Research Article: A Genomic-Based Approach Combining In Vivo Selection in Mice to Identify a Novel Virulence Gene in Leishmania

Date Published: June 11, 2008

Publisher: Public Library of Science

Author(s): Wen-Wei Zhang, Christopher S. Peacock, Greg Matlashewski, Dianne McMahon Pratt

Abstract: BackgroundInfection with Leishmania results in a broad spectrum of pathologies where L. infantum and L. donovani cause fatal visceral leishmaniasis and L. major causes destructive cutaneous lesions. The identification and characterization of Leishmania virulence genes may define the genetic basis for these different pathologies.Methods and FindingsComparison of the recently completed L. major and L. infantum genomes revealed a relatively small number of genes that are absent or present as pseudogenes in L. major and potentially encode proteins in L. infantum. To investigate the potential role of genetic differences between species in visceral infection, seven genes initially classified as absent in L. major but present in L. infantum were cloned from the closely related L. donovani genome and introduced into L. major. The transgenic L. major expressing the L. donovani genes were then introduced into BALB/c mice to select for parasites with increased virulence in the spleen to determine whether any of the L. donovani genes increased visceral infection levels. During the course of these experiments, one of the selected genes (LinJ32_V3.1040 (Li1040)) was reclassified as also present in the L. major genome. Interestingly, only the Li1040 gene significantly increased visceral infection in the L. major transfectants. The Li1040 gene encodes a protein containing a putative component of an endosomal protein sorting complex involved with protein transport.ConclusionsThese observations demonstrate that the levels of expression and sequence variations in genes ubiquitously shared between Leishmania species have the potential to significantly influence virulence and tissue tropism.

Partial Text: Leishmania protozoa are transmitted by the bite of an infected sandfly and cause a spectrum of diseases ranging from self-healing cutaneous lesions to fatal visceral infection [1],[2]. There are an estimated 12 million cases in over 80 countries, with an annual incidence of 0.5 million new cases of the visceral leishmaniasis and 2.0 million cases of the cutaneous leishmaniasis [3]. More than 20 different Leishmania species can infect humans. Host health status and genetic background can influence the outcome of infection [4],[5] and HIV co-infection has dramatically increased the incidence of visceral leishmaniasis [5]. The major factor that determines the tropism and pathology of Leishmania infection is however the species of Leishmania[1],[2]. For example, L. donovani, L. infantum and L. chagasi are closely related members of the L. donovani complex that cause visceral leishmaniasis, which is fatal if not treated. L. major and L. tropica infections usually result in cutaneous lesions that remain localized at the site of the sandfly bite. L. (Viannia) braziliensis causes cutaneous leishmaniasis but can also migrate from the site of initial infection to the nasopharyngeal area resulting in highly destructive mucocutaneous leishmaniasis. The Leishmania genome projects are expected to help identify the genetic differences between these parasites which govern the pathology and tropism of infection caused by the different Leishmania species [6]–[8].

In the present study we have developed an experimental approach to generate and follow transgenic L. major expressing L. donovani genes in vivo in BALB/c mice. Rational for this study comes from our previous observations where expression of the L. donovani specific A2 gene increased L. major survival in visceral organs [12] and we therefore anticipated that additional L. donovani/L. infantum specific genes could also increase L. major virulence in the visceral organs. L. major expressing the L. donovani Li1040 ortholog gene was selected for in the spleen of BALB/c mice and further shown to dramatically increase L. major parasite numbers in the liver and spleen and to a much lesser extent in the skin. This outcome was somewhat unexpected since the Li1040 ortholog gene was subsequently established to be present in L. major and L. braziliensis in addition to L. donovani and L. infantum. This revealed that genes ubiquitously present in different Leishmania species could also have a dramatic effect on parasite tropism and virulence.



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