Elastolytic Activity Research Articles

Genomic and phenotypic characterization of Pseudomonas aeruginosa isolates from two Mexican cystic fibrosis attention centers.

Thirty-nine clinical isolates of Pseudomonas aeruginosa collected from 11 cystic fibrosis (CF) patients at two CF attention centers over 10 years were subjected to whole genome sequencing (WGS). Phenotypic tests (i.e., elastase, motility, biofilm, growth rate, and antibiotic susceptibility) were performed to correlate results. A single strain of P. aeruginosa was found to persist over time in longitudinal isolates. No transmission between patients or centers was observed. A tendency to lack genes related to pyoverdine, flagellum, pili, and O-antigen was observed, whereas those related to biofilm, phenazine, and pyochelin were conserved among isolates. In a patient with a 10-year follow-up, a single strain of P. aeruginosa persisted and showed a gradual decrease in elastase activity and growth rate, demonstrating an adaptive phenotype.IMPORTANCEThis study investigates the genomic and phenotypic characteristics of Pseudomonas aeruginosa isolates from Mexican cystic fibrosis (CF) patients, an underrepresented group in CF research. To our knowledge, it is the first to use whole genome sequencing (WGS) to study longitudinally collected P. aeruginosa isolates from this population, evaluating both genomic features and clonal relationships. Remarkably, the study includes samples from one patient over 10 years, offering an extended observation time compared to existing literature. Unlike similar studies, which often lack phenotypic testing, this research incorporates various virulence-related phenotypic assays, enhancing our understanding of gene-to-phenotype correlations. Two potential mechanisms for the loss of elastolytic activity were identified. Furthermore, we conduct an in-depth mobilome analysis, an area that remains largely unexplored in CF contexts. Whole genome sequencing data are publicly available through the NCBI SRA database, facilitating further re-analysis for studies on P. aeruginosa in CF, as well as epidemiological and population structure research.

Скринінг продуцентів протеаз серед представників роду Bacillus, ізольованих з прибережної зони Кінбурнської коси

Protease-producing representatives of Bacillus have been isolated from various environments such as the rhizosphere, soil, sewage, food, waste, and saline environments (sea water, marine sediments, hypersaline lakes, salty food, soda lakes, etc.). However, today, little is known about the characteristics of proteases in representatives of the genus Bacillus, isolated from the coastal and aquatic environments. In this regard, the purpose of this work was to screen for the presence of different types of proteolytic activity in Bacillus cultures isolated from the coastal zone of the Kinburn split. Methods. The objects of the study were 15 cultures (L1-L15), isolated from the dry grass of the coastal zone of the Kinburn spit (Mykolaiv region). The cultures were grown under conditions of deep cultivation at 28 °С, with a mixing speed of the nutrient medium of 230 rpm for 2 days. Methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic, and collagenase) activity in the culture liquid supernatant were used. Results. Study of the spectrum of proteolytic activities of 15 strains of Bacillus sp. showed that L9, L1, L2 and L4 exhibited higher levels of proteolytic activity compared to the other strains tested. Bacillus sp. L9 and L2 showed the highest elastase activity (35.80 and 33.80 U/mL, respectively), L1, L2, and L4 − fibrinogenolytic (12.50, 11.20 and 10.33 U/mL, respectively), and L1 and L4 − fibrinolytic (13.00 and 10.90 U/mL, respectively) activity. Conclusions. According to its catalytic properties, a number of representatives of Bacillus sp. isolated from the dry grass of the coastal zone of the Kinburn split may be promising for further research as producers of enzymes with elastotic, fibrinolytic, and fibrinogenolytic activities.

