Raphaela Machado Campos Lopes1, Shaker Chuck Farah1
R-bodies were first observed in the cytoplasm of the bacterium Caedibacter taeniospiralis, an endosymbiont of Paramecium aurelia. An attack mechanism was identified in which P. aurelia carrying this endosymbiont exocytosed the bacterium, while protozoa that do not carry it died upon endocytosis.
R-bodies are polymers formed by low molecular weight proteins. Their ultrastructure varies significantly depending on the bacterium of origin. These polymers are sensitive to environmental conditions; for example, C. taeniospiralis R-bodies are sensitive to pH variations: in acidic conditions, R-bodies extend, while in neutral pH, they coil.
R-bodies have great biotechnological potential to be explored. Manipulating the conformational change mechanisms of these proteins may make them promising tools for membrane rupture and controlled release of substances.
To explore their potential and better characterize their structure, the E. coli BL21(DE3) strain was transformed with a plasmid containing the R-body operon. The bacteria are then cultivated in liquid medium, followed by induction of recombinant protein expression. The culture pellet was then subjected to a purification protocol based on the resistance of R-bodies to SDS and their insolubility. The final sample is used for transmission electron microscopy analysis. Subsequently, grids are prepared for cryotomography, and the data is processed using the Etomo software. The sample is also used to perform two-dimensional gel electrophoresis, and its spots are submitted to mass spectrometry. The results are analyzed using the FragPipe software
Preliminary results suggest successful imaging of R-bodies in different pH conditions. However, further optimization of purification and grid preparation is required. In addition, the preparation of grids for cryotomography needs to be improved, including the inclusion of fiducials in the samples to facilitate tomogram processing. Furthermore, mass spectrometry analysis of the two-dimensional gel bands enabled the identification of R-bodies' composition and suggested possible post-translational modifications that may affect the pI of them, highlighting that the polymerization process leading to R-body formation is a complex event. Nevertheless, further mass spectrometry analyses are still required to confirm these findings.
Agradecimentos: We thank Professor Adriana R. Lopes and Adrian Hand for their assistance with 2D-Gel; Gabriel G. Araujo for his assistance with cryo-EM sample preparation; Camila S. S. Santana, Ilda de S. Costa and Maria Griselda Perona for their assistance in routine laboratory procedures; the LNNANO-CNPEM staff.