Proteins Selected in Leishmania (Viannia) braziliensis by an Immunoproteomic Approach with Potential Serodiagnosis Applications for Tegumentary Leishmaniasis

Abstract

The serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationaryphase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, ␤-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the serodiagnosis of TL. L eishmaniasis consists of a spectrum of diseases caused by protozoan parasites of the genus Leishmania, which present high morbidity and mortality throughout the world (1). Approximately 350 million people in 98 countries are at risk of contracting the infection, while nearly 1.0 to 1.5 million cases of tegumentary leishmaniasis (TL) and 0.2 to 0.5 million cases of visceral leishmaniasis (VL) are registered annually (2). Although TL is not a fatal disease, it is endemic in more than 70 countries, and 90% of the cases have occurred in Afghanistan, Algeria, Brazil, Pakistan, Peru, Saudi Arabia, and Syria (3). The disease exhibits distinct clinical manifestations, such as cutaneous leishmaniasis (CL), diffuse cutaneous leishmaniasis (DCL), and mucosal leishmaniasis (ML) (4, 5). In Brazil, TL is caused mainly by infection with the species Leishmania (Viannia) braziliensis, Leishmania (V.) guyanensis, and Leishmania (Leishmania) amazonensis, although parasitological and molecular evidence has shown that L. braziliensis is the most important etiological agent of the disease (6, 7).At present, there is no gold standard test for TL diagnosis, and a combination of diagnostic methods is frequently needed to obtain more precise results (8). Parasitological diagnosis is definitive and consists of the microscopic examination of Giemsa-stained biopsy specimen smears, of histopathological examinati...

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 ].…”

“…Since amastigote form is the stage of parasite that survives inside mammalian host and is responsible for the development of active disease, researchers have been focused on the identification of immunoreactive proteins in amastigote proteome [ 10 , 65 ]. Interestingly, the immunodominant antigens to asymptomatic dog sera, enolase [ 15 , 55 ] and chaperonin HSP60 [ 56 , 66 ] were found to be abundantly expressed also in the amastigote form of the parasite. Subsequently, verification of these proteins in L .…”

Antileishmanial Activity, Cytotoxicity and Mechanism of Action of Clioquinol Against Leishmania infantum and Leishmania amazonensis Species

“…Stationary‐phase promastigotes were cultured as described elsewhere . To obtain the axenic amastigotes, a technical protocol was developed as described elsewhere, with few modifications . Briefly, 1 × 10 9 stationary‐phase promastigotes were washed three times in sterile phosphate buffer saline (PBS) 1× and incubated for 48 hr or 72 hr for L. amazonensis or L. infantum , respectively, in 5 mL FBS at 37°C.…”

Analysis of the Antigenic and Prophylactic Properties of the Leishmania Translation Initiation Factors eIF2 and eIF2B in Natural and Experimental Leishmaniasis

“…These families comprise tubulins (Abanades et al, 2012 ), heat shock proteins (Quijada et al, 1996 , 1998 ), histones (Soto et al, 1999 ; Maalej et al, 2003 ), or PUF proteins (Folgueira et al, 2010 ). Some of these proteins families are also antigenic in CL patients (Rafati et al, 2007 ; Souza et al, 2013 ; Duarte et al, 2015 ). The presence of high titers of anti- Leishmania antibodies is thought to be linked with pathology due to the adverse effects of deposition of the immune complexes in different tissues (García-Alonso et al, 1996 ; Jain et al, 2000 ).…”