Jinzhi Han

Purification and Characterization of Plantaricin JLA-9: A Novel Bacteriocin against Bacillus spp. Produced by Lactobacillus plantarum JLA-9 from Suan-Tsai, a Traditional Chinese Fermented Cabbage

Bacteriocins are ribosomally synthesized peptides with antimicrobial activity produced by numerous bacteria. A novel bacteriocin-producing strain, Lactobacillus plantarum JLA-9, isolated from Suan-Tsai, a traditional Chinese fermented cabbage, was screened and identified by its physiobiochemical characteristics and 16S rDNA sequence analysis. A new bacteriocin, designated plantaricin JLA-9, was purified using butanol extraction, gel filtration, and reverse-phase high-performance liquid chromatography. The molecular mass of plantaricin JLA-9 was shown to be 1044 Da by MALDI-TOF-MS analyses. The amino acid sequence of plantaricin JLA-9 was predicted to be FWQKMSFA by MALDI-TOF-MS/MS, which was confirmed by Edman degradation. This bacteriocin exhibited broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, especially Bacillus spp., high thermal stability (20 min, 121 °C), and narrow pH stability (pH 2.0-7.0). It was sensitive to α-chymotrypsin, pepsin, alkaline protease, and papain. The mode of action of this bacteriocin responsible for outgrowth inhibition of Bacillus cereus spores was studied. Plantaricin JLA-9 had no detectable effects on germination initiation over 1 h on monitoring the hydration, heat resistance, and 2,6-pyridinedicarboxylic acid (DPA) release of spores. Rather, germination initiation is a prerequisite for the action of plantaricin JLA-9. Plantaricin JLA-9 inhibited growth by preventing the establishment of oxidative metabolism and disrupting membrane integrity in germinating spores within 2 h. The results suggest that plantaricin JLA-9 has potential applications in the control of Bacillus spp. in the food industry.

iTRAQ-based proteomic analysis of LI-F type peptides produced by Paenibacillus polymyxa JSa-9 mode of action against Bacillus cereus

LI-F type peptides (AMP-jsa9) produced by Paenibacillus polymyxa JSa-9 are a group of cyclic lipodepsipeptide antibiotics that exhibit a broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi, especially Bacillus cereus and Fusarium moniliforme. In this study, to better understand the antibacterial mechanism of AMP-jsa9 against B. cereus, the ultrastructure of AMP-jsa9-treated B. cereus cells was observed by both atomic force microscopy and transmission electron microscopy, and quantitative proteomic analysis was performed on proteins extracted from treated and untreated bacterial cells by using isobaric tag for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to access differentially expressed proteins. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis, including determinations of ATP, NAD(+)H, NADP(+)H, reactive oxygen species (ROS), the activities of catalase (CAT) and superoxide dismutase (SOD), and the relative expression of target genes by quantitative real-time PCR. Bacterial cells exposed to AMP-jsa9 showed irregular surfaces with bleb projections and concaves; we hypothesize that AMP-jsa9 penetrated the cell wall and was anchored on the cytoplasmic membrane and that ROS accumulated in the cell membrane after treatment with AMP-jsa9, modulating the bacterial membrane properties and increasing membrane permeability. Consequently, the blebs were formed on the cell wall by the impulsive force of the leakage of intercellular contents. iTRAQ-based proteomic analysis detected a total of 1317 proteins, including 176 differentially expressed proteins (75 upregulated (fold >2) and 101 downregulated (fold <0.5)). Based on proteome analysis, the putative pathways of AMP-jsa9 action against B. cereus can be summarized as: (i) inhibition of bacterial sporulation, thiamine biosynthesis, energy metabolism, DNA transcription and translation, and cell wall biosynthesis, through direct regulation of protein levels; and (ii) indirect effects on the same pathways through the accumulation of ROS and the consequent impairment of cellular functions, resulting from downregulation of antioxidant proteins, especially CAT and SOD. BIOLOGICAL SIGNIFICANCE The mode of action of LI-F type antimicrobial peptides (AMP-jsa9) against B. cereus was elucidated at the proteomic level. Two pathways of AMP-jsa9 action upon B. cereus cells were identified and the mechanism of bleb formation on the surfaces of bacterial cells was predicted based on the results of ultrastructural observation and proteomic analysis. These results are helpful in understanding the mechanism of LI-F type peptides and in providing the theoretical base for applying AMP-jsa9 or its analogs to combat Gram-positive pathogenic bacteria in the food and feed industries.

