Screening and in Silico Validation of Anti-Microbial Peptides Derived from Lysostaphin, Entero and Endolysin

Introduction: Antimicrobial peptides (AMPs) are small molecules which are known to exert destructive effects upon pathogenic microorganisms. AMPs are designed from proteins obtained from various sources and tested under in vitro conditions to deduce their antimicrobial activity. Materials and Methods: A few of the peptidoglycan hydrolases such as lysostaphin (AAB53783.1), enterolysin (AGG79281.1), and endolysin (YP_009901016.1) were selected for the study based on an extensive text mining process. The protein sequences of the proteins were retrieved from the NCBI (National Centre for Biotechnology Information) database in the FASTA format (https://www.ncbi.nlm.nih.gov/protein/). Results and Discussion :In the antimicrobial protein lysostaphin, three antimicrobial peptide are been found, in which two is active and other is inactive, and one has antifungal property with a score of -0.15, and one having cell penetrating property, in which all are non toxic. Conclusion: The present study predicts AMPs from lysostaphin, entero and endolysins. These peptides were found to possess antifungal, anti-biofilm properties. Most of the peptides predicted were found to be non-cell penetrating and non-toxic.

An ancient antimicrobial protein co-opted by a fungal plant pathogen for in planta mycobiome manipulation

Microbes typically secrete a plethora of molecules to promote niche colonization. Soil-dwelling microbes are well-known producers of antimicrobials that are exploited to outcompete microbial coinhabitants. Also, plant pathogenic microbes secrete a diversity of molecules into their environment for niche establishment. Upon plant colonization, microbial pathogens secrete so-called effector proteins that promote disease development. While such effectors are typically considered to exclusively act through direct host manipulation, we recently reported that the soil-borne, fungal, xylem-colonizing vascular wilt pathogen Verticillium dahliae exploits effector proteins with antibacterial properties to promote host colonization through the manipulation of beneficial host microbiota. Since fungal evolution preceded land plant evolution, we now speculate that a subset of the pathogen effectors involved in host microbiota manipulation evolved from ancient antimicrobial proteins of terrestrial fungal ancestors that served in microbial competition prior to the evolution of plant pathogenicity. Here, we show that V. dahliae has co-opted an ancient antimicrobial protein as effector, named VdAMP3, for mycobiome manipulation in planta. We show that VdAMP3 is specifically expressed to ward off fungal niche competitors during resting structure formation in senescing mesophyll tissues. Our findings indicate that effector-mediated microbiome manipulation by plant pathogenic microbes extends beyond bacteria and also concerns eukaryotic members of the plant microbiome. Finally, we demonstrate that fungal pathogens can exploit plant microbiome-manipulating effectors in a life stage–specific manner and that a subset of these effectors has evolved from ancient antimicrobial proteins of fungal ancestors that likely originally functioned in manipulation of terrestrial biota.

Extended ɛ34 Phage TSP Renatures After Urea-acid Unfolding

In antimicrobial-peptide/protein engineering, understanding the peptide/protein’s adaptability to harsh environmental conditions such as urea, proteases, fluctuating temperatures, high salts provide enormous insight into the pharmacokinetics and pharmacodynamics of the engineered peptide/protein and its ability to survive the harsh internal environment of the human body such as the gut or the harsh external environment to which they are applied. A previous work in our laboratory demonstrated that our cloned Eɛ34 TSP showed potent antimicrobial activity against Salmonella newington, and more so, could prevent biofilm formation on decellularized tissue. In this work, the effects of urea-acid on the Eɛ34 stability is studied, and the results demonstrates that at lower pHs of 3 and 4 with urea the protein was denatured into monomeric species. However, the protein withstood urea denaturation above pH of 5 and thus remained as trimeric protein. The mechanism of denaturation of Eɛ34 TSP seems to show that urea denatures proteins by depleting hydrophobic core of the protein by directly binding to the amide units via hydrogen bonds. The results of our in-silico investigation determined that urea binds with Eɛ34 TSP with relative free energies range of -3.4 to -2.9 kcal/mol at the putative globular head binding domain of the protein. The urea molecules interacts with with the protein’s predicted hydrophobic core, thus, disrupting and exposing the shielded hydrophobic moieties of Eɛ34 TSP to the solvent. We further showed that after the unfolding of Eɛ34 TSP via urea-acid, renaturation of the protein to its native conformation was possible within few hours. This unique characteristic of refolding of Eɛ34 TSP which is similar to that of the P22 phage tailspike protein is of special interest to protein scientists and can also be exploited in antimicrobial-protein engineering.