A42 A HIGH SALT DIET INCREASES ELASTOLYTIC ACTIVITY AND WORSENS COLITIS IN GNOTOBIOTIC MICE

Abstract Background Recent evidence has linked ultra-processed foods, which are high in salt, to increased risk of inflammatory bowel disease (IBD). A high salt diet (HSD) was also shown to be colitogenic in specific pathogen-free mice, in part, through modulation of the microbiota. We recently described high microbial proteolytic activity (PA) in feces from IBD patients, that when transferred to germ-free mice, increased acute colitis severity. However, the drivers of high microbial PA are unknown. Aims To determine whether HSD increases proteolytic activity, thereby exacerbating colitis. Methods Adult germ-free (GF) C57BL/6 mice were colonized with feces from a patient with UC in flare (n=13). Mice were fed either a control diet (CD; 7004, Teklad) or a HSD (7004 supplemented with 4% NaCl) plus 1% NaCl in drinking water. Three weeks following colonization, chronic colitis was induced in half of the mice by three cycles (five days each; 2.0%, 1.5% and 1.5%, respectively) of DSS in drinking water with a five-day wash-out period between cycles. Fecal PA and predicted microbial protease profiles were measured before colitis induction. Weight loss and disease activity (e.g., occult blood in feces and stool consistency) was evaluated for the duration of colitis and microscopic colitis was evaluated at endpoint. Results In mice colonized with UC microbiota, a HSD increased elastolytic activity (p=.03 versus CD) and induced a unique predicted protease and peptidase profile. After colitis induction, mice consuming a HSD lost more weight than mice consuming the CD (p=.0001) and had higher disease activity (p=.0004). Additionally, HSD induced more severe microscopic colitis scores compared with the CD (p=.0001). Conclusions These results raise the hypothesis that a HSD drives microbial proteolytic activity of IBD microbiota. However, whether this shift is causally associated with the worsening of colitis remains unclear. Mechanistic insight underlying this HSD-microbial proteolytic function could lead to specific dietary modifications or anti-proteolytic therapies for IBD in patients with proteolytic-driven inflammation. Funding Agencies CCC, CIHR

Diclofenac Sodium‐Encapsulated Poly(lactic‐co‐glycolic acid) Nanoparticles: A Non‐antibiotic Formulation to Attenuate the Proteolytic Activity of P. aeruginosa Clinical Isolates

AbstractEncapsulating drugs in poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles can provide a gradual and consistent drug release. Herein, diclofenac sodium‐encapsulated PLGA nanoparticles (DS‐PLGA NPs) were synthesized, characterized, and their effects on bacterial proteolysis and elastolytic activity were evaluated using skim milk agar plate, elastin‐congo red, and staphylolysin disk susceptibility assays. Quantitative PCR was employed to examine the effect of nanoparticles on the expression of lasA/B genes. The DS‐PLGA NPs displayed spherical morphology with an average size of 86 nm, zeta potential of ‐15.2 mV, and DLS size of 124.6 nm. At a sub‐inhibitory concentration, DS‐PLGA NPs inhibited bacterial elastolytic activity by 41.6 to 62.1% during 30–180 min of incubation. The elastolytic curve slopes of samples from PLGA and DS‐PLGA treated groups were respectively 0.0714 and 0.0380 activity/min, indicating a significant reduction of bacterial extracellular elastases by DS‐PLGA NPs. Exposure of bacteria to a sub‐inhibitory concentration of the nanoparticles attenuated the expression of the lasA and lasB genes by 0.21 and 0.31 folds, respectively. Attenuation of the proteolytic activity of P. aeruginosa suggests that the antivirulence potential of the DS‐PLGA NPs along with the anti‐inflammatory properties of diclofenac sodium, can be considered for the treatment of acute and chronic P. aeruginosa infections.

Proteolytic Activity of Bacillus Strains Isolated from Soil of Rice Agrocenosis

Microorganisms are the most common sources of commercial enzymes due to their physiological and biochemical properties, facile culture conditions, and ease of cell manipulation. Among microbial enzymes, proteases are ubiquitous in nature and have been found in all living forms encompassing the eukaryotes like plants, animals, fungi, and protists as well as the prokaryotic domains of bacteria and archaea. Proteases are the most important for the industry and constitute approximately 60% of the total industrial enzyme market. Among the bacteria, the genus Bacillus has a very prominent place in terms of the commercial production of proteases. Earlier from the water and bottom sediments of the Black Sea, we have isolated a number of producers of proteolytic enzymes from Bacillus species. The aim of this work was to investigate the ability of representatives of a number of soil bacilli species to synthesize enzymes that hydrolyze such protein substrates as elastin, fibrin, fibrinogen, and keratin. Methods. The objects of the study were 8 cultures (KS 1 — KS 8) isolated from the soil of the rice agrocenosis. Cultures were grown under conditions of deep cultivation at 28 °С, with a mixing speed of for the nutrient medium of 230 rpm for 4 days. Methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic, and keratinase) activity in the culture liquid supernatant were used. Disulfide reductase activity was measured spectrophotometrically at 412 nm by evaluating the yellow sulfide formed during the reduction of 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB). Results. The study of the spectrum of proteolytic activities of 8 freshly isolated strains showed that only KS 6 under experimental conditions did not show the ability to hydrolyze any of the studied substrates (casein, elastin, fibrin, fibrinogen, and keratin). Strains KS 1, KS 2, KS 7, and KS 8 showed higher levels of activity compared to other strains studied. The most interesting for further research are: І) strain KS 1, which showed the highest fibrinolytic activity, ІІ) strain KS 2 as the most effective producer with elastase and fibrinogenolytic activity, III) KS 7 and KS 8, which simultaneously showed the highest rates as keratinase (7 U/mL and 9 U/mL) and sulfate reductase (33 μmol/min and 31 μmol/min) activity, respectively. Conclusions. According to the catalytic properties, a number of representatives of Bacillus, isolated from the soil of the rice agrocenosis may be promising for further research as an enzyme producer with proteolytic activity.

Isolation and characterization of Bacillus sp. IMV B-7883 proteases

The representatives of Bacillus are some of the best protease producers studied so far since they exhibit broad substrate specificity, significant activity, stability, simple downstream purification, short period of fermentation and low cost. Earlier, we showed that Bacillus sp. IMV B-7883 strain synthesizes an extracellular proteases, which exhibit elastolytic and fibrinogenolytic activity. The aim of the work was to isolate and purify these enzymes from the culture liquid of the Bacillus sp. IMV B-7883 strain, as well as to study their properties. Isolation and purification of proteases was carried out by precipitation of the culture liquid with ammonium sulfate, gel permeation and ion exchange chromatography and rechromatography on Sepharose 6B. As a result, proteases with elastolytic and fibrinogenolytic activity with a molecular weight of 23 and 20 kDa respectively were isolated with elastase activity increased by 63.6 and fibrinogenolytic activity by 44.1 times. The enzyme with elastase activity had a pH-optimum of 7.0 and hydrolyzed only elastin, while the enzyme with fibrinogenolytic activity was an alkaline protease with a pH-optimum of 8.0 and in addition to fibrinogen, showed specificity for fibrin and, in trace amounts, for collagen. Keywords: Bacillus sp. IMV B-7883, elastase, fibrinogenase, pH optimum, substrate specificity

Proteolitic Activity of Marine Strain Bacillus sp. 051

The main interest in the study of marine microorganisms is due to their ability to produce a wide range of unique enzymes, including peptidases with different specificities. In recent years, interest has increased in peptidases that are able to cleave elastin as a specific substrate. Streptomyces fradiae and Bacillus thermoproteolyticus elastases are among the most potent elastolytic proteinases discovered to date because they are 4-8-fold more effective than pancreatic elastases. The disadvantages of these producers include the fact that most of them are pathogenic for humans, and the elastase enzyme secreted from them is directly involved in the initiation of the pathogenetic process. All this significantly limits the scope of their practical application. Therefore, the search for new, more effective, safe for humans’ producers continues to be an urgent question, taking into account the fact that there are no highly active elastase producers in Ukraine. Previously we found elastase activity in only 4 of the 10 studied isolates of bacteria from the Black Sea. Since among them, the elastase activity of the Bacillus sp. 051 was the highest, the aim of this work was to study the physicochemical properties and substrate specificity of the enzyme. Methods. We used methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic) activity. Protein concentration was determined by the Lowry method. The study of the effect of temperature on the enzymatic activity was carried out in the range from 4 to 70 °C and pH values from 2.0 to 12.0, created using 0.01 M phosphate-citrate buffer. Results. It has been shown that the growing temperature of 12°C is the most optimal for biosynthesis of enzyme by the culture of Bacillus sp. 051. The complex enzyme preparation capable of hydrolyzing elastin, casein and fibrinogen. The enzyme showed maximum activity in relation to elastin (3.65 U/mg). The optimum pH of the enzyme action is 8.0, the thermal optimum is 40°C. The rate of casein hydrolysis compared to elastin was 2.7 times lower and amounted to 1.35 U/mg. The complex enzyme preparation also hydrolyzed fibrinogen (1.16 U/mg). Conclusions. According to its physicochemical and catalytic properties, the representative of the Black Sea, Bacillus sp. 051 is promising for further research as an enzyme producer with elastolytic activity.

Effect of some antiseptics on elastase production from Pseudomonas aeruginosa.

The present study was carried out to investigate the effect of clinically used antiseptics on elastase production from Pseudomonas aeruginosa. 39 clinical isolates were collected from wounds 10 (25.64%) and burns 29 (74.35%) from hospitalized patients in Baghdad city. Elastin preparation by the autoclaving method yielded 10.5 gm of elastin powder from (250 gm) of sheep's lungs, 3.6 gm from (50 gm) of sheep's bladder, and sheep's ligamentum nuchae (65 gm) yielded 15 gm. All P. aeruginosa isolates were tested for their ability to produce elastase by being cultivated on elastin nutrient agar and observed for the enzyme's activity. The bacteria that make elastase grew, and a clear border emerged surrounding the growth after 24 hours. 32 (82.05%) of P. aeruginosa isolates produced the enzyme on the elastin nutrient agar. Elastase-producing P. aeruginosa was tested quantitatively using the ELISA reader and spectrophotometer at (A495) to detect the released amount of Congo red dye from the degradation of the elastin Congo red. P. aeruginosa (P41) showed the highest elastolytic activity; thus, it was selected to determine the effect of the sub-MIC of the antiseptics on elastase production. The results showed that acetic acid was the best agent to inhibit elastase production, followed by silver nitrate, hydrogen peroxide and ethanol in descending order. Keywords: Elastase; Elastin; Pseudolysin; Antiseptics; Acetic acid; Pseudomonas aeroginosa

Facile Production of the Pseudomonas aeruginosa Virulence Factor LasB in Escherichia coli for Structure-Based Drug Design.

The human pathogen Pseudomonas aeruginosa has a number of virulence factors at its disposal that play crucial roles in the progression of infection. LasB is one of the major virulence factors and exerts its effects through elastolytic and proteolytic activities aimed at dissolving connective tissue and inactivating host defense proteins. LasB is of great interest for the development of novel pathoblockers to temper the virulence, but access has thus far largely been limited to protein isolated from Pseudomonas cultures. Here, we describe a new protocol for high-level production of native LasB in Escherichia coli. We demonstrate that this facile approach is suitable for the production of mutant, thus far inaccessible LasB variants, and characterize the proteins biochemically and structurally. We expect that easy access to LasB will accelerate the development of inhibitors for this important virulence factor.

Antibiofilm and antimicrobial activity of Lactobacillus cell free supernatant against Pseudomonas aeruginosa isolated from burn wounds.

The present study investigated the antimicrobial and anti-biofilm effects of indigenous Lactobacillus probiotic strains on Pseudomonas aeruginosa isolated from burn wound infection in laboratory conditions. The effect of 7 probiotic strains isolated from infant faeces on the pathogenicity factors of P. aeruginosa, including protease, elastase, antibiofilm and antipyocyanin was measured. Also, diffusion methods in the well and micro broth dilution were used to evaluate the antimicrobial activity of probiotics. All tests were performed in triplicate. A negative control and a positive control were used for each test. SPSS version 22 software was used for statistical analysis, and a p < 0.05 was considered statistically significant. A total of 30 clinical isolates of P. aeruginosa were isolated. The elastolytic activity of P. aeruginosa isolates decreased after adding Cell free supernatant (CFS) of each Lactobacillus. L1, L4, L5, and L6 strains had a 100% inhibitory effect on pathogen isolates. L3 and L7 strains had the lowest inhibitory effect. The inhibitory effect of CFS extracted from lactobacilli on protease production by P. aeruginosa. L1, L4, L5, and L6 strains had an inhibitory effect on all tested isolates. L2, L3, and L7 strains had a less inhibitory effect. L4 strain had the highest inhibitory effect on pyocyanin production by P. aeruginosa (50%), followed by L5 (43.3%), L1 (40%), and L6 (23.3%) strains. L3 and L7 strains had no inhibitory effect on the pyocyanin production of P. aeruginosa isolates. It was found that the CFS of 4 isolates (L1, L4, L5, and L6) was the most active extract and had a 100% inhibitory effect against biofilm formation of all P. aeruginosa strains. The L3 strain had the least inhibitory effect against the biofilm formation of pathogens. Overall, this study showed that probiotics could be promising alternatives to combat the pathogenicity of P. aeruginosa in burn wounds.

Crohn’s disease proteolytic microbiota enhances inflammation through PAR2 pathway in gnotobiotic mice

ABSTRACT Emerging evidence implicates microbial proteolytic activity in ulcerative colitis (UC), but whether it also plays a role in Crohn’s disease (CD) remains unclear. We investigated the effects of colonizing adult and neonatal germ-free C57BL/6 mice with CD microbiota, selected based on high (CD-HPA) or low fecal proteolytic activity (CD-LPA), or microbiota from healthy controls with LPA (HC-LPA) or HPA (HC-HPA). We then investigated colitogenic mechanisms in gnotobiotic C57BL/6, and in mice with impaired Nucleotide-binding Oligomerization Domain-2 (NOD2) and Protease-Activated Receptor 2 (PAR2) cleavage resistant mice (Nod2 −/−; R38E-PAR2 respectively). At sacrifice, total fecal proteolytic, elastolytic, and mucolytic activity were analyzed. Microbial community and predicted function were assessed by 16S rRNA gene sequencing and PICRUSt2. Immune function and colonic injury were investigated by inflammatory gene expression (NanoString) and histology. Colonization with HC-LPA or CD-LPA lowered baseline fecal proteolytic activity in germ-free mice, which was paralleled by lower acute inflammatory cell infiltrate. CD-HPA further increased proteolytic activity compared with germ-free mice. CD-HPA mice had lower alpha diversity, distinct microbial profiles and higher fecal proteolytic activity compared with CD-LPA. C57BL/6 and Nod2 −/− mice, but not R38E-PAR2, colonized with CD-HPA had higher colitis severity than those colonized with CD-LPA. Our results indicate that CD proteolytic microbiota is proinflammatory, increasing colitis severity through a PAR2 pathway.

A3 CROHN’S DISEASE PROTEOLYTIC MICROBIOTA ENHANCES INFLAMMATION THROUGH PAR2 PATHWAY IN GNOTOBIOTIC MICE

Abstract Background An imbalance in host proteases has been implicated in inflammatory bowel disease (IBD). Recent evidence implicates microbial proteolytic activity (PA) in ulcerative colitis but whether it also plays a role in Crohn’s disease (CD) remains unclear. Purpose We therefore investigated the colitogenic potential and underlying pathways of proteolytic CD microbiota. Method Adult germ-free (GF) C57BL/6 mice were colonized with CD microbiota selected based on high (CD-HPA) or low fecal proteolytic activity (CD-LPA), and from healthy controls with LPA (HC-LPA), after which total fecal proteolytic, elastolytic and mucolytic activity were analyzed in the mice. Microbial community was assessed by 16S rRNA gene sequencing. Immune function and colonic injury were investigated by inflammatory gene expression (NanoString) and histology. Colitis severity and underlying pathways were investigated in C57BL/6, Nucleotide-binding Oligomerization Domain-2 knock-out (Nod2-/-), and Protease-Activated Receptor 2 (PAR2) cleavage resistant mice (R38E-PAR2) subjected to 2% dextran sodium sulfate in drinking water for 5 days followed by 2 days on water. Result(s) Colonization with HC-LPA or CD-LPA lowered baseline fecal proteolytic activity compared with GF mice, which was paralleled by lower acute inflammatory cell infiltrate. CD-HPA further increased proteolytic activity compared with GF mice. Fecal supernatants from CD-LPA or HC-LPA colonized mice had lower in vitro PAR2 cleavage compared to supernatants from GF and CD-HPA colonized mice. Several genes, such as Map kinases, Rhoa, Myd88, and Tollip, were increased in GF mice compared to colonized mice. 18 genes related to inflammation and barrier function (e.g., Mapk2k6, Tnf, Claudin1) were differentially expressed between CD-LPA and CD-HPA. CD-HPA mice had lower alpha diversity, distinct microbial profiles, and higher fecal proteolytic activity compared with CD-LPA. Abundance of several beneficial species (e.g., Akkermansia muciniphilia) was decreased while other taxa were increased (e.g., Hungattella hathewayi) in CD-HPA compared to CD-LPA. H. hathewayi as well as the serine protease K04772 were transcriptionally increased in fecal samples from CD-HPA colonized mice. C57BL/6 and Nod2-/- mice, but not R38E-PAR2 mice, colonized with CD-HPA developed earlier and more severe colitis compared with mice colonized with CD-LPA. Conclusion(s) CD proteolytic microbiota is proinflammatory through a PAR2 pathway. H. hathewayi correlates with the proinflammatory phenotype through the serine protease K04772 in this model. The results support a role of microbial PA in CD, which could constitute a biomarker for identifying patients who would benefit from anti-proteolytic therapies. Disclosure of Interest None Declared

Cathepsin S Levels and Survival Among Patients With Non-ST-Segment Elevation Acute Coronary Syndromes

BackgroundPatients with non–ST-segment elevation acute coronary syndromes (NSTE-ACS) are at high residual risk for long-term cardiovascular (CV) mortality. Cathepsin S (CTSS) is a lysosomal cysteine protease with elastolytic and collagenolytic activity that has been involved in atherosclerotic plaque rupture. ObjectivesThe purpose of this study was to determine the following: 1) the prognostic value of circulating CTSS measured at patient admission for long-term mortality in NSTE-ACS; and 2) its additive value over the GRACE (Global Registry of Acute Coronary Events) risk score. MethodsThis was a single-center cohort study, consecutively recruiting patients with adjudicated NSTE-ACS (n = 1,112) from the emergency department of an academic hospital. CTSS was measured in serum using enzyme-linked immunosorbent assay. All-cause mortality at 8 years was the primary endpoint. CV death was the secondary endpoint. ResultsIn total, 367 (33.0%) deaths were recorded. CTSS was associated with increased risk of all-cause mortality (HR for highest vs lowest quarter of CTSS: 1.89; 95% CI: 1.34-2.66; P < 0.001) and CV death (HR: 2.58; 95% CI: 1.15-5.77; P = 0.021) after adjusting for traditional CV risk factors, high-sensitivity C-reactive protein, left ventricular ejection fraction, high-sensitivity troponin-T, revascularization and index diagnosis (unstable angina/ non–ST-segment elevation myocardial infarction). When CTSS was added to the GRACE score, it conferred significant discrimination and reclassification value for all-cause mortality (Delta Harrell’s C: 0.03; 95% CI: 0.012-0.047; P = 0.001; and net reclassification improvement = 0.202; P = 0.003) and CV death (AUC: 0.056; 95% CI: 0.017-0.095; P = 0.005; and net reclassification improvement = 0.390; P = 0.001) even after additionally considering high-sensitivity troponin-T and left ventricular ejection fraction. ConclusionsCirculating CTSS is a predictor of long-term mortality and improves risk stratification of patients with NSTE-ACS over the GRACE score.

Phytochemical composition and antimicrobial, and anti-quorum sensing activities of Punica granatum L. methanolic extract.

In this work, our aims were to investigate the antimicrobial resistance, and anti-quorum sensing activities of Punica granatum L. methanolic extract. Antibacterial and antifungal activities were performed against thirteen bacteria and five fungal pathogens. Ultra-high performance liquid chromatography was used to identify the polyphenolic extract. The inhibition of pyocyanin production, proteolytic and elastolytic activity and swarming motility in Pseudomonas aeruginosa PAO1 test strain were estimated. The methanolic extract from P. granatum L. was dominated by chlorogenic acid (34.028 mg/g), rutin (26.05 mg/g), epicatechin (12.207 mg/g), gallic acid (11.157 mg/g), and caffeic acid 9.768 mg/g). Results showed antibacterial activities against almost all tested microorganisms with mean diameter of growth inhibition zone ranging from 6 ± 0 to 30 ± 0 mm for Candida species and from 6 ± 0 to 22.66 ± 0.57 for bacterial strains. The lowest minimal inhibitory concentrations were recorded for Listeria monocytogenes ATCC 19115 and Salmonella enterica CECT 529 (0.14 mg/ml, respectively). The anti-quorum sensing activity of methanolic extract against P. aeruginosa showed a significant inhibition of swarming motility and an attenuation in virulence factors like pyocyanin production at low concentrations. The obtained results indicates that P. granatum L. extracts is a rich source of phenolic compounds and highlighted the possibilities uses of pomegranate to attenuate the expression of quorum sensing controlled factors in P. aeruginosa PAO1 strain.

Novel Quorum Quenching YtnP Lactonase From Bacillus paralicheniformis Reduces Pseudomonas aeruginosa Virulence and Increases Antibiotic Efficacy in vivo.

Bacterial infections have become increasingly difficult to treat due to the occurrence of antibiotic-resistant strains. A promising strategy to increase the efficacy of therapy is to combine antibacterials with agents that decrease pathogen virulence via the modulation of the quorum sensing (QS). Lactonases inhibit acylated homoserine lactone (AHL)-mediated QS in Gram-negative bacteria, including the leading nosocomial pathogen Pseudomonas aeruginosa. Here we describe the characteristics of heterologously expressed YtnP lactonase from Bacillus paralicheniformis ZP1 (YtnP-ZP1) isolated from agricultural soil using the culture enrichment method. Purified YtnP-ZP1 hydrolyzed different AHLs with preference to substrates with long acyl residues as evaluated in assays with biosensors and HPLC. The enzyme showed good thermostability and activity in a wide temperature range. YtnP-ZP1 in 50 μg mL–1 concentration reduced the amount of P. aeruginosa-produced long-chain AHLs by 85%, while it hydrolyzed 50% of short-chain AHLs. Incubation of P. aeruginosa PAO1 with YtnP-ZP1 reduced its swarming motility and elastolytic activity without bactericidal effect. YtnP-ZP1 caused the inhibition of biofilm formation and disintegration of mature biofilms in P. aeruginosa PAO1 and multiresistant clinical strain BR5H that was visualized by crystal violet staining. The treatment with YtnP-ZP1 in concentrations higher than 25 μg mL–1 improved the survival of P. aeruginosa PAO1-infected zebrafish (Danio rerio), rescuing 80% of embryos, while in combination with tobramycin or gentamicin survival rate increased to 100%. The treatment of P. aeruginosa PAO1 biofilms on infected zebrafish tail wounds with 50 μg mL–1 YtnP-ZP1 and 2 × MIC tobramycin led to infection clearing in 2 days. The extensive toxicity studies proved YtnP-ZP1 was non-toxic to human cells and zebrafish. In conclusion, novel YtnP-ZP1 lactonase with its effective anti-virulence activity could be used to increase the efficacy of clinically approved antibiotics in clearing both systemic and biofilm-associated P. aeruginosa infections.

Sequence preference and scaffolding requirement for the inhibition of human neutrophil elastase by ecotin peptide.

Human neutrophil elastase (hNE) is an abundant serine protease that is a major constituent of lung elastolytic activity. However, when secreted in excess, if not properly attenuated by selective inhibitor proteins, it can have detrimental effects on host tissues, leading to chronic lung inflammation and non-small cell lung cancer. To improve upon the design of inhibitors against hNE for therapeutic applications, here, we report the crystal structure of hNE in complex with an ecotin (ET)-derived peptide inhibitor. We show that the peptide binds in the nonprime substrate binding site. Unexpectedly, compared with full-length (FL) ET, we find that our short linear peptides and circular amide backbone-linked peptides of ET are incapable of efficient hNE inhibition. Our structural insights point to a preferred amino acid sequence and the potential benefit of a scaffold for optimal binding and function of the peptide inhibitor, both of which are retained in the FL ET protein. These findings will aid in the development of effective peptide-based inhibitors against hNE for targeted therapy.

Quorum quenchers affect the virulence regulation of non-mucoid, mucoid and heavily mucoid biofilms co-cultured on cell lines

Biofilm formation conferring pathogenicity is a survival strategy for Pseudomonas aeruginosa. P. aeruginosa’s virulence may differ due to differences in host-microbe interactions and the growth environment. The epithelial cell line within the respiratory system and the keratinocytes on the skin form the first physical barrier of defence. P. aeruginosa spp. biofilm formation and virulence factor secretion with and without quorum quenching (QQ) treatment was studied in co-culture using A549 and HaCaT cell lines; pyocyanin and rhamnolipid productions and elastolytic activity as virulence factors were quantified by independent assays. Biofilm formation was evaluated under dynamic conditions by quantifying total carbohydrates, alginate, proteins and eDNA. A sandwich ELISA was performed to study IL-8 secretion by the epithelial cells. The difference in gene expression of the quorum sensing (QS) and virulence factors between strains during individual and combination treatments was analysed by qPCR. Combination treatment by farnesol and tyrosol was more effective against P. aeruginosa biofilms when grown in co-cultures. The strain RBHi was found to be 3 to 4 times more virulent compared to PAO1 and NCTC 10,662, respectively, and combination treatment was more effective against RBHi strain when grown in co-culture with A549 cell line. The addition of quorum quenchers (QQs) individually and in combination reduced IL-8 secretion by A549 cells. Relative mRNA expression showed upregulation of the QS genes and virulence factors. Co-culture of P. aeruginosa and HaCaT cell line showed a general decrease in gene expression, especially in the case of P. aeruginosa RBHi when treated with farnesol and tyrosol combination.Key points• Differentiating the interactions of biofilm formed by different phenotypes of P. aeruginosa, NCTC 10,662 (non-mucoid), PAO1 (semi mucoid) and RBHi (heavily mucoid).• Biofilm formed by these P. aeruginosa strains on two commonly afflicted tissues represented by A549 (lung) and HaCaT (skin) cell lines.• Anti-biofilm/anti-virulence roles of quorum quenchers, tyrosol and farnesol in co-cultures.

