- Email: [email protected]
Analysis of a malaria sporozoite protein family required for gliding motility and cell invasion
COMMENT
Analysis of a malaria sporozoite protein family required for gliding motility and cell invasion Victor Nussenzweig and Robert Menard
M
alaria infection begins when sporozoites, found in the salivary glands of Anopheles mosquitoes, are injected into mammalian hosts. Sporozoites enter the bloodstream and rapidly invade hepatocytes, where they multiply. Cell invasion by these parasites is an active process dependent on their actin–myosin system, and is linked with the ability to glide on the surface of solid substrates. Malaria sporozoites, and other important human apicomplexan parasites such as Toxoplasma and Cryptosporidium spp., do not have flagella or cilia, and do not extend pseudopods. Nevertheless, when deposited on glass slides, they move rapidly in circles without any apparent changes in morphology. The molecular mechanisms underlying this unusual type of locomotion are being investigated by several groups. Recent findings have highlighted the essential role of members of the thrombospondinrelated adhesive protein (TRAP) family of molecules in gliding and cell invasion. Null mutations of two genes, encoding TRAP (Ref. 1) and the circumsporozoiteand TRAP-related protein, CTRP (Refs 2,3), respectively, have been engineered in the rodent malaria parasite Plasmodium berghei. The genes encoding TRAP and CTRP are differentially expressed in sporozoites and ookinetes, respectively, two motile forms of Plasmodium spp. found exclusively during the life cycle of the parasite in the mosquito. Although TRAPknockout sporozoites develop normally in the insect midgut, they do not glide or invade mosquito salivary glands or rodent livers.
The life cycle of CTRP knockouts appears to be interrupted even earlier as the ookinetes fail to invade the mosquito midgut. TRAPping motility Members of the TRAP family are typical type 1 transmembrane molecules, containing a well defined carboxy-terminal transmembrane domain and a short cytoplasmic domain with some conserved acidic residues. Recent observations have indicated that the cytoplasmic domains of the TRAP family are interchangeable, indicating that they perform similar functions4. At the amino termini, TRAP-family proteins contain single or multiple copies of two adhesive motifs: the I domain of integrins and the type 1 thrombospondin domain. In eukaryotic cells, these motifs mediate cell–cell and cell–matrix interactions. The phenotype of the TRAP and CTRP knockouts1–3, the structural features of the TRAP family and other observations made in Toxoplasma gondii5, provide strong support for the mechanistic model for gliding motility proposed by Russell and Sinden6, and King7. According to this model, gliding and cell invasion by apicomplexan parasites are consequences of the ‘capping’ of molecules on the parasite surface. The members of the TRAP family are good candidates for playing a central role in this V. Nussenzweig* and R. Menard are in the New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA. *tel:11 212 263 5337, fax: 11 212 263 8179, e-mail: [email protected]
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
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process. In the case of malaria sporozoites, capping would depend on the attachment of the extracellular domains of TRAP, directly or indirectly, to the substrate. The backwards movement of TRAP would be empowered by the interaction of its acidic cytoplasmic tail with parasite motor proteins and, as the substrate is fixed, this would drive the parasites forward. TRAP structure and function Further support for this model will require genetic and/or biochemical verification of the postulated roles of the domains of the TRAP family proteins, and the identification of the extracellular and intracellular partners of the TRAP molecules. Recent work by Crisanti’s group8 addressed some of these important issues by introducing mutations in the adhesive domains of TRAP and analysing the phenotypes of the mutant parasites in mosquitoes. However, we disagree with the conclusions of this paper because, in our opinion, the authors took an unorthodox approach to structure–function analysis. Rather than introducing mutations directly in the endogenous TRAP gene, they took a circuitous route. They first generated transgenic P. berghei parasites in which the TRAP gene (PbTRAP) was substituted with that of Plasmodium falciparum (PfTRAP). Subsequently, they introduced mutations in PfTRAP and analysed the corresponding phenotypes. This was a daring decision because P. berghei infects rodents and P. falciparum infects humans, and the extracellular domains of the PbTRAP and PfTRAP proteins differ substantially in size and amino acid PII: S0966-842X(00)01700-5 MARCH 2000
