RESUMO
Brucellosis is a major zoonotic disease, and Brucella melitensis is the species most often associated with human infection. Vaccination is the most efficient tool for controlling animal brucellosis, with a consequent decrease of incidence of human infections. Commercially available live attenuated vaccines provide some degree of protection, but retain residual pathogenicity to human and animals. In this study, Brucella ovis ∆abcBA (Bo∆abcBA), a live attenuated candidate vaccine strain, was tested in two formulations (encapsulated with alginate and alginate plus vitelline protein B [VpB]) to immunize mice against experimental challenge with B. melitensis strain 16M. One week after infection, livers and spleens of immunized mice had reduced numbers of the challenge strain B. melitensis 16M when compared with those of nonimmunized mice, with a reduction of approximately 1-log10 of B. melitensis 16M count in the spleens from immunized mice. Moreover, splenocytes stimulated with B. melitensis antigens in vitro secreted IFN-γ when mice had been immunized with Bo∆abcBA encapsulated with alginate plus VpB, but not with alginate alone. Body and liver weights were similar among groups, although spleens from mice immunized with Bo∆abcBA encapsulated with alginate were larger than those immunized with Bo∆abcBA encapsulated with alginate plus VpB or nonimmunized mice. This study demonstrated that two vaccine formulations containing Bo∆abcBA protected mice against experimental challenge with B. melitensis.
Assuntos
Vacina contra Brucelose/imunologia , Brucella melitensis/imunologia , Brucella ovis/imunologia , Brucelose/imunologia , Brucelose/prevenção & controle , Animais , Citocinas , Modelos Animais de Doenças , Feminino , Imunização , Fígado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Vacinação , Vacinas Atenuadas/imunologiaRESUMO
Brucella spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR) sensor IRE1α (inositol-requiring enzyme 1) and ER-associated autophagy confer susceptibility to Brucella melitensis and Brucella abortus intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from Ern1 (encoding IRE1) conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to Brucella intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of Brucella intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of Brucella.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Brucella melitensis/patogenicidade , Brucelose/patologia , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Autofagia/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteína Beclina-1/genética , Brucelose/microbiologia , Linhagem Celular , Drosophila melanogaster , Endorribonucleases/genética , Interações Hospedeiro-Patógeno/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/genética , MAP Quinase Quinase Quinase 5/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Células RAW 264.7 , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Proteínas de Transporte Vesicular/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Intracellular bacterial pathogens secrete a repertoire of effector proteins into host cells that are required to hijack cellular pathways and cause disease. Despite decades of research, the molecular functions of most bacterial effectors remain unclear. To address this gap, we generated quantitative genetic interaction profiles between 36 validated and putative effectors from three evolutionarily divergent human bacterial pathogens and 4,190 yeast deletion strains. Correlating effector-generated profiles with those of yeast mutants, we recapitulated known biology for several effectors with remarkable specificity and predicted previously unknown functions for others. Biochemical and functional validation in human cells revealed a role for an uncharacterized component of the Salmonella SPI-2 translocon, SseC, in regulating maintenance of the Salmonella vacuole through interactions with components of the host retromer complex. These results exhibit the power of genetic interaction profiling to discover and dissect complex biology at the host-pathogen interface.