Elastase Production Research Articles

Dexamethasone and IFN-γ primed mesenchymal stem cells conditioned media immunomodulates aberrant NETosis in SLE via PGE2 and IDO.

Systemic Lupus Erythematosus (SLE) is characterized by dysregulated immune responses, with neutrophil extracellular traps (NETs) playing a significant role. NETs are recognized by autoantibodies in SLE patients, exacerbating pathology. Both excessive NET formation and impaired degradation contribute to SLE pathophysiology. To investigate the immunomodulatory effects of Dexamethasone-primed Wharton's jelly (WJ) derived MSCs CM (DW) and IFN-γ-primed WJ-MSCs-CM (IW) on NETosis and associated protein markers in SLE patients' LPS or ribonucleoprotein immune complexes (RNP ICs) induced neutrophils and in pristane induced lupus (PIL) model. And to elucidate the mechanism involved therein. We investigated the immunomodulatory effects of DW and IW on NETosis in SLE. Utilizing ex vivo and in vivo models, we assessed the impact of preconditioned media on NET formation and associated protein markers neutrophil elastase (NE), citrullinated histone (citH3), myeloperoxidase (MPO), cytoplasmic and mitochondrial ROS production. We also examined the involvement of key immunomodulatory factors present in DW and IW, including prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO), and transforming growth factor-beta (TGF-β). Preconditioned media effectively suppressed NETosis and reduced ROS generation in SLE neutrophils, indicating their immunomodulatory potential. Inhibition studies implicated IDO and PGE2 in mediating this effect. Combined treatment with DW or IW together with hydroxychloroquine (HCQ) demonstrated superior efficacy over HCQ alone, a standard SLE medication. In PIL mouse model, DW and IW treatments reduced NETosis, ROS generation, as evidenced by decreased NET-associated protein expression in vital organs. Our study highlights the multifaceted impact of IW and DW on NETosis, ROS dynamics, and lupus severity in SLE. These findings underscore the potential of preconditioned media for the development of targeted, personalized approaches for SLE treatment.

Molecular characterization of Pseudomonas aeruginosa from diabetic foot infections in Tunisia.

Background. Pseudomonas aeruginosa is an invasive organism that frequently causes severe tissue damage in diabetic foot ulcers.Gap statement. The characterisation of P. aeruginosa strains isolated from diabetic foot infections has not been carried out in Tunisia.Purpose. The aim was to determine the prevalence of P. aeruginosa isolated from patients with diabetic foot infections (DFIs) in Tunisia and to characterize their resistance, virulence and molecular typing.Methods. Patients with DFIs admitted to the diabetes department of the International Hospital Centre of Tunisia, from September 2019 to April 2021, were included in this prospective study. P. aeruginosa were obtained from the wound swabs, aspiration and soft tissue biopsies during routine clinical care and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing, serotyping, integron and OprD characterization, virulence, biofilm production, pigment quantification, elastase activity and molecular typing were analysed in all recovered P. aeruginosa isolates by phenotypic tests, specific PCRs, sequencing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.Results. Sixteen P. aeruginosa isolates (16.3 %) were recovered from 98 samples of 78 diabetic patients and were classified into 6 serotypes (O:11 the most frequent), 11 different PFGE patterns and 10 sequence types (three of them new ones). The high-risk clone ST235 was found in two isolates. The highest resistance percentages were observed to netilmicin (69 %) and cefepime (43.8 %). Four multidrug-resistant (MDR) isolates (25 %) were detected, three of them being carbapenem-resistant. The ST235-MDR strain harboured the In51 class 1 integron (intI1 +aadA6+orfD+qacED1-sul1). According to the detection of 14 genes involved in virulence or quorum sensing, 5 virulotypes were observed, including 5 exoU-positive, 9 exoS-positive and 2 exoU/exoS-positive strains. The lasR gene was truncated by ISPpu21 insertion sequence in one isolate, and a deletion of 64 bp in the rhlR gene was detected in the ST235-MDR strain. Low biofilm, pyoverdine and elastase production were detected in all P. aeruginosa; however, the lasR-truncated strain showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity, high production of phenazines and high biofilm formation.Conclusions. Our study demonstrated for the first time the prevalence and the molecular characterization of P. aeruginosa strains from DFIs in Tunisia, showing a high genetic diversity, moderate antimicrobial resistance, but a high number of virulence-related traits, highlighting their pathological importance.

