Selective Membrane Disruption: Mode of Action of C16G2, a Specifically Targeted Antimicrobial Peptide

ABSTRACT The specifically targeted antimicrobial peptide (STAMP) C16G2 was developed to target the cariogenic oral pathogen Streptococcus mutans. Because the design of this peptide was novel, we sought to better understand the mechanism through which it functioned. Compared to antimicrobial peptides (AMPs) with wide spectra of activity, the STAMP C16G2 has demonstrated specificity for S. mutans in a mixed-culture environment, resulting in the complete killing of S. mutans while having minimal effect on the other streptococci. In the current study, we sought to further confirm the selectivity of C16G2 and also compare its membrane activity to that of melittin B, a classical toxic AMP, in order to determine the STAMP's mechanism of cell killing. Disruption of S. mutans cell membranes by C16G2 was demonstrated by increased SYTOX green uptake and ATP efflux from the cells similar to those of melittin B. Treatment with C16G2 also resulted in a loss of membrane potential as measured by DiSC(3)5 fluorescence. In comparison, the individual moieties of C16G2 demonstrated no specificity and limited antimicrobial activity compared to those of the STAMP C16G2. The data suggest that C16G2 has a mechanism of action similar to that of traditional AMPs and kills S. mutans through disruption of the cell membrane, allowing small molecules to leak out of the cell, which is followed by a loss of membrane potential and cell death. Interestingly, this membrane activity is rapid and potent against S. mutans, but not other noncariogenic oral streptococci.

[1]  A. Whittaker,et al.  Structure and orientation of the pore-forming peptide, melittin, in lipid bilayers. , 1994, Journal of molecular biology.

[2]  B. Bechinger,et al.  Structure and Functions of Channel-Forming Peptides: Magainins, Cecropins, Melittin and Alamethicin , 1997, The Journal of Membrane Biology.

[3]  C. Evans,et al.  The Surgeon General's report on America's oral health: opportunities for the dental profession. , 2000, Journal of the American Dental Association.

[4]  W. Shi,et al.  Systematic Approach to Optimizing Specifically Targeted Antimicrobial Peptides against Streptococcus mutans , 2010, Antimicrobial Agents and Chemotherapy.

[5]  W. Shi,et al.  Achieving Probiotic Effects via Modulating Oral Microbial Ecology , 2009, Advances in dental research.

[6]  M. T. Parker,et al.  Streptococci and aerococci associated with systemic infection in man. , 1976, Journal of medical microbiology.

[7]  A. Waring,et al.  Impact of single-residue mutations on the structure and function of ovispirin/novispirin antimicrobial peptides. , 2002, Protein engineering.

[8]  Y. Shai,et al.  Selective lysis of bacteria but not mammalian cells by diastereomers of melittin: structure-function study. , 1997, Biochemistry.

[9]  D. Beighton,et al.  The complex oral microflora of high-risk individuals and groups and its role in the caries process. , 2005, Community dentistry and oral epidemiology.

[10]  H. Inaba,et al.  Detection of oral bacteria in cardiovascular specimens. , 2009, Oral microbiology and immunology.

[11]  W. Shi,et al.  Adding Selectivity to Antimicrobial Peptides: Rational Design of a Multidomain Peptide against Pseudomonas spp , 2006, Antimicrobial Agents and Chemotherapy.

[12]  Tianlei Liu,et al.  A Method for Structure–Activity Analysis of Quorum-Sensing Signaling Peptides from Naturally Transformable Streptococci , 2009, Biological Procedures Online.

[13]  Ramesh Rathinakumar,et al.  Broad-spectrum antimicrobial peptides by rational combinatorial design and high-throughput screening: the importance of interfacial activity. , 2009, Journal of the American Chemical Society.

[14]  S. Goodman,et al.  Peptide pheromone induced cell death of Streptococcus mutans. , 2005, FEMS microbiology letters.

[15]  M. H. Anderson,et al.  A probiotic approach to caries management. , 2006, Pediatric dentistry.

[16]  H. Black,et al.  Single-dose pharmacokinetics and antibacterial activity of daptomycin, a new lipopeptide antibiotic, in healthy volunteers , 1992, Antimicrobial Agents and Chemotherapy.

[17]  T. Zendo,et al.  Lactococcal membrane-permeabilizing antimicrobial peptides , 2010, Applied Microbiology and Biotechnology.

[18]  Maxwell H. Anderson,et al.  Interspecies Interactions within Oral Microbial Communities , 2007, Microbiology and Molecular Biology Reviews.

[19]  E Maier,et al.  Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. , 1999, Biochemistry.

[20]  H. Sahl,et al.  The co-evolution of host cationic antimicrobial peptides and microbial resistance , 2006, Nature Reviews Microbiology.

[21]  L. J. Cole,et al.  Antibacterial action of a bee venom fraction (melittin) against a penicillin-resistant staphylococcus and other microorganisms. USNRDL-TR-67-101. , 1967, Research and development technical report. United States. Naval Radiological Defense Laboratory, San Francisco.

[22]  G. van den Bogaart,et al.  On the Mechanism of Pore Formation by Melittin* , 2008, Journal of Biological Chemistry.

[23]  Y. Shai.  Molecular recognition between membrane-spanning polypeptides. , 1995, Trends in biochemical sciences.

[24]  J. A. Aas,et al.  Microbial Risk Indicators of Early Childhood Caries , 2005, Journal of Clinical Microbiology.

[25]  R. Hancock.  Peptide antibiotics , 1997, The Lancet.

[26]  W. Shi,et al.  Novel Synthetic Antimicrobial Peptides against Streptococcus mutans , 2007, Antimicrobial Agents and Chemotherapy.

[27]  W. Shi,et al.  Targeted Antimicrobial Therapy Against Streptococcus mutans Establishes Protective Non-cariogenic Oral Biofilms and Reduces Subsequent Infection , 2010, International Journal of Oral Science.

[28]  Y. Shai,et al.  Mode of action of linear amphipathic α-helical antimicrobial peptides , 1998 .

[29]  Y. Shai,et al.  Thermodynamics of melittin binding to lipid bilayers. Aggregation and pore formation. , 2009, Biochemistry.

[30]  C H Wang,et al.  Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. , 1974, Biochemistry.

[31]  N. D. Cowell,et al.  The in vivo effect of nisin on the microflora of the oral cavity. , 1971, The Journal of applied bacteriology.

[32]  Jian He,et al.  Design and Characterization of an Acid‐Activated Antimicrobial Peptide , 2010, Chemical biology & drug design.

[33]  J. Kreth,et al.  Streptococcal Antagonism in Oral Biofilms: Streptococcus sanguinis and Streptococcus gordonii Interference with Streptococcus mutans , 2008, Journal of bacteriology.

[34]  R. Hancock,et al.  Cationic peptides: a new source of antibiotics. , 1998, Trends in biotechnology.

[35]  B. Bechinger,et al.  The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy. , 1999, Biochimica et biophysica acta.

[36]  R. Benz,et al.  The peptide antibiotic subtilin acts by formation of voltage-dependent multi-state pores in bacterial and artificial membranes. , 1989, European journal of biochemistry.

[37]  W. Shi,et al.  Targeted Killing of Streptococcus mutans by a Pheromone-Guided “Smart” Antimicrobial Peptide , 2006, Antimicrobial Agents and Chemotherapy.