Exploiting Peptide Chirality and Transport to Dissect the Complex Mechanism of Action of Host Peptides on Bacteria

Elucidation of the complex mechanisms of action of antimicrobial peptides (AMPs) is critical for improving their efficacy. A major challenge in AMP research is distinguishing AMP effects resulting from various protein interactions from those caused by membrane disruption. Moreover, since AMPs often act in multiple cellular compartments, it is challenging to pinpoint where their distinct activities occur. Nodule-specific cysteine-rich (NCR) peptides secreted by legumes, including NCR247, have evolved from AMPs to regulate differentiation of their nitrogen-fixing bacterial partner during symbiosis as well as to exert antimicrobial actions. At sub-lethal concentrations, NCR247 exhibits strikingly pleiotropic effects on Sinorhizobium meliloti. We used the L- and D-enantiomeric forms of NCR247 to distinguish between phenotypes resulting from stereospecific, protein-targeted interactions and those caused by achiral interactions such as membrane disruption. In addition, we utilized an S. meliloti strain lacking BacA, the transporter that imports NCR peptides into the cytoplasm. BacA plays critical symbiotic roles by reducing periplasmic peptide accumulation and fine-tuning symbiotic signaling. Use of the BacA-deficient strain made it possible to distinguish between phenotypes resulting from peptide interactions in the periplasm and those occurring in the cytoplasm. At high concentrations, both L- and D-NCR247 permeabilize bacterial membranes, consistent with nonspecific cationic AMP activity. In the cytoplasm, both NCR247 enantiomers sequester heme and trigger iron starvation in an achiral but BacA-dependent manner. However, only L-NCR247 activates bacterial two-component systems via stereospecific periplasmic interactions. By combining stereochemistry and genetics, this work disentangles the spatial and molecular complexity of NCR247 action. This approach provides critical mechanistic insights into how host peptides with pleiotropic functions modulate bacterial physiology. Author SummaryMany organisms produce antimicrobial peptides (AMPs) to fight infections, but legumes have uniquely co-opted these molecules to control their symbiotic partners. During symbiosis between Medicago truncatula and Sinorhizobium meliloti, the plant secreted Nodule-specific Cysteine-Rich (NCR) peptides, transforms free-living bacteria into differentiated bacteroids that fix nitrogen but cannot reproduce outside the host. One such peptide, NCR247, exerts pleiotropic effects on the bacteria, acting on different subcellular locations, including membrane, heme, and proteins. Using a mirror-image (D-form) peptide, we disentangled peptide effects arising from generic physiochemical interactions versus stereospecific binding. The inner membrane protein BacA is known to play a protective role by importing NCR peptides into the cytoplasm. Using a bacterium lacking BacA, we were able to distinguish the effects of the peptide within and outside the cytoplasm. It was thought that BacA safeguards symbiotic bacteria by internalizing NCR peptides, thereby limiting their toxic membrane lytic effects, yet this has not been demonstrated. We show that BacA prevents lethal overstimulation of signaling pathways in the periplasm by internalizing the peptides. Our methods provide a framework for testing mechanism of action of new peptide-based antibiotics to combat multidrug-resistant bacteria.