Effect of phenylalanine arginyl β-naphthylamide on the imipenem resistance, elastase production, and the expression of quorum sensing and virulence factor genes in Pseudomonas aeruginosa clinical isolates.

Pseudomonas aeruginosa is one of the most important nosocomial pathogens that possess the ability to produce multiple antibiotic resistance and virulence factors. Elastase B (LasB) is the major factor implicated in tissue invasion and damage during P. aeruginosa infections, whose synthesis is regulated by the quorum sensing (QS) system. Anti-virulence approach is now considered as potential therapeutic alternative and/or adjuvant to current antibiotics' failure. The aim of this study is primarily to find out the impact of the efflux pump inhibitor (EPI) phenylalanine arginyl β-naphthylamide (PAβN) on the production of elastase B and the gene expression of lasI quorum sensing and lasB virulence factor in clinical isolates of P. aeruginosa. Five P. aeruginosa isolates recovered from patients with respiratory tract infections were examined in this study. Antimicrobial susceptibility of isolates was performed by the disk agar diffusion method. Effect of the PAβN on imipenem susceptibility, bacterial viability, and elastase production was evaluated. The expression of lasB and lasI genes was measured by quantitative real-time PCR in the presence of PAβN. All isolates were identified as multidrug-resistant (MDR) and showed resistance to carbapenem (MIC = 64-256µg/mL). Susceptibility of isolates to imipenem was highly increased in the presence of efflux inhibitor. PAβN significantly reduced elastase activity in three isolates tested without affecting bacterial growth. In addition, the relative expression of both lasB and lasI genes was diminished in all isolates in the presence of inhibitor. Efflux inhibition by using the EPI PAβN could be a potential target for controlling the P. aeruginosa virulence and pathogenesis. Furthermore, impairment of drug efflux by PAβN indicates its capability to be used as antimicrobial adjuvant that can decrease the resistance and lower the effective doses of current drugs.

The role of bacterial genital infections in spontaneous preterm delivery: a case-control study.

Spontaneous preterm delivery is defined as the beginning of the birth process before the 37th week of pregnancy. The presence of microorganisms in the fetal membranes is accompanied by an increase in the production of prostaglandin, one of the important factors associated with the prevalence of preterm birth. The invasion of microorganisms leads to the production of protease, coagulase, and elastase, which directly stimulate the onset of childbirth. We investigated the role of genital infections in women with preterm birth. The present case-control study was conducted in the west of Iran on 100 women with spontaneous preterm delivery (following 24 weeks of gestation and before 36 weeks and 6 days) as the case group and 100 women with normal delivery as controls. A questionnaire was applied to collect the data. Polymerase chain reaction and pathological examination of the placenta were performed. The average age in women with normal delivery (30.92 ± 5.10) in women with spontaneous preterm delivery (30.27 ± 4.93). The prevalence of Chlamydia trachomatis, Neisseria gonorrhea, Listeria monocytogenes, and Mycoplasma genitalium infections was zero in both groups. The highest prevalence of Gardnerella vaginalis was 19 (19%) in the case group and Ureaplasma parvum 15 (15%) in the control group. Also, Placental inflammation was zero in controls and 7(7%) in the patient group. There was a significant relationship between Gardnerella vaginalis bacteria and spontaneous preterm delivery. The results of our study showed that except for Gardnerella vaginalis bacteria, there is no significant relationship between the above bacterial infections and spontaneous preterm birth. Moreover, despite the significant reduction in the prevalence of many sexually transmitted infections in this research, it is still suggested to increase the awareness of people, including pregnant women, about the ways it can be transmitted by gynecologists and health and treatment centers.

The Long-Acting Serine Protease Inhibitor mPEG-SPA-MDSPI16 Alleviates LPS-Induced Acute Lung Injury.