COMMENT
sequence (P. falciparum is closer in evolution to avian malaria parasites). The sequence homology between the I domains (~200 residues) of PbTRAP and PfTRAP is 40%, probably reflecting the selective evolutionary pressures in the different mammalian hosts (a tree rat in P. berghei and humans in P. falciparum), and in different species of Anopheles mosquitoes. This does not imply that heterologous complementation is not informative. For example, we have exchanged the cytoplasmic tail of PfTRAP for that of the homologous T. gondii MIC2, and have shown that the phenotypes of the mutant and wild type are indistinguishable. Nevertheless, to identify the functional amino acids in the tail, we introduced subtle mutations in the wild-type TRAP rather than in the hybrid TRAP (Ref. 4). The results of Crisanti’s paper do not support the conclusion that P. berghei sporozoites carrying the PfTRAP gene (referred to here as PfTRAP sporozoites) develop normally. In fact, they glide and infect the salivary glands of mosquitoes poorly, and appear to be ~10–100 times less infective to mice compared with wild-type P. berghei sporozoites. Only 2–10% of PfTRAP sporozoites in the salivary gland glide, and even those that glide leave short trails and stop gliding after two turns. By contrast, 70% or more of wild-type sporozoites in the salivary gland can glide for extended periods of time, and leave long circular trails. This observation was a reason for concern for the authors and they suggested that the poor motility of PfTRAP sporozoites was the result of a non-defined, non-specific, toxic effect of salivary gland debris, which must have been specifically elicited by the presence of the mutants. Nevertheless, they concluded that PfTRAP sporozoites and wild-type P. berghei sporozoites had identical phenotypes. Because motility is required for cell invasion, the poor infectivity of PfTRAP sporozoites is only to be expected. This is reflected in the very low ratios between the number of sporozoites found in mosquito salivary glands and in
TRENDS
IN
the midgut, and in the increase of one to two days in the prepatent periods of infection when the PfTRAP sporozoites were injected into mice. A prepatent period is defined as the number of days between infection with sporozoites and the first appearance of parasites in peripheral blood. Because the dose-response curves relating prepatent periods to the number of infective sporozoites are very flat, a delay of a single day in patency implies at least a ten times difference in the innoculum1,9. The causes of the abnormal properties of the PfTRAP sporozoites are not known. They might reflect abnormal recognition of the extracellular TRAP domains by host ligands and/or anomalous targeting of the polypeptide to the parasite secretory organelles and plasma membrane. The possibility that a lower concentration of TRAP is present in PfTRAP sporozoites cannot be excluded because the authors relied exclusively on indirect immunofluorescence (IFA) methodology to document its expression. It would have been more appropriate instead to use semiquantitative methods such as western blots. IFA performed by light microscopy is not quantitative and interpretation of results can be misleading. For example, in a previous paper Crisanti and his collaborators reported that, as judged by IFA, PfTRAP was present in 1.5% of sporozoites from oocysts, and in 98.5% of sporozoites from salivary glands10. This led to the conclusion that the signals for PfTRAP synthesis might reside in the ‘micro-environment of the salivary glands’9. A few years later, however, TRAP was shown to be expressed in oocyst sporozoites, and to be required for the invasion of the salivary glands1. The authors have now reconsidered the prior assessment of PfTRAP distribution8. Using the same monoclonal antibodies, and IFA methodology, they have documented the presence of PfTRAP in the oocyst sporozoites (although expression is ‘very weak’). Conclusions As PfTRAP sporozoites do not glide normally, and are much less
