Proteomic Adaptation of Streptococcus pneumoniae to the Antimicrobial Peptide Human Beta Defensin 3 (hBD3) in Comparison to Other Cell Surface Stresses

The antimicrobial peptide human Beta defensin 3 (hBD3) is an essential part of the innate immune system and is involved in protection against respiratory pathogens by specifically permeabilizing bacterial membranes. The Gram-positive bacterium Streptococcus pneumoniae causes serious diseases including pneumonia, meningitis, and septicemia, despite being frequently exposed to human defense molecules, including hBD3 during colonization and infection. Thus, the question arises how pneumococci adapt to stress caused by antimicrobial peptides. We addressed this subject by analyzing the proteome of S. pneumoniae after treatment with hBD3 and compared our data with the proteomic changes induced by LL-37, another crucial antimicrobial peptide present in the human respiratory tract. As antimicrobial peptides usually cause membrane perturbations, the response to the membrane active cationic detergent cetyltrimethylammonium bromide (CTAB) was examined to assess the specificity of the pneumococcal response to antimicrobial peptides. In brief, hBD3 and LL-37 induce a similar response in pneumococci and especially, changes in proteins with annotated transporter and virulence function have been identified. However, LL-37 causes changes in the abundance of cell surface modification proteins that cannot be observed after treatment with hBD3. Interestingly, CTAB induces unique proteomic changes in S. pneumoniae. Though, the detergent seems to activate a two-component system that is also activated in response to antimicrobial peptide stress (TCS 05). Overall, our data represent a novel resource on pneumococcal adaptation to specific cell surface stresses on a functional level. This knowledge can potentially be used to develop strategies to circumvent pneumococcal resistance to antimicrobial peptides.

Download Full-text

Heterologous Expression and Purification of the Antimicrobial Peptide Buforin II

Bulletin of Medical Sciences ◽

10.2478/orvtudert-2019-0010 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Boda Ferenc-András ◽

Szabó Zoltán-István ◽

Szőcs Erika ◽

Salamon Pál ◽

Orbán Csongor ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Heterologous Expression ◽

Antimicrobial Peptide ◽

Solid Phase ◽

Host Cells ◽

Fusion Partner ◽

Monitoring And Control ◽

Expression And Purification ◽

Recombinant Peptide ◽

Bacterial Membranes

AbstractAntimicrobial peptides are natural substances that have played a role in the development of the adaptive immune system, and are currently involved in the prevention of infections, through their direct antimicrobial and immunomodulatory properties. While the amino acid composition and spatial structure vary, most antibacterial peptides have a positive surface charge, which allows them to bind to the negative bacterial membranes. Buforin II is a widely studied antimicrobial peptide first obtained through the structural modification of buforin I, a peptide isolated from Bufo gargarizans. The peptide showed significant antibacterial activity against Gram-positive and Gram-negative bacterial strains. The mechanism of action of buforin II differs from that of other antimicrobial peptides, as it binds directly to bacterial DNA and RNA. The aim of our study was to obtain recombinant buforin II with a ubiquitin fusion partner, through heterologous expression in Escherichia coli Rosetta™ (DE3)pLysS cells, using a laboratory scale biore-actor. The incubation of expression host cells in a bioreactor allowed the constant monitoring and control of the process parameters, leading to high biomass levels and an increased production rate of the peptide. The parameters used during incubation were: 37°C, pH=6.9 and dissolved oxygen level above 40%. Purification of the recombinant protein was accomplished by affinity chromatography using a Ni-chelate solid phase to which the 10xHistag of our construct showed affinity. Method optimisation consisted in the use of gradient and linear elution, of which the latter was found to be more effective. Digestion of the fusion partner from the target peptide was performed with ubiquitin carboxyl-terminal hydrolase enzyme. The expression and purification protocols developed in our experiment allow the production of a significant amount of buforin II, allowing its use for further research. Furthermore, the presented methods could be suitable for industrial production of the recombinant peptide..

Download Full-text

Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability and viability of Escherichia coli

Biochemical Journal ◽

10.1042/bj20031975 ◽

2004 ◽

Vol 380 (3) ◽

pp. 859-865 ◽

Cited By ~ 103

Author(s):

Maria Luisa MANGONI ◽

Niv PAPO ◽

Donatella BARRA ◽

Maurizio SIMMACO ◽

Argante BOZZI ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Membrane Permeability ◽

