Cloning and expression of a Leishmania donovani gene instructed by a peptide isolated from major histocompatibility complex class II molecules of infected macrophages

Antonio Campos-Neto, Lynn Soong, José L. Cordova, Derek Sant'Angelo, Yasir A W Skeiky, Nancy H. Ruddle, Steven G. Reed, Charles Janeway, Diane McMahon-Pratt

Research output: Contribution to journalArticle

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Abstract

The studies reported here describe the isolation of peptides from MHC class II molecules of murine macrophages infected with Leishmania donovani, and the use of the derived peptide sequences to rescue the pathogen peptide donor protein. The isolation of the peptides was carried out by comparing the RP HPLC profile of peptides extracted from infected macrophages with the peptides extracted from noninfected cells. Several distinct HPLC peaks unique to infected macrophages were sequenced. One of the peptides that was not homologous to any known protein was used to instruct the designing of an oligonucleotide sense primer that was used in combination with an oligo dT nucleotide (anti-sense primer) to amplify by PCR a DNA fragment from L. donovani cDNA. The amplified DNA fragment was cloned and used as a probe to screen L. donovani cDNA library. The cloned gene (Ld peptide gene) has an open reading frame of 525 bp and has no homology with any known protein/gene sequence. Northern blot analyses indicated that the Ld peptide/gene is broadly distributed and expressed among species of the Leishmania genus, in both the amastigote and promastigote life cycle forms. Using the pGEX 2T vector, the gene was expressed and the relationship of the purified recombinant protein with L. donovani was confirmed using both antibody and T cell responses from immunized or infected animals. The gene encodes a 23-kD molecule (Ldp 23) associated with the cell surface of L. donovani promastigotes. In addition, T cells purified from the lymph nodes of BALB/c mice immunized with L. donovani or infected with L. major, and from CBA/J mice infected with L. amazonensis were stimulated to proliferate by the recombinant Ldp 23 and produced high levels of IFN-γ and no IL 4. This observation suggests that the Ldp 23 is an interesting parasite molecule for the studies concerning the host/parasitic interaction because the Th1 pattern of cytokine response that it induces is correlated with resistance to Leishmania infections. These results clearly point to an alternative strategy for the purification of proteins useful for the development of both vaccines and immunological diagnostic tools not only against leishmaniasis but also for other diseases caused by intracellular pathogens.

Original languageEnglish (US)
Pages (from-to)1423-1433
Number of pages11
JournalJournal of Experimental Medicine
Volume182
Issue number5
DOIs
StatePublished - Nov 1 1995
Externally publishedYes

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Leishmania donovani
Major Histocompatibility Complex
Organism Cloning
Macrophages
Peptides
Genes
Leishmania
Proteins
High Pressure Liquid Chromatography
T-Lymphocytes
Inbred CBA Mouse
DNA Primers
Leishmaniasis
DNA
Life Cycle Stages
Gene Library
Recombinant Proteins
Interleukin-4
Northern Blotting
Open Reading Frames

ASJC Scopus subject areas

  • Immunology

Cite this

Cloning and expression of a Leishmania donovani gene instructed by a peptide isolated from major histocompatibility complex class II molecules of infected macrophages. / Campos-Neto, Antonio; Soong, Lynn; Cordova, José L.; Sant'Angelo, Derek; Skeiky, Yasir A W; Ruddle, Nancy H.; Reed, Steven G.; Janeway, Charles; McMahon-Pratt, Diane.

In: Journal of Experimental Medicine, Vol. 182, No. 5, 01.11.1995, p. 1423-1433.

Research output: Contribution to journalArticle

Campos-Neto, Antonio ; Soong, Lynn ; Cordova, José L. ; Sant'Angelo, Derek ; Skeiky, Yasir A W ; Ruddle, Nancy H. ; Reed, Steven G. ; Janeway, Charles ; McMahon-Pratt, Diane. / Cloning and expression of a Leishmania donovani gene instructed by a peptide isolated from major histocompatibility complex class II molecules of infected macrophages. In: Journal of Experimental Medicine. 1995 ; Vol. 182, No. 5. pp. 1423-1433.
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AU - Skeiky, Yasir A W

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