Protein-Engineered Polymers Functionalized with Antimicrobial Peptides for the Development of Active Surfaces

Antibacterial resistance is a major worldwide threat due to the increasing number of infections caused by antibiotic-resistant bacteria with medical devices being a major source of these infections. This suggests the need for new antimicrobial biomaterial designs able to withstand the increasing pressure of antimicrobial resistance. Recombinant protein polymers (rPPs) are an emerging class of nature-inspired biopolymers with unique chemical, physical and biological properties. These polymers can be functionalized with antimicrobial molecules utilizing recombinant DNA technology and then produced in microbial cell factories. In this work, we report the functionalization of rPBPs based on elastin and silk-elastin with different antimicrobial peptides (AMPs). These polymers were produced in Escherichia coli, successfully purified by employing non-chromatographic processes, and used for the production of free-standing films. The antimicrobial activity of the materials was evaluated against Gram-positive and Gram-negative bacteria, and results showed that the polymers demonstrated antimicrobial activity, pointing out the potential of these biopolymers for the development of new advanced antimicrobial materials.

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Microfactories-Bioproduction of Chemicals and Pharmaceuticals by using Microbes

Asian Journal of Biology ◽

10.9734/ajob/2021/v12i230161 ◽

2021 ◽

pp. 44-53

Author(s):

Attiya Rasool

Keyword(s):

Natural Products ◽

Recombinant Dna ◽

Biological Activities ◽

Carbon Sources ◽

Chemical Production ◽

Recombinant Dna Technology ◽

Fine Chemical ◽

Chemical Structures ◽

Microbial Cell Factories ◽

Cell Factories

A variety of organisms, such as bacteria, fungi, and plants, produce secondary metabolites, also known as natural products. Natural products have been a prolific source and an inspiration for numerous medical agents with widely divergent chemical structures and biological activities, including antimicrobial, immunosuppressive, anticancer, and anti-inflammatory activities, many of which have been developed as treatments and have potential therapeutic applications for human diseases. Aside from natural products, the recent development of recombinant DNA technology has sparked the development of a wide array of biopharmaceutical products, such as recombinant proteins, offering significant advances in treating a broad spectrum of medical illnesses and conditions. Fine chemicals that are physiologically active, such as pharmaceuticals, cosmetics, nutritional supplements, flavoring agents as well as additives for foods, feed, and fertilizer are produced by enzymatically or through microbial fermentation. The identification of enzymes that catalyze the target reaction makes possible to synthesis of the desired fine chemical. The genes encoding these enzymes are then introduced into suitable microbial hosts that are cultured with inexpensive, naturally abundant carbon sources, and other nutrients. Metabolic engineering create efficient microbial cell factories for producing chemicals at higher yields. In the present review, we summarize recent studies on bio-based fine chemical production and assess the potential of synthetic bioengineering for further improvement their productivity.

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Nanostructured Antimicrobial Peptides: Crucial Steps of Overcoming the Bottleneck for Clinics

Frontiers in Microbiology ◽

10.3389/fmicb.2021.710199 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zhanyi Yang ◽

Shiqi He ◽

Hua Wu ◽

Ting Yin ◽

Lili Wang ◽

...

Keyword(s):

