Role of Cysteine Residues and Disulfide Bonds in the Activity of a Legume Root Nodule-specific, Cysteine-rich Peptide

Monica Sani,Renato Longhi,Gail P Ferguson,Matteo Zanda,Peter Mergaert,Bernhard Kerscher,Sergio Dall'Angelo,Mikhail Baloban,Andreas F Haag,Heather M Wilson

doi:10.1074/jbc.m111.311316

Abstract

The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.

Highlights

Legume antimicrobial peptides (AMPs) mediate Sinorhizobium meliloti bacteroid differentiation
Unlike NCR2471–2,3–4, which was capable of increasing S. meliloti cell size at 2 ␮M, NSR247 only started to increase the cell size of S. meliloti at 4 ␮M and to a lower extent than NCR2471–2,3– 4 (Fig. 1C)
We found using higher concentrations of each peptide (10 ␮M), that the NSR247 peptide had substantially reduced antibacterial activity against S. meliloti in vitro relative to NCR2471–2,3–4 (Fig. 1E). These findings showed that the cysteine residues are important for the full activity of NCR247 against S. meliloti

Summary

Introduction

Legume antimicrobial peptides (AMPs) mediate Sinorhizobium meliloti bacteroid differentiation. Results: Cysteine replacements and disulfide bond modifications influence the antimicrobial activity of a legume AMP and its ability to mediate S. meliloti bacteroid differentiation. Conclusion: Specific changes to legume AMPs influence their activity against S. meliloti. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S–S) bridges. We found that either cysteine replacements or S–S bond modifications influenced the activity of NCR247 against S. meliloti. Either substitution of cysteines for serines, changing the S–S bridges from cysteines 1–2, 3– 4 to 1–3, 2– 4 or oxidation of NCR247 lowered its activity against S. meliloti. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides

Objectives

Results

Conclusion