r s aureus atcc 25923 s epidermidis rp62a s aureus atcc 43300b e  (ATCC)

Journal: JACS Au

Article Title: Discovery of Encrypted Peptides in a Human Matrix Metallopeptidase

doi: 10.1021/jacsau.5c00947

Figure Lengend Snippet: Identification, production and antimicrobial activity of MMP-19-derived encrypted peptides. (A) Novel antimicrobial peptides were identified within the MMP-19 precursor protein through a previously reported in silico prediction strategy. All computational tools are provided in the Supporting Information of the original publication (Files S1.xls and S2.doc). Two regions of MMP-19 were selected as a source of putative antimicrobial peptides. Two-dimensional plots showing AS values as a function of residue position for selected window lengths are provided in Supporting Figures S1 and S2 . (B) Recombinant expression of peptides in bacterial cells. The first identified region (residues 1-33) was recombinantly produced in two forms: a shorter peptide (residues 1-19), termed r­(P)­YLL19, and a longer peptide (residues 1-33), termed r­(P)­YLL33. In all cases, recombinant peptides were expressed as chimeric proteins fused to the carrier protein onconase. (C) Antimicrobial activity of MMP-19-derived peptides against a panel of 18 bacterial strains. Experiments were performed using an initial bacterial inoculum of ∼2 × 10 6 CFU mL –1 . Reported data represent the mean ± SD from three independent experiments. (D) Time-kill curves of S. epidermidis ATCC 35984 and A. baumannii ATCC 19606 treated over 0–24 h with MMP-19-derived peptides at their respective MICs (10 μM r­(P)­YLL19, 5 μM r­(P)­YLL33, 10 μM r­(P)­PRT33 for S. epidermidis ATCC 35984; 5 μM r­(P)­YLL19, 5 μM r­(P)­YLL33, 5 μM r­(P)­PRT33 for A. baumannii ATCC 19606). Colistin (2.4 μM) was used as a positive control, while untreated cells served as negative controls.

Article Snippet: Notably, treatment of S. epidermidis ATCC 35984 with r (P) YLL33 combined with colistin or polymyxin B, as wells as r (P) PRT33 combined with colistin, resulted in >99.9 % bacterial cell death within 15 min, far faster than either agent alone ( B).

Techniques: Activity Assay, Derivative Assay, In Silico, Residue, Recombinant, Expressing, Produced, Positive Control

Journal: JACS Au

Article Title: Discovery of Encrypted Peptides in a Human Matrix Metallopeptidase

doi: 10.1021/jacsau.5c00947

Figure Lengend Snippet: Antimicrobial activity of MMP-19-derived EPs. (A) Morphological analyses of S. epidermidis ATCC 35984 and A. baumannii ATCC 19606 by SEM. Representative images are shown. Sixty cells were analyzed per condition in two independent experiments. Scale bars: 1 and 2 μm. (B) Time-kill curves showing the effects of combinatorial treatments using MMP-19-derived peptides with colistin or polymyxin B on S. epidermidis ATCC 35984. Results are compared with single-agent treatments at equivalent sub-MIC concentrations. The initial bacterial inoculum was approximately 2 × 10 6 CFU mL –1 . Peptides, polymyxin B, and colistin were tested at the sub-MIC levels used in the combination assays. The first panel represents a combination with an FICI of 0.71 (2.5 μM r­(P)­YLL33 + 4 μM colistin), while the second and third panels correspond to combinations with FICI values of 1.0 (2.5 μM r­(P)­YLL33 + 2 μM polymyxin B; 2.5 μM r­(P)­PRT33 + 2 μM polymyxin B).

Article Snippet: Notably, treatment of S. epidermidis ATCC 35984 with r (P) YLL33 combined with colistin or polymyxin B, as wells as r (P) PRT33 combined with colistin, resulted in >99.9 % bacterial cell death within 15 min, far faster than either agent alone ( B).

Techniques: Activity Assay, Derivative Assay

Journal: JACS Au

Article Title: Discovery of Encrypted Peptides in a Human Matrix Metallopeptidase