Strawberry WRKY Transcription Factor WRKY50 Is Required for Resistance to Necrotrophic Fungal Pathogen Botrytis cinerea

WRKY protein is one of the largest plant-specific transcription factors that plays critical roles in plant stress responses, but few WRKY transcription factors have been functionally analyzed in strawberry. In this study, a Botrytis cinerea response WRKY gene, FvWRKY50, was isolated from the woodland strawberry. Expression analysis indicated that the transcript of FvWRKY50 was gradually decreased with fruit ripening, but was significantly induced by B. cinerea infection in mature strawberry fruit. Subcellular localization assay revealed that FvWRKY50 was localized in the nucleus. Several cis-elements related to pathogen responses were observed in the promoter region of FvWRKY50. Pathogen infection assay indicated that overexpression of FvWRKY50 in strawberry fruit significantly enhanced their resistance against B. cinerea, while the silencing of FvWRKY50 dramatically compromised their disease-resistant ability. The expression levels of several genes involved in jasmonic acid (JA) biosynthesis, signaling transduction, and antimicrobial protein biosynthesis were regulated to diverse extents in FvWRKY50 overexpressed and silenced fruit. Collectively, our study inferred that FvWRKY50 is a positive regulator that mediates resistance against B. cinerea through regulating some JA pathway and defense-related genes.

Experimental Evaluation of an Antimicrobial Protein from Bacillus Amyloliquefaciens MBL27 for Wound Healing Potential in Rats

Objective: This study was aimed at assessing the ability of the antimicrobial protein (AMP) produced by Bacillus amyloliquefaciens MBL27 as a potent wound healant. Methods: Rat models were used to study the efficacy of AMP and AMP incorporated chitosan sheet along with control groups.Results: AMP and AMP incorporated chitosan sheet significantly improved wound contraction when compared to controls. Rate of wound contraction (97.23%), decreased period of epithelialization (14 days) and the levels of biochemical markers such as hydroxyproline (collagen), hexosamine, uronic acid and total protein in the granulation tissue on various days of wound healing revealed the wound healing efficacy of the AMP. The histological examinations also correlated well with the biochemical findings, confirming the wound healing efficacy of the AMP. Conclusion: The results indicate the beneficial effects of AMP from B.amyloliquefaciens MBL27 and its potential to be developed into new therapeutic agent for dermal wound healing.

The Function of Aβ in the Healthy Brain: Beta Amyloid as an Antimicrobial Protein

There is no question as to whether or not the beta amyloid (Aβ) peptide plays a role in the exacerbation and onset of AD. There are very evident correlations between the volume of Aβ deposition in the AD brain and the severity of the symptoms of AD-typical neurodegeneration. There are certainly many factors involved in the progression of AD, of which the aggregation of Aβ peptides is only one. While many have ruminated on the mechanism by which Aβ serves to impair synaptic function and contribute to neurodegeneration, the role of this protein has yet to be fully uncovered, not just in the AD brain, but in the normally functioning brain as well. Recent research has shed light on the role of Aβ in the normal brain, offering evidence for the fact that beta amyloid functions as an antimicrobial protein, with its primary objective being to serve an immunoresponsive purpose. This text will highlight some of the critical studies on the topic, and illuminate the role that Aβ most likely plays in the functioning of the normal brain, and how this influences its pathological deposition in the AD brain.

Neutrophil Granule Proteins Inhibit Amyloid Beta Aggregation and Neurotoxicity

Background: A role for neutrophils in the pathogenesis of Alzheimer’s disease (AD) is emerging. We previously showed that the neutrophil granule proteins cationic antimicrobial protein of 37 kDa (CAP37), cathepsin G (CG), and neutrophil elastase (NE) directly bind the amyloid-beta peptide Aβ1-42, a central player in AD pathogenesis. CAP37, CG, and NE are serine proteases that can cleave Aβ1-42 at different sites and with different catalytic activities. Objective: In this study, we compared the effects of these three proteins on Aβ1-42 fibrillation and neurotoxicity. Methods: Using mass spectrometry and in vitro aggregation assay, we found that NE and CG effi- ciently cleave Aβ1-42. This cleavage correlates well with the inhibition of Aβ1-42 aggregation into fi- brils. In contrast, CAP37 did not efficiently cleave Aβ1-42, but was still able to inhibit its fibrillation, most likely through a quenching effect. Inhibition of Aβ1-42 aggregation by NE and CG neutralized its toxicity measured in cultured neurons. In contrast, inhibition of Aβ1-42 aggregation by CAP37 did not inhibit its neurotoxicity. Results: We found that a peptide derived from CAP37 could mimic the quenching and inhibition of Aβ1-42 aggregation effects of the full-length protein. Additionally, this peptide was able to inhibit the neurotoxicity of the most toxic Aβ1-42 aggregate, an effect that was not found with the full-length CAP37. Conclusion: These results shed light on the mechanisms of action of neutrophil granule proteins with regard to inhibition of Aβ1-42 aggregation and neurotoxicity and open up a possible strategy for the discovery of new disease-modifying drugs for AD.