Insights into the Antimicrobial Activity and Cytotoxicity of Engineered α-Helical Peptide Amphiphiles

Antimicrobial peptides (AMPs) have gained increasing attention, as they can overcome recurring microbial invasions. However, their poor antimicrobial activity and potential cytotoxicity remain impediments to their clinical applications as novel therapeutic agents. To enhance the antimicrobial activity and cell selectivity of AMPs, a series of amphiphilic peptides based on leucocin A were designed by substituting noncharged hydrophilic residues with arginine and leucine. Of the engineered peptides, peptide 7 (WRL3) (WLRAFRRLVRRLARGLRR-NH2) exhibited the highest cell selectivity toward bacterial cells over erythrocytes and macrophages. Fluorescent measurements and microscopic observations demonstrated that 7 increased cell membrane permeability and disrupted membrane envelope integrity, and eventually led to whole cell lysis. Additionally, flow cytometry analysis and subcellular localization studies revealed that 7 showed potent cytotoxicity against human hepatoma cells HepG2. In summary, the data indicate that these engineered peptides, in particular 7, have enormous promise for antibacterial and/or antitumor therapeutics.

Translocation of the thioesterase domain for the redesign of plipastatin synthetase

Non-ribosomal peptide synthetases (NRPSs) are large enzymatic complexes that catalyse the synthesis of biologically active peptides in microorganisms. Genetic engineering has recently been applied to reprogram NRPSs to produce lipopeptides with a new sequence. The carboxyl-terminal thioesterase (TE) domains from NRPSs catalyse cleavage products by hydrolysis or complex macrocyclization. In this study, we modified plipastatin synthetase by moving the intrinsic TE region to the end of the internal thiolation (T) domains, thus generating Bacillus subtilis strains that could produce new truncated cyclic or linear peptides of the predicted sequence, which further provided an important insight into the regioselectivity of plipastatin TE. The TE was capable of recognizing and catalysing the lactone formation between L-Try3 with the last few residues L-Pro7 and L-Gln8 at the C-terminus. Additionally, the unmatched linkers connecting the TE region and T domain resulted in nonproduction strains, suggesting that the native T–TE linker is necessary and sufficient for the TE domain to release the products from the hybrid enzymes. This is the first report to demonstrate truncated cyclic lipopeptides production and module skipping by simply moving the TE domain forward in an NRPS system.

Biocombinatorial Synthesis of Novel Lipopeptides by COM Domain-Mediated Reprogramming of the Plipastatin NRPS Complex

Both donors and acceptors of communication-mediating (COM) domains are essential for coordinating intermolecular communication within nonribosomal peptides synthetases (NRPSs) complexes. Different sets of COM domains provide selectivity, allowing NRPSs to utilize different natural biosynthetic templates. In this study, novel lipopeptides were synthesized by reprogramming the plipastatin biosynthetic machinery. A Thr-to-Asp point mutation was sufficient to shift the selectivity of the donor COM domain of ppsB toward that of ppsD. Deletion and/or interchangeability established donor and acceptor function. Variations in acceptor COM domain did not result in novel product formation in the presence of its partner donor, whereas plipastatin formation was completely abrogated by altering donor modules. Five novel lipopeptides (cyclic pentapeptide, linear hexapeptide, nonapeptide, heptapeptide, and cyclic octapeptide) were identified and verified by high-resolution LC-ESI-MS/MS. In addition, we demonstrated the potential to generate novel strains with the antimicrobial activity by selecting compatible COM domains, and the novel lipopeptides exhibited antimicrobial activity against five of the fungal species at a contention of 31.25–125 μg/ml.

Membrane-Active Amphipathic Peptide WRL3 with in Vitro Antibiofilm Capability and in Vivo Efficacy in Treating Methicillin-Resistant Staphylococcus aureus Burn Wound Infections

Methicillin-resistant Staphylococcus aureus (MRSA) has become increasingly prevalent in hospitals, clinics, and the community. MRSA can cause significant and even lethal infections, especially in skin burn wounds. The currently available topical agents have largely failed to eliminate MRSA infections due to resistance. Therefore, there is an urgent need for new and effective approaches for treating MRSA. Here, we show that a novel engineered amphipathic peptide, WRL3 (WLRAFRRLVRRLARGLRR-NH2), exhibits potent antimicrobial activity against MRSA, even in the presence of various salts or serum. The cell selectivity of WRL3 was demonstrated by its ability to specifically eliminate MRSA cells over host cells in a coculture model. Additionally, WRL3 showed a synergistic effect against MRSA when combined with ceftriaxone and effectively inhibited sessile biofilm bacteria growth leading to a reduction in biomass. Fluorescent measurements and microscopic observations of live bacterial cells and artificial membranes revealed that WRL3 exerted its bactericidal activity possibly by destroying the bacterial membrane. In vivo studies indicate that WRL3 is able to control proliferation of MRSA in wound tissue and reduce bioburden and provides a more favorable environment for wound healing. Collectively, our data suggest that WRL3 has enormous potential as a novel antimicrobial agent for the treatment of clinical MRSA infections of skin burn wounds.

Optimizing the Maximum Recovery of Dihydromyricetin from Chinese Vine Tea, Ampelopsis grossedentata, Using Response Surface Methodology

This work provides an optimized extraction approach intended to maximize the recovery of dihydromyricetin (DHM) from Chinese vine tea (Ampelopsis grossedentata) leaves. The presented work adopts a Box-Behnken design as a response surface methodology to understand the role and influence of specific extraction parameters including: time, temperature, and solvent composition/ethanol (%) on DHM final yields. Initially, single factor experiments were used to delineate the role of above factors (temperature, time, and solvent composition) before proceeding with three factors-three levels Box-Behnken design with 17 separate runs to assess the effect of multifactorial treatments on DHM recovery rates. The collected data shows that independent variables (solvent composition, time, and temperature) can significantly affect DHM recovery rates with maximum yields resulting from a combined 60 °C, 60% aqueous ethanol, and 180 min treatment. From the empirical point of view, the above optimized extraction protocol can substantially enhance processing and profitability margins with a minimum need of interventions or associated costs.

The antibacterial activity and modes of LI‐F type antimicrobial peptides against Bacillus cereus in vitro

LI‐Fs are a family of highly potent cyclic lipodepsipeptide antibiotics with a broad antimicrobial spectrum (Gram‐positive bacteria and fungi). In this study, LI‐F‐type antimicrobial peptides (AMP‐jsa9) composing of LI‐F03a, LI‐F03b, LI‐F04a, LI‐F04b and LI‐F05b were isolated from Paenibacillus polymyxa JSA‐9. To better understand the antimicrobial mechanism of AMP‐jsa9, the potency and action(s) of AMP‐jsa9 against Bacillus cereus were examined.

Mechanism of action of AMP-jsa9, a LI-F-type antimicrobial peptide produced by Paenibacillus polymyxa JSa-9, against Fusarium moniliforme

LI-F type peptides (AMP-jsa9) are a group of cyclic lipodepsipeptides that exhibit broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi. We sought to assess the toxicity of AMP-jsa9 and the mechanism of AMP-jsa9 action against Fusarium moniliforme. AMP-jsa9 exhibited weak hemolytic activity and weak cytotoxicity at antimicrobial concentrations (32μg/ml). Confocal laser microscopy, SEM, and TEM indicated that AMP-jsa9 primarily targets the cell wall, plasma membrane, and cytoskeleton, increases membranepermeability, and enhances cytoplasm leakage (e.g., K+, protein). Quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) detected a total of 162 differentially expressed proteins (59 up-regulated and 103 down-regulated) following treatment of F. moniliforme with AMP-jsa9. AMP-jsa9 treatment also led to reductions in chitin, ergosterol, NADH, NADPH, and ATP levels. Moreover, fumonisin B1 expression and biosynthesis was suppressed in AMP-jsa9-treated F. moniliforme. Our results provide a theoretical basis for the application of AMP-jsa9 as a natural and effective antifungal agent in the agricultural, food, and animal feed industries.

The antibacterial activity of LI-F type peptide against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and inhibition of infections in murine scalded epidermis

LI-F type peptides are a family of cyclic lipodepsipeptide antibiotics isolated from Paenibacillus polymyxa and display potent activities against positive bacteria including methicillin-resistant S. aureus (MRSA). In this study, we investigated the mechanism of action of LI-F type peptide AMP-jsa9 against a MRSA (S. aureus CICC10790), which is resistant to ciprofloxacin, gentamicin, kanamycin, chloramphenicol, methicillin, and tetracycline. It was found that AMP-jsa9 mainly targets the cell membrane of MRSA and is able to inhibit biofilm formation through killing planktonic bacteria cells. Moreover, AMP-jsa9 can bind to DNA in vitro, which represents another pathway for the action on MRSA. Furthermore, in vivo treatment of scalded mice with AMP-jsa9 resulted in inhibiting MRSA infections and healing of the scalded wound. In addition, it was demonstrated that AMP-jsa9 can effectively inhibit MRSA infections in scalded murine epidermis and that inflammatory cytokines including IL-8, IL-6, tumor necrosis factor alpha (TNF-α), and monocyte chemotactic factor-1 (MCP-1) were reduced; moreover, both protein and gene expression levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (e-NOS) were enhanced, which promote neovascularization and proliferation of new granulation tissue.