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The C-terminal stretch of glycine-rich proline-rich protein (SbGPRP1) from Sorghum bicolor serves as an antimicrobial peptide by targeting the bacterial outer membrane protein

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The C-terminal stretch in SbGPRP1 (Sorghum glycine-rich proline-rich protein) acts as an antimicrobial peptide in the host innate defense mechanism.

Abstract

Cationic antimicrobial proteins or peptides can either bind to the bacterial membrane or target a specific protein on the bacterial membrane thus leading to membrane perturbation. The 197 amino acid polypeptide of SbGPRP1 showed disordered structure at the N-terminal end and ordered conformation at the C-terminal end. In the present study, the expression of N-SbGPRP1, C-SbGPRP1, and ∆SbGPRP1 followed by antimicrobial assays showed potential antimicrobial property of the C-terminal peptide against gram-positive bacteria Bacillus subtilis and phytopathogen Rhodococcus fascians. The SbGPRP1 protein loses its antimicrobial property when the 23 amino acid sequence (GHGGHGVFGGGYGHGGYGHGYGG) from position 136 to 158 is deleted from the protein. Thus, it can be concluded that the 23 amino acid sequence is vital for the said antimicrobial property. NPN assay, SEM analysis, and electrolyte leakage assays showed potent antimicrobial activity for C-SbGPRP1. Overexpression of the C-SbGPRP1 mutant protein in tobacco followed by infection with Rhodococcus fascians inhibited bacterial growth as shown by SEM analysis. To determine if C-SbGPRP1 might target any protein on the bacterial membrane we isolated the bacterial membrane protein from both Bacillus subtilis and Rhodococcus fascians. Bacterial membrane protein that interacted with the column-bound C-SbGPRP1 was eluted and subjected to LC–MS/MS. LC–MS/MS data analysis showed peptide hit with membrane protein YszA from Bacillus subtilis and a membrane protein from Rhodococcus fascians. Isolated bacterial membrane protein from Bacillus subtilis or Rhodococcus fascians was able to reduce the antimicrobial activity of C-SbGPRP1. Furthermore, BiFC experiments showed interactions between C-SbGPRP1 and YszA protein from Bacillus subtilis leading to the conclusion that bacterial membrane protein was targeted in such membrane perturbation leading to antimicrobial activity.

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Acknowledgements

We sincerely express our wholehearted thanks to DST- FIST and UGC CAS for instrumental facilities of Department of Botany, University of Calcutta. We thank CRNN-University of Calcutta for their support regarding scanning electron microscopy. We are grateful to Souvik Roy, Bose Institute for his support with the LC-MS/MS facility.This work is supported by grants to SR from the Council of Scientific and Industrial Research, Government of India (38(1402)/15/EMR-II dated 14.08.2015)).ShRandAD thank the University Grants Commission, Government of India for Research Fellowship (Sanction No.(2061530629 dated 10/12/2015), (813/(CSIR-UGC NET DEC. 2016) respectively). TA and TH thank the Council of Scientific and Industrial Research Institute Fellowship (Sanction No.(09/0028(11361)/2021-EMR-I), (09/ 0028(11939)/2021-EMR-I) respectively). GU thanks the Department of Science and Technology, Government of India for research fellowship.(Sanction No. DST/INSPIRE Fellowship/2015/IF150503).

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Council for Scientific and Industrial Research,38(1402)/15/EMR-II dated 14.08.2015),Sudipta Ray

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  1. Plant Functional Genomics Laboratory, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, West Bengal, India

    Shuddhanjali Roy, Tanushree Agarwal, Arup Das, Tanmoy Halder, Gouranga Upadhyaya & Sudipta Ray

  2. Functional Genomics Laboratory, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, West Bengal, India

    Binay Chaubey

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  1. Shuddhanjali Roy
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SR abstracted and administrated the experiments; SR, TA, AD, TH, and GU performed most of the experiments; SR and TA designed the experiments and analyzed the data; SR conceived the project; SR and TA wrote the manuscript. SR supervised and complemented the writing. All authors read and approved the manuscript.

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Roy, S., Agarwal, T., Das, A. et al. The C-terminal stretch of glycine-rich proline-rich protein (SbGPRP1) from Sorghum bicolor serves as an antimicrobial peptide by targeting the bacterial outer membrane protein. Plant Mol Biol 111, 131–151 (2023). https://doi.org/10.1007/s11103-022-01317-1

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