Characterization of the A2-A2rel gene cluster in Leishmania donovani: involvement of A2 in visceralization during infection

The A2 gene family is present in Leishmania donovani, which causes fatal vi sceral leishmaniasis in human patients, but is not present in Leishmania ma jor, which causes cutaneous leishmaniasis infections. The A2 genes in L. do novani are stage specific and are expressed at high levels in the amastigot e stage in the mammalian host, but are not expressed in the promastigote st age in the insect sandfly vector. The A2 genes are tandem repeated with a d istinct gene family termed the A2rel genes. In order to characterize the st ructure and function of the A2-A2rel gene clusters, the 5 ' and 3 ' DNA seq uences flanking the A2-A2rel cluster were isolated, sequenced and used to g enerate mutants through gene targeting. Although it was possible to generat e partial A2-A2rel gene clusters knock-out mutants, it was not possible to delete all the A2-A2rel gene clusters completely from the L. donovani genom e, suggesting that, within this cluster, there are genes that are essential for survival in culture. Characterization of these mutants revealed that A 2 and A2rel gene expression was compensated by amplifying the remaining int act A2 and A2rel genes, and the proliferation of these mutants in culture a nd their virulence in BALB/c mice were compromised. In order to explore fur ther the biological role of A2, the L. donovani A2 gene was introduced into L. major. In comparison with the control L. major, the A2-expressing L. ma jor parasites demonstrated an increased ability to survive in the spleen of BALB/c mice. These data suggest that A2 plays a role in the visceralizatio n of infection associated with L. donovani.