Cloning, sequencing, and expression of the PSA genes from Leishmania infantum

Abstract

We describe the molecular cloning of the PSA genes from the Leishmania infantum parasite, which show high sequence similarity with the L. major PSA-2 and L. amnzonensis GP46/M2 genes. The PSA genes in L. infantum are arrayed in tandem with a repetition unit of 6 kb. A single-size class of PSA mRNA of 4 kb was detected. The characterised L. infantum PSA genes code for a protein lacking the glycosylphosphatidylinositol addition signal described in other Leishmania species due to the presence of a stop codon located upstream from the DNA sequence coding for the signal. The data obtained after immunoprecipitation of PSA indicate that the protein is present as a water-soluble form, but that also a membrane-anchored form can be detected. The amino acid sequence derived from the isolated PSA gene shows that 60% of the deduced protein is formed by 13 leucine-rich repeats, each one of which is 24 amino acids long. The analysis of the consensus sequence of the repeats revealed that the L. infantum PSA as well as the described L. major PSA-2 and L. amnzonensis GP46/M2 proteins may be classified as new members of the leucine-rich repeat-containing protein superfamily. The number of leucine-rich motifs, however, varies considerably between the PSA protein from L. infantum and from the other Leishmania species. The PSA protein is a major antigen determinant during L. infantum infections since 87% of the sera from naturally infected dogs recognise the recombinant PSA purified from Escherichia coli.