Purification and Antimicrobial Activity of Antimicrobial Protein from Brown-spotted Grouper, Epinephelus fario

Antimicrobial proteins and peptides had been found from a wide variety of organisms in the last few years These molecules have attracted much research interest because of their biochemical diversity, broad specificity on anti-viral, anti-bacterial, anti-fungi, anti-protozoan parasites, anti-tumoural, and wound-healing effects. Antimicrobial proteins and peptides play key roles in innate immunity. They interact directly with bacteria and kill them. The brown-spotted grouper, Epinephelusfario, is an important marine fish cultured in southem China. Recently, bacteria and virus have caused high mortality in E. fario cultures, but its endogenous antimicrobial peptides and proteins have not been explored. An antimicrobial component was found from the skin homogenate of E. fario. After the skin homogenate was digested with trypsin, its antimicrobial activity was lost, which showed that the antimicrobial component is a protein. The antimicrobial protein （Efap） was purified from the skin homogenate of E. fario by successive ion-exchange and gel filtration chromatography. Efap was demonstrated to be single protein band by SDS-PAGE, with the apparent molecular weight of 41 kD. Efap exhibited antimicrobial activity both for the Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis, and for the Gram-negative bacteria, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio fluvialis, Pasteurella multocida, Aeromonas hydrophila, Eschrrichiu coli, and Pseudomonas aeruginosa. Except A. hydrophila, P. aeruginosa, and E. coli （MIC〉20 mol/L）, most of the tested Gram-negative bacteria were sensitive to Efap （MIC〈20 mol/L）. Interestingly, Efap showed potent antimicrobial activity against Gram-positive bacteria S. aureus （MIC 5-10 mol/L） but comparatively weak antimicrobial activity against M. luteus and B. subtilis. The broad antimicrobial activities of Efap suggest that it contributes to the innate host defence of E. fario.

Purification and Antimicrobial Assay of an Antimicrobial Protein from a Biocontrol Bacterium Strain K2-1 against Aquatic Pathogens

[Objective] The aim of this study was to purify an antimicrobial protein from a biocontrol bacterium strain K2-1 and analyze its antimicrobial activity in vitro against some typical aquatic pathogens. [Method] The antimicrobial protein was ob- tained by using ammonium sulfate precipitation and Sephadex chromatography combined with hot water bath. The antimicrobial assay was conducted by means of agar diffusion technique, using Vibrio alginolyticus, Aeromonas hydrophila, Aeromonas. Sobria, Pseudomonas fluorescens, Vibrio Parahaemolyticus, Vibrio har- veyi and Vibrio anguillarum as test bacteria. [Result] Antimicrobial protein APK2 can be derived from fermentation broth of strain K2-1 and purified to the chromatogra- phy pure level by the methods provided, the final yield of the antimicrobial compo- nent is approximately 0.08%. This antimicrobial protein had a strong antimicrobial activity against the growth of most those bacteria. [Conclusion] The results show that APK2 could be a potential alternative to replace chemical antimicrobial agent in the control and prevention of aquatic diseases.

Cationic antimicrobial protein of Mr 37 kDa:a multifunctional inflammatory protein