Dependent Manner ◽

Inner Membrane ◽

Tetrazolium Chloride ◽

Bacterial Membranes ◽

Microbial Invasion

Antimicrobial peptides are produced by all organisms in response to microbial invasion and are considered as promising candidates for future antibiotics. There is a wealth of evidence that many of them interact and increase the permeability of bacterial membranes as part of their killing mechanism. However, it is not clear whether this is the lethal step. To address this issue, we studied the interaction of the antimicrobial peptide temporin L with Escherichia coli by using fluorescence, confocal and electron microscopy. The peptide previously isolated from skin secretions of the frog Rana temporaria has the sequence FVQWFSKFLGRIL-NH2. With regard to fluorescence microscopy, we applied, for the first time, a triple-staining method based on the fluorochromes 5-cyano-2,3-ditolyl tetrazolium chloride, 4´,6-diamidino-2-phenylindole and FITC. This technique enabled us to identify, in the same sample, both living and total cells, as well as bacteria with altered membrane permeability. These results reveal that temporin L increases the permeability of the bacterial inner membrane in a dose-dependent manner without destroying the cell's integrity. At low peptide concentrations, the inner membrane becomes permeable to small molecules but does not allow the killing of bacteria. However, at high peptide concentrations, larger molecules, but not DNA, leak out, which results in cell death. Very interestingly, in contrast with many antimicrobial peptides, temporin L does not lyse E. coli cells but rather forms ghost-like bacteria, as observed by scanning and transmission electron microscopy. Besides shedding light on the mode of action of temporin L and possibly that of other antimicrobial peptides, the present study demonstrates the advantage of using the triple-fluorescence approach combined with microscopical techniques to explore the mechanism of membrane-active peptides in general.

Download Full-text

Romo1-Derived Antimicrobial Peptide Is a New Antimicrobial Agent against Multidrug-Resistant Bacteria in a Murine Model of Sepsis

mBio ◽

10.1128/mbio.03258-19 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 2

Author(s):

Hye-Ra Lee ◽

Deok-gyun You ◽

Hong Kyu Kim ◽

Jang Wook Sohn ◽

Min Ja Kim ◽

...

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Murine Model ◽

Multidrug Resistant ◽

Drug Candidate ◽

Resistant Bacteria ◽

Human Beings ◽

Defense Proteins ◽

Bacterial Membranes

ABSTRACT To overcome increasing bacterial resistance to conventional antibiotics, many antimicrobial peptides (AMPs) derived from host defense proteins have been developed. However, there are considerable obstacles to their application to systemic infections because of their low bioavailability. In the present study, we developed an AMP derived from Romo1 (AMPR-11) that exhibits a broad spectrum of antimicrobial activity. AMPR-11 showed remarkable efficacy against sepsis-causing bacteria, including multidrug-resistant strains, with low toxicity in a murine model of sepsis after intravenous administration. It seems that AMPR-11 disrupts bacterial membranes by interacting with cardiolipin and lipid A. From the results of this study, we suggest that AMPR-11 is a new class of agent for overcoming low efficacy in the intravenous application of AMPs and is a promising candidate to overcome multidrug resistance. IMPORTANCE Abuse of antibiotics often leads to increase of multidrug-resistant (MDR) bacteria, which threatens the life of human beings. To overcome threat of antibiotic resistance, scientists are developing a novel class of antibiotics, antimicrobial peptides, that can eradicate MDR bacteria. Unfortunately, these antibiotics have mainly been developed to cure bacterial skin infections rather than others, such as life-threatening sepsis. Major pharmaceutical companies have tried to develop antiseptic drugs; however, they have not been successful. Here, we report that AMPR-11, the antimicrobial peptide (AMP) derived from mitochondrial nonselective channel Romo1, has antimicrobial activity against Gram-positive and Gram-negative bacteria comprising many clinically isolated MDR strains. Moreover, AMPR-11 increased the survival rate in a murine model of sepsis caused by MDR bacteria. We propose that AMPR-11 could be a novel antiseptic drug candidate with a broad antimicrobial spectrum to overcome MDR bacterial infection.

Download Full-text

Antimicrobial Peptides: A Promising Avenue for Human Healthcare

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666191011121722 ◽

2020 ◽

Vol 21 (2) ◽

pp. 90-96 ◽

Cited By ~ 2

Author(s):

Girish M. Bhopale

Keyword(s):

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Antimicrobial Agents ◽

Current Status ◽

Antimicrobial Drugs ◽

Spectrum Activity ◽

Low Levels ◽

Human Healthcare ◽

Broad Spectrum Activity ◽

Promising Avenue

Antimicrobial drugs resistant microbes have been observed worldwide and therefore alternative development of antimicrobial peptides has gained interest in human healthcare. Enormous progress has been made in the development of antimicrobial peptide during the last decade due to major advantages of AMPs such as broad-spectrum activity and low levels of induced resistance over the current antimicrobial agents. This review briefly provides various categories of AMP, their physicochemical properties and mechanism of action which governs their penetration into microbial cell. Further, the recent information on current status of antimicrobial peptide development, their applications and perspective in human healthcare are also described.

