Gene deletion suggests a role for Trypanosoma Cruzi surface glycoprotein gp72 in the insect and mammalian stages of the life cycle

Trypanosoma cruzi, the etiologic agent of Chagas’ disease, has a complex life cycle consisting of four major stages. The parasite undergoes distinct biochemical and morpho-logical 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 try-panosomes (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 fla-gellum 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 ham-pered by the lack of sexual exchange in this species and, 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 estab-lish the function of GP72 had suggested that it might con-trol the differentiation of the non-infective epimastigote into the infective metacyclic trypomastigote (Sher and Snary, 1982). Knowledge of the amino acid sequence of GP72 pro-vided 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 mor-phology: 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, biochemi-cal and functional parameters.

T. cruzi YNIH and Dm28c (obtained from Dr Samuel Golden-berg, Instituto Oswaldo Cruz) strain epimastigotes were grown in liver infusion tryptose (LIT) medium (Nogueira et al., 1981) at 26-28°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).

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 72-96 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 1 ×10⁷ ml⁻¹ in 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 hemocy-tometer.

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 puri-fied 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 5×10⁷ 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 FITC-conjugated goat antimouse 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 0.0025% Hoechst 33258 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 450-490 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.

Mouse macrophages were obtained by peritoneal lavage of CD1 mice with PBS plus heparin. The cells were adjusted to 10⁶ml⁻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 5×10⁶ 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 0.0025% Hoechst 33258 stain, and visualized using a Nikon Optiphot microscope with an excitation wavelength of 330-380 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 L6E9 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 cov-erslips 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.

Female C3H mice (6-to 8-weeks-old) were injected intraperi-toneally with mixtures of 10⁵, 10⁶, or 10⁷ trypomastigotes and amastigotes derived from L6E9 cells. At intervals of 4 to 7 days, blood was collected from the tail veins and parasitemias deter-mined by viewing 100 fields at ×400 magnification of Giemsa-stained thin smears.

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 critical-point dried (maximum temperature 34°C; maximum pressure 110 pounds per square inch (750 kPa)) using a Samdri apparatus (Tou-simis 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.

Cells, at a concentration of 10⁹ ml⁻¹, were washed once in PBS and lysed in PBS, 0.5% NP40, 1mM PMSF, 0.5 μg ml⁻¹ leu-peptin. Then 10 μl of each lysate was tested for sialidase activity in duplicate, by measuring the hydrolysis of the fluorogenic sub-strate 2′-(4-methylumbelliferyl) α-D-N-acetylneuraminic acid (4MU-NANA) as described (Schenkman et al., 1992). The reac-tion 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 10.4. 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).

Fifth-instar larvae of Triatoma infestans were reared synchronously from eggs (Garcia and Azambuja, 1991). The experimen-tal insects, weighing 34.7±2.2 mg (n=40), which had been starved for 25-30 days, were allowed to feed on citrated and de-comple-mented rabbit blood containing 8×10⁷ 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.

We evaluated the ability of the GP72 null mutant, YNIH D5C3, to differentiate from epimastigotes to metacyclic try-pomastigotes 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 72-96 hours. Samples of these parasites and mid-log-phase parasites obtained directly from LIT medium, were incubated with 30% of fresh or heat-inactivated 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.

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 epi-mastigotes, WIC 29.26 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 wild-type metacyclic trypomastigotes, mAb WIC 29.26 reaction was much weaker and more distinctly restricted to the fla-gellum and an area at the posterior end (Fig. 2A-C). In the null mutant epimastigote, mAb WIC 29.26 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 29.26 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 29.26 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 try-pomastigotes. 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 29.26 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).

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.

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 amastig-otes 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 (meso-mastigotes, 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 super-natants was performed on different days. We observed a variation in the percentage of trypomastigotes or meso-mastigotes, 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 (1.9-9.5%) (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 sub-cultures 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 wild-type and GP72 null mutant strains in vivo. A highly sus-ceptible strain of mouse, C3H, was inoculated with mix-tures of trypomastigotes and amastigotes obtained from L6E9 cultures. However, despite several attempts with dif-ferent inocula, we were not able to establish significant parasitemias with either parasite.

