RESUMEN

Rhodococcus equi is a facultative intracellular bacterium causing severe pyogranulomatous pneumonia, ulcerative enterocolitis, and mesenteric lymphadenopathy in foals aged less than 6 months. Less frequently, this pathogen affects various other species, such as pigs, cattle, cats, and even humans. Although rhodococcosis is treated with a combination of antimicrobial agents, resistance is developed in some cases, and thus, antimicrobial susceptibility must be monitored and managed. Considering these limitations of the current therapy and unavailability of a vaccine to prevent the disease, research is particularly focused on the development of an effective vaccine against rhodococcosis. Most vaccines undergoing development utilize the virulence-associated protein (Vap) A antigen, which was identified previously as a key virulence factor of R. equi. Nevertheless, other proteins, such as VapG, present in most virulent R. equi strains, are also encoded by vap genes located on the R. equi bacterial virulence plasmid. In the present study, we evaluated the effect of VapG immunization on the survival of R. equi-challenged mice. We used attenuated Salmonella as a carrier for VapG (Salmonella-vapG+), a procedure previously adopted to develop a VapA-based vaccine. We observed that vaccination with Salmonella-vapG+ induced both an increased IFN-γ, IL-12, and TNF-α production, and a decreased bacterial burden in organs of the R. equi-challenged mice. Nevertheless, Salmonella-vapG+ vaccination protected only 50% of the mice challenged with a lethal dose of R. equi. Interestingly, we observed an increased frequency of B cells in the spleen of Salmonella-vapG+-vaccinated mice and showed that Salmonella-vapG+-vaccinated R. equi-challenged B-cell-knockout mice did not reduce the bacterial burden. Given these results, we discussed the potential role of the humoral immune response induced by Salmonella-vapG+ vaccination in conferring protection against R. equi infection, as well as the employment of VapG antigen for obtaining hyperimmune plasma to prevent rhodoccocosis in young foals.

RESUMEN

Conventional vaccines to prevent the pneumonia caused by Rhodococcus equi have not been successful. We have recently demonstrated that immunization with Salmonella enterica Typhimurium expressing the VapA antigen protects mice against R. equi infection. We now report that oral vaccination of mice with this recombinant strain results in high and persistent fecal levels of antigen-specific IgA, and specific proliferation of the spleen cells of immunized mice in response to the in vitro stimulation with R. equi antigen. After in vitro stimulation, spleen cells of immunized mice produce high levels of Th1 cytokines and show a prominent mRNA expression of the Th1 transcription factor T-bet, in detriment of the Th2 transcription factor GATA-3. Following R. equi challenge, a high H2O2, NO, IL-12, and IFN-gamma content is detected in the organs of immunized mice. On the other hand, TNF-alpha and IL-4 levels are markedly lower in the organs of vaccinated mice, compared with the non-vaccinated ones. The IL-10 content and the mRNA transcription level of TGF-beta are also higher in the organs of immunized mice. A greater incidence of CD4+ and CD8+ T cells and B lymphocytes is verified in vaccinated mice. However, there is no difference between vaccinated and non-vaccinated mice in terms of the frequency of CD4+CD25+Foxp3+ T cells. Finally, we show that the vaccination confers a long-term protection against R. equi infection. Altogether, these data indicate that the oral vaccination of mice with S. enterica Typhimurium expressing VapA induces specific and long-lasting humoral and cellular responses against the pathogen, which are appropriately regulated and allow tissue integrity after challenge.

Asunto(s)

Infecciones por Actinomycetales/prevención & control , Vacunas Bacterianas/administración & dosificación , Inmunidad Mucosa , Rhodococcus equi/inmunología , Salmonella enterica/inmunología , Células TH1/inmunología , Infecciones por Actinomycetales/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Vacunas Bacterianas/inmunología , Secuencia de Bases , Proliferación Celular , Citocinas/metabolismo , Cartilla de ADN , Citometría de Flujo , Factor de Transcripción GATA3/metabolismo , Peróxido de Hidrógeno/metabolismo , Inmunidad Celular , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas de Dominio T Box/metabolismo , Células TH1/citología

RESUMEN

Protein glycosylation represents one of the most important post-translational events, and is a mean of diversifying a protein without recourse to the genome. The venoms produced by snakes contain an abundance of glycoproteins with N-linked carbohydrates. N-linked glycosylation can ensure the correct folding of important functional domains. Characterization of carbohydrates structures aids in development of human therapeutics by snake venom toxins.

