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Exploring bioactive peptides from bacterial secretomes using PepSAVI-MS: identification and characterization of Bac-21 from Enterococcus faecalis pPD1.

ABSTRACT: As current methods for antibiotic drug discovery are being outpaced by the rise of antimicrobial resistance, new methods and innovative technologies are necessary to replenish our dwindling arsenal of antimicrobial agents. To this end, we developed the PepSAVI-MS pipeline to expedite the search for natural product bioactive peptides. Herein we demonstrate expansion of PepSAVI-MS for the discovery of bacterial-sourced bioactive peptides through identification of the bacteriocin Bac-21 from Enterococcus faecalis pPD1. Minor pipeline modifications including implementation of bacteria-infused agar diffusion assays and optional digestion of peptide libraries highlight the versatility and wide adaptability of the PepSAVI-MS pipeline. Additionally, we have experimentally validated the primary protein sequence of the active, mature Bac-21 peptide for the first time and have confirmed its identity with respect to primary sequence and post-translational processing. Successful application of PepSAVI-MS to bacterial secretomes as demonstrated herein establishes proof-of-principle for use in novel microbial bioactive peptide discovery.

SUBMITTER: Kirkpatrick CL

PROVIDER: S-EPMC6116741 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Exploring bioactive peptides from bacterial secretomes using PepSAVI-MS: identification and characterization of Bac-21 from Enterococcus faecalis pPD1.

Kirkpatrick Christine L CL Parsley Nicole C NC Bartges Tessa E TE Wing Casey E CE Kommineni Sushma S Kristich Christopher J CJ Salzman Nita H NH Patrie Steven M SM Hicks Leslie M LM

Microbial biotechnology 20180716 5

As current methods for antibiotic drug discovery are being outpaced by the rise of antimicrobial resistance, new methods and innovative technologies are necessary to replenish our dwindling arsenal of antimicrobial agents. To this end, we developed the PepSAVI-MS pipeline to expedite the search for natural product bioactive peptides. Herein we demonstrate expansion of PepSAVI-MS for the discovery of bacterial-sourced bioactive peptides through identification of the bacteriocin Bac-21 from Entero ...[more]

PMID: 30014612

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Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Dataset Information

Exploring bioactive peptides from bacterial secretomes using PepSAVI-MS: identification and characterization of Bac-21 from Enterococcus faecalis pPD1.

Publications

Exploring bioactive peptides from bacterial secretomes using PepSAVI-MS: identification and characterization of Bac-21 from Enterococcus faecalis pPD1.

Similar Datasets

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