Immunoproteomic and bioinformatic approaches to identify secreted Leishmania amazonensis, L. braziliensis, and L. infantum proteins with specific reactivity using canine serum

Abstract

Leishmania spp have a wide range of hosts, and each host can harbor several Leishmania species. Dogs, for example, are frequently infected by Leishmania infantum, where they constitute its main reservoir, but they also serve as hosts for L. braziliensis and L. amazonensis. Serological tests for antibody detection are valuable tools for diagnosis of L. infantum infection due to the high levels of antibodies induced, unlike what is observed in L. amazonensis and L. braziliensis infections. Likewise, serology-based antigen-detection can be useful as an approach to diagnose any Leishmania species infection using different corporal fluid samples. Immunogenic and secreted proteins constitute powerful targets for diagnostic methods in antigen detection. As such, we performed immunoproteomic (2-DE, western blot and mass spectrometry) and bioinformatic screening to search for reactive and secreted proteins from L. amazonensis, L. braziliensis, and L. infantum. Twenty-eight non-redundant proteins were identified, among which, six were reactive only in L. amazonensis extracts, 10 in L. braziliensis extracts, and seven in L. infantum extracts. After bioinformatic analysis, seven proteins were predicted to be secreted, two of which were reactive only in L. amazonensis extracts (52kDa PDI and the glucose-regulated protein 78), one in L. braziliensis extracts (pyruvate dehydrogenase E1 beta subunit) and three in L. infantum extracts (two conserved hypothetical proteins and elongation factor 1-beta). We propose that proteins can be suitable targets for diagnostic methods based on antigen detection.

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

“…Also, non-infective promastigotes can be lysed through the activity of neutrophil extracellular traps although metacyclic promastigotes are resistant to this innate immunity mechanism (Guimaraes-Costa et al, 2009 , 2014 ; Hurrell et al, 2016 ). The release of different intracellular antigenic proteins contained in secreted vesicles (Silverman et al, 2008 ; Cuervo et al, 2009 ; Torrecilhas et al, 2012 ) may be also an alternative form of presentation of those antigens to the host immune system, since Leishmania is able to release microvesicles in the mammalian host (Silverman and Reiner, 2012 ) and their contents have been found to induce humoral responses in murine susceptible hosts (Hernández-Chinea, 2007 ), canine (Lima et al, 2016 ), and human patients (Soares et al, 2015 ) as well as inhibitory signaling for dendritic cells activation (Markikou-Ouni et al, 2015 ; Iborra et al, 2016 ; von Stebut and Tenzer, 2018 ). Interestingly, whereas in patients with the active form of the disease they induce strong humoral responses (Requena et al, 2000a ; Maalej et al, 2003 ; Rafati et al, 2007 ; Costa et al, 2012 ; Souza et al, 2013 ) as well as IL-10 mediated responses (Bottrel et al, 2001 ; de Carvalho et al, 2003 ; Antonelli et al, 2004 ; Carvalho et al, 2005 ) in asymptomatic or in cured patients these proteins usually induce Th1-like responses (Probst et al, 2001 ; Bourreau et al, 2003 ; Baharia et al, 2014 ; Jaiswal et al, 2014 ; Cecilio et al, 2017 ).…”

Proteomics and Leishmaniasis: Potential Clinical Applications

“…Retrotransposon hot spot reacted with sera from chagasic patients in different stages of the disease, but not with sera from patients with malaria, leishmaniasis, African trypanosomiasis or toxoplasmosis, indicating high specificity . A recent study analyzed immunoreactive proteins secreted by Leishmania that could be applied to the diagnosis of leishmaniasis . The immunoreactivity of patients' sera to protein extracts obtained from L. braziliensis ., L. amazonensis , and L. infantum chagasi was investigated by 2D Western blot.…”

“…The immunoreactivity of patients' sera to protein extracts obtained from L. braziliensis ., L. amazonensis , and L. infantum chagasi was investigated by 2D Western blot. Seven of the most recognized proteins were predicted to be secreted, two of which were exclusive of L. amazonensis , one of L. braziliensis , and three of L. infantum chagasi . HSP70 and β‐tubulin were reactive for all the three species.…”

Biomarkers in Leishmaniasis: From Basic Research to Clinical Application

“…Traditionally the intrinsic intracellular and surface proteins of the parasites were targeted as primary source of antigens. Recently, secreted proteins are considered an untapped source of possible antigens and are being exploited using combinations of bioinformatic and immunoproteomic approaches [87,88] .…”