Identification of genetic markers associated with virulence of New World Leishmania species

Leishmaniasis constitutes a major threat to human health by affecting some 12 million people, mostly children and young adults, in 88 countries. 350 million people are exposed to the risk of infection and the annual incidence of new cases is about 2 million. The leishmaniases are caused by some twenty different species of the protozoan parasite Leishmania. These parasites are the causative agents of a large spectrum of diseases, varying from disfiguring cutaneous and mucocutaneous lesions to visceral dissemination, the latter of which is often fatal if not treated.
Prevalent in Central and South America, mucocutaneous leishmaniasis commences with a skin ulcer which, years later, spreads to the mucous membranes of the nose, mouth and pharynx, destroying tissue and causing severe damage. Little is known about the pathogenesis of mucocutaneous leishmaniasis, especially concerning the dissemination of infection from the site of inoculation to secondary sites (metastasis). The identification and characterisation of markers involved in the dissemination of Leishmania parasites (such as Leishmania guyanensis) would significantly contribute to the understanding of the disease progression, and provide tools for the early detection of this virulence trait, thereby potentially preventing metastasis from occuring. Furthermore, the characterisation of such markers could lead to the discovery of gene products for use in the development of a diagnostic test or which could be included in the composition of a therapeutic or preventive vaccine cocktail.
In this collaborative project involving groups in Nicaragua, Colombia and Switzerland, we aim to elucidate the genetic basis of this metastatic process in New World Leishmania species. More precisely, we are addressing the following question, What is the molecular basis of metastasis in Leishmania Viannia guyanensis species? To answer this question we aim to define which protein(s) and which gene(s) are associated with the metastatic phenotype. To achieve this goal, we have commenced our analysis at the protein expression level. We are presently analysing different Leishmania guyanensis clones exhibiting different metastatic phenotypes classified as highly metastatic clones (e.g. clone 13), or little or non-metastatic clones (e.g. clone 17). To date, we have used the 2-D gel electrophoresis system to produce comparative 2-D maps of the soluble proteins present in clones 13 and 17. A number of variables in the multi-step 2-DE protocol (including the conditions used for preparation of soluble protein extracts, electrophoresis and staining of gels) have been optimised to achieve maximum separation and resolution of proteins. Comparative 2-DE analyses of soluble protein extracts have consistently revealed exclusive presence of the potential metastasis factor Met30 in extracts of clone 13 (M+) but not clone 17 (M-). We are analysing peptidic products from 2-DE protein spots by mass spectrometry. These products are then compared with in silico peptide fingerprints present in databases. To date, these studies have enabled us to identify a protein which could be involved in Leishmania disease progression. Additional experiments are required to demonstrate the role of this protein in the metastatic process and/or disease progression.
Key words: Mucocutaneous leishmaniasis, Leishmania, metastasis, proteomics

Prof. Nicolas Fasel
Institute of Biochemistry
University of Lausanne
Ch. des Boveresses 155
1066 Epalinges
Switzerland
Tel. 21-6925732
Fax. 21-6925705
E-mail :Nicolas.Fasel@ib.unil.ch

Dr. Nancy Gore Saravia, PhD.
Executive Director
Fundación CIDEIM
Avenida 1 Norte No 3-03
Cali, Valle
Colombia
Tel: +57(2) 668 2164. Fax: +57(2) 667 2989.
E-mail: cideim@cali.cetcol.net.co

Dr Alcides Gonzales, MD, PhD
General Director
Centro Nacional de Diagnostico y Referencia
Ministerio de Salud de Nicaragua
Complejo Conception Palacios
Colonia Primero de Mayo
Managua
Nicaragua