Characterising Mycobacterium tuberculosis Rv1510c protein and determining its sequences that specifically bind to two target cell lines.

The process of Mycobacterium tuberculosis infection of the macrophage implies a very little-known initial recognition and adherence step, important for mycobacterial survival; many proteins even remain like hypothetical. The Rv1510c gene, encoding a putatively conserved membrane protein, was investigated by analysing the M. tuberculosis genome sequence data reported by Cole et al. and a previous report that used PCR assays to show that the Rv1510 gene was only present in M. tuberculosis. This article confirmed all the above and identified the transcribed gene in M. tuberculosis, Mycobacterium africanum, and in M. tuberculosis clinical isolates. Antibodies raised against peptides from this protein recognised a 44 kDa band, corresponding to Rv1510c theoretical mass (44,294 Da). Assays involving synthetic peptides covering the whole protein binding to U937 and A549 cell lines led to recognising five high activity binding peptides in the Rv1510 protein: 11094, 11095, 11105, 11108, and 11111. Their affinity constants and Hill coefficients were determined by using U937 cells. Cross-linking assays performed with some of these HABPs showed that they specifically bound to a U937 cell line 51 kDa protein, but not to Hep G2 or red blood cell proteins, showing this interaction's specificity.

Amino terminal peptides from the Plasmodium falciparum EBA-181/JESEBL protein bind specifically to erythrocytes and inhibit in vitro merozoite invasion.

Several EBA-175 paralogues (EBA-140, EBA-165, EBA-175, EBA-181, and EBL-1) have been described among the Plasmodium falciparum malaria parasite proteins, which are important in the red blood cell (RBC) invasion process. EBA-181/JESEBL is a 181 kDa protein expressed in the late schizont stage and located in the micronemes; it belongs to the Plasmodium Duffy binding-like family and is able to interact with the erythrocyte surface. Here, we describe the synthesis of 78, 20-mer synthetic peptides derived from the reported EBA-181/JESEBL sequence and their ability to bind RBCs in receptor-ligand assays. Five peptides (numbered 30030, 30031, 30045, 30051, and 30060) displayed high specific binding to erythrocytes; their equilibrium binding parameters were then determined. These peptides interacted with 53 and 33 kDa receptor proteins on the erythrocyte surface, this binding being altered when RBCs were pretreated with enzymes. They were able to inhibit P. falciparum merozoite invasion of RBCs when tested in in vitro assays. According to these results, these five EBA-181/JESEBL high specific erythrocyte binding peptides, as well as the entire protein, were seen to be involved in the molecular machinery used by the parasite for invading RBCs. They are thus suggested as potential candidates in designing a multi-sub-unit vaccine able to combat the P. falciparum malaria parasite.

Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes.

Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to the vascular endothelium is a critical event in the pathology of Plasmodium falciparum malaria. It has been suggested that the clag gene family is essential in cytoadherence to endothelial receptors. Primers used in PCR and RT-PCR assays allowed us to determine that the gene encoding CLAG 3 (GenBank accession no. NP_473155) is transcribed in the Plasmodium falciparum FCB2 strain. Western blot showed that antisera produced against polymerized synthetic peptides from this protein recognized a 142-kDa band in P. falciparum schizont lysate. Seventy-one 20-amino-acid-long nonoverlapping peptides, spanning the CLAG 3 (cytoadherence-linked asexual protein on chromosome 3) sequence were tested in C32 cell and erythrocyte binding assays. Twelve CLAG peptides specifically bound to C32 cells (which mainly express CD36) with high affinity, hereafter referred to as high-affinity binding peptides (HABPs). Five of them also bound to erythrocytes. HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. Affinity constants were between 100 nM and 800 nM. Cross-linking and SDS-PAGE analysis allowed two erythrocyte binding proteins of around 26 kDa and 59 kDa to be identified, while proteins of around 53 kDa were identified as possible receptor sites for C-32 cells. The HABPs' role in Plasmodium falciparum invasion inhibition was determined. Such an approach analyzing various CLAG 3 regions may elucidate their functions and may help in the search for new antigens important for developing antimalarial vaccines.

MAEBL Plasmodium falciparum protein peptides bind specifically to erythrocytes and inhibit in vitro merozoite invasion.

MAEBL is an erythrocyte binding protein located in the rhoptries and on the surface of mature merozoites, being expressed at the beginning of schizogony. The structure of MAEBL originally isolated from rodent malaria parasites suggested a molecule likely to be involved in invasion. We thus became interested in identifying possible MAEBL functional regions. Synthetic peptides spanning the MAEBL sequence were tested in erythrocyte binding assays to identify such possible MAEBL functional regions. Nine high activity binding peptides (HABPs) were identified: two were found in the M1 domain, one was found between the M1 and M2 regions, five in the erythrocyte binding domain (M2), and one in the protein's repeat region. The results showed that peptide binding was saturable; some HABPs inhibited in vitro merozoite invasion and specifically bound to a 33kDa protein on red blood cell membrane. HABPs' possible function in merozoite invasion of erythrocytes is also discussed.