8 research outputs found
Analysis of Proteasomal Proteolysis during the In Vitro Metacyclogenesis of Trypanosoma cruzi
Proteasomes are large protein complexes, whose main function is to degrade unnecessary or damaged proteins. The inhibition of proteasome activity in Trypanosoma cruzi blocks parasite replication and cellular differentiation. We demonstrate that proteasome-dependent proteolysis occurs during the cellular differentiation of T. cruzi from replicative non-infectious epimastigotes to non-replicative and infectious trypomastigotes (metacyclogenesis). No peaks of ubiquitin-mediated degradation were observed and the profile of ubiquitinated conjugates was similar at all stages of differentiation. However, an analysis of carbonylated proteins showed significant variation in oxidized protein levels at the various stages of differentiation and the proteasome inhibition also increased oxidized protein levels. Our data suggest that different proteasome complexes coexist during metacyclogenesis. The 20S proteasome may be free or linked to regulatory particles (PA700, PA26 and PA200), at specific cell sites and the coordinated action of these complexes would make it possible for proteolysis of ubiquitin-tagged proteins and oxidized proteins, to coexist in the cell
Desenvolvimento e avaliação de uma cepa knockout de Brucella abortus obtida pela deleção do gene virB10
Brucella spp. são bactérias gram-negativas, intracelulares facultativas que são patogênicas para muitas espécies de mamíferos causando a brucelose, uma zoonose difundida mundialmente. Por isso a busca de alternativas de controle mais eficientes se faz necessário como o desenvolvimento de novas cepas que possam ser testadas como potenciais imunógenos. Neste estudo realizou-se a deleção do gene virB10 da cepa S2308 de Brucella abortus gerando uma cepa knockout provavelmente incapaz de produzir a proteína nativa correspondente. O gene virB10 faz parte de um operon que codifica para um sistema de secreção do tipo IV, essencial para a sobrevivência intracelular e multiplicação da bactéria em células hospedeiras. A deleção foi realizada pela construção do plasmídeo suicida pBlue:virB10:kan e eletroporação deste em células eletrocompetentes de B. abortus S2308, ocorrendo a troca do gene selvagem pelo gene interrompido, com o gene de resistência a canamicina, por recombinação homóloga dupla. Camundongos BALB/c foram inoculados com as cepas S19, RB-51, ΔvirB10 de B. abortus e B. abortus S2308 selvagem; os resultados demonstraram que camundongos BALB/c inoculados com S19 e camundongos BALB/c inoculados com S2308 apresentaram queda mais rápida de linha de tendência, quando comparadas aos demais grupos, para recuperação bacteriana (RB) e peso esplênico (PE) respectivamente. Os grupos que receberam ΔvirB10 S2308 de B. abortus e RB-51 demonstraram comportamento semelhante para ambas as características. Na sexta semana após a inoculação, os resultados para RB (log de UFC ± desvio padrão) e PE (peso esplênico ± desvio padrão), respectivamente, mostraram: grupos inoculados com as cepas S2308 (4,44±1,97 e 0,44±0,11), S19 (1,83±2,54 e 0,31±0,04), RB-51 (0,00±0,00 e 0,20±0,01) e ΔvirB10 S2308 (1,43±1,25 e 0,19±0,03). Considerado o clearance bacteriano, todos os grupos diferiram estatisticamente do grupo que recebeu S2308 (p<0,0001), o grupo inoculado com ΔvirB10 S2308 de B. abortus foi semelhante ao grupo S19 (p=0,4302) e diferente do grupo RB-51 (p=0,0063). A avaliação da persistência revelou que o gene virB10 é essencial para a manutenção da virulência da bactéria. Os resultados obtidos possibilitarão que outras pesquisas sejam realizadas avaliando o potencial imunogênico desta cepa mutante
IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu
Anaplasma marginale is an important vector-borne rickettsia of
ruminants in tropical and subtropical regions of the world.
Immunization with purified outer membranes of this organism induces
protection against acute anaplasmosis. Previous studies, with proteomic
and genomic approach identified 21 proteins within the outer membrane
immunogen in addition to previously characterized major surface
protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9,
VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered
part of the type IV secretion system (TFSS), which mediates secretion
or cell-to-cell transfer of macromolecules, proteins, or DNA-protein
complexes in Gram-negative bacteria. EF-Tu can be located in the
bacterial surface, mediating bacterial attachment to host cells, or in
the bacterial cytoplasm for protein synthesis. However, the roles of
VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9,
VirB10, and EF-Tu have not been explored as vaccine antigens. In this
study, we demonstrate that sera of cattle infected with A. marginale,
with homologous or heterologous isolates recognize recombinant VirB9,
VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with
these proteins. Recognition of epitopes by total IgG and by IgG2 from
infected cattle with A. marginale support the inclusion of these
proteins in recombinant vaccines against this rickettsia
Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale
Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle