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International Journal of Molecular... Jul 2021The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from or LytM from . Recently, enzymes with broad specificities...
The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from or LytM from . Recently, enzymes with broad specificities were reported, such as EnpA from , that cleaves D,L peptide bond between the stem peptide and a cross-bridge. Previously, the activity of EnpA was demonstrated only on isolated peptidoglycan fragments. Herein we report conditions in which EnpA lyses bacterial cells live with very high efficiency demonstrating great bacteriolytic potential, though limited to a low ionic strength environment. We have solved the structure of the EnpA H109A inactive variant and analyzed it in the context of related peptidoglycan hydrolases structures to reveal the bases for the specificity determination. All M23 structures share a very conserved β-sheet core which constitutes the rigid bottom of the substrate-binding groove and active site, while variable loops create the walls of the deep and narrow binding cleft. A detailed analysis of the binding groove architecture, specificity of M23 enzymes and D,L peptidases demonstrates that the substrate groove, which is particularly deep and narrow, is accessible preferably for peptides composed of amino acids with short side chains or subsequent L and D-isomers. As a result, the bottom of the groove is involved in interactions with the main chain of the substrate while the side chains are protruding in one plane towards the groove opening. We concluded that the selectivity of the substrates is based on their conformations allowed only for polyglycine chains and alternating chirality of the amino acids.
Topics: Amino Acid Sequence; Bacterial Proteins; Catalytic Domain; Endopeptidases; Enterococcus faecalis; N-Acetylmuramoyl-L-alanine Amidase; Peptide Hydrolases; Peptidoglycan; Prophages; Protein Binding; Staphylococcus; Staphylococcus aureus; Substrate Specificity
PubMed: 34281200
DOI: 10.3390/ijms22137136 -
AIMS Microbiology 2019The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan...
The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study, we constructed a 2 kb-triple-acting fusion gene () encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (D-glutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The gene was expressed in plants using the non-replicating (CPMV)-based vector pEAQ-HT and the replicating AltMV)-based pGD5TGB123-MCS-CP3 vector, and in using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a gene codon-optimized for expression in tobacco plants, resulting in an improved codon adaptation index (CAI) from 0.79 ( gene) to 0.93 ( gene). Incorporation of the nt gene into the pEAQ-HT vector, followed by transient expression in , led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against 305, showing 14% of growth inhibition, while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains, showing 68 (IC 25 µg/ml) and 60% (IC 71 µg/ml) growth inhibition against 305 and Newman, respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants, the most functionally active antimicrobial protein was obtained using the prokaryotic expression system.
PubMed: 31384710
DOI: 10.3934/microbiol.2019.2.158 -
Journal of Dairy Science Dec 2017The aim of the study was to evaluate the concentrations of cytokines IL-4, IL-6, and IL-10 and acute phase protein amyloid A in milk and in serum from cows with...
The aim of the study was to evaluate the concentrations of cytokines IL-4, IL-6, and IL-10 and acute phase protein amyloid A in milk and in serum from cows with subclinical mastitis caused by coagulase-negative staphylococci and from healthy cows. The blood and milk samples were obtained from 35 midlactation, multiparous (between parities 2 and 4) Holstein-Friesian cows. In the milk samples from 20 cows with subclinical mastitis, the following species of Staphylococcus were detected: Staphylococcus xylosus (8 samples), Staphylococcus chromogenes (6 samples), Staphylococcus haemolyticus (2 samples), Staphylococcus simulans (2 samples), and Staphylococcus sciuri (2 samples). The results of the present study indicate that the level of IL-6 in cows suffering from subclinical mastitis tended to be high in both serum and milk (432.09 and 254.32 pg/mL) compared with the level in healthy cows (164.47 and 13.02 pg/mL, respectively). Amyloid A value also was significantly higher in milk of unhealthy cows compared with cows without subclinical mastitis (790.2 and 360.5 ng/mL). No significant differences were found in levels of amyloid A in serum of both tested groups of cows (2,680.0 and 2,720.0 ng/mL). In contrast, concentration of IL-4 was significantly lower both in serum and in milk of cows with staphylococcal mastitis (86.1 and 123.17 pg/mL) compared with control animals (413.5 and 670.2 pg/mL). The level of IL-10 also was significantly higher in milk of healthy cows than in infected cows (39.78 and 22.5 pg/mL); however, differences in serum levels of this cytokine between tested groups were significantly less important (220.6 and 175.1 pg/mL).
Topics: Animals; Asymptomatic Infections; Cattle; Coagulase; Female; Interleukin-10; Interleukin-4; Interleukin-6; Mastitis, Bovine; Milk; Serum Amyloid A Protein; Staphylococcal Infections; Staphylococcus
PubMed: 28964518
DOI: 10.3168/jds.2017-13552 -
Iranian Journal of Microbiology Apr 2023secretes an antimicrobial compound called lysostaphin, which has bactericidal properties. It destroys staphylococci through the hydrolysis of peptidoglycan in the cell...
BACKGROUND AND OBJECTIVES
secretes an antimicrobial compound called lysostaphin, which has bactericidal properties. It destroys staphylococci through the hydrolysis of peptidoglycan in the cell wall. Therefore, this unique property indicates the high ability of lysostaphin in the treatment of staphylococcal infections and is considered as an anti-staphylococcal agent.
MATERIALS AND METHODS
BL21 (DE3) competent cells were transformed with pET32a-lysostaphin clone and induced by isopropyl-β-D-thio-galactoside (IPTG). The recombinant protein was purified by affinity chromatography. Recombinant lysostaphin -A-based ointment was used for external wound healing in animal model. activity of ointment was evaluated by clinical evidences and cytological microscopic assessment.
RESULTS
Our results showed the recombinant protein was produced exactly. The results of checkerboard tests showed MIC, MBC and antibacterial activity test an acute reduction of cell viability during the use of lysostaphin, and SEM results approved the intense wrecking effects of lysostaphin in combination on bacterial cells. Macroscopic findings and microscopic data showed that the recombinant lysostaphin ointment was effective on excisional wound healing.
CONCLUSION
Our findings proved that the recombinant lysostaphin ointment was effective on wound healing due to infection.
PubMed: 37193239
DOI: 10.18502/ijm.v15i2.12476 -
Biochimica Et Biophysica Acta.... Aug 2023Antimicrobial peptides (AMPs) commonly target bacterial membranes and show broad-spectrum activity against microorganisms. In this research we used three AMPs (nisin,...
Antimicrobial peptides (AMPs) commonly target bacterial membranes and show broad-spectrum activity against microorganisms. In this research we used three AMPs (nisin, epilancin 15×, [R4L10]-teixobactin) and tested their membrane effects towards three strains (Staphylococcus simulans, Micrococcus flavus, Bacillus megaterium) in relation with their antibacterial activity. We describe fluorescence and luminescence-based assays to measure effects on membrane potential, intracellular pH, membrane permeabilization and intracellular ATP levels. The results show that our control peptide, nisin, performed mostly as expected in view of its targeted pore-forming activity, with fast killing kinetics that coincided with severe membrane permeabilization in all three strains. However, the mechanisms of action of both Epilancin 15× as well as [R4L10]-teixobactin appeared to depend strongly on the bacterium tested. In certain specific combinations of assay, peptide and bacterium, deviations from the general picture were observed. This was even the case for nisin, indicating the importance of using multiple assays and bacteria for mode of action studies to be able to draw proper conclusions on the mode of action of AMPs.
Topics: Nisin; Antimicrobial Peptides; Antimicrobial Cationic Peptides; Bacteria; Anti-Bacterial Agents
PubMed: 37100361
DOI: 10.1016/j.bbamem.2023.184160 -
Journal of Dairy Science Jul 2024Several studies have described variations in lactose content (LC) in dairy cows during udder quarter health disorder or negative energy balance (NEB). However, their...
Several studies have described variations in lactose content (LC) in dairy cows during udder quarter health disorder or negative energy balance (NEB). However, their joint effects on LC have never been described. This was the aim of a longitudinal observational study performed on 5 Quebec dairy farms using automatic milking systems. Quarter milk samples were collected every 14 d from 5 to 300 DIM. Quarter health status was described by combining SCC level (SCC or SCC: < or ≥100,000 cells/mL, respectively) and infectious status (Patho or Patho: absence or presence of pathogens on a milk culture, respectively). Cows with NEB in early lactation (DIM <70) were identified using milk BHB content: <0.15 mM = BHB-; 0.15 to 0.19 mM = BHB+; >0.19 mM = BHB++. A total of 14,505 quarter cisternal milk samples were collected from 380 lactating cows. The quarter LC was analyzed using a mixed linear regression model with the following fixed effects: quarter health status, parity, time interval between last milking and sampling, quarter milk yield (in kg/d), DIM, and herd. A random quarter intercept with a repeated measures correlation structure and a cow random intercept were also specified. The LC of SCC quarters was lower (-0.17 ± 0.013 percentage points) compared with LC of SCC quarters for both primiparous and multiparous cows. Of the 162 bacterial species identified, only 8 species had a prevalence greater than 4.0%, and just 5 of them were associated with a reduction in LC: Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus epidermidis, and Staphylococcus simulans. Cows identified as BHB+ and BHB++ in early lactation had a lower LC (-0.05 ± 0.019 and -0.13 ± 0.020 percentage points, respectively) compared with BHB- cows. For BHB++ cows, in both parity groups the decrease in LC (-0.20 ± 0.025 percentage points) was higher in SCC quarters compared with SCC quarters. Moreover, the additive effect of the quarter health status and NEB on milk LC was greater with larger increases in BHB. Our findings highlight the necessity to jointly take into consideration both quarter health status and milk BHB concentration when using LC as a biomarker for NEB.
Topics: Animals; Cattle; Female; Milk; Lactation; Lactose; Mastitis, Bovine; Mammary Glands, Animal; Longitudinal Studies; Dairying; Energy Metabolism; Cattle Diseases
PubMed: 38428490
DOI: 10.3168/jds.2023-24134 -
Case Reports in Infectious Diseases 2018is a coagulase-negative organism, mainly an animal pathogen. Reports of human infection have been infrequent, mainly in patients with repeated animal contact. We report...
is a coagulase-negative organism, mainly an animal pathogen. Reports of human infection have been infrequent, mainly in patients with repeated animal contact. We report the first case of pleural empyema in an elderly woman. tends to cause more severe infection because of a biofilm layer which helps in adherence and colonization of smooth surfaces, especially prosthetic devices, shunts, and catheters. The challenging problem even after CoNS isolation and identification is the assessment of their clinical relevance. Major factors that inhibit the penetration of antibiotics is the large-sized effusions/empyema, thickness of pleura, and the nature of antibiotic itself. Source control for septic patients remains the cornerstone of treatment along with optimal antimicrobial coverage. , a coagulase-negative staphylococcus, is emerging as an important cause of virulent infections with high mortality in humans. Given its propensity for multidrug resistance, including vancomycin, there is an imperative for early and accurate identification of the isolate. Despite aggressive treatment, the patient succumbed to her illness.
PubMed: 30405924
DOI: 10.1155/2018/7831284 -
Journal of Global Antimicrobial... Mar 2015The broth microdilution method for telavancin susceptibility testing was revised and now utilises DMSO as solvent for stock solution preparation and diluent for stock...
Telavancin activity when tested by a revised susceptibility testing method against uncommonly isolated Gram-positive pathogens responsible for documented infections in hospitals worldwide (2011-2013).
The broth microdilution method for telavancin susceptibility testing was revised and now utilises DMSO as solvent for stock solution preparation and diluent for stock solution dilution, following CLSI guidelines for water-insoluble agents. The revised method also incorporates polysorbate 80 in the test medium to mitigate drug binding to plastics. This revised methodology provides more accurate and reproducible MIC determinations, which results in values lower than the previously established method. This study was conducted to re-establish telavancin potencies and susceptibility profiles (using updated interpretive criteria) against a collection of uncommon clinical pathogens (3821 isolates). Telavancin showed MIC values of 0.06mg/L against tested staphylococcal species (MIC, 0.03/0.06mg/L; 98.1-100.0% susceptible), with lower results for Staphylococcus hominis (MIC, ≤0.015mg/L), Staphylococcus lugdunensis (MIC, ≤0.015mg/L) and Staphylococcus simulans (MIC, 0.03mg/L). Vancomycin (MIC, 1mg/L), daptomycin (MIC, 0.12-1mg/L) and linezolid (MIC, 0.25-1mg/L) had MIC results at least four-fold higher than telavancin against CoNS. Streptococci (99.2-100.0% susceptible) displayed telavancin MIC values of ≤0.015-0.03mg/L. Vancomycin (MIC, 0.25-0.5mg/L) and linezolid (MIC, 0.5-1mg/L) had higher MIC results against streptococci, whilst daptomycin MIC values varied from ≤0.06mg/L to 0.5mg/L. Micrococcus, Listeria and Corynebacterium spp. were inhibited by telavancin at ≤0.015, ≤0.03 and ≤0.06mg/L, respectively. Telavancin exhibited potent in vitro activity against this collection, greater than comparators (daptomycin, linezolid, vancomycin). This study provides new baseline MIC results for telavancin and confirms the spectrum and potency of telavancin against less commonly encountered Gram-positive species.
PubMed: 27873648
DOI: 10.1016/j.jgar.2014.12.003 -
Food Chemistry: X Mar 2024The aim of this study was to investigate and compare the effects of different mixed starter cultures ( and ) on the bacterial communities and flavor of fermented...
The aim of this study was to investigate and compare the effects of different mixed starter cultures ( and ) on the bacterial communities and flavor of fermented sausages. The results indicated that native starters grew well in fermented sausages and became dominant at the end of ripening. Among them, spp. had the highest relative abundance, followed by spp. In addition, the inoculation of the mixed starters promoted the formation of taste and aroma compounds that contribute to the overall flavor of the fermented sausages. Among them, the CQ01107 + CD207 (CCA) treatment was found to have the highest umami amino acid, nucleotide, lactic acid, fatty acid and ketone contents ( < 0.05), as well as excellent sensory properties. In conclusion, the CCA starter may be a desirable starter culture to enhance the flavor of fermented sausages.
PubMed: 38389578
DOI: 10.1016/j.fochx.2024.101225 -
Frontiers in Microbiology 2018The present study explored the possible prebiotic application of potato peel and bamboo shoot extracts for the proliferation of lactic acid bacteria (LAB) from diverse...
The present study explored the possible prebiotic application of potato peel and bamboo shoot extracts for the proliferation of lactic acid bacteria (LAB) from diverse niches and their tolerance ability to simulated gastrointestinal tract (GIT) conditions was also examined. Initially, the complete 16S rDNA sequencing of selected isolates revealed them as (6), (2), and (1). Higher cell densities and rapid pH change were obtained from cultured media supplemented with BS (2%) and PP (2%) as a carbon source. Their higher tolerance and the lowest reducing sugar abilities were obtained for BS at pH 2.5 and 9.0, while at pH 3.5 and 8.0 for PP. The isolates were screened for additional functional and technological properties to harvest the most appropriate starter. The selected isolates harbored promising functional properties such as amylase presence, cell surface hydrophobicity, autoaggregation, proteolytic and lipolytic activity, antifungal action, as well as exopolysaccharide production. On the basis of these attributes, microencapsulated strain K3 was found resistant to gastrointestinal conditions after 2 h, resulting in significantly ( ≤ 0.05) improved survival compared to non-capsulated strain. The current approach presents an interesting economical strategy to modulate LAB through supplementation of plant-derived carbon sources as well as to enhance their survival under GIT.
PubMed: 30233560
DOI: 10.3389/fmicb.2018.02114