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 com-pared 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 wild-type parasites, micromastigotes were dominant (>90%) in the null mutant. The null mutant flagellated forms (meso-mastigotes) did not have the normal morphology of a true trypomastigote (Fig. 4J-L): there was no undulating mem-brane and the flagellum emerged from the middle of the cell, having a much longer free end (compare with a typi-cal 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 trypomastig-ote-specific surface antigens were used in indirect immuno-fluorescence assays to assess whether the few flagellated parasites observed were biochemically differentiated. A try-pomastigote-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 trypomastig-otes (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 try-pomastigotes 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 ante-rior 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 wild-type trypomastigote, it is always hard to discern the fla-gellum, due to its attachment to the parasite cell body (Fig. 5A,B,E). In the null mutants, several morphological variants 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 middle of the cell (Fig. 5K). Forms that seemed to be inter-mediate 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 trypo-mastigotes/mesomastigotes and amastigotes/micromastig-otes expressed the epitope recognized by mAb WIC 29. 26 (data not shown).

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). Sial-idase 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 mesomastig-otes showed activities of 0.27±0.005, and 0.48±0.085 μU per 10⁷ cells, respectively, for wild-type and null mutant in two independent experiments. A null mutant micromastig-ote population, which contained <1% mesomastigotes, showed very low activity (0.024±0.001 μU per 10⁷ 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 0.4-3.48 μU per 10⁷ cells in the wild-type parasite, and from 0.68-3.38 μU per 10⁷ cells in the null mutant. The percentages of fla-gellated forms in these cultures (Fig. 6) did not correlate with enzyme activity. The null mutant appeared to have higher activity per mesomastigote. One possible explana-tion for this finding is the problem of the aberrant mor-phology of mutant parasites, which resulted in an under-estimation 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.

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 par-asites 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 3×10⁷ or 1.75×10⁷ YNIH and YNIH D5 parasites, respectively, the number of surviving parasites declined to an average value of 4.2×10⁵ and 7.1×10⁵, 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). There-fore, 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 per-centage 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 approx-imately 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 metacy-clogenesis 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.

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 con-firm this in wild-type parasites by RNA analysis. Northern analysis of total RNA indicated the presence of GP72 tran-scripts in trypomastigotes (data not shown), but confirmatory experiments will be necessary to show whether these lower RNA levels are translated into protein.

These results extend our previous observations on the mor-phological 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 abnor-mal flagellar morphology in the epimastigote stage. Here we show that effects of the mutation are evident through-out the life cycle.

Morphological changes were seen in the infective meta-cyclic mutant (mesomastigote), where the flagellum remains detached from the cell body and the kinetoplast does not migrate completely to the posterior end. Comple-ment-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 mor-phological abnormalities, the GP72 null mutant metacyclics are complement resistant, express sialidase activity and are able to infect L6E9 cells, differentiate into amastigotes and reproduce to an extent that is indistinguishable from the parental parasites.

Likewise, although they displayed morphological abnor-malities, 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 pop-ulation 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 flagel-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 out-side. When they were observed, their morphology was aberrant in the point of flagellum emergence and the kine-toplast 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, which are frequently observed after cloning T. cruzi, could 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 sur-face 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 29.26 (Chapman et al., 1984), it appeared that GP72 was distrib-uted 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 29.26 epitope is present on multiple molecules (Cooper et al., 1993). In the present work, indi-rect immunofluorescence using mAb WIC 29.26 in the YNIH wild-type strain showed that the glycan epitope rec-ognized 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 29.26 epitope is only expressed on GP72. This epitope was not detected in tissue culture amastigotes or try-pomastigotes. 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 29.26. 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 flagellum 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 Leishmania (Warburg et al., 1989), in T. cruzi (Garcia and Azambuja, 1991) and in T. brucei (Vickerman et al., 1988), the fla-gellum is the attachment point. Although the mechanisms involved in parasite-vector relationships are not under-stood, parasite attachment seems to be important for meta-cyclogenesis. 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 29.26. The present paper shows that the GP72 null mutant undergoes meta-cyclogenesis, as detected by expression of some biochem-ical 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 blot-ting 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. In vitro measurements of T. cruzi adhesion to the vector gut, as has been used to study the interaction between Leishmania and the sandfly (Pimenta et al., 1992) and the derivation of mutants lacking the WIC 29.26 epitope, may be necessary to resolve the role of GP72 and its attached glycans.

We thank Nadia Nogueira for excellent technical help in the invasion studies and for stimulating discussions. We thank Alan Sher, Enrique Medina-Acosta and Gloria Orge, for helpful sug-gestions and Norma Andrews for monoclonal antibodies. A.R.de J. was supported by the Fogarty International Center (D43 TW00018), S.P. by a Physician-Scientist Award (AI01071) and M.E. is the recipient of a stipend from CONACYT, Mexico, and thanks Bibiana Chavez for help with the scanning electron microscopy. This work was supported by grant AI26197 from the National Institutes of Health.