Anti-inflammatory drugs have become the second-largest class of common drugs after anti-infective drugs in animal clinical care worldwide and are often combined with other drugs to treat fever and viral diseases caused by various factors. In our previous study, a novel serine protease inhibitor-encoding gene (MDSPI16) with improved anti-inflammatory activity was selected from a constructed suppressive subducted hybridization library of housefly larvae. This protein could easily induce an immune response in animals and had a short half-life, which limited its wide application in the clinic. Thus, in this study, mPEG-succinimidyl propionate (mPEG-SPA, Mw = 5 kDa) was used to molecularly modify the MDSPI16 protein, and the modified product mPEG-SPA-MDSPI16, which strongly inhibited elastase production, was purified. It had good stability and safety, low immunogenicity, and a long half-life, and the IC50 for elastase was 86 nM. mPEG-SPA-MDSPI16 effectively inhibited the expression of neutrophil elastase and decreased ROS levels. Moreover, mPEG-SPA-MDSPI16 exerted anti-inflammatory effects by inhibiting activation of the NF-κB signaling pathway and the MAPK signaling pathway in neutrophils. It also exerted therapeutic effects on a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. In summary, mPEG-SPA-MDSPI16 is a novel anti-inflammatory protein modified with PEG that has the advantages of safety, nontoxicity, improved stability, and strong anti-inflammatory activity in vivo and in vitro and is expected to become an effective anti-inflammatory drug.

Dimethyl α-Ketoglutarate Promotes the Synthesis of Collagen and Inhibits Metalloproteinases in HaCaT Cells.

We observed that treatment with dimethyl α-ketoglutarate (DMK) increased the amount of intracellular α-ketoglutarate significantly more than that of α-ketoglutarate in HaCaT cells. DMK also increased the level of intracellular 4-hydroxyproline and promoted the production of collagen in HaCaT cells. In addition, DMK decreased the production of collagenase and elastase and down-regulated the expression of selected matrix metalloproteinases (MMPs), such as MMP-1, MMP-9, MMP-10, and MMP-12, via transcriptional inhibition. The inhibition of MMPs by DMK was mediated by the suppression of the IL-1 signaling cascade, leading to the attenuation of ERK1/2 phosphorylation and AP-1 transactivation. Our study results illustrate that DMK, an alkylated derivative of α-ketoglutarate, increased the level of 4-hydroxyproline, promoted the production of collagen, and inhibited the expression of selected MMPs by affecting the IL-1 cascade and AP-1 transactivation in HaCaT cells. The results suggest that DMK might be useful as an anti-wrinkle ingredient.

Quercetin: a promising virulence inhibitor of Pseudomonas aeruginosa LasB in vitro

With the inappropriate use of antibiotics, antibiotic resistance has emerged as a major dilemma for patients infected with Pseudomonas aeruginosa. Elastase B (LasB), a crucial extracellular virulence factor secreted by P. aeruginosa, has been identified as a key target for antivirulence therapy. Quercetin, a natural flavonoid, exhibits promising potential as an antivirulence agent. We aim to evaluate the impact of quercetin on P. aeruginosa LasB and elucidate the underlying mechanism. Molecular docking and molecular dynamics simulation revealed a rather favorable intermolecular interaction between quercetin and LasB. At the sub-MICs of ≤256 μg/ml, quercetin was found to effectively inhibit the production and activity of LasB elastase, as well as downregulate the transcription level of the lasB gene in both PAO1 and clinical strains of P. aeruginosa. Through correlation analysis, significant positive correlations were shown between the virulence gene lasB and the QS system regulatory genes lasI, lasR, rhlI, and rhlR in clinical strains of P. aeruginosa. Then, we found the lasB gene expression and LasB activity were significantly deficient in PAO1 ΔlasI and ΔlasIΔrhlI mutants. In addition, quercetin significantly downregulated the expression levels of regulated genes lasI, lasR, rhlI, rhlR, pqsA, and pqsR as well as effectively attenuated the synthesis of signaling molecules 3-oxo-C12-HSL and C4-HSL in the QS system of PAO1. Quercetin was also able to compete with the natural ligands OdDHL, BHL, and PQS for binding to the receptor proteins LasR, RhlR, and PqsR, respectively, resulting in the formation of more stabilized complexes. Taken together, quercetin exhibits enormous potential in combating LasB production and activity by disrupting the QS system of P. aeruginosa in vitro, thereby offering an alternative approach for the antivirulence therapy of P. aeruginosa infections.Key points• Quercetin diminished the content and activity of LasB elastase of P. aeruginosa.• Quercetin inhibited the QS system activity of P. aeruginosa.• Quercetin acted on LasB based on the QS system.

