9 cells, differentiate into amastigotes and

8 1030 A. Ribeiro de Jesus and others Fig. 5. Indirect immunofluorescence of YNIH wild-type and GP72 null mutant tissue culture-derived parasites (mixtures of flagellated and amastigote forms), using the stagespecific antibodies. (A-F) Wild-type; (G-N) null mutant. (A-D and G-L) Labelling with trypomastigotespecific mab 2H11 and 2A1 mixture; (F,N) labelling with amastigote-specific mab 2C2. (E,M) Phase-contrast. Antibodies visualised with FITC alone (A,C,G) or combined with Hoechst DNA stain (B,D,H,I-L,N). (C,D) An intermediate form (left), and a folded trypomastigote (right). (E,F) An amastigote (left) and trypomastigote (right). The positions of the nucleus (n) and kinetoplast (k) are indicated on representative panels. In amastigotes the kinetoplast is close to or superimposed on the nucleus. Bar, 10 µm.

9 Trypanosoma cruzi developmental mutant 1031 Fig. 6. Sialidase activity measured in lysates of the YNIH wildtype and GP72 null mutants. Epimastigotes, metacyclics produced in vitro and a mixture of tissue culture amastigotes and trypomastigotes were tested from parental and null mutant parasites. Isolated amastigotes (<1% trypomastigotes) were tested only in the null mutant. The results with epimastigotes and with amastigotes and the amastigote/trypomastigote mixtures (Amast/TCT) represent the means ± s.d. of duplicate experiments. The result with metacyclics represent the mean ± s.d. of duplicates from two independent transformations. Amastigote/ trypomastigote mixtures were isolated from the supernatant of L6E9 cells infected with 10 parasites per L6E9 cell and collected on 3 occasions (1 or 2 days apart). The percentage values indicate the proportion of trypomastigotes (wild type) or mesomastigotes (null mutant) in the populations. reproduce to an extent that is indistinguishable from the parental parasites. Likewise, although they displayed morphological abnormalities, mutant trypanosomes propagated efficiently and differentiated biochemically in tissue culture. They expressed appropriate stage-specific surface markers and normal sialidase levels. More puzzling, however, was the change in the morphology of the extracellular parasite population after several subcultures. In the begining, the null mutant cells were remarkably distinct from the parental forms. The most striking feature of the null mutant amastigote was the uniform presence of a short fla g e l- lum (micromastigote), which we almost never saw on the parental amastigotes, even though this feature has been described in normal T. cruzi amastigotes (see, for example, Andrews et al., 1987). Flagellated null mutant forms were never seen inside the host cells and rarely outside. When they were observed, their morphology was aberrant in the point of flagellum emergence and the kinetoplast location (mesomastigote). The fact that both of these characters were altered serves to emphasize the close association between the basal body and kinetoplast, as has been elegantly explored in T. brucei (Robinson and Gull, 1991). After some subcultures, however, the proportion of mesomastigotes increased and they could also be seen inside the host cells, although their morphology remained aberrant. Such changes in behaviour during cultivation, Fig. 7. Kinetics of infection of YNIH wild type (YNIH), GP72 single deletion (YNIH D5), and GP72 null mutant (YNIH D5C3) in Triatoma infestans. The data represent the mean ± s.d. of the number of parasites isolated from the digestive tract of groups of six insects, counted in a hemocytometer. which are frequently observed after cloning T. cruzi, c o u l d be caused by additional genetic changes or other adaptations, and are probably not directly attributable to GP72 deletion. Previous studies showed that the epitope recognised by mab WIC29.26 was evenly distributed over the entire surface of the epimastigote, including the flagellum (Kirchhoff et al., 1984). Using both indirect immunofluorescence and immunoelectron microscopy, with two additional mabs that apparently recognize the same epitope as mab WIC (Chapman et al., 1984), it appeared that GP72 was distributed throughout the cell surface membrane, including the flagellar pocket and the cytostome (Harth et al., 1992). However, deletion of the GP72 genes showed, for the first time, that mab WIC epitope is present on multiple molecules (Cooper et al., 1993). In the present work, indirect immunofluorescence using mab WIC in the YNIH wild-type strain showed that the glycan epitope recognized by this mab is abundant in the flagellar region, although the entire cell surface is reactive. The observation that almost the same fluorescence pattern was seen in the GP72 null mutant epimastigote, except for a weaker fluorescence in the flagella region (especially in the third closer to the cell body) argues in favor of a preferential localization of GP72 in the flagellar attachment region. The data are even clearer in metacyclic trypomastigotes produced in vitro. The wild-type parasite showed exclusive fluorescence in the flagellum and a small region at the posterior end. The GP72 null mutant showed fluorescence only in the cell body and a very weak fluorescence in the flagellum in the early stages of differentiation, which disappeared in the later stages. The absence of fluorescence from the GP72 null mutant metacyclics demonstrates that, in this stage, the WIC epitope is only expressed on GP72. This epi-

10 1032 A. Ribeiro de Jesus and others tope was not detected in tissue culture amastigotes or trypomastigotes. However, the morphological abnormalities in the GP72 null mutant in these stages of its life cycle argue in favor of the expression of GP72 in these stages, with a loss of the epitope recognized by mab WIC The preliminary RNA data are consistent with this but need to be verified by more discriminating methods, since the levels appeared to be very low. Infection of Triatoma infestans suggested that the viability of GP72 null mutant was drastically decreased, but we only made static measurements of parasite numbers and have no information on growth and excretion kinetics. After 30 days, only 1 of 6 insects remained infected and the number of parasites isolated was much lower than in any insects infected withynih wild-type or GP72 single mutants. In this single insect, some metacyclics were seen, but far fewer than in insects infected with the wild-type or GP72 single mutant. This significant impairment in survival of the GP72 null mutant could be due to the fla g e l- lum abnormality interfering with parasite mobility within the digestive tract of the insect, perhaps leading to excessive excretion of parasites, or with the attachment of the parasite in the insect gut surface. All trypanosomes studied attach to some organ surface at certain stages in the insect vector (Garcia and Azambuja, 1991). In L e i s h m a n i a (Warburg et al., 1989), in T. cruzi (Garcia and Azambuja, 1991) and in T. brucei (Vickerman et al., 1988), the flagellum is the attachment point. Although the mechanisms involved in parasite-vector relationships are not understood, parasite attachment seems to be important for metacyclogenesis. Because of its preferential localization in the flagellar zone, GP72 could be one of the molecules involved in this attachment mechanism. It is not clear why but, even in vitro, attachment is considered to be necessary for differentiation (Goldenberg et al., 1987). Previous work (Sher and Snary, 1982) has indicated that GP72 may play a role in the control of cellular differentiation, based on inhibition studies using mab WIC The present paper shows that the GP72 null mutant undergoes metacyclogenesis, as detected by expression of some biochemical markers, in spite of suffering incomplete morphological changes. The presence of other proteins expressing the same glycan epitope as GP72, as is evident in western blotting of extracts from the null mutant (Cooper et al., 1993) and also demonstrated by immunofluorescence in this paper, suggest that these might also contribute to cell differentiation. Alternatively, the glycan epitope itself could be the responsible structure, controlling differentiation either directly or indirectly by promoting parasite attachment. 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