Recombinant Cysteine Proteinase B from Leishmania braziliensis and Its Domains: Promising Antigens for Serodiagnosis of Cutaneous and Visceral Leishmaniasis in Dogs.

Leishmaniasis represents a group of parasitic diseases caused by a protozoan of the genus Leishmania and is widely distributed in tropical and subtropical regions. Leishmaniasis is one of the major tropical neglected diseases, with 1.5 to 2 million new cases occurring annually. Diagnosis remains a challenge despite advances in parasitological, serological, and molecular methods. Dogs are an important host for the parasite and develop both visceral and cutaneous lesions. Our goal was to contribute to the diagnosis of canine cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) using the recombinant cysteine proteinase B (F-CPB) from Leishmania braziliensis and its N- and C-terminal domains (N-CPB and C-CPB) as antigens in an enzyme-linked immunosorbent assay (ELISA). Sera from dogs from Northwest Argentina diagnosed with CL were tested by ELISA against a supernatant of L. braziliensis lysate, the F-CPB protein, and its domains. We found values of sensitivity (Se) of 90.7%, 94.4%, and 94.3% and specificity (Sp) of 95.5%, 90.9%, and 91.3% for F-CPB and its N- and C-terminal domains, respectively. In sera from dogs diagnosed with VL from Northeast Argentina, we found Se of 93.3%, 73.3%, and 66.7% and Sp of 92.3%, 76.9%, and 88.5% for F-CPB and its N- and C-terminal domains, respectively. These results support CPB as a relevant antigen for canine leishmaniasis diagnosis in its different clinical presentations. More interestingly, the amino acid sequence of CPB showed high percentages of identity in several Leishmania species, suggesting that the CPB from L. braziliensis qualifies as a good antigen for the diagnosis of leishmaniasis caused by different species.

Dynamic identification of H2 epitopes from Leishmania (Leishmania) amazonensis cysteine proteinase B with potential immune activity during murine infection.

Peptides from the COOH-terminal extension of cysteine proteinase B from Leishmania (Leishmania) amazonensis (cyspep) can modulate immune responses in vertebrate hosts. With this hypothesis as base, we used the online analysis tool SYFPEITHI to predict seven epitopes from this region with potential to bind H2 proteins. We performed proliferation tests and quantified reactive T lymphocytes applying a cytometry analysis, using samples from draining lymph node of lesions from L. (L.) amazonensis-infected mice. To define reactivity of T cells, we used complexes of DimerX (H2 D(b):Ig and H2 L(d):Ig) and the putative epitopes. Additionally, we applied surface plasmon resonance to verify real time interactions between the putative epitopes and DimerX proteins. Five peptides induced blastogenesis in BALB/c cells, while only two presented the same property in C57BL/6 mouse cells. In addition, our data indicate the existence of CD8+ T lymphocyte populations able to recognize each tested peptide in both murine strains. We observed an overlapping of results between the peptides that induced lymphocyte proliferation and those capable of binding to the DimerX in the surface plasmon resonance assays thus indicating that using these recombinant proteins in biosensing analyses is a promising tool to study real time molecular interactions in the context of major histocompatibility complex epitopes. The data gathered in this study reinforce the hypothesis that cyspep-derived peptides are important factors in the murine host infection by L. (L.) amazonensis.

Reactivity of sera from dogs living in a leishmaniasis-endemic area to the COOH-terminal region of cysteine proteinase B.

Cysteine proteinases are well-known virulence factors of Leishmania spp. with demonstrated actions in both experimental mouse infection and human infection. However, studies on these enzymes in canine leishmaniasis are scarce. Here, we show, for the first time, the reactivity of sera from dogs living in an endemic area to a recombinant protein from the COOH-terminal region of cysteine B protease. In this work, enzyme-linked immunosorbent assays were performed using a 14kDa rcyspep protein obtained through a pET28-a expression system in Escherichia coli. First, 96-well plates were coated with rcyspep (500ng/well) and incubated with sera from dogs (1:100). Subsequently, IgG antibody detection was performed using rabbit anti-dog IgG antibodies conjugated with peroxidase. Sera from dogs (n=114), including suspect (n=30) and positive (n=50) dogs from a leishmaniasis-endemic area and dogs from a nonendemic area, (n=34), negative for leishmaniasis, were assessed. The results showed that sera from the suspect (42%) and positive (68%) groups responded differently to the antigen titers tested above the cut-off (Optical Density=0.166). This finding suggests that the immune response detected against cyspep may be related to clinical disorders present in these animals. Collectively, the data gathered here suggest that cyspep can sensitize the immune systems of dogs from a leishmaniasis-endemic area to elicit a humoral response, an immunological parameter indicating the contribution of this protein in host-parasite interaction.

Structural and dynamic insights into the C-terminal extension of cysteine proteinase B from Leishmania amazonensis.

Cysteine proteinase B (CPB) is a significant virulence factor for Leishmania infections. Upon processing from its zymogen form, it happens a release of the immunomodulatory CPB C-terminal extension (cyspep) into the cytoplasm of the macrophage. Epitopes derived from this fragment were shown to influence the proportion of lymphocytes CD8+ upon infection, favoring the parasite escaping from the host́s immune system. At present, there is no available structural data of cyspep, which impairs a proper understanding of its biological functions. Here, we attempted to build molecular models for this fragment and subsequently evaluate their stabilities in aqueous solution from molecular dynamics simulations analysis. Characterization of our models obtained with distinct techniques (comparative modeling, threading, and ab initio) indicates a prevalence of ß-sheets in agreement with consensus secondary structure predictions. Simulation data supported this finding since the formation of new strands, along with a rapid disruption of helical content, were observed. Overall, this study provides a rationalization of epitope mapping data and an improved understanding of cyspep antigenicity.

Reactivity of sera from dogs living in a leishmaniasis-endemic area to the COOH-terminal region of cysteine proteinase B

ABSTRACT Cysteine proteinases are well-known virulence factors of Leishmania spp. with demonstrated actions in both experimental mouse infection and human infection. However, studies on these enzymes in canine leishmaniasis are scarce. Here, we show, for the first time, the reactivity of sera from dogs living in an endemic area to a recombinant protein from the COOH-terminal region of cysteine B protease. In this work, enzyme-linked immunosorbent assays were performed using a 14 kDa rcyspep protein obtained through a pET28-a expression system in Escherichia coli. First, 96-well plates were coated with rcyspep (500 ng/well) and incubated with sera from dogs (1:100). Subsequently, IgG antibody detection was performed using rabbit anti-dog IgG antibodies conjugated with peroxidase. Sera from dogs (n = 114), including suspect (n = 30) and positive (n = 50) dogs from a leishmaniasis-endemic area and dogs from a nonendemic area, (n = 34), negative for leishmaniasis, were assessed. The results showed that sera from the suspect (42%) and positive (68%) groups responded differently to the antigen titers tested above the cut-off (Optical Density = 0.166). This finding suggests that the immune response detected against cyspep may be related to clinical disorders present in these animals. Collectively, the data gathered here suggest that cyspep can sensitize the immune systems of dogs from a leishmaniasis-endemic area to elicit a humoral response, an immunological parameter indicating the contribution of this protein in host-parasite interaction.