Production of a kinesin-related recombinant protein (Lbk39) from Leishmania braziliensis by Leishmania tarentolae promastigotes and its application in the serodiagnosis of leishmaniasis

Abstract

The leishmaniases are multifactorial zoonotic diseases requiring a multidisciplinary One Health approach for diagnosis and control. For leishmaniasis diagnosis, here we describe production of a new recombinant protein based on a kinesin-related gene of Leishmania braziliensis (Lbk39), which shows 59% amino acid identity to the L. infantum homologue. The Lbk39 gene was synthesized, inserted into the pLEXSY-sat2 vector and transfected into L. tarentolae cells by electroporation. Culturing was carried out, and the secreted recombinant protein with a C-terminal histidine tag purified using nickel affinity chromatography on the culture supernatant, yielding a final product at 0.4 mg/mL. An indirect enzyme linked immunosorbent assay (ELISA) was standardised using sera from 74 Brazilian patients with cutaneous leishmaniasis and 11 with visceral leishmaniasis. Optimal ELISA conditions were established for the Lbk39 antigen in comparison with a crude extract from L. braziliensis. The sensitivity, specificity analysis and receiver operating characteristic (ROC) curve were determined with a significance level of 5%. The ROC curve showed a good accuracy with an area under curve (AUC) = 0.967, p < 0.001 (0.941–0.993) for CL patients and an AUC = 100 (100-100) for VL patients. The values of sensitivity and specificity were 88 and 98% for CL and 100 and 100% for VL, respectively. The study showed good production and expression of the target protein and has generated a potential new antigen for the diagnosis of leishmaniasis.

Glycosylation of Trypanosoma cruzi TcI antigen reveals recognition by chagasic sera

“…The L. tarentolae expression system has been used previously to produce Trypanosoma brucei gambiense surface antigens, as a low cost alternative to harvesting diagnostic antigens from T. b. gambiense grown in vitro 29 . Expression in L. tarentolae has also been applied in attempts to improve diagnostic antigens for the trypanosomatids Leishmania braziliensis 35 and Leishmania donovani 36 .…”

The efficacy of recombinant protein lbk39 for the diagnosis of leishmaniosis in dogs

“…Lbk39 recombinant protein was produced essentially as previously described (De Souza et al, 2019). Briefly, the Lbk39 fragment of a kinesin-related gene of L. braziliensis is composed of 843 base pairs and encodes repetitive immunological amino acids.…”

“…The protein is related to the kinesin-related gene of L. infantum (Burns et al, 1993), which has 6.5 repetitions of 39 amino acids. The synthetic gene Lbk39 was cloned with a 6xHis-tag in the recombinant vector pLEXSY-sat2 and inserted into the eukaryotic host L. tarentolae (De Souza et al, 2019).…”

“…Cross-reactions are less likely to occur when using recombinant proteins in ELISA, such as rA2, rK9, rK26 and rK39 (Solano-Gallego et al, 2009). Considering the need for new antigens for improved serodiagnosis of CVL that show both high specificity and sensibility, and no cross-reaction with other diseases, we undertook this study, in which we investigated the use of the recently described Lbk39 recombinant protein (De Souza et al, 2019) as an antigen in CVL diagnosis by the ELISA method.…”

Comparative Transcriptomic Analysis of Antimony Resistant and Susceptible Leishmania Infantum Lines

“…In L. donovani, kinesin plays an important role in cell division, cytoskeleton lament formation, agellar beating and intracellular transport of vesicles and organelles [65]. The kinesin motor domain region of L. donovani induces a strong Th1type immune response, important for protection, being a potential vaccine candidate against visceral leishmaniasis [66][67][68][69].…”