Su-Chi Chiang

Expression and cellular localization of ribonucleotide reductase small subunit M2 protein in hydroxyurea-resistant Leishmania mexicana amazonensis

We raised a specific antiserum against the recombinant M2 subunit protein of ribonucleotide reductase of Leishmania mexicana amazonensis in rabbit. This antiserum was used to study the expression and cellular location of the M2 protein in wildtype as well as hydroxyurea-resistant variants (HuR) of the parasite. The protein increased with increasing dose of the drug used for selection of resistance. The increase in protein level was accompanied by an increase in the copy numbers of mRNA of the M2 gene in the variants. In contrast to mammalian cells, the M2 protein of Leishmania is located in the nucleus rather than in the cytoplasm. The number of cells expressing M2 protein is also different in mammalian cells versus Leishmania. In mammalian cells, expression of M2 protein is a strictly S-phase-correlated event and in exponentially growing cells only approximately 50% of the cells are in S-phase and only these cells synthesize M2 protein. In L. m. amazonensis, however, almost all exponentially growing cells are positive for M2 protein. This makes it unlikely that M2 protein expression in Leishmania is S-phase dependent. In view of these findings, a fresh look in the future into the regulatory mechanisms of synthesis and the site of action of RNR in L. m. amazonensis is warranted.

ICAM-L gene is conserved only in Leishmania species in the family of kinetoplastida.

Attachment of Leishmania to its host macrophage is a ligand-receptor mediated event. Two abundant surface molecules, a complex carbohydrate lipophosglycan (LPG) and a glycosylphosphotidylinositol (GPI) anchored GP63 protease have been shown to independently play a critical role in this event. LPG and GP63 are widely conserved among members of the kinetoplastida family. An intercellular adhesive molecule from Leishmania (ICAM-L) that acts as a ligand of Leishmania for the macrophage has recently been described by our lab. When assessed by molecular determinations including Southern, Northern and sequencing, ICAM-L appears to be conserve only among Leishmania species but not in other closely related members of the kinetoplastida. However using a polyclonal antibody specific to ICAM-L, the molecule appears to be conserved among all the kinetoplastids. The detention by antibody of ICAM-L molecules among other kinetoplastids may due to conservation in these parasites of certain immunoreactive epitopes at the amino acid level, while the nucleotide sequence is divergent enough to preclude detection by methods employed.

Molecular, cellular and functional characterizations of a novel ICAM-like molecule of the immunoglobulin superfamily from Leishmania mexicana amazonensis.

A molecule with two immunoglobulin (Ig) domains cloned from Leishmania mexicana amazonensis was characterized to have a sequence homology to the Ig domains of an ICAM-like molecule telencephalin, cloned from the brain of mammals, as well as to the variable domains of human immunoglobulin lambda light chain. The molecule therefore appears to be an ICAM-like molecule as well as a member of the immunoglobulin superfamily. We thus named it ICAM-L for Leishmania ICAM. The gene was coamplified with the ribonucleotide reductase M(2) subunit gene responsible for hydroxyurea resistance from hydroxyurea (Hu)-resistant Leishmania variants. As expected, an increase of the ICAM-L protein as well as an increase of the specific ICAM-L transcript of 2.1 kb was detected in the Hu-resistant variants with increasing doses of the drug used for resistance selection. Structurally, ICAM-L is more similar to the secretory adhesive molecules, such as 1Bgp and the link protein of the immunoglobulin superfamily, in that it lacks a transmembrane region and a GPI anchor sequence. Although ICAM-L was mainly localized in the nucleus of the parasite by confocal microscopy, however, detailed studies by electron microscopy and FACS analysis indicated that the protein was also localized on the surface of the parasite. The surface localization of the protein was furthered strengthened by the observations that anti-ICAM-L or ICAM-L itself can significantly block the binding of the parasite to macrophages. The blocking of the attachment of parasite to macrophages may indicate that ICAM-L functions as an intercellular adhesive molecule.

An improved method for detection of Leishmania amastigotes by an antibody probe against the small subunit of leishmanial ribonucleotide reductase

Abstract By taking advantage of an antibody raised against the small M2 subunit of ribonucleotide reductase of Leishmania that reacts with the enzyme in the nucleus of the parasite but does not cross-react with the same enzyme of the host macrophage, an improved fluorescence-staining method is developed for enumeration of leishmanial amastigotes inside the macrophage. The method offers an accurate and easy way of counting, compared with Giemsa staining.