Anti-methicillin-resistant Staphylococcus aureus and antibiofilm activity of new peptides produced by a Brevibacillus strain.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is listed as a highly prioritized pathogen by the World Health Organization (WHO) to search for effective antimicrobial agents. Previously, we isolated a soil Brevibacillus sp. strain SPR19 from a botanical garden, which showed anti-MRSA activity. However, the active substances were still unknown. Methods: The cell-free supernatant of this bacterium was subjected to salt precipitation, cation exchange, and reversed-phase chromatography. The antimicrobial activity of pure substances was determined by broth microdilution assay. The peptide sequences and secondary structures were characterized by tandem mass spectroscopy and circular dichroism (CD), respectively. The most active anti-MRSA peptide underwent a stability study, and its mechanism was determined through scanning electron microscopy, cell permeability assay, time-killing kinetics, and biofilm inhibition and eradication. Hemolysis was used to evaluate the peptide toxicity. Results: The pure substances (BrSPR19-P1 to BrSPR19-P5) were identified as new peptides. Their minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) against S. aureus and MRSA isolates ranged from 2.00 to 32.00 and 2.00 to 64.00 µg/mL, respectively. The sequence analysis of anti-MRSA peptides revealed a length ranging from 12 to 16 residues accompanied by an amphipathic structure. The physicochemical properties of peptides were predicted such as pI (4.25 to 10.18), net charge at pH 7.4 (-3 to +4), and hydrophobicity (0.12 to 0.96). The CD spectra revealed that all peptides in the water mainly contained random coil structures. The increased proportion of α-helix structure was observed in P2-P5 when incubated with SDS. P2 (NH2-MFLVVKVLKYVV-COOH) showed the highest antimicrobial activity and high stability under stressed conditions such as temperatures up to 100 °C, solution of pH 3 to 10, and proteolytic enzymes. P2 disrupted the cell membrane and caused bacteriolysis, in which its action was dependent on the incubation time and peptide concentration. Antibiofilm activity of P2 was determined by which the half-maximal inhibition of biofilm formation was observed at 2.92 and 4.84 µg/mL for S. aureus TISTR 517 and MRSA isolate 2468, respectively. Biofilm eradication of tested pathogens was found at the P2 concentration of 128 µg/mL. Furthermore, P2 hemolytic activity was less than 10% at concentrations up to 64 µg/mL, which reflected the hemolysis index thresholds of 32. Conclusion: Five novel anti-MRSA peptides were identified from SPR19. P2 was the most active peptide and was demonstrated to cause membrane disruption and cell lysis. The P2 activity was dependent on the peptide concentration and exposure time. This peptide had antibiofilm activity against tested pathogens and was compatible with human erythrocytes, supporting its potential use as an anti-MRSA agent in this post-antibiotic era.

Purification and Characterization of Novel Anti-MRSA Peptides Produced by Brevibacillus sp. SPR-20.

Methicillin-resistant Staphylococcus aureus (MRSA) is listed as a high-priority pathogen because its infection is associated with a high mortality rate. It is urgent to search for new agents to treat such an infection. Our previous study isolated a soil bacterium (Brevibacillus sp. SPR-20), showing the highest antimicrobial activity against S. aureus TISTR 517 and MRSA strains. The present study aimed to purify and characterize anti-MRSA substances produced by SPR-20. The result showed that five active substances (P1-P5) were found, and they were identified by LC-MS/MS that provided the peptide sequences of 14-15 residues. Circular dichroism showed that all peptides contained ß-strand and disordered conformations as the major secondary structures. Only P1-P4 adopted more α-helix conformations when incubated with 50 mM SDS. These anti-MRSA peptides could inhibit S. aureus and MRSA in concentrations of 2-32 µg/mL. P1 (NH2-VVVNVLVKVLPPPVV-COOH) had the highest activity and was identified as a novel antimicrobial peptide (AMP). The stability study revealed that P1 was stable in response to temperature, proteolytic enzymes, surfactant, and pH. The electron micrograph showed that P1 induced bacterial membrane damage when treated at 1× MIC in the first hour of incubation. The killing kinetics of P1 was dependent on concentration and time. Mechanisms of P1 on tested pathogens involved membrane permeability, leakage of genetic material, and cell lysis. The P1 peptide at a concentration up to 32 µg/mL showed hemolysis of less than 10%, supporting its safety for human erythrocytes. This study provides promising anti-MRSA peptides that might be developed for effective antibiotics in the post-antibiotic era.

In Vitro Enhanced Sensitivity to Cisplatin in D67Y BRCA1 RING Domain Protein.

