RESUMO
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
Caprine arthritis encephalitis is a worldwide, multisystemic disease caused by a small ruminant lentivirus. Although the main route of transmission is oral, detection of proviral DNA of the caprine arthritis encephalitis virus (CAEV) in caprine semen has been previously described. However, the presence of viral antigens in the male reproductive tract has apparently never been reported. The objective was to study lesions in the buck reproductive system and to detect, in these tissues, the presence of proviral DNA, viral RNA and CAEV antigens. Tissues from eight CAEV-infected bucks (one naturally and seven experimentally infected) were analyzed by histopathology, nested polymerase chain reaction, reverse transcriptase-polymerase chain reaction, and immunohistochemistry. Interstitial pneumonia, synovitis, and lesions in the male reproductive tract were detected in some of the bucks. Proviral DNA was detected in the lungs and joints as well as in the reproductive systems of all animals, whereas viral RNA was detected only in the genital tract of the naturally infected buck. Viral antigens were immunostained in most of the organs of the male reproductive tract. This report was apparently the first to clearly demonstrate CAEV antigen expression in the male reproductive tract, which indicates the possibility of venereal transmission of CAEV.
