RESUMO
Trypanosomes evade host immunity by exchanging variant surface glycoprotein (VSG) coats. VSG genes are transcribed from telomeric expression sites, which contain a diverse family of expression-site-associated genes (ESAGs). We have discovered that the mRNAs for one ESAG family, ESAG9, are strongly developmentally regulated, being enriched in stumpy forms, a life-cycle stage in the mammalian bloodstream that is important for the maintenance of chronic parasite infections and for tsetse transmission. ESAG9 gene sequences are highly diverse in the genome and encode proteins with weak similarity to the massively diverse MASP proteins in Trypanosoma cruzi. We demonstrate that ESAG9 proteins are modified by N-glycosylation and can be shed to the external milieu, this being dependent upon coexpression with at least one other family member. The expression profile and extracellular release of ESAG9 proteins represents a novel and unexpected aspect of the transmission biology of trypanosomes in their mammalian host. We suggest that these molecules might interact with the external environment, with possible implications for infection chronicity or parasite transmission.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Estágios do Ciclo de Vida , Trypanosoma brucei brucei/fisiologia , Tripanossomíase Africana/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/biossíntese , Animais , Patógenos Transmitidos pelo Sangue/isolamento & purificação , Linhagem Celular , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Glicosilação , Estágios do Ciclo de Vida/genética , Camundongos , Engenharia de Proteínas , Transgenes/genética , Trypanosoma brucei brucei/isolamento & purificação , Trypanosoma brucei brucei/patogenicidade , Tripanossomíase Africana/genética , Tripanossomíase Africana/parasitologia , Tripanossomíase Africana/transmissão , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , VirulênciaRESUMO
Life cycle differentiation of African trypanosomes entails developmental regulation of mitochondrial activity. This requires regulation of the nuclear genome and the kinetoplast, the trypanosome's unusual mitochondrial genome. To investigate the potential cross talk between the nuclear and mitochondrial genome during the events of differentiation, we have 1) disrupted expression of a nuclear-encoded component of the cytochrome oxidase (COX) complex; and 2) generated dyskinetoplastid cells, which lack a mitochondrial genome. Using RNA interference (RNAi) and by disrupting the nuclear COX VI gene, we demonstrate independent regulation of COX component mRNAs encoded in the nucleus and kinetoplast. However, two independent approaches (acriflavine treatment and RNA interference ablation of mitochondrial topoisomerase II) failed to establish clonal lines of dyskinetoplastid bloodstream forms. Nevertheless, dyskinetoplastid forms generated in vivo could undergo two life cycle differentiation events: transition from bloodstream slender to stumpy forms and the initiation of transformation to procyclic forms. However, they subsequently arrested at a specific point in this developmental program before cell cycle reentry. These results provide strong evidence for a requirement for kinetoplast DNA in the bloodstream and for a kinetoplast-dependent control point during differentiation to procyclic forms.
Assuntos
Diferenciação Celular/fisiologia , Genoma de Protozoário , Estágios do Ciclo de Vida/fisiologia , Mitocôndrias/genética , Trypanosoma/fisiologia , Acriflavina/farmacologia , África , Animais , Anti-Infecciosos Locais/farmacologia , Antimetabólitos/farmacologia , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA de Cinetoplasto , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Marcação de Genes , Glicoproteínas de Membrana/metabolismo , Mitocôndrias/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA/metabolismo , Trypanosoma/citologia , Trypanosoma/efeitos dos fármacosRESUMO
African trypanosomes (Trypanosoma brucei) have a digenetic lifecycle that alternates between the mammalian bloodstream and the tsetse fly vector. In the bloodstream, replicating long slender parasites transform into non-dividing short stumpy forms. Upon transmission into the fly midgut, short stumpy cells differentiate into actively dividing procyclics. A hallmark of this process is the replacement of the bloodstream-stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procyclin. Pre-existing VSG is shed by a zinc metalloprotease activity (MSP-B) and glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). We now provide a detailed analysis of the coordinate and inverse regulation of these activities during synchronous differentiation. MSP-B mRNA and protein levels are upregulated during differentiation at the same time as proteolysis whereas GPI-PLC levels decrease. When transcription or translation is inhibited, VSG release is incomplete and a substantial amount of protein stays cell-associated. Both modes of release are still evident under these conditions, but GPI hydrolysis plays a quantitatively minor role during normal differentiation. Nevertheless, GPI biosynthesis shifts early in differentiation from a GPI-PLC sensitive structure to a resistant procyclic-type anchor. Translation inhibition also results in a marked increase in the mRNA levels of both MSP-B and GPI-PLC, consistent with negative regulation by labile protein factors. The relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host.