doi: 10.1021/jacsau.5c00947

Figure Lengend Snippet: MMP-19-derived EPs affect membrane polarization without promoting resistance development. (A) Membrane depolarization in response to increasing concentrations of MMP-19-derived peptides, assessed via fluorescence changes of the membrane potential-sensitive dye DiSC 3 (5). Bacterial cells were treated for 1 h with peptides at 2.5, 5, and 10 μM. Colistin (8 μM) was used as a positive control, and untreated cells served as negative controls. Bacteria were incubated at an optical density (OD 600 nm ) of 0.03–0.06, corresponding to approximately 5 × 10 7 CFU mL –1 . Data represent the mean ± SD from three independent experiments. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Assessment of resistance phenotype development in A. baumannii ATCC 19606 and S. epidermidis ATCC 35984 following serial exposure to colistin (4 or 8 μM), polymyxin B (2 or 4 μM), ciprofloxacin (1.5 or 6 μM), r­(P)­YLL19 (5 μM), r­(P)­YLL33 (5 μM), or r­(P)­PRT33 (5 μM). The initial bacterial inoculum was approximately 2 × 10 6 CFU mL –1 in all cases. (C) SEM analysis of A. baumannii ATCC 19606 and S. epidermidis ATCC 35984 cells after 21 serial treatments with ciprofloxacin, compared to untreated control cells. Bacteria were incubated at an optical density (OD 600 nm ) of 0.1, corresponding to approximately 1 × 10 8 CFU mL –1 . Scale bars: 2 μm.

Article Snippet: Notably, treatment of S. epidermidis ATCC 35984 with r (P) YLL33 combined with colistin or polymyxin B, as wells as r (P) PRT33 combined with colistin, resulted in >99.9 % bacterial cell death within 15 min, far faster than either agent alone ( B).

Techniques: Derivative Assay, Membrane, Fluorescence, Positive Control, Bacteria, Incubation, Control

Journal: JACS Au

Article Title: Discovery of Encrypted Peptides in a Human Matrix Metallopeptidase

doi: 10.1021/jacsau.5c00947

Figure Lengend Snippet: Anti-biofilm activity of MMP-19-derived EPs. Effects of MMP-19 derived peptides on S. epidermidis ATCC 35984 (left panels) and A. baumannii ATCC 19606 (right panels) biofilm attachment, formation and detachment, as analyzed by CLSM. Bacteria were treated with sub-MIC concentrations of the peptides and stained with Syto9 (total cells) and Propidium Iodide (PI; dead cells). (A) Fluorescence main intensity reported in arbitrary units, (B) biofilm thickness, and (C) representative 3D reconstructions. Data refer to three independent experiments, each comprising at least three image acquisitions. Statistical significance was determined using Student’s t -test, with comparisons made against the corresponding control groups. Significance levels are indicated as follows: * p < 0.05, ** p < 0.01, *** or p < 0.001.

Article Snippet: Notably, treatment of S. epidermidis ATCC 35984 with r (P) YLL33 combined with colistin or polymyxin B, as wells as r (P) PRT33 combined with colistin, resulted in >99.9 % bacterial cell death within 15 min, far faster than either agent alone ( B).

Techniques: Activity Assay, Derivative Assay, Bacteria, Staining, Fluorescence, Control

Journal: JACS Au

Article Title: Discovery of Encrypted Peptides in a Human Matrix Metallopeptidase

doi: 10.1021/jacsau.5c00947

Figure Lengend Snippet: In vitro antimicrobial activity and biocompatibility of synthetic peptide D­(P)­YLL19. (A) Schematic representation of the design strategy showing the substitution of natural l -amino acids with D -amino acids to generate the synthetic peptide D­(P)­YLL19. (B) Antimicrobial activity of D­(P)­YLL19 against six bacterial strains. Bacterial inocula were approximately 2 × 10 6 CFU mL –1 . Experiments were performed in triplicate, and data are reported as mean values. MIC and MBC values of D­(P)­YLL19 were determined and compared with those of the parent peptide r­(P)­YLL19. (C) Antimicrobial activity of D­(P)­YLL19 in combination with conventional antibiotics against S. epidermidis ATCC 35984 and A. baumannii ATCC 19606. FIC indexes were determined from a minimum of three independent experiments, each performed in triplicate. (D) Time-kill kinetics of D­(P)­YLL19 in combination with colistin or polymyxin B against A. baumannii ATCC 19606 (first graph) and S. epidermidis ATCC 35984 (second and third graph). The combination of colistin and D­(P)­YLL19 corresponds to an FIC index of 1.0 against A. baumannii ATCC 19606 and 0.59 against S. epidermidis ATCC 35984; the combination of polymyxin B and D­(P)­YLL19 corresponds to an FIC index of 0.62 against S. epidermidis ATCC 35984.

Article Snippet: Notably, treatment of S. epidermidis ATCC 35984 with r (P) YLL33 combined with colistin or polymyxin B, as wells as r (P) PRT33 combined with colistin, resulted in >99.9 % bacterial cell death within 15 min, far faster than either agent alone ( B).

Techniques: In Vitro, Activity Assay