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infective to cells than wild-type P. berghei sporozoites are, it is very difficult to assess the significance of the phenotypes resulting from the introduction of mutations in the I and thrombospodin domains of PfTRAP. Even if this approach did reveal a further impairment in function, does this mean that the same would occur if the mutations had been introduced in the wildtype P. berghei TRAP gene, and thus in a properly functioning parasite? It would be imprudent to guess. A case in point is the finding that PfTRAP sporozoites bearing a deletion in the thrombospondin domain did not glide (3000 were examined), but were nevertheless infective to rats. This finding, which is not discussed in the paper, contradicts the generally accepted idea linking motility with invasion, and will require independent confirmation. A few years after the introduction of gene targeting in Plasmodium, it is now possible to use structure–function analysis to study protein function. If a decision is made to embark on such a laborious project, the correct methodology should be carefully chosen if significant conclusions are to be reached. References 1 Sultan, A.A. et al. (1997) TRAP is necessary for gliding motility and infectivity of Plasmodium sporozoites. Cell 90, 511–522 2 Yuda, M. et al. (1999) Targeted disruption of the Plasmodium berghei CTRP gene reveals its essential role in malaria infection of the vector mosquito. J. Exp. Med. 190, 1711–1715 3 Dessens, J.T. et al. (1999) CTRP is essential for mosquito infection by malaria ookinetes. EMBO J. 18, 6221–6227 4 Kappe, S. et al. (1999) Conservation of a gliding motility and cell invasion machinery in apicomplexan parasites. J. Cell Biol. 147, 937–944 5 Dobrowolski, J.M. and Sibley, L.D. (1996) Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 84, 933–939 6 Russell, D.G. and Sinden R.E. (1981) The role of the cytoskeleton in the motility of coccidian sporozoites. J. Cell Sci. 50, 345–359 7 King, C.A. (1988) Cell motility of sporozoan protozoa. Parasitol. Today 4, 315–319
MARCH 2000
COMMENT
8 Wengelnik, K. et al. (1999) The A-domain and the thrombospondinrelated motif of Plasmodium falciparum TRAP are implicated in the invasion process of mosquito salivary glands.
EMBO J. 19, 5195–5204 9 Schmidt, L.H. et al. (1982) The characteristics of untreated sporozoite-induced and trophozoite-induced infections. Am. J. Trop. Med. Hyg. (Suppl.) 31, 612–645
Analysis of a malaria sporozoite protein family required for gliding motility and cell invasion: Response Kai Wengelnik, Roberta Spaccapelo, Silvia Naitza and Andrea Crisanti
W
e were surprised to read such strong objections to the experimental strategy that we developed to investigate the functional significance of the amino acid residues shared by the thrombospondin-related adhesive proteins (TRAP) found in Plasmodium berghei (PbTRAP) and Plasmodium falciparum (PfTRAP)1. Not only have complementation experiments, using heterologous host systems, been widely used to elucidate gene function in a variety of organisms, but Drs Nussenzweig and Menard themselves have used such an approach to assess the function of conserved residues in the TRAP cytoplasmic tail: ‘cytoplasmic domains of this family (TRAP) are interchangeable indicating that they perform a similar function’2.What Drs Nussenzweig and Menard omit to say is that MIC2, the homologue of PbTRAP in Toxoplasma gondii, is less than 30% homologous to PbTRAP in the cytoplasmic tail. We wonder what makes it appropriate for them to use an experimental procedure that they regard as daring and unorthodox when used by us. As far as the vertebrate host range of P. berghei is concerned, we are aware that this parasite species does not infect humans. However, in vitro, P. berghei sporozoites are perfectly capable of invading human hepatocytes, where they can complete their development; this provided the rationale behind the study of P. falciparum molecules, that were implicated in the
recognition and invasion process, in P. berghei. We fail to understand how Drs Nussenzweig and Menard have reached the conclusion that P. berghei parasites carrying the PfTRAP gene (referred to as PfTRAP parasites) are 10–100 times less infective than wild-type P. berghei parasites. Our data clearly indicate that PfTRAP parasites are fully capable of completing the sporogonic cycle and are infective for mosquito salivary glands and mice. We have demonstrated that a dose of 100 sporozoites per mouse is sufficient to start an infection in 66% and 63% of mice injected with wildtype and PfTRAP parasites, respectively1. The group of mice injected with PfTRAP parasites showed a prepatent period that was six to 12 hours longer than that of mice injected with wild-type parasites. Although the prepatent period is a function of the number of sporozoites invading the hepatocytes, this index is also influenced by the growth rate of blood-stage parasites. In this respect, it should be taken into account that PfTRAP and wild-type parasites also differ in the expression of the selectable marker DHFR-TS from T. gondii which, in the absence of selection pressure, could represent a growth disadvantage. It would be somewhat arbitrary to utilize a short delay in the prepatent period to infer quantitative differences in the infectivity of PfTRAP and wildtype sporozoites. Drs Nussenzweig and Menard suggest that we should have
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
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NO. 3
10 Robson, K.J.H. et al. (1995) Thrombospondin-related adhesive protein (TRAP) of Plasmodium falciparum: expression during sporozoite ontogeny and binding to human hepatocytes. EMBO J. 14, 3883–3894
analysed the expression of PfTRAP by immunoblot, as the indirect immunofluorescence analysis (IFA) could be misleading. They inform the reader that we have previously reported in P. falciparum that, as judged by IFA, ‘PfTRAP was present in 1.5% of sporozoites from oocysts, and in 98.5% of sporozoites from salivary glands’3. They say that this observation led us to the conclusion that the signals for PfTRAP synthesis might reside in the ‘micro-environment of the salivary glands’3. However, in this paper, after discussing the possibility of the salivary gland microenvironment we stated that ‘The presence of few TRAP1 CS protein1 parasites among hemocoel sporozoites would suggest that sporozoites may express TRAP before penetrating into the salivary glands, thus TRAP expression may be time rather than site dependently regulated’3. In our latest paper1, we have shown that a substitution in a conserved residue of the A-domain or a deletion in region II1 of PfTRAP impairs the ability of sporozoites to invade mosquito salivary glands. Oocyst sporozoites from these transgenic parasites are still able to infect mice. Sporozoites carrying a mutation in the A-domain of PfTRAP were motile, whereas no gliding motility could be detected in sporozoites with a region II1 deletion1. On the basis of these results, we concluded that ‘TRAP is implicated in recognition and invasion of salivary glands by the sporozoite and that this process is functionally distinct from its involvement in parasite motility’. It is argued that it would be imprudent to guess whether a similar phenotype could be observed after introducing the mutations in wildtype PbTRAP. The answer to this question can be found in reading what Drs Nussenzweig and Menard have written in an abstract reporting unpublished results: ‘The rate of PII: S0966-842X(00)01701-7 MARCH 2000
Analysis of a malaria sporozoite protein family required for gliding motility and cell invasion Victor Nussenzweig and Robert Menard
M
alaria infection begins when sporozoites, found in the salivary glands of Anopheles mosquitoes, are injected into mammalian hosts. Sporozoites enter the bloodstream and rapidly invade hepatocytes, where they multiply. Cell invasion by these parasites is an active process dependent on their actin–myosin system, and is linked with the ability to glide on the surface of solid substrates. Malaria sporozoites, and other important human apicomplexan parasites such as Toxoplasma and Cryptosporidium spp., do not have flagella or cilia, and do not extend pseudopods. Nevertheless, when deposited on glass slides, they move rapidly in circles without any apparent changes in morphology. The molecular mechanisms underlying this unusual type of locomotion are being investigated by several groups. Recent findings have highlighted the essential role of members of the thrombospondinrelated adhesive protein (TRAP) family of molecules in gliding and cell invasion. Null mutations of two genes, encoding TRAP (Ref. 1) and the circumsporozoiteand TRAP-related protein, CTRP (Refs 2,3), respectively, have been engineered in the rodent malaria parasite Plasmodium berghei. The genes encoding TRAP and CTRP are differentially expressed in sporozoites and ookinetes, respectively, two motile forms of Plasmodium spp. found exclusively during the life cycle of the parasite in the mosquito. Although TRAPknockout sporozoites develop normally in the insect midgut, they do not glide or invade mosquito salivary glands or rodent livers.