PURPOSE: To investigate the role of cationic antimicrobial protein of Mr 37 kDa (CAP37) a neutrophil-derived inflammatory mediator on endothelial cell function. DATA SOURCES: Endothelial cells used in this study were obtained from human lung microvessels and rat aorta. The latter was a kind gift of Dr. Paula Grammas. The mono-mac 6 cell line used in this study was the generous gift of Dr. H.W. Loms Ziegler-Heitbrock. STUDY SELECTION AND DATA EXTRACTION: Endothelial cell proteins kinase C activity was determined by measuring calcium- and phospholipid-dependent phosphorylation of histone. Endothelial cell migration was determined using Costar Transwell apparatus. Cell surface expression of adhesion molecules, ICAM-1 and PECAM-1 was determined using flow cytometry. RT-PCR was used to amplify the CAP37 from endothelial cells treated with LPS. RESULTS: We demonstrated that CAP37 which was originally identified as having potent antimicrobial activity and chemotactic activity for monocytes was capable of modulating endothelial cell functions. CAP37 activated endothelial cell protein kinase C in a dose- and time-dependent fashion. Importantly CAP37 increased the adhesive properties of the endothelium for monocytes. CAP37 upregulated the well known adhesion molecules, ICAM-1 and PECAM-1 in a dose- and time-dependent manner. In addition, CAP37 promoted endothelial cell migration. Further investigations indicated that CAP37 was induced in endothelial cells in response to pro-inflammatory cytokines such as tumor necrosis factor-alpha and interleukin-1 alpha as well as inflammatory mediators such as lipopolysaccharide. Unstimulated endothelial cells did not constitutively express CAP37. The cDNA sequence of endothelial CAP37 was determined and found to be highly homologous to the sequence obtained for neutrophil-derived CAP37. CONCLUSIONS: Our studies strongly suggest that CAP37 plays a pivotal role in monocyte-endothelial interactions and the transmigration of monocytes from the vasculature into extravascular tissues.

Purification and Characterization of an Antibacterial Protein from the Cultured Mycelia of Cordyceps sinensis

An antibacterial protein was isolated from the cultured mycelia of Cordyceps sinensis, and was designated as Cordyceps sinensis Antibacterial Protein （CSAP）. CSAP was single-chained, with an apparent molecular mass of 35 × 10^3 revealed by SDS-PAGE and a novel hydrophobic N-terminal sequence N-ALATQHGAP. The antimicrobial assays showed CSAP could inbibit the growth of Gram-positive and Gram-negative bacteria but no significant inhibition against fungi or yeasts. Further more, the antibacterial activity of CSAP was not bactericidal but bacteriostatic. It was the first time that an antibacterial protein was described in the Cordyceps species, which might involve in the chemical defense mechanism of the hosts.

Bactericidal/permeability-increasing protein originates in both the testis and the epididymis and localizes in mouse spermatozoa

Bactericidal/permeability-increasing protein （BPI） is an endogenous antibiotic protein with activity against gram-negative bacteria. In the present study, we examined the expression of BPI in postnatal mouse testes and epididymides as well as the subcellular localization within epididymal spermatozoa. Our results showed that, BPI mRNA was expressed in testis and epididymis independently. Throughout the epididymis, the BPI protein level gradually decreased in the epididymal epithelium in a spatial manner, specialized within the cytoplasm of clear cells in the cauda part. We detected BPI proteins in intact acrosome, implying its testicular origin; on the other hand, after the acrosome reaction, BPI proteins were observed dispersed across the entire sperm head, especially enriched at the equatorial segment. Our findings suggested a dual origin of the BPI that generated both in the testis and epididymis, and associated with mouse spermatozoa. BPI protein might be involved in the dynamics modification of the sperm plasma membrane and also the fertilization process.

Antimicrobial Activity in Kombucha Fermentation Broth

[Objective] The aim was to study if the fermentation broth of Kombucha has protein with antimicrobial activity. [Method] The effects of different cultivation time on cell concentration, pH, total protein concentration and inhibition zone were studied. The fermentation broth of Kombucha on the sixth day was adjusted to different pH. Fermentation broth treated with protease was put into the plates of Escherichia co/i, Bacillus cereue and Staphylococcus aureus as control, as well as ampenicilin. The zone of broth inhibition was measured. [ Result] The concentration of general protein was of positive relevance to the antimicrobial activity of Kombucha fermentation broth. The zone of inhibition decreased with rising pH and was the lowest when pH was 7. Later, as pH increasing, it enlarged. The inhibition effect of processed protease reduced remarkably. [ Conclusion] The fermentation broth of Kombucha revealed antimicrobial proteins.