Antimicrobial Activity ◽

Controlled Release ◽

Antimicrobial Peptides ◽

Biological Activities ◽

Antimicrobial Properties ◽

Multidrug Resistant ◽

Great Promise ◽

Resistant Bacteria ◽

Biological Stability ◽

Security Issue

The security issue of human health is faced with dispiriting threats from multidrug-resistant bacteria infections induced by the abuse and misuse of antibiotics. Over decades, the antimicrobial peptides (AMPs) hold great promise as a viable alternative to treatment with antibiotics due to their peculiar antimicrobial mechanisms of action, broad-spectrum antimicrobial activity, lower drug residue, and ease of synthesis and modification. However, they universally express a series of disadvantages that hinder their potential application in the biomedical field (e.g., low bioavailability, poor protease resistance, and high cytotoxicity) and extremely waste the abundant resources of AMP database discovered over the decades. For all these reasons, the nanostructured antimicrobial peptides (Ns-AMPs), based on a variety of nanosystem modification, have made up for the deficiencies and pushed the development of novel AMP-based antimicrobial therapies. In this review, we provide an overview of the advantages of Ns-AMPs in improving therapeutic efficacy and biological stability, reducing side effects, and gaining the effect of organic targeting and drug controlled release. Then the different material categories of Ns-AMPs are described, including inorganic material nanosystems containing AMPs, organic material nanosystems containing AMPs, and self-assembled AMPs. Additionally, this review focuses on the Ns-AMPs for the effect of biological activities, with emphasis on antimicrobial activity, biosecurity, and biological stability. The “state-of-the-art” antimicrobial modes of Ns-AMPs, including controlled release of AMPs under a specific environment or intrinsic antimicrobial properties of Ns-AMPs, are also explicated. Finally, the perspectives and conclusions of the current research in this field are also summarized.

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Limacus flavus yellow slug: bioactive molecules in the mucus

10.1101/2021.05.06.442857 ◽

2021 ◽

Author(s):

Patricia Yumi Hayashida ◽

Pedro Ismael Silva Junior

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Water Solubility ◽

Micrococcus Luteus ◽

Chemical Characterization ◽

New Drugs ◽

Bioactive Molecules ◽

Resistant Bacteria ◽

Drug Resistant Bacteria ◽

Wide Range

Background: Snails and slugs were used as a treatment for many health problems therefore ancient times. Since the antimicrobial resistance became a major global thread, antimicrobial peptides have been considered as a potential source for development of new drugs, especially for drug-resistant bacteria. Nowadays reports confirm that the mucous secretions have antimicrobial, antiviral and antifungal properties. Methods: The present study has the objective to characterize and evaluate antimicrobial peptides of Limacus flavus mucus. The mucus was obtained by thermal shock and submitted to RP-HPLC. Fractions were used to perform the antimicrobial activity and hemolytic assays, electrophoresis (SDS-Page Gel) and submitted to mass spectrometry (LC-MS / MS). Identification and characterization was performed by PeaksX+ software. The physicochemical parameters were evaluated with bioinformatics tools, which predicted water solubility, iso-electric point, charge net and its primary structure. Results: Three fractions were isolated from the mucus of L. flavus and presented antifungal and antibacterial activity. The mucus showed greater inhibition for filamentous fungi (Aspergillus niger), yeast (Cryptococcus neoformans), Gram positive bacteria (Bacillus subtilis, Micrococcus luteus) and Gram negative bacteria (Enterobacter cloacae). These fractions also did not show hemolytic activity for human blood cells (erythrocytes). Fractions sequences were identified and presents Mw <3kDa, WLGH, DLQW, YLRW, respectively. Conclusion: This study revealed three antimicrobial peptides of L. flavus mucus with a wide range of antimicrobial activity and its physic-chemical characterization. Keywords: Limacus flavus, mucus, slug, antimicrobial peptide, bioactive molecules, resistance, microorganisms.

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Conjugation of Silver Nanoparticles with De Novo Engineered Cationic Antimicrobial Peptides: Proposal for Study (Preprint)

10.2196/preprints.28307 ◽

2021 ◽

Author(s):

Alvin Hu

Keyword(s):

