Proteomic Approach for Characterization of Immunodominant Membrane-Associated 30- to 36-Kilodalton Fraction Antigens ofLeishmania infantumPromastigotes, Reacting with Sera from Mediterranean Visceral Leishmaniasis Patients

Abstract

The aim of the present study was to identify and characterize proteins of a 30-to 36-kDa fraction of Leishmania infantum promastigote membranes previously shown to be an immunodominant antigen(s) in Mediterranean visceral leishmaniasis (MVL) and a consistent and reliable serological marker of this disease. By the first approach, Coomassie-stained protein bands (32-and 33-kDa fractions) that specifically reacted by immunoblotting with sera from MVL patients were excised from the gel and submitted to enzymatic digestion to generate peptides. Four peptides were sequenced, three of which were shown to be definitely associated with MVL-reactive antigens and ascribed to a mitochondrial integral ADP-ATP carrier protein from L. major, a putative NADH cytochrome b 5 reductase, and a putative mitochondrial carrier protein, respectively. The second approach combined two-dimensional gel electrophoresis of membrane antigens and mass spectrometry (liquid chromatography-mass spectrometry/mass spectrometry) by using a quadrupole time-of-flight analysis. Six immunoreactive spots that resolved within a molecular mass range of 30 to 36 kDa and a pH range of 6.7 to 7.4 corresponded to four Leishmania products. The sequences derived from two spots were ascribed to a beta subunit-like guanine nucleotide binding protein, known as the activated protein kinase C receptor homolog antigen LACK, and to a probable member of the aldehyde reductase family. One spot was identified as a probable ubiquinol-cytochrome c reductase (EC 1.10.2.2) Rieske iron-sulfur protein precursor. The remaining three spots were identified as truncated forms of elongation factor 1␣. These antigens correspond to conserved proteins ubiquitously expressed in eukaryotic cells and represent potential candidates for the design of a reliable tool for the diagnosis of this disease.

Identification of Immunoreactive Leishmania infantum Protein Antigens to Asymptomatic Dog Sera through Combined Immunoproteomics and Bioinformatics Analysis

“…Additionally, the identified proteins included 2 hypothetical conserved proteins and 3 proteins with unknown function (ribonucleoprotein p18, protein transport protein Sec13, and i/6 autoantigen-like protein). As expected, some of the proteins identified in the present work have been previously associated with humoral responses in VL and tegumentary leishmaniasis (TL) and are considered important candidate antigens for diagnosis, such as enolase [ 55 ], chaperonin HSP60 [ 56 ] and LACK [ 57 , 58 , 59 ]. Interestingly, enolase, LACK, cyclophilin, eukaryotic intiation factor 4a and chaperonin HSP60, have been identified in the secretome of different Leishmania parasites, further enhancing their use in diagnostic applications or as potential immunoprophylactic antigens [ 60 , 61 , 62 ].…”

Comparative two-dimensional gel electrophoresis maps for promastigotes of Leishmania amazonensis and Leishmania major

“…For example, a direct MS-based approach has been used successfully in Plasmodium falciparum [23]. A combination of the narrow-pH 2-DE system and Western blot detection system would be a powerful approach for identifying Leishmania proteins that are preferentially recognized by sera for human patients [24], as well as healing versus non-healing animals (our unpublished results).…”

Reacción de antígenos de Leishmania (Leishmania) mexicana con sueros de pacientes con leishmaniosis cutánea de Sinaloa, México

“…Kamoun-Essghaier y colaboradores 10 reportan un grupo de seis antígenos derivados de promastigotes de L. (L.) infantum evidenciados mediante Western blot de 2-D con pesos moleculares cercanos a los detectados en este trabajo, en el rango de 30-36 kDa, pero con diferente pI. 10 Esto sugiere que los antígenos derivados de L. (L.) mexicana reportados en el presente estudio son novedosos. Sin embargo, es necesario continuar con los estudios a fin de incluir un mayor número de pacientes, así como secuenciar los diferentes antígenos para identificar su naturaleza bioquímica y diseñar un ELISA con alguno de estos antígenos o con combinaciones de los mismos.…”