The life cycle of CTRP knockouts appears to be interrupted even earlier as the ookinetes fail to invade the mosquito midgut. TRAPping motility Members of the TRAP family are typical type 1 transmembrane molecules, containing a well defined carboxy-terminal transmembrane domain and a short cytoplasmic domain with some conserved acidic residues. Recent observations have indicated that the cytoplasmic domains of the TRAP family are interchangeable, indicating that they perform similar functions4. At the amino termini, TRAP-family proteins contain single or multiple copies of two adhesive motifs: the I domain of integrins and the type 1 thrombospondin domain. In eukaryotic cells, these motifs mediate cell–cell and cell–matrix interactions. The phenotype of the TRAP and CTRP knockouts1–3, the structural features of the TRAP family and other observations made in Toxoplasma gondii5, provide strong support for the mechanistic model for gliding motility proposed by Russell and Sinden6, and King7. According to this model, gliding and cell invasion by apicomplexan parasites are consequences of the ‘capping’ of molecules on the parasite surface. The members of the TRAP family are good candidates for playing a central role in this V. Nussenzweig* and R. Menard are in the New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA. *tel:11 212 263 5337, fax: 11 212 263 8179, e-mail: [email protected]
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
IN
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NO. 3
process. In the case of malaria sporozoites, capping would depend on the attachment of the extracellular domains of TRAP, directly or indirectly, to the substrate. The backwards movement of TRAP would be empowered by the interaction of its acidic cytoplasmic tail with parasite motor proteins and, as the substrate is fixed, this would drive the parasites forward. TRAP structure and function Further support for this model will require genetic and/or biochemical verification of the postulated roles of the domains of the TRAP family proteins, and the identification of the extracellular and intracellular partners of the TRAP molecules. Recent work by Crisanti’s group8 addressed some of these important issues by introducing mutations in the adhesive domains of TRAP and analysing the phenotypes of the mutant parasites in mosquitoes. However, we disagree with the conclusions of this paper because, in our opinion, the authors took an unorthodox approach to structure–function analysis. Rather than introducing mutations directly in the endogenous TRAP gene, they took a circuitous route. They first generated transgenic P. berghei parasites in which the TRAP gene (PbTRAP) was substituted with that of Plasmodium falciparum (PfTRAP). Subsequently, they introduced mutations in PfTRAP and analysed the corresponding phenotypes. This was a daring decision because P. berghei infects rodents and P. falciparum infects humans, and the extracellular domains of the PbTRAP and PfTRAP proteins differ substantially in size and amino acid PII: S0966-842X(00)01700-5 MARCH 2000
COMMENT
sequence (P. falciparum is closer in evolution to avian malaria parasites). The sequence homology between the I domains (~200 residues) of PbTRAP and PfTRAP is 40%, probably reflecting the selective evolutionary pressures in the different mammalian hosts (a tree rat in P. berghei and humans in P. falciparum), and in different species of Anopheles mosquitoes. This does not imply that heterologous complementation is not informative. For example, we have exchanged the cytoplasmic tail of PfTRAP for that of the homologous T. gondii MIC2, and have shown that the phenotypes of the mutant and wild type are indistinguishable. Nevertheless, to identify the functional amino acids in the tail, we introduced subtle mutations in the wild-type TRAP rather than in the hybrid TRAP (Ref. 4). The results of Crisanti’s paper do not support the conclusion that P. berghei sporozoites carrying the PfTRAP gene (referred to here as PfTRAP sporozoites) develop normally. In fact, they glide and infect the salivary glands of mosquitoes poorly, and appear to be ~10–100 times less infective to mice compared with wild-type P. berghei sporozoites. Only 2–10% of PfTRAP sporozoites in the salivary gland glide, and even those that glide leave short trails and stop gliding after two turns. By contrast, 70% or more of wild-type sporozoites in the salivary gland can glide for extended periods of time, and leave long circular trails. This observation was a reason for concern for the authors and they suggested that the poor motility of PfTRAP sporozoites was the result of a non-defined, non-specific, toxic effect of salivary gland debris, which must have been specifically elicited by the presence of the mutants. Nevertheless, they concluded that PfTRAP sporozoites and wild-type P. berghei sporozoites had identical phenotypes. Because motility is required for cell invasion, the poor infectivity of PfTRAP sporozoites is only to be expected. This is reflected in the very low ratios between the number of sporozoites found in mosquito salivary glands and in
