Assessment of Antimicrobial Activity of Substance P and Neuropeptide Y

Inflammation and infection of mucosal tissue may induce the production of neuropeptides, specifically Substance P and Neuropeptide Y. These neuropeptides have similar amino acid composition, amphipathic design, cationic charge, and size to antimicrobial peptides. Objective: It was our goal to determine if Substance P and Neuropeptide Y had antimicrobial activity against a panel of common bacteria including oral microorganisms. Methods: A radial diffusion assay was used to assess the antimicrobial activity of Substance P and Neuropeptide Y against the following microorganisms: S. mutans, A. actinomycetemcomitans, E. coli, S. aureus, P. aeruginosa, K. pneumoniae, S. marcescens, and C. albicans. Cultures were grown in TSB, pelleted by centrifugation, resuspended in sodium phosphate buffer, adjusted to 1.0 X 10⁸ CFU/ml and added to underlay agar. Synthetic Neuropeptide Y and Substance P were diluted from 500 to 18 ug/ml and added to holes punched in underlay agar. The plates were incubated at 37°C for 3 hours and then covered with a nutrient-rich overlay and incubated overnight at 37°C. Zones of inhibition were measured and plotted against the concentrations of the neuropeptides to obtain the MIC value (y-intercept). All assays were run in triplicate. Results: Neuropeptide Y and Substance P demonstrated antimicrobial activity against E. coli (MIC's = 20.64 ug/ml and 71.5 ug/ml, respectively), but did not have activity against S. mutans, A. actinomycetemcomitans, S. aureus, P. aeruginosa, K. pneumoniae, S. marcescens, and C. albicans (MIC >500 ug/ml). Conclusions: Neuropeptides had antimicrobial activity against E. coli. While Substance P and Neuropeptide Y did not have direct antimicrobial activity against the other microorganisms tested including the oral pathogens, they may still have indirect effects by stimulating local epithelial cells, i.e. keratinocytes, to produce other innate antimicrobial peptides. This hypothesis remains to be tested. Supported by NIH/NIDCR T32 DE014678 and R01 DE014390.