Download Full-text

A mixed chirality α-helix in a stapled bicyclic and a linear antimicrobial peptide revealed by X-ray crystallography

RSC Chemical Biology ◽

10.1039/d1cb00124h ◽

2021 ◽

Author(s):

Stéphane Baeriswyl ◽

Hippolyte Personne ◽

Ivan Di Bonaventura ◽

Thilo Köhler ◽

Christian van Delden ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Crystal Structures ◽

X Ray ◽

X Ray Crystallography ◽

Α Helix

We report the first X-ray crystal structures of mixed chirality α-helices comprising only natural residues as the example of bicyclic and linear membrane disruptive amphiphilic antimicrobial peptides containing seven l- and four d-residues.

Download Full-text

Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Marine Drugs ◽

10.3390/md19050239 ◽

2021 ◽

Vol 19 (5) ◽

pp. 239

Author(s):

Anguo Li ◽

Ruihao Huang ◽

Chaogang Wang ◽

Qunju Hu ◽

Hui Li ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Chlamydomonas Reinhardtii ◽

Penaeus Monodon ◽

Total Soluble Protein ◽

Cell Factory ◽

Gram Positive Bacteria ◽

Resistance Selection ◽

Expression Cluster ◽

Potential Strategy

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.

Download Full-text

Structural elucidation upon binding of antimicrobial peptides into binary mixed lipid monolayers mimicking bacterial membranes

Journal of Colloid and Interface Science ◽

10.1016/j.jcis.2021.04.037 ◽

2021 ◽

Author(s):

Daniela Ciumac ◽

Haoning Gong ◽

Richard A. Campbell ◽

Mario Campana ◽

Hai Xu ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Structural Elucidation ◽

Lipid Monolayers ◽

Bacterial Membranes

Download Full-text

Beneficial Impacts of Incorporating the Non-Natural Amino Acid Azulenyl-Alanine into the Trp-Rich Antimicrobial Peptide buCATHL4B

Biomolecules ◽

10.3390/biom11030421 ◽

2021 ◽

Vol 11 (3) ◽

pp. 421

Author(s):

Areetha R. D’Souza ◽

Matthew R. Necelis ◽

Alona Kulesha ◽

Gregory A. Caputo ◽

Olga V. Makhlynets

Keyword(s):

Amino Acid ◽

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Mechanism Of Action ◽

Bactericidal Activity ◽

Antimicrobial Agents ◽

Human Cells ◽

Side Chains ◽

Natural Amino Acid ◽

Proteolytic Stability

Antimicrobial peptides (AMPs) present a promising scaffold for the development of potent antimicrobial agents. Substitution of tryptophan by non-natural amino acid Azulenyl-Alanine (AzAla) would allow studying the mechanism of action of AMPs by using unique properties of this amino acid, such as ability to be excited separately from tryptophan in a multi-Trp AMPs and environmental insensitivity. In this work, we investigate the effect of Trp→AzAla substitution in antimicrobial peptide buCATHL4B (contains three Trp side chains). We found that antimicrobial and bactericidal activity of the original peptide was preserved, while cytocompatibility with human cells and proteolytic stability was improved. We envision that AzAla will find applications as a tool for studies of the mechanism of action of AMPs. In addition, incorporation of this non-natural amino acid into AMP sequences could enhance their application properties.

Download Full-text

Long-term effects of the proline-rich antimicrobial peptide Oncocin112 on the Escherichia coli translation machinery

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013587 ◽

2020 ◽

Vol 295 (38) ◽

pp. 13314-13325

Author(s):

Yanyu Zhu ◽

James C. Weisshaar ◽

Mainak Mustafi

Keyword(s):

Escherichia Coli ◽

Antimicrobial Peptides ◽

Binding Site ◽

Elongation Factor ◽

Microscopy Study ◽

Single Step ◽

Long Term Effects ◽

Bacterial Membranes

Proline-rich antimicrobial peptides (PrAMPs) are cationic antimicrobial peptides unusual for their ability to penetrate bacterial membranes and kill cells without causing membrane permeabilization. Structural studies show that many such PrAMPs bind deep in the peptide exit channel of the ribosome, near the peptidyl transfer center. Biochemical studies of the particular synthetic PrAMP oncocin112 (Onc112) suggest that on reaching the cytoplasm, the peptide occupies its binding site prior to the transition from initiation to the elongation phase of translation, thus blocking further initiation events. We present a superresolution fluorescence microscopy study of the long-term effects of Onc112 on ribosome, elongation factor-Tu (EF-Tu), and DNA spatial distributions and diffusive properties in intact Escherichia coli cells. The new data corroborate earlier mechanistic inferences from studies in vitro. Comparisons with the diffusive behavior induced by the ribosome-binding antibiotics chloramphenicol and kasugamycin show how the specific location of each agent's ribosomal binding site affects the long-term distribution of ribosomal species between 30S and 50S subunits versus 70S polysomes. Analysis of the single-step displacements from ribosome and EF-Tu diffusive trajectories before and after Onc112 treatment suggests that the act of codon testing of noncognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of noncognate TCs on a sub-ms timescale is essential to enable incorporation of the rare cognate amino acids into the growing peptide chain at a rate of ∼20 aa/s.

Download Full-text