Repurposing albendazole as a potent inhibitor of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa: Novel prospects of a classical drug

Pseudomonas aeruginosa has emerged as a critical superbug that poses a serious threat to public health. Owing to its virulence and multidrug resistance profiles, the pathogen demands immediate attention for devising alternate intervention strategies. In an attempt to repurpose drugs against P. aeruginosa, this preclinical study was aimed at investigating the antivirulence prospects of albendazole (AbZ), an FDA-approved anti-helminthic drug, recently predicted to disrupt quorum sensing (QS) in Chromobacterium violaceum. AbZ was scrutinized for its quorum quenching (QQ) prospects, effect on bacterial virulence, different motility phenotypes, and biofilm formation in vitro. Additionally, in silico analysis was employed to predict the molecular interactions between AbZ and QS receptors. At sub-inhibitory levels, AbZ demonstrated anti-QS activity and significantly abrogated AHL biosynthesis in P. aeruginosa. Moreover, AbZ significantly downregulated the transcript levels of QS- (lasI/lasR, rhlI/rhlR, and pqsA/pqsR) and QS-dependent virulence (aprA, lasA, lasB, plcH, and toxA) genes in P. aeruginosa. This coincided with reduced hemolysin, alginate, pyocyanin, rhamnolipids, total protease, and elastase production, thereby lowering phenotypic virulence. Molecular docking with AbZ further revealed strong associations and high binding energies with LasR (−8.8 kcal/mol), RhlR (−6.5 kcal/mol), and PqsR (−6.3 kcal/mol) receptors. AbZ also impeded bacterial motility and abolished EPS production, severely compromising pseudomonal biofilm formation. For the first time, AbZ was shown to interfere with QS circuitry and consequently disarming pseudomonal virulence. Hence, AbZ can be exploited for its antivirulence properties against P. aeruginosa.

Absence of Neutropenia in Patient with an Exon 2 Nonsense Mutation in ELANE: Clinical Evidence to Support Gene Therapy Efforts for Severe Congenital Neutropenia

Introduction Severe congenital neutropenia (SCN) is an inherited bone marrow failure condition most often caused by heterozygous mutations in the ELANE gene. While granulocyte colony stimulating factor (G-CSF) is effective in increasing neutrophil counts in the majority of patients, approximately 15% of patients do not respond, necessitating the creation of alternative treatment strategies. Due to its association with significant, life-threatening infections and heightened risk of progression to myelodysplastic syndrome and/or acute myeloid leukemia, SCN has been the focus of recent gene therapy efforts by multiple groups (Nasri et al., 2020; Rao et al., 2021). Pathogenic variants in ELANE most often cause missense mutations or in-frame indels which lead to the production and expression of mutant neutrophil elastase (NE) proteins. Frameshift mutations resulting in out-of-frame stop codons or nonsense mutations are also seen in the third through fifth (final) exons of ELANE due to their ability to circumvent nonsense-mediated decay (NMD) and generate mutant NE. In contrast, frameshift and nonsense mutations in the first and second exons have not been shown to cause neutropenia, most likely because NMD prevents the translation of mutant NE (Rao et al., 2021; Horwitz, 2021). Collectively, these observations suggest that the pathogenicity of ELANE mutations may rely on gain-of-function alterations in mutant NE rather than loss-of-function alterations in normal NE. Because the absence of one copy of ELANE does not appear to be deleterious, one theory posits that therapeutic inactivation of mutant copies of ELANE may have corrective potential (Horwitz et al., 2019; Nasri et al., 2020; Rao et al., 2021). Thus, the focus of several gene editing efforts has been CRISPR/Cas9-mediated knockout in early exons of ELANE in hematopoietic stem and progenitor cells (HSPCs) of patients with ELANE-mutated SCN for allogeneic stem cell transplant (Nasri et al., 2020; Rao et al., 2021). With this study, we sought to understand whether an inherited ELANE mutation analogous to those utilized in gene therapy would cause neutropenia. Case Presentation Here, we present a six-month-old Caucasian male with an ELANE mutation whose clinical findings support this model. The patient was born at full term to a 35-year-old G6P2 mother. The patient's constellation of complex symptoms, including polymicrogyria, corpus callosum dysgenesis, hypotonia, seizures, and feeding difficulties prompted clinical trio whole genome sequencing (WGS) at approximately two months of age. The patient was incidentally found to have a de novo nonsense variant in exon 2 of the ELANE gene (NM_001972.2:c.151G>T, p.[G51*]), predicted to result in protein truncation or NMD. WGS also identified variants of uncertain significance in CHRNB2 and HFE. Importantly, although the ELANE variant was classified as likely pathogenic in the clinical genetics report, the patient has not had clinically significant neutropenia. Absolute neutrophil counts have ranged from 1.27 - 10.95 K cells/μL, with mean and median values of 4.09 and 2.95 K cells/μL, respectively (n=10). The patient has not had a significant infectious history, nor has he required any treatment for hematologic concerns. Discussion The absence of a neutropenia pathology in this patient, at least in his first six months of life, provides evidence in support of gene therapy efforts which seek to ameliorate pathogenic ELANE mutations with gene editing to produce frameshifts in early exons. Most likely, in this patient, NMD of mutant ELANE mRNA prevented its translation, thus allowing for ample production and maturation of neutrophils. A similar pattern was observed in another report which examined patients with whole gene deletions of ELANE (Horwitz et al., 2019), but this is the first report of a nonsense variant analogous to those proposed to be introduced by gene or base editing for gene therapy of SCN. Thus, the present case adds to a growing body of evidence which suggests that ELANE knockout may have therapeutic potential for SCN.

