TY - JOUR
T1 - Identification and elucidation of proline-rich antimicrobial peptides with enhanced potency and delivery
AU - Lai, Pin Kuang
AU - Tresnak, Daniel T.
AU - Hackel, Benjamin J.
N1 - Publisher Copyright:
© 2019 Wiley Periodicals, Inc.
PY - 2019/10/1
Y1 - 2019/10/1
N2 - Proline-rich antimicrobial peptides (PrAMPs) kill bacteria via a nonlytic mechanism in which they permeate through the outer membrane, utilize protein-mediated transport across the inner membrane, and target the ribosome to inhibit protein synthesis. We previously reported that substitutions of oncocin (VDKPPYLPRPRPPRRIYNR–NH2) with a pair of cationic residues improved the antimicrobial activity. In this study, we applied the design protocol to three other PrAMPs: apidaecin-1b, pyrrhocoricin, and bactenecin 7(1–16) and found that the substitutions (R4K and I8K/R) for apidaecin-1b improve the activity by twofold (p <.05) against nonpathogenic Escherichia coli. Moreover, the substitutions (L7K/R and R14K) for pyrrhocoricin improve the activity by 2–10-fold (p <.05) against some strains of E. coli and Salmonella Typhimurium. We also performed activity tests against inner membrane protein (SbmA or YgdD) knockout strains. The result is consistent with previous studies that SbmA is the major transporter for apidaecin-1b and pyrrhocoricin derivatives. However, bactenecin 7(1–16) functions independently of these transporters. In addition, several apidaecin-1b derivatives exhibit enhanced activity relative to wild-type only in the absence of SbmA, which is consistent with mutations that enhance transport across the inner membrane. A high performance liquid chromatography-based kinetic assay for cellular association and internalization demonstrates that the selected cationic mutations can improve cellular association in minimal media, but this enhanced association is not required for increased activity, which suggests the importance of inner membrane transport. These functional studies on cationic mutants of PrAMPs advance understanding of potency and mechanism and advance the ability to engineer improved antimicrobials as evidenced by the identification of the pyrrhocoricin mutant (L7R and R14K) with 10-fold elevated potency against pathogenic E. coli.
AB - Proline-rich antimicrobial peptides (PrAMPs) kill bacteria via a nonlytic mechanism in which they permeate through the outer membrane, utilize protein-mediated transport across the inner membrane, and target the ribosome to inhibit protein synthesis. We previously reported that substitutions of oncocin (VDKPPYLPRPRPPRRIYNR–NH2) with a pair of cationic residues improved the antimicrobial activity. In this study, we applied the design protocol to three other PrAMPs: apidaecin-1b, pyrrhocoricin, and bactenecin 7(1–16) and found that the substitutions (R4K and I8K/R) for apidaecin-1b improve the activity by twofold (p <.05) against nonpathogenic Escherichia coli. Moreover, the substitutions (L7K/R and R14K) for pyrrhocoricin improve the activity by 2–10-fold (p <.05) against some strains of E. coli and Salmonella Typhimurium. We also performed activity tests against inner membrane protein (SbmA or YgdD) knockout strains. The result is consistent with previous studies that SbmA is the major transporter for apidaecin-1b and pyrrhocoricin derivatives. However, bactenecin 7(1–16) functions independently of these transporters. In addition, several apidaecin-1b derivatives exhibit enhanced activity relative to wild-type only in the absence of SbmA, which is consistent with mutations that enhance transport across the inner membrane. A high performance liquid chromatography-based kinetic assay for cellular association and internalization demonstrates that the selected cationic mutations can improve cellular association in minimal media, but this enhanced association is not required for increased activity, which suggests the importance of inner membrane transport. These functional studies on cationic mutants of PrAMPs advance understanding of potency and mechanism and advance the ability to engineer improved antimicrobials as evidenced by the identification of the pyrrhocoricin mutant (L7R and R14K) with 10-fold elevated potency against pathogenic E. coli.
KW - SbmA receptor
KW - apidaecin-1b
KW - bactenecin
KW - proline-rich antimicrobial peptides
KW - pyrrhocoricin
UR - http://www.scopus.com/inward/record.url?scp=85071753258&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85071753258&partnerID=8YFLogxK
U2 - 10.1002/bit.27092
DO - 10.1002/bit.27092
M3 - Article
C2 - 31209863
AN - SCOPUS:85071753258
SN - 0006-3592
VL - 116
SP - 2439
EP - 2450
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
IS - 10
ER -