Asunto(s)

Glicoproteínas/química , Proteínas de Reptiles/química , Venenos de Serpiente/química , Serpientes , Secuencia de Aminoácidos , Animales , Glicosilación , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Serina Endopeptidasas/química , Venenos de Serpiente/enzimología

RESUMEN

Crotoxin is the main toxic component of the South American rattlesnake Crotalus durissus terrificus venom. It is composed of two different subunits: CA, crotapotin, and CB (basic subunit of cortoxin isolated from C. d. terrificus), a weakly toxic phospholipase A(2) with high enzymatic activity. The phospholipases A(2) are abundant in snake venoms and are responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. However, in addition to their normal digestive action, a wide range of pharmacological activities, such as neurotoxic, myotoxic, oedema-inducing, hypotensive, platelet-aggregating, cardiotoxic, and anticoagulant effects have been attributed to venom phospholipases A(2). In this study, we used a non-immune human single-chain fragment variable library, Griffin.1 (Medical Research Council, Cambridge, UK) for selection of recombinant antibodies against antigens present in C. d. terrificus venom and identification of specific antibodies able to inhibit the phospholipase activity. Two clones were identified as capable of inhibiting partially this activity in vitro. These clones were able to reduce in vivo the myotoxic and oedema-inducing activity of CB and the lethality of C. d. terrificus venom and crotoxin, but had no effect on the in vitro anticoagulant activity of CB. These results demonstrate the potential of using recombinant single-chain fragment variable libraries in the production of antivenoms.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Venenos de Crotálidos/antagonistas & inhibidores , Fragmentos de Inmunoglobulinas/farmacología , Inhibidores de Fosfolipasa A2 , Animales , Venenos de Crotálidos/toxicidad , Crotoxina/antagonistas & inhibidores , Crotoxina/toxicidad , Humanos , Masculino , Ratones , Proteínas Recombinantes/farmacología

RESUMEN

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M(r)=34,000 under reducing conditions and pI approximately 6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (-70 to 37 degrees C), pH values (3-9) or in the presence of divalent metal ions (Ca(2+), Mg(2+), Zn(2+) and Mn(2+)). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom.

Asunto(s)

Venenos de Crotálidos/enzimología , Trombina/metabolismo , Secuencia de Aminoácidos , Animales , Bothrops , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica , Masculino , Ratones , Datos de Secuencia Molecular , Trombina/química , Trombina/aislamiento & purificación

RESUMEN

Galectin-3 is a beta-galactoside-binding lectin implicated in the fine-tuning of innate immunity. Rhodococcus equi, a facultative intracellular bacterium of macrophages, causes severe granulomatous bronchopneumonia in young horses and immunocompromised humans. The aim of this study is to investigate the role of galectin-3 in the innate resistance mechanism against R. equi infection. The bacterial challenge of galectin-3-deficient mice (gal3-/-) and their wild-type counterpart (gal3+/+) revealed that the LD50 for the gal3(-/-) mice was about seven times higher than that for the gal3+/+ mice. When challenged with a sublethal dose, gal3(-/-) mice showed lower bacteria counts and higher production of IL-12 and IFN-gamma production, besides exhibiting a delayed although increased inflammatory reaction. Gal3(-/-) macrophages exhibited a decreased frequency of bacterial replication and survival, and higher transcript levels of IL-1beta, IL-6, IL-10, TLR2 and MyD88. R. equi-infected gal3+/+ macrophages showed decreased expression of TLR2, whereas R. equi-infected gal3(-/-) macrophages showed enhanced expression of this receptor. Furthermore, galectin-3 deficiency in macrophages may be responsible for the higher IL-1beta serum levels detected in infected gal3(-/-) mice. Therefore galectin-3 may exert a regulatory role in innate immunity by diminishing IL-1beta production and thus affecting resistance to R. equi infection.

Asunto(s)

Infecciones por Actinomycetales/inmunología , Citocinas/inmunología , Galectina 3/metabolismo , Inmunidad Innata , Interleucina-1beta/inmunología , Macrófagos/microbiología , Infecciones por Actinomycetales/microbiología , Animales , Citocinas/biosíntesis , Citocinas/genética , Galectina 3/deficiencia , Galectina 3/inmunología , Técnicas de Silenciamiento del Gen , Interleucina-12/biosíntesis , Interleucina-1beta/biosíntesis , Hígado/citología , Hígado/inmunología , Hígado/microbiología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rhodococcus equi/inmunología , Bazo/citología , Bazo/inmunología , Bazo/microbiología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo

RESUMEN

A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M(r)=61,000, pI approximately 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans to improve thrombin-like activity of BjussuSP-I toxin.

Asunto(s)

Factores de Coagulación Sanguínea/química , Bothrops , Venenos de Crotálidos/enzimología , Serina Endopeptidasas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Coagulación Sanguínea/aislamiento & purificación , Factores de Coagulación Sanguínea/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Calicreínas/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Trombina/química , Tiempo de Trombina