Carbon quantum dots derived from resveratrol enhances anti-virulence activity against Pseudomonas aeruginosa

Resveratrol (Res), one phenolic compound derived from many kinds of fruits, has been shown to possess anti-virulence activity against the food spoilage bacterium Pseudomonas aeruginosa. However, the high usage and low activity limit its widespread application in food industry. In this study, Res was prepared into resveratrol carbon quantum dots (CQDRes) and the anti-virulence activity and anti-virulence mechanism of CQDRes were revealed for the first time. Exposure to CQDRes at 100 µg/mL significantly inhibited chemotaxis and the production of elastase, pyocyanin, rhamnolipid, and biofilms on the interface, and this inhibitory effect was higher than that of Res. Comprehensive transcriptome, proteomics, and parallel reaction monitoring analyses indicated that the pqs system of P. aeruginosa PAO1 was seriously disrupted after treatment with CQDRes while Res exposure had no such disrupted effect. The disrupted pqs system notably down-regulated the expressions of virulence-related genes and reduced the secretion of PQS-related signal molecules and pathogenicity of P. aeruginosa PAO1. The pqsC-knockout assay presented the reduced production of virulence factors and further confirmed the dysfunction of pqs system induced by CQDRes. These data indicated that CQDRes has the potential to function as an anti-virulence agent to defending against the foodborne pathogen P. aeruginosa.

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

Bioactive Compounds from P. pertomentellum That Regulate QS, Biofilm Formation and Virulence Factor Production of P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many nosocomial infections. This bacterium uses Quorum Sensing (QS) to generate antimicrobial resistance (AMR) so its disruption is considered a novel approach. The current study describes the antibiofilm and QS inhibitory potential of extract and chemical components from Piper pertomentellum. The methodo- logy included the phytochemical study on the aerial part of the species, the determination of QS inhibition efficacy on Chromobacterium violaceum and the evaluation of the effect on biofilm formation and virulence factors on P. aeruginosa. The phytochemical study led to the isolation and identification of a new piperamide (ethyltembamide 1), together with four known amides (tembamide acetate 2, cepharadione B 3, benzamide 4 and tembamide 5). The results indicated that the ethanolic extract and some fractions reduced violacein production in C. violaceum, however, only the ethanolic extract caused inhibition of biofilm formation of P. aeruginosa on polystyrene microtiter plates. Finally, the investigation determined that molecules (1-5) inhibited the formation of biofilms (50% approximately), while compounds 2-4 can inhibit pyocyanin and elastase production (30-50% approximately). In this way, the study contributes to the determination of the potential of extract and chemical constituents from P pertomentellum to regulate the QS system in P. aeruginosa.

3-(Bromoacetyl) coumarin is a potential therapeutic agent against neonatal sepsis-associated Pseudomonas extremorientalis.