TRENDS
IN
the midgut, and in the increase of one to two days in the prepatent periods of infection when the PfTRAP sporozoites were injected into mice. A prepatent period is defined as the number of days between infection with sporozoites and the first appearance of parasites in peripheral blood. Because the dose-response curves relating prepatent periods to the number of infective sporozoites are very flat, a delay of a single day in patency implies at least a ten times difference in the innoculum1,9. The causes of the abnormal properties of the PfTRAP sporozoites are not known. They might reflect abnormal recognition of the extracellular TRAP domains by host ligands and/or anomalous targeting of the polypeptide to the parasite secretory organelles and plasma membrane. The possibility that a lower concentration of TRAP is present in PfTRAP sporozoites cannot be excluded because the authors relied exclusively on indirect immunofluorescence (IFA) methodology to document its expression. It would have been more appropriate instead to use semiquantitative methods such as western blots. IFA performed by light microscopy is not quantitative and interpretation of results can be misleading. For example, in a previous paper Crisanti and his collaborators reported that, as judged by IFA, PfTRAP was present in 1.5% of sporozoites from oocysts, and in 98.5% of sporozoites from salivary glands10. This led to the conclusion that the signals for PfTRAP synthesis might reside in the ‘micro-environment of the salivary glands’9. A few years later, however, TRAP was shown to be expressed in oocyst sporozoites, and to be required for the invasion of the salivary glands1. The authors have now reconsidered the prior assessment of PfTRAP distribution8. Using the same monoclonal antibodies, and IFA methodology, they have documented the presence of PfTRAP in the oocyst sporozoites (although expression is ‘very weak’). Conclusions As PfTRAP sporozoites do not glide normally, and are much less
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infective to cells than wild-type P. berghei sporozoites are, it is very difficult to assess the significance of the phenotypes resulting from the introduction of mutations in the I and thrombospodin domains of PfTRAP. Even if this approach did reveal a further impairment in function, does this mean that the same would occur if the mutations had been introduced in the wildtype P. berghei TRAP gene, and thus in a properly functioning parasite? It would be imprudent to guess. A case in point is the finding that PfTRAP sporozoites bearing a deletion in the thrombospondin domain did not glide (3000 were examined), but were nevertheless infective to rats. This finding, which is not discussed in the paper, contradicts the generally accepted idea linking motility with invasion, and will require independent confirmation. A few years after the introduction of gene targeting in Plasmodium, it is now possible to use structure–function analysis to study protein function. If a decision is made to embark on such a laborious project, the correct methodology should be carefully chosen if significant conclusions are to be reached. References 1 Sultan, A.A. et al. (1997) TRAP is necessary for gliding motility and infectivity of Plasmodium sporozoites. Cell 90, 511–522 2 Yuda, M. et al. (1999) Targeted disruption of the Plasmodium berghei CTRP gene reveals its essential role in malaria infection of the vector mosquito. J. Exp. Med. 190, 1711–1715 3 Dessens, J.T. et al. (1999) CTRP is essential for mosquito infection by malaria ookinetes. EMBO J. 18, 6221–6227 4 Kappe, S. et al. (1999) Conservation of a gliding motility and cell invasion machinery in apicomplexan parasites. J. Cell Biol. 147, 937–944 5 Dobrowolski, J.M. and Sibley, L.D. (1996) Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 84, 933–939 6 Russell, D.G. and Sinden R.E. (1981) The role of the cytoskeleton in the motility of coccidian sporozoites. J. Cell Sci. 50, 345–359 7 King, C.A. (1988) Cell motility of sporozoan protozoa. Parasitol. Today 4, 315–319
MARCH 2000
COMMENT
8 Wengelnik, K. et al. (1999) The A-domain and the thrombospondinrelated motif of Plasmodium falciparum TRAP are implicated in the invasion process of mosquito salivary glands.