Antimicrobial Activity ◽

Silver Nanoparticles ◽

Minimum Inhibitory Concentration ◽

Antimicrobial Peptides ◽

Inhibitory Concentration ◽

De Novo ◽

Resistant Bacteria ◽

Drug Resistant ◽

Cationic Antimicrobial Peptides ◽

Drug Resistant Bacteria

BACKGROUND Cationic antimicrobial peptides have broad antimicrobial activity and provide a novel way of targeting multi drug resistant bacteria in an era of increasing antimicrobial resistance. Current developments show positive prospects for both antimicrobial peptides and silver nanoparticles individually. OBJECTIVE The primary objective is to propose another method of enhancing antimicrobial activity by conjugating silver nanoparticles with cationic antimicrobial peptides for a subsequent preliminary assessment on studying the minimum inhibitory concentration of multi drug resistant bacteria. The secondary objective would be to evaluate the safety of the conjugated compound to assess viability for in vivo use. METHODS The proposition is planned for approximately 3 overarching stages. Firstly, I propose synthesis of wlbu2c, a modified version of antimicrobial peptide wlbu2 with an added cysteine group, using standard Fmoc procedure. This will subsequently be attempted to stably conjugate with silver nanoparticles ideally through photochemical means. Secondly, the conjugate wlbu2c-AgNP will be tested for antimicrobial activity following Clinical & Laboratory Standards Institute Manual on standard minimum inhibitory concentration testing. If all of the above is completed the experiment can progress to the assessment of cytotoxicity using cell lysis assays. RESULTS I-TASSER simulation revealed that our modified peptide wlbu2c has similar secondary structure to original wlbu2 peptide. No other results have been obtained at this time other than aforementioned theoretical propositions. CONCLUSIONS The addition of silver nanoparticles to already developing de novo engineered antimicrobial peptides provide a second degree of freedom toward the development of potent antimicrobials. Future prospects include emergency last line therapy, treatment for current difficult to eradicate bacterial colonization such as in cystic fibrosis, implantable medical devices, cancer and immunotherapy. This proposal is intended to be provided to the public as I do not anticipate funding at this time.

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Rational Designed Hybrid Peptides Show up to a 6-fold Increase in Antimicrobial Activity and Demonstrate Different Ultrastructural Changes as the Parental Peptides measured by BioSAXS

10.1101/2021.11.05.467440 ◽

2021 ◽

Author(s):

Kai Hilpert ◽

Jurnorain Gani ◽

Christoph Rumancev ◽

Nathan Simpson ◽

Paula Matilde Lopez-Perez ◽

...

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Fold Increase ◽

Ultrastructural Changes ◽

Therapeutic Window ◽

Resistant Bacteria ◽

Haemolytic Activity ◽

Hybrid Peptide ◽

E Coli ◽

Hybrid Peptides

Antimicrobial peptides (AMPs) are a promising class of compounds being developed against multi-drug resistant bacteria. Hybridization has been reported to increase antimicrobial activity. Here, two proline-rich peptides (consP1: VRKPPYLPRPRPRPL-CONH2 and Bac5-v291: RWRRPIRRRPIRPPFWR-CONH2) were combined with two arginine-isoleucine-rich peptides (optP1: KIILRIRWR-CONH2 and optP7: KRRVRWIIW-CONH2). Proline-rich antimicrobial peptides (PrAMPs) are known to inhibit the bacterial ribosome, shown also for Bac5-v291, whereas it is hypothesized a dirty drug model for the arginine-isoleucine-rich peptides. That hypothesis was underpinned by transmission electron microscopy and biological small-angle X-ray scattering (BioSAXS). The hybrid peptides showed a stronger antimicrobial activity compared to the proline-rich peptides, except when compared to Bac5-v291 against E. coli. The increase in activity compared to the arginine-isoleucine-rich peptides was up to 6-fold, however, it was not a general increase but was dependent on the combination of peptides and bacteria. BioSAXS experiments revealed that proline-rich peptides and arginine-isoleucine-rich peptides induce very different ultrastructural changes in E. coli, whereas a hybrid peptide (hyP7B5GK) shows changes, different to both parental peptides and the untreated control. These different ultrastructural changes indicated that the mode of action of the parental peptides is different from each other as well as from the hybrid peptide hyP7B5GK. All peptides showed very low haemolytic activity, some of them showed a 100-fold or larger therapeutic window, demonstrating the potential for further drug development.

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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.

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Rational Designed Hybrid Peptides Show up to a 6-Fold Increase in Antimicrobial Activity and Demonstrate Different Ultrastructural Changes as the Parental Peptides Measured by BioSAXS

Frontiers in Pharmacology ◽

10.3389/fphar.2021.769739 ◽

2021 ◽

Vol 12 ◽

Author(s):

Kai Hilpert ◽

Jurnorain Gani ◽

Christoph Rumancev ◽

Nathan Simpson ◽

Paula Matilde Lopez-Perez ◽

...