Increased CAP37 Expression in Stable Chronic Obstructive Pulmonary Disease

Objective Cationic antimicrobial protein of 37 kDa(CAP37),a neutrophil-derived protein originally identified for its antimicrobial activity,is now known to have many regulatory effects on host cells.However,its role in the pathogenesis of chronic obstructive pulmonary disease(COPD)has not been studied.We therefore investigated the expression of CAP37 in COPD and its effects on airway structural cells,including bronchial epithelial cells,smooth muscle cells,and fibroblasts.Methods CAP37 was detected in the lung tissue,sputum,and plasma of COPD patients and the control subjects,as well as in the neutrophils stimulated by cigarette smoke extract(CSE).BEAS-2B cells,human bronchial smooth muscle cells(HBSMCs),and MRC-5 cells were treated with CAP37 or an anti-CAP37 antibody plus CAP37.Interleukin(IL)-6 and IL-8 were detected in the BEAS-2B cells.The cell proliferation was analyzed in the HBSMCs.Collagens were also detected in the MRC-5 cells.Results The expression of CAP37 was increased in the lung tissue and sputum supernatant of the COPD patients compared with the control subjects.The sputum supernatant CAP37 levels were inversely correlated with the forced expiratory volume in the first second percentage predicted in COPD.CAP37 was induced by CSE stimulation in the peripheral blood neutrophils from healthy non-smokers.CAP37 induced expression of IL-6 and IL-8 in BEAS-2B cells,and collagen expression of lung fibroblasts(MRC-5 cells).However,CAP37 did not significantly alter the proliferation of the HBSMCs.Conclusion Our findings indicated that neutrophil-derived CAP37 may be involved in airway inflammation and fibrosis in COPD via affecting the bronchial epithelial cells,and fibroblasts,thus suggesting a possible role of CAP37 in the development and progression of COPD.

Cloning and Analysis of TasA Gene in Bacillus subtilis HAS

In order to clarify the mechanism for the inhibitory action of the Bacillus subtilis strain HAS on Sporisorium scitamineum （ Syd. ）, which can cause sugarcane smut, the full-length TasA gene which encodes a protein with broad-spectrum antimicrobial activity, was PCR-amplified from HAS, and cloned into pMD18-T vector. Sequence analysis indicated that the full-length TasA cloned from HAS consisted of 786 nucleotides, and shared 99% homology in nucleotide sequence with the TasA gene sequence published in Genbank （AJ871386.1）. It was predicted that the molecular weight of TasA protein was about 28 kD. Base transitions or transversions ~curred at positions 104, 164, 169,250, 399,623 and 627, at the 2nd, 2nd, 3rd , 1st , 3rd, 2nd and 3rd bases of TasA codons. The mutations in the seven bases may cause the missense mutations of the polypeptide chain. Compared with the amino acid sequences of the TasA protein encoded by Bacillus aubtilis subsp, subtilis str. 168, mutations in two amino acids at positions 150 and 209 of the protein encoded by the cloned TasA gene were found, and as a result, an ala- nine was replaced with a threonine.

ANTIVIRAL EFFECTS OF BACTERIOCIN AGAINST ANIMAL-TO-HUMAN TRANSMITTABLE MUTATED SARS-COV-2:A SYSTEMATIC REVIEW

The COVID-19 caused by SARS-CoV-2 has resulted in millions of people being infected and thousands of deaths globally since November 2019.To date,no unique therapeutic agent has been developed to slow the progression of this pandemic.Despite possessing antiviral traits the potential of bacteriocins to combat SARS-CoV-2 infection has not been fully investigated.This review summarizes the mechanisms by which bacteriocins can be manipulated and implemented as effective virus entry blockers with infection suppression potential properties to highly transmissible viruses through comprehensive immune modulations that are potentially effective against COVID-19.These antimicrobial peptides have been suggested as effective antiviral therapeutics and therapeutic supplements to prevent rapid virus transmission.This review also provides a new insight into the cellular and molecular alterations which have made SARS-CoV-2 self-modified with diversified infection patterns.In addition,the possible applications of antimicrobial peptides through both natural and induced mechanisms in infection prevention perspectives on changeable virulence cases are comprehensively analyzed.Specific attention is given to the antiviral mechanisms of the molecules along with their integrative use with synthetic biology and nanosensor technology for rapid detection.Novel bacteriocin based therapeutics with cutting-edge technologies might be potential substitutes for existing time-consuming and expensive approaches to fight this newly emerged global threat.