HrpY protein of Ralstonia solanacearum exhibits spontaneous formation of pilus like assembly: analysis of its stability

Abstract

Type 3 secretory system (T3SS), a complex protein machinery has a unique virulence mechanism that involves injecting effector proteins directly into host cells. The T3SS effector proteins are transported through an extracellular long hollow needle made up of multiple copies of a small protein. In T3SS of the plant pathogen Ralstonia solanacearum, the 8.6 kDa HrpY protein assembles into a large needle like apparatus (pilus) for transporting effector proteins. To study structural details of HrpY, we recombinantly expressed and purified HrpY in E. coli. The dynamic light scattering (DLS) analysis showed that rHrpY has spontaneously formed oligomers of large order (>100 nm). Transmission electron microscopy of rHrpY samples revealed that the large structures are tube like assembly having dimensions 86.3–166.6 nm and 5.8–6.8 nm in length and width respectively. Different molecular sizes of the purified rHrpY hindered the crystallization of the protein. The stability of oligomer assembly was studied with denaturants and surfactants. Denaturants like urea and guanidine HCl could not break them apart; however, detergents like SDS, sarkosyl, Octyl-β-Glucoside, CHAPS, Tween 20, Tween 80 and Triton X-100 showed disassembly of the oligomer. rHrpY assembly was found to withstand up to 50 °C and the circular dichroism analysis revealed that there is no significant change in the secondary structural composition with increase in temperature. However, change in the secondary structure was observed with the addition of SDS.

Communicated by Ramaswamy H. Sarma

Keywords:

• T3SS
• pilus
• needle assembly
• pilus disassembly
• protein oligomer and detergents

Acknowledgements

KP thanks DST-FIST, Government of India for the DLS equipment sanctioned to the department (No: SR/FST/LSII-037/2014). KP thanks DST, Government of India for the BLI equipment sanctioned to him. VL and NA thank the DBT for the research fellowship.

Author contribution

NA & VL, performed experiments, analysed data, and contributed to writing. RN, performed experiments. KP, Conceptualization, Methodology, Resources, Writing – Review & Editing, Supervision, Project administration, Funding acquisition.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The study was supported by funds of the Department of Biotechnology (DBT), Government of India in the form of grant [BT/PR23649/BPA/118/313/2017 dated 11-Sep-2018].