BRCA1 is a tumor suppressor protein involved in maintaining genomic integrity through multiple functions in DNA damage repair, transcriptional regulation, cell cycle checkpoint, and protein ubiquitination. The BRCA1-BARD1 RING complex has an E3 ubiquitin ligase function that plays essential roles in response to DNA damage repair. BRCA1-associated cancers have been shown to confer a hypersensitivity to chemotherapeutic agents. Here, we have studied the functional consequence of the in vitro E3 ubiquitin ligase activity and cisplatin sensitivity of the missense mutation D67Y BRCA1 RING domain. The D67Y BRCA1 RING domain protein exhibited the reduced ubiquitination function, and was more susceptible to the drug than the D67E or wild-type BRCA1 RING domain protein. This evidence emphasized the potential of using the BRCA1 dysfunction as an important determinant of chemotherapy responses in breast cancer.

The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by the platinum-based anticancer drugs.

The breast cancer susceptibility protein 1 (BRCA1) participates in the maintenance of cells genomic integrity through DNA repair, cell cycle checkpoint, protein ubiquitination, and transcriptional regulation. The N-terminus of BRCA1 contains a RING domain that preferentially forms a heterodimeric complex with BARD1. The BRCA1-BARD1 RING complex has an E3 ubiquitin ligase activity that plays an essential role in response to DNA damage. Preclinical and clinical studies have recently revealed that structural changes to the heterodimer result in alterations to the BRCA1-mediated DNA repair pathways in cancer cells, and lead to hypersensitivity to several chemotherapeutic agents. It is of interest to approach the BRCA1 RING domain as a potentially molecular target for platinum-based drugs for cancer therapy. A previous study has shown that the anticancer drug cisplatin formed intramolecular and intermolecular BRCA1 adducts in which His117 was the primary platinum-binding site, and conferred conformational changes and induced thermostability. Here, we have studied the functional consequence of the in vitro platination of the BRCA1 RING domain by a number of platinum complexes. The BRCA1 ubiquitin ligase activity was inhibited by transplatin > cisplatin > oxaliplatin > carboplatin in that order. The consequences of the binding of the platinum complexes on the reactivity of the BRCA1 were also discussed. The data raised the possibility of selectively targeting the BRCA1 DNA repair for cancer therapy.

Substitution of aspartic acid with glutamic acid at position 67 of the BRCA1 RING domain retains ubiquitin ligase activity and zinc(II) binding with a reduced transition temperature.

Breast cancer susceptibility protein 1 (BRCA1) participates in genomic integrity maintenance through DNA repair, cell cycle checkpoint, protein ubiquitination, and transcriptional regulation. The N-terminus of BRCA1 contains a RING domain which forms two Zn(2+) binding sites in an interleaved fashion. A number of deleterious BRCA1 missense mutations, which predispose an individual to a subset of hereditary breast and ovarian cancers, have been identified in the RING domain. Disruption of Zn(2+) binding sites and protein structure results in the inactivation of BRCA1 tumor suppression function. An unprecedented D67E BRCA1 mutation, identified in Thai familial breast cancer patients, is located in the vicinity of Zn(2+) binding site II, and its pathogenic significance remains elusive. The present study revealed that the D67E BRCA1 RING protein assumes a preformed structure in the absence of Zn(2+). The Zn(2+)-bound mutant protein was more folded, resulting in enhanced proteolytic resistance and dimerization. This indicated that the mutation retained Zn(2+) binding, and barely perturbed the native global structure of the BRCA1 RING domain. The complex between D67E BRCA1 and BARD1 RING domains exhibited a substantial ubiquitin ligase activity compared with a defective complex containing the C61G BRCA1 mutation. However, the D67E mutation was slightly less stable toward thermal denaturation. This implies that the D67E mutation might be a neutral or mild cancer-risk modifier of other defective mechanisms underlying BRCA1-mutation-related breast cancer.

Cisplatin affects the conformation of apo form, not holo form, of BRCA1 RING finger domain and confers thermal stability.

The breast cancer suppressor protein 1 (BRCA1) has been shown to participate in genomic integrity maintenance. Preclinical and clinical studies have recently revealed that the inactivation of BRCA1 in cancer cells leads to chemosensitivity. Approaching the BRCA1 RING protein as a potentially molecular target for a platinum-based drug might be of interest in cancer therapy. In the present study, the in vitro platination of the BRCA1 RING protein by the anticancer drug cisplatin was observed. The protein contained a preformed structure in the apo form with structural changes and resistance to limited proteolysis after Zn2+ binding. SDS-PAGE and mass-spectrometric analyses revealed that cisplatin preferentially formed monofunctional and bifunctional BRCA1 adducts. Tandem mass spectrometry (MS/MS) of the 656.29(2+) ion indicated that the ion arose from [Pt(NH3)2(OH)]+ bound to the BRCA1 peptide (111)ENNSPEHLK(119). The product-ion spectrum revealed the Pt-binding site on His117. Circular dichroism showed that the apo form, not holo form, of BRCA1 underwent more folded structural rearrangement upon cisplatin binding. Cisplatin-bound protein exhibited an enhanced thermostability by 13 degrees , resulting from the favorably intermolecular cross-links driven by the free energy. Our findings demonstrated the first conformational and thermal evidences for a direct binding of cisplatin to the BRCA1 RING domain and could raise a possibility of selectively targeted treatment of cancer with less toxicity or improved response to conventional regimens.