Neonatal sepsis is a severe bacterial infection that can lead to life-threatening complications in newborns. Pseudomonas extremorientalis is a Gram-negative bacterium andthese Gram-negative organismshave been identified as a major cause of neonatal sepsis. The virulence factors produced by this bacterium play a crucial role in its pathogenicity. Therefore, targeting these virulence factors could be a potential strategy to treat neonatal sepsis caused by P. extremorientalis. In this study, we investigated the efficacy of 3-(bromoacetyl) coumarin (3-BC) in reducing the virulence factors of P. extremorientalis strains isolated from neonatal sepsis. Our results showed that 3-BC effectively reduced the production of various virulence factors, including protease, elastase, siderophore, and exopolysaccharide in these strains. Furthermore, at a concentration of 125µg/ml, 3-BC also inhibited the biofilm formation ability of these strains in combination with ciprofloxacin. It was discovered that 3-BC was functionally effective in protecting C. elegans against bacterial infection. Moreover, the in vitro and in vivo outcomes were strongly correlated with docking studies of various activator proteins. Overall, our findings suggest that 3-BC could be a potential therapeutic agent for the treatment of neonatal sepsis caused by P. extremorientalis. Further studies are needed to explore the mechanism of action of 3-BC and its potential use in clinical settings.

Antibacterial and anticancer activities of (free and immobilized) elastase produced by Klebsiella pneumoniae

Elastase is a type of protease that degrades explicitly elastin. The elastase produced by Klebsiella pneumoniae isolates was purified by three steps: ammonium sulfate precipitation, ion-exchange chromatography, and Sephadex G-150 chromatography. The optimal condition for elastase production showed high specific activity with starch (3.8 U/mg protein) and casein as a nitrogen source with a specific activity reaching (3.3 U/mg protein). The maximum elastase production was obtained when the pH value was (7.5) with specific activity (4.4 U/mg protein). Elastase (free and immobilized on TiO2- NPs) was used in application as antibacterial and anticancer, and results showed high antibacterial activity against pathogenic isolates, especially Lactobacillus acidophilus and Pseudomonas aeruginosa were affected by immobilized elastase. Free and immobilized elastase have anticancer activity against lung cancer using the A549 cell line, and immobilized elastase had the potent cytotoxic effect on A549 cells with IC50 142.8 µg/ ml compared with IC50 of normal cell line HdFn on 655.0 µg /ml. Key Words: Klebsiella pneumoniae, Elastase, Immobilization, TiO2-Nps- Antibacterial, Anticancer.

Anti-virulence prospects of Metformin against Pseudomonas aeruginosa: A new dimension to a multifaceted drug

Metformin (MeT) is an FDA-approved drug with a myriad of health benefits. Besides being used as an anti-diabetic drug, MeT is also effective against various cancers, liver-, cardiovascular-, and renal diseases. This study was undertaken to examine its unique potential as an anti-virulence drug against an opportunistic bacterial pathogen, Pseudomonas aeruginosa. Due to the menace of multidrug resistance in pathogenic microorganisms, many novel or repurposed drugs with anti-virulence prospects are emerging as next-generation therapies with the aim to overshadow the application of existing antimicrobial regimens. The quorum sensing (QS) mechanisms of P. aeruginosa are an attractive drug target for attenuating bacterial virulence. In this context, the anti-QS potential of MeT was scrutinized using biosensor assays. MeT was comprehensively evaluated for its effects on different motility phenotypes, virulence factor production (phenotypic and genotypic expression) along with biofilm development in P. aeruginosa in vitro. At sub-lethal concentrations, MeT displayed prolific quorum quenching (QQ) ability and remarkably inhibited AHL biosynthesis in P. aeruginosa. Moreover, MeT (1/8 MIC) effectively downregulated the expression levels of various QS- and virulence genes in P. aeruginosa, which coincided with a notable reduction in the levels of alginate, hemolysin, pyocyanin, pyochelin, elastase, and protease production. In silico analysis through molecular docking also predicted strong associations between MeT and QS receptors of P. aeruginosa. MeT also compromised the motility phenotypes and successfully abrogated biofilm formation by inhibiting EPS production in P. aeruginosa. Hence, MeT may be repurposed as an anti-virulence drug against P. aeruginosa in clinical settings.