EMBO J. 19, 5195–5204 9 Schmidt, L.H. et al. (1982) The characteristics of untreated sporozoite-induced and trophozoite-induced infections. Am. J. Trop. Med. Hyg. (Suppl.) 31, 612–645
Analysis of a malaria sporozoite protein family required for gliding motility and cell invasion: Response Kai Wengelnik, Roberta Spaccapelo, Silvia Naitza and Andrea Crisanti
W
e were surprised to read such strong objections to the experimental strategy that we developed to investigate the functional significance of the amino acid residues shared by the thrombospondin-related adhesive proteins (TRAP) found in Plasmodium berghei (PbTRAP) and Plasmodium falciparum (PfTRAP)1. Not only have complementation experiments, using heterologous host systems, been widely used to elucidate gene function in a variety of organisms, but Drs Nussenzweig and Menard themselves have used such an approach to assess the function of conserved residues in the TRAP cytoplasmic tail: ‘cytoplasmic domains of this family (TRAP) are interchangeable indicating that they perform a similar function’2.What Drs Nussenzweig and Menard omit to say is that MIC2, the homologue of PbTRAP in Toxoplasma gondii, is less than 30% homologous to PbTRAP in the cytoplasmic tail. We wonder what makes it appropriate for them to use an experimental procedure that they regard as daring and unorthodox when used by us. As far as the vertebrate host range of P. berghei is concerned, we are aware that this parasite species does not infect humans. However, in vitro, P. berghei sporozoites are perfectly capable of invading human hepatocytes, where they can complete their development; this provided the rationale behind the study of P. falciparum molecules, that were implicated in the
recognition and invasion process, in P. berghei. We fail to understand how Drs Nussenzweig and Menard have reached the conclusion that P. berghei parasites carrying the PfTRAP gene (referred to as PfTRAP parasites) are 10–100 times less infective than wild-type P. berghei parasites. Our data clearly indicate that PfTRAP parasites are fully capable of completing the sporogonic cycle and are infective for mosquito salivary glands and mice. We have demonstrated that a dose of 100 sporozoites per mouse is sufficient to start an infection in 66% and 63% of mice injected with wildtype and PfTRAP parasites, respectively1. The group of mice injected with PfTRAP parasites showed a prepatent period that was six to 12 hours longer than that of mice injected with wild-type parasites. Although the prepatent period is a function of the number of sporozoites invading the hepatocytes, this index is also influenced by the growth rate of blood-stage parasites. In this respect, it should be taken into account that PfTRAP and wild-type parasites also differ in the expression of the selectable marker DHFR-TS from T. gondii which, in the absence of selection pressure, could represent a growth disadvantage. It would be somewhat arbitrary to utilize a short delay in the prepatent period to infer quantitative differences in the infectivity of PfTRAP and wildtype sporozoites. Drs Nussenzweig and Menard suggest that we should have
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
IN
MICROBIOLOGY
96
VOL. 8
NO. 3
10 Robson, K.J.H. et al. (1995) Thrombospondin-related adhesive protein (TRAP) of Plasmodium falciparum: expression during sporozoite ontogeny and binding to human hepatocytes. EMBO J. 14, 3883–3894
analysed the expression of PfTRAP by immunoblot, as the indirect immunofluorescence analysis (IFA) could be misleading. They inform the reader that we have previously reported in P. falciparum that, as judged by IFA, ‘PfTRAP was present in 1.5% of sporozoites from oocysts, and in 98.5% of sporozoites from salivary glands’3. They say that this observation led us to the conclusion that the signals for PfTRAP synthesis might reside in the ‘micro-environment of the salivary glands’3. However, in this paper, after discussing the possibility of the salivary gland microenvironment we stated that ‘The presence of few TRAP1 CS protein1 parasites among hemocoel sporozoites would suggest that sporozoites may express TRAP before penetrating into the salivary glands, thus TRAP expression may be time rather than site dependently regulated’3. In our latest paper1, we have shown that a substitution in a conserved residue of the A-domain or a deletion in region II1 of PfTRAP impairs the ability of sporozoites to invade mosquito salivary glands. Oocyst sporozoites from these transgenic parasites are still able to infect mice. Sporozoites carrying a mutation in the A-domain of PfTRAP were motile, whereas no gliding motility could be detected in sporozoites with a region II1 deletion1. On the basis of these results, we concluded that ‘TRAP is implicated in recognition and invasion of salivary glands by the sporozoite and that this process is functionally distinct from its involvement in parasite motility’. It is argued that it would be imprudent to guess whether a similar phenotype could be observed after introducing the mutations in wildtype PbTRAP. The answer to this question can be found in reading what Drs Nussenzweig and Menard have written in an abstract reporting unpublished results: ‘The rate of PII: S0966-842X(00)01701-7 MARCH 2000