Elastolytic activity is associated with inflammation in bladder cancer.

Cancer development and progression is often associated with inflammation. Late diagnosis of inflammation that directly leads to the development of neoplasm-cancer is associated with a reduction in the chance of successful treatment or is associated with therapeutic difficulties. A panel of chromogenic substrates was used for the qualitative determination of the specific activity of enzymes in urine of patients with confirmed inflammatory reaction and/or epithelial neoplasms in particular tumours at various stages of development. Urine of people with excluded inflammation was used as a control group. Proteolytic activity was determined in urine samples collected from patients with epithelial neoplasms and/or inflammation. What is more, we determine human neutrophil elastase activity-related inflammation based on the examination of urine samples. We suspect that the proteolytical activity of urine samples is due to neutrophil response to inflammation, which is directly related to cancer. This is the first study to determine elastolytic activity in bladder cancer urine samples. It supports wider use of urine for inflammation screening.

Human Single-Chain Antibodies That Neutralize Elastolytic Activity of Pseudomonas aeruginosa LasB.

LasB (elastase/pseudolysin) is an injurious zinc-metalloprotease secreted by the infecting Pseudomonas aeruginosa. LasB is recognized as the bacterial key virulence factor for establishment of successful infection, acquisition of nutrients, dissemination, tissue invasion, and immune modulation and evasion. LasB digests a variety of the host tissue proteins, extracellular matrices, as well as components of both innate and adaptive immune systems, including immunoglobulins, complement proteins, and cytokines. Thus, this enzyme is an attractive target for disarming the P. aeruginosa. This study generated human single-chain antibodies (HuscFvs) that can neutralize the elastolytic activity of native LasB by using phage display technology. Gene sequences coding HuscFvs (huscfvs) isolated from HuscFv-displaying phage clones that bound to enzymatically active LasB were sub-cloned to expression plasmids for large scale production of the recombinant HuscFvs by the huscfv-plasmid transformed Escherichia coli. HuscFvs of two transformed E. coli clones, i.e., HuscFv-N42 and HuscFv-N45, neutralized the LasB elastolytic activities in vitro. Computer simulation by homology modeling and molecular docking demonstrated that antibodies presumptively formed contact interfaces with the LasB residues critical for the catalytic activity. Although the LasB neutralizing mechanisms await elucidation by laboratory experiments, the HuscFvs should be tested further towards the clinical application as a novel adjunctive therapeutics to mitigate severity of the diseases caused by P. aeruginosa.

Studies on Elastase and Elastase Inhibitor from Aspergillus flavus

The biological properties of elastase and Aspergillus flavus elastase inhibitor (AFLEI) from A. flavus were examined. Pathogenicity of elastase was investigated in mice immunocompromised with cyclophosphamide, cyclosporine, prednisolone and carrageenan. Compared to cyclophosphamide immunocompromised mice treated with the spores of elastase nonproducing strain, cyclophosphamide immunocompromised mice treated with the spores of elastase producing strain had a significantly shorter survival rate. Molecular mass of AFLEI was determined to be 7525.8 Da. The elastolytic activity of elastases from A. flavus, and human leukocytes were inhibited by AFLEI. The primary structure of AFLEI was determined by the Edman sequencing procedure. The search for amino acid homology with other proteins demonstrated that amino acid residues 1 to 68 of AFLEI are 100% identical to residues 20 to 87 of the hypothetical protein AFUA_3G14940 of A. fumigatus. When immunocompromised mice administered of cyclophosphamide were infected by inhalation of A. flavus then administered amphotericin B (AMPH) alone or in combination with AFLEI, survival rate tended to be higher with combination treatment than with AMPH alone. Moreover, although extensive bleeding was seen in pathology sections taken from rat lung resected 24 h after elastase was administered to the lung via the bronchus, this bleeding was inhibited by AFLEI. The X-ray analysis has revealed that the structure of this inhibitor was wedge shaped and composed of a binding loop and a scaffold protein core. As synthetic-inhibitor strongly inhibited cytotoxicity induced by elastase in human-derived cells, it could prove beneficial for the treatment of pulmonary aspergillosis.