The merozoite surface protein 6 gene codes for a 36 kDa protein associated with the Plasmodium falciparum merozoite surface protein-1 complex.

A complex of non-covalently bound polypeptides is located on the surface of the merozoite form of the human malaria parasite Plasmodium falciparum. Four of these polypeptides are derived by proteolytic processing of the merozoite surface protein 1 (MSP-1) precursor. Two components, a 22 and a 36 kDa polypeptide are not derived from MSP-1. The N-terminal sequence of the 36 kDa polypeptide has been determined, the corresponding gene cloned, and the protein characterised. The 36 kDa protein consists of 211 amino acids and is derived from a larger precursor of 371 amino acids. The precursor merozoite surface protein 6 (MSP-6) has been designated, and the 36 kDa protein, MSP-6(36). Mass spectrometric analysis of peptides released from the polypeptide by tryptic digestion confirmed that the gene identified codes for MSP-6(36). Antibodies were produced to a recombinant protein containing the C-terminal 45 amino acid residues of MSP-6(36). In immunofluorescence studies these antibodies bound to antigen at the parasite surface or in the parasitophorous vacuole within schizonts, with a pattern indistinguishable from that of antibodies to MSP-1. MSP-6(36) was present in the MSP-1 complex immunoprecipitated from the supernatant of in vitro parasite cultures, but was also immunoprecipitated from this supernatant in a form not bound to MSP-1. Examination of the MSP-6 gene in three parasite lines detected no sequence variation. The sequence of MSP-6(36) is related to that of the previously described merozoite surface protein 3 (MSP-3). The MSP-6(36) amino acid sequence has 50% identity and 85% similarity with the C-terminal region of MSP-3. The proteins share a specific sequence pattern (ILGWEFGGG-[AV]-P) and a glutamic acid-rich region. The remainder of MSP-6 and MSP-3 are unrelated, except at the N-terminus. Both MSP-6(36) and MSP-3 are partially associated with the parasite surface and partially released as soluble proteins on merozoite release. MSP-6(36) is a hydrophilic negatively charged polypeptide, but there are two clusters of hydrophobic amino acids at the C-terminus, located in two amphipathic helical structures identified from secondary structure predictions. It was suggested that this 35 residue C-terminal region may be involved in MSP-6(36) binding to MSP-1 or other molecules; alternatively, based on the secondary structure and coil formation predictions, the region may form an intramolecular anti-parallel coiled-coil structure.

Plasmodium yoelii: effects of red blood cell modification and antibodies on the binding characteristics of the 235-kDa rhoptry protein.

The 235-kDa rhoptry protein of the rodent malaria parasite Plasmodium yoelii yoelii was shown to bind to the surface of mouse red blood cells in a calcium-independent process, using a erythrocyte-binding assay. This binding is affected by modification of the surface of the red blood cells by enzymatic treatment. Chymotrypsin and trypsin but not neuraminidase treatment of the erythrocytes significantly reduced the binding of the 235-kDa proteins. The binding of an unrelated 135-kDa protein was abolished by treatment with chymotrypsin. Although the 235-kDa proteins bind to both reticulocytes and mature red blood cells, the binding to mature cells was more pronounced. In the presence of hyperimmune infection serum or specific polyclonal antibodies to the 235-kDa protein its binding to erythrocytes was reduced, further demonstrating the specificity of this ligand-receptor interaction.

Passive immunization with antibodies against three distinct epitopes on Plasmodium yoelii merozoite surface protein 1 suppresses parasitemia.

We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an approximately 42-kDa fragment (MSP-142) that is derived from the C terminus of MSP-1. MSP-142 is further cleaved to an N-terminal approximately 33-kDa polypeptide (MSP-133) and a C-terminal approximately 19-kDa polypeptide (MSP-119) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-142 but not with either of the constituents MSP-133 and MSP-119, B4 recognized an epitope within the N terminus of MSP-133, and B6, B10, F5, and G3 bound to MSP-119. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes on P. yoelii MSP-1 are recognized by antibodies that suppress parasitemia in vivo.

Plasmodium knowlesi: secondary processing of the malaria merozoite surface protein-1.

