Search

We have recently characterized a novel Leishmania major gene encoding a polypeptide of 30 kDa that was homologous to mammalian ribosomal protein S3a and was named LmS3a-related protein (LmS3arp). The protein was found to be expressed by all the Leishmania species so far examined (L. infantum, L. amazonensis, and L. mexicana). In the present study we have extended our approach to the analysis of LmS3arp activity on T- and B-cell functions in a murine model. The results presented in this report show that LmS3arp plays a dual role in the regulation of T- and B-cell reactivity. Indeed, we found that injection of the LmS3arp recombinant protein (rLmS3arp) into BALB/c mice induces preferential activation of B cells, as shown by the following criteria: (i) increased expression of CD69 molecules on immunoglobulin M (IgM)-secreting spleen cells, (ii) a considerable increase of IgM-secreting B cells, and (iii) elevated levels of IgM antibodies in the sera of injected animals. Moreover, the IgM antibodies are not specific to the Leishmania antigens but preferentially recognize heterologous antigens like myosin, thyroglobulin, DNA, and keyhole limpet hemocyanin. Furthermore, the strong polyclonal expansion of nonspecific, non-parasite-directed B-cell clones induced by rLmS3arp is concomitant with a marked inhibition of T-cell proliferation. Analysis of cytokine production revealed a significant downregulation of gamma interferon, interleukin-2 (IL-2), and IL-12 secretion. Taken together, our data suggest that rLmS3arp, through direct or indirect action toward B and T cells and cytokine secretion, could participate in the immunoregulatory processes that play a role in the balance of the Th1 and Th2 immune response.

Following purification by affinity chromatography, a Leishmania major S-hexylglutathione- binding protein of molecular mass 66kDa was isolated. The immune serum against the parasite 66kDa polypeptide when used to screen a L. major cDNA library could identify clones encoding for the human v-fos transformation effector homologue, namely ribosomal protein S3a, and thus was named LmS3a-related protein (LmS3arp). A 1027bp cDNA fragment was found to contain the entire parasite gene encoding for a highly basic protein of 30kDa calculated molecular mass sharing homology to various ribosomal S3a proteins from different species. Using computer methods for a multiple alignment and sequence motif search, we found that LmS3arp shares a sequence homology to class theta glutathione S-transferase mainly in a segment containing critical residues involved in glutathione binding. These new findings are discussed in the light of recent published data showing multiple function(s) of the ribosomal proteins S3a.

In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms. In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (e.g. logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes). The reactive antigen was also detected in Trypanosoma cruzi extracts. Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used. No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti-LmSIR2rp immune serum against parasite nuclear extracts. Furthermore, immunoprecipitation of [35S]methionine-labeled promastigote antigens after pulse chase experiments, using anti-LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted-secreted antigens (ESA). Moreover, immunofluorescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface. The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues.