The proteome and transcriptome of the infectious metacyclic form of Trypanosoma brucei define quiescent cells primed for mammalian invasion

Abstract

Summary The infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic forms. We used an in vitro system based on expressing the RNA binding protein 6 to obtain infectious metacyclics and determined their protein and mRNA repertoires by mass-spectrometry (MS) based proteomics and mRNA sequencing (RNA-Seq) in comparison to non-infectious procyclic trypanosomes. We showed that metacyclics are quiescent cells, and propose this influences the choice of a monocistronic variant surface glycoprotein expression site. Metacyclics have a largely bloodstream-form type transcriptome, and thus are programmed to translate a bloodstream-form type proteome upon entry into the mammalian host and resumption of cell division. Genes encoding cell surface components showed the largest changes between procyclics and metacyclics, observed at both the transcript and protein levels. Genes encoding metabolic enzymes exhibited expression in metacyclics with features of both procyclic and bloodstream forms, suggesting that this intermediate-type metabolism is dictated by the availability of nutrients in the tsetse fly vector.

The crystal structure and localization ofTrypanosoma bruceiinvariant surface glycoproteins suggest a more permissive VSG coat in the tsetse-transmitted metacyclic stage

“…In addition, we cannot rule out that the undetermined VSG repertoire of the TSW-196 strain 77 (used in this study) could be different to that from other strains 78 . Importantly, previous studies have found similar results although this aspect has been overlooked 34,36,64,74 (Supplementary Table 3). For example, at least two BSF VSGs were identified in saliva from flies infected with T. b. brucei EATRO 1125 34 ; one BSF vsg was found to be highly transcribed in a T. b. brucei RUMP503 SG infection 64 ; and 15 BSF vsg s had similar transcription levels than mvsg s (and a few were detected as protein) in T. b. brucei Lister 427 MCF overexpressing the RNA-binding protein 6 74 .…”

“…The identification of mVSG peptides in infected saliva is not surprising given that infected SGs contain thousands of quiescent metacyclic cells, which may release mVSGs into the SG lumen. mVSG release may occur partly by the action of the parasite GPI-PLC, which is highly expressed in MCFs 21,74 and/or by the action of tsetse salivary proteases. Interestingly, among the VSG species detected, only one corresponds to a canonical mVSG (mVAT4), which has not been previously detected as protein, but its MES has been well characterized 75 .…”

“…Surprisingly, however, we identified at least four BSF VSGs, including MiTat 1.2 (identified by three unique peptides). This suggests that either (1) MCF could have active BES (unlikely since MCFs do not appear to express ESAG proteins 74 ) or 2) there is recombination at the MES replacing the mvsg by a vsg gene, or 3) there is recombination between mvsg and vsg genes leading to the formation of mosaics. In addition, we cannot rule out that the undetermined VSG repertoire of the TSW-196 strain 77 (used in this study) could be different to that from other strains 78 .…”

The zinc finger proteins ZC3H20 and ZC3H21 stabilise mRNAs encoding membrane proteins and mitochondrial proteins in insect-formTrypanosoma brucei

“…The 3'-UTRs of these mRNAs have, respectively, two, five, and seven copies of the RBP10 consensus recognition motif ( Fig 1A). All three mRNAs are more abundant, and considerably better translated, in procyclic forms than in growing bloodstream forms , Dejung et al, 2016, Jensen et al, 2014, Antwi et al, 2016, and their expression is also lower in metacyclic forms than in procyclic forms (Christiano et al, 2017). When ZC3H20 or ZC3H21 are tethered (via a lambdaNpeptide -boxB interaction) to a reporter mRNA in bloodstream forms, expression is stimulated, whereas tethering of ZC3H22 leads to repression (Lueong et al, 2016.…”

The zinc finger proteins ZC3H20 and ZC3H21 stabilise mRNAs encoding membrane proteins and mitochondrial proteins in insect‐form Trypanosoma brucei

“…The 3′‐UTRs of these mRNAs have, respectively, two, five and seven copies of the RBP10 consensus recognition motif (Figure a). All three mRNAs are more abundant, and considerably better translated, in procyclic forms than in growing bloodstream forms (Antwi et al, ; Dejung et al, ; Fadda et al, ; Jensen et al, ), and their expression is also lower in metacyclic forms than in procyclic forms (Christiano et al, ). When ZC3H20 or ZC3H21 are tethered (via a lambdaN‐peptide–boxB interaction) to a reporter mRNA in bloodstream forms, expression is stimulated, whereas tethering of ZC3H22 leads to repression (Erben, Fadda, Lueong, Hoheisel, & Clayton, ; Lueong, Merce, Fischer, Hoheisel, & Erben, ).…”