Secondary processing of the Plasmodium falciparum malaria merozoite surface protein-1 (MSP-1) is defined as a single proteolytic cleavage within the carboxy-terminal membrane-bound component of the MSP-1 protein complex on the free merozoite surface. The N-terminal cleavage product (MSP-1(33)) is shed from the parasite surface along with a number of other polypeptides, whereas the C-terminal processing product remains bound to the merozoite surface and is the only part of MSP-1 detectable in the newly invaded host cell. We report that secondary processing of MSP-1 takes place in a similar manner on invasive merozoites of the simian malaria parasite Plasmodium knowlesi. Processing can take place to a limited extent in pure isolated merozoites; however, within 10 min of the addition of purified invasive merozoites to rhesus erythrocytes, processing and shedding of MSP-1 has gone to completion only in those parasites which have undergone invasion; residual free merozoites remain uniformly reactive with antibodies against MSP-1(33). Successful invasion is therefore associated with complete shedding of MSP-1(33) from the merozoite surface. The nucleotide sequence of the 3' domain of the P. knowlesi MSP-1 gene is also presented.

Antibodies inhibit the protease-mediated processing of a malaria merozoite surface protein.

When merozoites of the malaria parasite Plasmodium falciparum are released from infected erythrocytes and invade new red cells, a component of a protein complex derived from the merozoite surface protein 1 (MSP-1) precursor undergoes a single proteolytic cleavage known as secondary processing. This releases the complex from the parasite surface, except for a small membrane-bound fragment consisting of two epidermal growth factor (EGF)-like domains, which is the only part of MSP-1 to be carried into invaded erythrocytes. We report that, a group of monoclonal antibodies specific for epitopes within the EGF-like domains, some interfere with secondary processing whereas others do not. Those that most effectively inhibit processing have previously been shown to prevent invasion. Other antibodies, some of which can block this inhibition, not only do not prevent invasion but are carried into the host cell bound to the merozoite surface. These observations unequivocally demonstrate that the binding of antibody to the COOH-terminal region of MSP-1 on the merozoite surface may not be sufficient to prevent erythrocyte invasion, and show that the interaction of different antibodies with adjacent epitopes within the EGF-like domains of MSP-1 can have distinct biochemical effects on the molecule. Inhibition of MSP-1 processing on merozoites may be a mechanism by which protective antibodies interrupt the asexual cycle of the malaria parasite.

Drug synergy in experimental African trypanosomiasis.

The possibilities of trypanocidal drug synergy have been examined in vitro and in vivo using a monomorphic laboratory strain of Trypanosoma rhodesiense. Twenty-seven different drug pair combinations were chosen from among 12 representative trypanocides on the basis of surmized or reported synergy, evidence of collateral sensitivity in resistant strains, and known differences in modes of action or in field use. In vitro synergy test were made with a transfer plate technique which allows direct isobol determination by microtest tray superimposition. All pairs showing synergy in vitro were tested further in mice. Statistically significant synergy was shown only by suramin and tryparsamide, suramin and Puromycin, and suramin and Berenil. Although six other pairs, of which three contained suramin, showed higher than addictive cure rates, these rates fell short of statistical significance.

A compact semi-automated continuous cultivation system for Plasmodium falciparum.

A compact inexpensive semi-automated continuous cultivation system for Plasmodium falciparum is described. The system is simple to operate and allows cultures to proceed unattended for several days. A particularly useful feature is a portable cultivation module that is freely transferable from the incubator to a tissue culture cabinet for manual operations such as sampling or addition of red cells. Each cultivation module holds plastic flasks which can give a combined culture surface area of up to 500 cm2. Parasitaemias of 12% are attained routinely at red cell densities of 1 to 7 x 10(8) per ml, if the medium is changed daily.

Trypanocidal activity of antitumor antibiotics and other metabolic inhibitors.

A microtest has been devised for the rapid preliminary assay in vitro of the effect of over 100 drugs and inhibitors on African trypanosomes (Trypanosoma brucei and T. rhodesiense). Parasite motility and infectivity for mice are indexes, respectively, of respiration and glycolysis and of cell division; trypanocidal titers based on these indexes can show primary metabolic areas of drug attack. Various specific inhibitors have also been tested to detect metabolic sites which might be therapeutically vulnerable. RNA synthesis inhibitors are highly active (adenine nucleosides, daunorubicin, doxorubicin, chromomycin, actinomycin D, mitomycin C); the activity of the nucleoside cordycepin was increased in vitro and in vivo by an adenosine deaminase inhibitor. In view of the polyanionic nature of the trypanocide suramin, a series of polyanions was tested; several showed activity but only poly-d-glutamic acid was active in vivo. Among various miscellaneous inhibitors, quercetin, disulfiram, and the Ca-complexing agents arsenazo I and III showed marked activity, the latter exclusively on the arsenical-resistant T. brucei. The implications of these results for combination chemotherapy and depot prophylaxis (with polyanions) are indicated.