Leishmania major Friedlin chromosome 1 has an unusual distribution of protein-coding genes.

Leishmania are evolutionarily ancient protozoans (Kinetoplastidae) and important human pathogens that cause a spectrum of diseases ranging from the asymptomatic to the lethal. The Leishmania genome is relatively small [ approximately 34 megabases (Mb)], lacks substantial repetitive DNA, and is distributed among 36 chromosomes pairs ranging in size from 0.3 Mb to 2.5 Mb, making it a useful candidate for complete genome sequence determination. We report here the nucleotide sequence of the smallest chromosome, chr1. The sequence of chr1 has a 257-kilobase region that is densely packed with 79 protein-coding genes. This region is flanked by telomeric and subtelomeric repetitive elements that vary in number and content among the chr1 homologs, resulting in an approximately 27.5-kilobase size difference. Strikingly, the first 29 genes are all encoded on one DNA strand, whereas the remaining 50 genes are encoded on the opposite strand. Based on the gene density of chr1, we predict a total of approximately 9,800 genes in Leishmania, of which 40% may encode unknown proteins.

A study of antibody and T cell recognition of rhoptry-associated protein-1 (RAP-1) and RAP-2 recombinant proteins and peptides of Plasmodium falciparum in migrants and residents of the state of Rondonia, Brazil.

Humoral and cellular responses were examined among natives and migrants in an area of the Amazon region of Brazil. Rhoptry-associated protein-1 (RAP-1) and RAP-2 expressed in Escherichia coli expression systems, a peptide corresponding to the epitope bound by inhibitory anti-RAP-1 antibodies, and four other RAP-1 and RAP-2 synthetic peptides were used in these studies. Plasma from the native population had greater IgG reactivity to the N-terminal third of RAP-1 than the migrant population; both populations had low levels of IgM to this region of RAP-1. The IgG reactivity to RAP-2 and to the C-terminal third of RAP-1, as well as for all the peptides, including the peptide from the inhibitory domain, were low or absent in both populations. In contrast, there were a high number of subjects with an IgM response to the peptides. Cellular responses were measured by proliferation of peripheral blood mononuclear cells (PBMC) and, in some subjects, by reverse transcription-polymerase chain reaction for interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-4, and IL-10. Proliferation of PBMC was low when stimulated by recombinant proteins, peptides, or parasite lysate. Both RAP-1 and RAP-2 stimulated cytokine production by donor T cells; IL-2, IL-4, and IFN-gamma RNA transcripts were observed in response to recombinant proteins and parasite lysate, but with no uniform trends. From the observed antibody responses, RAP-1 appears to be more immunogenic than RAP-2.

Analysis of inhibitory epitopes in the Plasmodium falciparum rhoptry protein RAP-1 including identification of a second inhibitory epitope.

Immune responses to Plasmodium falciparum rhoptry-associated protein 1 (RAP-1), RAP-2, and RAP-3 appear to contribute to protection against infection by this human malarial parasite. This conclusion is suggested by results of monkey immunization trials and of cell culture studies showing antibody-dependent inhibition of erythrocyte invasion. In the present study, splenectomized owl monkeys were infected with P. falciparum in order to monitor anti-RAP-1 antibody production as antiparasite immunity developed. The monkeys responded to a primary infection with the production of antibodies to a fragment of RAP-1 containing amino acids 1 to 294 (RAP-1(1-294)). After drug cure and reinfection, the monkeys had a prolonged prepatent period, indicating they had already developed partial immunity to the parasite. Sera from these animals showed major increases in anti-RAP-1(1-294) antibodies. In contrast, only low levels of antibodies to inhibitory B-cell epitope 1 (iB-1), an inhibitory epitope in RAP-1(1-294) with the sequence N200TLTPLEELYPT211, was observed after the initial parasite infection, and the anti-iB-1 antibodies were not readily boosted upon reinfection. These results suggest that iB-1 is an immunogenic but not immunodominant epitope and that anti-iB-1 antibodies do not substantially contribute to early stages of naturally acquired immunity in the owl monkey model. To identify additional epitopes bound by inhibitory antibodies, mouse monoclonal antibodies were produced with a recombinant fusion protein containing RAP-1(1-294). Monoclonal antibody 1D6 inhibited parasite invasion of erythrocytes in vitro. 1D6 did not bind peptide iB-1 but rather bound a second inhibitory epitope called iB-2. iB-2, like iB-1, is found near the amino terminus of p67, a RAP-1 processing product thought to be involved in merozoite invasion of erythrocytes. Since anti-iB-1 antibodies were not readily produced during parasite infection, it may be desirable to direct antibody responses to particular epitopes in RAP-1, such as iB-1 and iB-2.