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Fold Increase ◽

Ultrastructural Changes ◽

Therapeutic Window ◽

Resistant Bacteria ◽

Haemolytic Activity ◽

Hybrid Peptide ◽

E Coli ◽

Hybrid Peptides

Antimicrobial peptides (AMPs) are a promising class of compounds being developed against multi-drug resistant bacteria. Hybridization has been reported to increase antimicrobial activity. Here, two proline-rich peptides (consP1: VRKPPYLPRPRPRPL-CONH2 and Bac5-v291: RWRRPIRRRPIRPPFWR-CONH2) were combined with two arginine-isoleucine-rich peptides (optP1: KIILRIRWR-CONH2 and optP7: KRRVRWIIW-CONH2). Proline-rich antimicrobial peptides (PrAMPs) are known to inhibit the bacterial ribosome, shown also for Bac5-v291, whereas it is hypothesized a “dirty drug” model for the arginine-isoleucine-rich peptides. That hypothesis was underpinned by transmission electron microscopy and biological small-angle X-ray scattering (BioSAXS). The strength of BioSAXS is the power to detect ultrastructural changes in millions of cells in a short time (seconds) in a high-throughput manner. This information can be used to classify antimicrobial compounds into groups according to the ultrastructural changes they inflict on bacteria and how the bacteria react towards that assault. Based on previous studies, this correlates very well with different modes of action. Due to the novelty of this approach direct identification of the target of the antimicrobial compound is not yet fully established, more research is needed. More research is needed to address this limitation. The hybrid peptides showed a stronger antimicrobial activity compared to the proline-rich peptides, except when compared to Bac5-v291 against E. coli. The increase in activity compared to the arginine-isoleucine-rich peptides was up to 6-fold, however, it was not a general increase but was dependent on the combination of peptides and bacteria. BioSAXS experiments revealed that proline-rich peptides and arginine-isoleucine-rich peptides induce very different ultrastructural changes in E. coli, whereas a hybrid peptide (hyP7B5GK) shows changes, different to both parental peptides and the untreated control. These different ultrastructural changes indicated that the mode of action of the parental peptides might be different from each other as well as from the hybrid peptide hyP7B5GK. All peptides showed very low haemolytic activity, some of them showed a 100-fold or larger therapeutic window, demonstrating the potential for further drug development.

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Impact of a Single Point Mutation on the Antimicrobial and Fibrillogenic Properties of Cryptides from Human Apolipoprotein B

Pharmaceuticals ◽

10.3390/ph14070631 ◽

2021 ◽

Vol 14 (7) ◽

pp. 631

Author(s):

Rosa Gaglione ◽

Giovanni Smaldone ◽

Angela Cesaro ◽

Mariano Rumolo ◽

Maria De Luca ◽

...

Keyword(s):

Antimicrobial Activity ◽

Point Mutation ◽

Apolipoprotein B ◽

Single Point ◽

Biological Properties ◽

Resistant Bacteria ◽

Single Point Mutation ◽

Scanning Calorimetry ◽

Host Defense Peptides ◽

Human Apolipoprotein

Host defense peptides (HDPs) are gaining increasing interest, since they are endowed with multiple activities, are often effective on multidrug resistant bacteria and do not generally lead to the development of resistance phenotypes. Cryptic HDPs have been recently identified in human apolipoprotein B and found to be endowed with a broad-spectrum antimicrobial activity, with anti-biofilm, wound healing and immunomodulatory properties, and with the ability to synergistically act in combination with conventional antibiotics, while being not toxic for eukaryotic cells. Here, a multidisciplinary approach was used, including time killing curves, differential scanning calorimetry, circular dichroism, ThT binding assays, and transmission electron microscopy analyses. The effects of a single point mutation (Pro → Ala in position 7) on the biological properties of ApoB-derived peptide r(P)ApoBLPro have been evaluated. Although the two versions of the peptide share similar antimicrobial and anti-biofilm properties, only r(P)ApoBLAla peptide was found to exert bactericidal effects. Interestingly, antimicrobial activity of both peptide versions appears to be dependent from their interaction with specific components of bacterial surfaces, such as LPS or LTA, which induce peptides to form β-sheet-rich amyloid-like structures. Altogether, obtained data indicate a correlation between ApoB-derived peptides self-assembling state and their antibacterial activity.

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Characterization of the antimicrobial activity of the cell-penetrating peptide TAT-RasGAP317-326

10.1101/2020.10.02.321802 ◽

2020 ◽

Author(s):

Maria Georgieva ◽

Tytti Heinonen ◽

Alessandra Vitale ◽

Simone Hargraves ◽

Senka Causevic ◽

...

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Bacterial Resistance ◽

Divalent Cations ◽

Low Ph ◽

Resistant Bacteria ◽

Cell Penetrating Peptide ◽

Cell Penetrating ◽

Emergence Of Resistance ◽

Insight Into

AbstractThe efficiency of classical antibiotics is undermined by the emergence of resistance. Antimicrobial peptides (AMPs) may represent an alternative to antibiotics currently used in the clinic. TAT-RasGAP317-326 is one of such AMP. It is a cell-penetrating peptide with broad antimicrobial activity against Gram-positive and Gram-negative bacteria. However, the mechanisms allowing this peptide to kill bacteria are not characterized. Here, we aim to provide a better understanding of the factors influencing TAT-RasGAP317-326 antimicrobial activity.In this article, we show that divalent cations, serum proteins, and low pH levels modulate TAT-RasGAP317-326 activity against bacteria. We demonstrate that low pH and divalent cations reduce the binding of the peptide to bacteria. Using transcriptomics and screening of bacterial mutant libraries, we mapped the transcriptional response of TAT-RasGAP317-326-exposed bacteria and highlighted cellular pathways that may play a role in bacterial resistance to this peptide. We tested combinations of TAT-RasGAP317-326 with other AMPs and detected an inhibitory effect of melittin on TAT-RasGAP317-326 activity. Finally, we performed a resistance selection screen that revealed differences between bacterial strains with respect to their rate of resistance emergence against TAT-RasGAP317-326.Our study provides the first investigation of factors that impact the antimicrobial activity of TAT-RasGAP317-326 and brings insights into the mechanisms underlying the peptide’s activity.ImportanceAntibiotic resistance is an increasing concern for modern medicine. Resistant bacteria cause life-threatening infections that are very challenging to treat. Antimicrobial peptides (AMPs) could be a promising alternative to classical antibiotics because of their antibacterial properties and low propensity for the development of bacterial resistance. To maximize the efficiency of AMPs and avoid the emergence of resistance, it is important to understand the factors affecting their activity. In this study, we investigate the antimicrobial activity of a recently described AMP, TAT-RasGAP317-326. We outline a subset of extracellular factors that influence its antimicrobial activity and characterize the bacterial response to this AMP. Our findings here also provide vital insight into the potential of bacteria developing resistance to this TAT-RasGAP317-326 peptide. Overall, this study provides important insight into the basic mechanism of action of TAT-RasGAP317-326 and a better understanding of the interaction between TAT-RasGAP317-326 and its bacterial targets.

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Enzymatic Synthesis of 15N-L-aspartic Acid Using Recombinant Aspartase from Escherichia Coli K12

Revista de Chimie ◽

10.37358/rc.08.11.2004 ◽

2008 ◽

Vol 59 (11) ◽

Cited By ~ 1

Author(s):

Iulia Lupan ◽

Sergiu Chira ◽

Maria Chiriac ◽

Nicolae Palibroda ◽

Octavian Popescu

Keyword(s):

Amino Acids ◽

Aspartic Acid ◽

Enzymatic Synthesis ◽

Large Scale ◽

Recombinant Dna ◽

Industrial Enzymes ◽

Recombinant Dna Technology ◽

Bacterial Fermentation ◽

Natural Protein ◽

Novel Method

Amino acids are obtained by bacterial fermentation, extraction from natural protein or enzymatic synthesis from specific substrates. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to enzymatic synthesis of amino acids. Aspartase (L-aspartate ammonia-lyase) catalyzes the reversible deamination of L-aspartic acid to yield fumaric acid and ammonia. It is one of the most important industrial enzymes used to produce L-aspartic acid on a large scale. Here we described a novel method for [15N] L-aspartic synthesis from fumarate and ammonia (15NH4Cl) using a recombinant aspartase.

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