BI 1291583: a novel selective inhibitor of cathepsin C with superior in vivo profile for the treatment of bronchiectasis

BackgroundAirway inflammation in chronic inflammatory lung diseases (e.g. bronchiectasis) is partly mediated by neutrophil-derived serine protease (NSP)/antiprotease imbalance. NSPs are activated during neutrophil myelopoiesis in bone marrow by cathepsin C (CatC; DPP1). CatC is therefore an attractive target to reduce NSP activity in the lungs of patients with bronchiectasis, restoring the protease/antiprotease balance. We report results from the preclinical pharmacological assessment of the novel CatC inhibitor BI 1291583.MethodsBinding kinetics of BI 1291583 to human CatC were determined by surface plasmon resonance. In vitro inhibition of human CatC activity was determined by CatC-specific fluorescent assay, and selectivity was assessed against related cathepsins and unrelated proteases. Inhibition of NSP neutrophil elastase (NE) production was assessed in a human neutrophil progenitor cell line. In vivo inhibition of NE and NSP proteinase 3 (PR3) in bronchoalveolar lavage fluid (BALF) neutrophils after lipopolysaccharide (LPS) challenge and distribution of BI 1291583 was determined in a mouse model.ResultsBI 1291583 bound human CatC in a covalent, reversible manner, selectively and fully inhibiting CatC enzymatic activity. This inhibition translated to concentration-dependent inhibition of NE activation in U937 cells and dose-dependent, almost-complete inhibition of NE and PR3 activity in BALF neutrophils in an in vivo LPS-challenge model in mice. BI 1291583 exhibited up to 100 times the exposure in the target tissue bone marrow compared with plasma.ConclusionBI 1291583-mediated inhibition of CatC is expected to restore the protease–antiprotease balance in the lungs of patients with chronic airway inflammatory diseases such as bronchiectasis.

Biosurfactant derived from probiotic Lactobacillus acidophilus exhibits broad-spectrum antibiofilm activity and inhibits the quorum sensing-regulated virulence.

Antimicrobial resistance by pathogenic bacteria has become a global risk to human health in recent years. The most promising approach to combating antimicrobial resistance is to target virulent traits of bacteria. In the present study, a biosurfactant derived from the probiotic strain Lactobacillus acidophiluswas tested against three Gram-negative bacteria to evaluate its inhibitory potential on their biofilms, and whether it affected the virulence factors controlled by quorum sensing (QS). A reduction in the virulence factors of Chromobacterium violaceum (violacein production), Serratia marcescens (prodigiosin production) and Pseudomonas aeruginosa (pyocyanin, total protease, LasB elastase and LasA protease production) was observed at different sub-MIC concentrations in a dose-dependent manner. Biofilm development was reduced by 65.76%, 70.64% and 58.12% at the highest sub-MIC levels for C. violaceum, P. aeruginosa and S. marcescens, respectively. Biofilm formation on glass surfaces exhibited significant reduction, with less bacterial aggregation and reduced formation of extracellular polymeric materials. Additionally, swimming motility and exopolysaccharides (EPS) production were shown to be reduced in the presence of the L. acidophilus-derived biosurfactant. Furthermore, molecular docking analysis performed on compounds identified through gas chromatography-mass spectrometry (GC-MS) analysis of QS and biofilm proteins yielded further insights into the mechanism underlying the anti-QS activity. Therefore, the present study has clearly demonstrated that a biosurfactant derived from L. acidophilus can significantly inhibit virulence factors of Gram-negative pathogenic bacteria. This could provide an effective method to inhibit the formation of biofilms and QS in Gram-negative bacteria.

Biosynthesized Silver Nanoparticles Derived from Probiotic Lactobacillus rhamnosus (AgNPs-LR) Targeting Biofilm Formation and Quorum Sensing-Mediated Virulence Factors.

In recent years, bacterial pathogens have developed resistance to antimicrobial agents that have created a global threat to human health and environment. As a novel approach to combating antimicrobial resistance (AMR), targeting bacteria's virulent traits that can be explained by quorum sensing (QS) is considered to be one of the most promising approaches. In the present study, biologically synthesized silver nanoparticles derived from Lactobacillus rhamnosus (AgNPs-LR) were tested against three Gram-negative bacteria to determine whether they inhibited the formation of biofilms and triggered the virulence factors controlled by QS. In C. violaceum and S. marcescens, a remarkable inhibition (>70%) of QS-mediated violacein and prodigiosin production was recorded, respectively. A dose-dependent decrease in virulence factors of P. aeruginosa (pyocyanin, pyoverdine, LasA protease, LasB elastase and rhamnolipid production) was also observed with AgNPs-LR. The biofilm development was reduced by 72.56%, 61.70%, and 64.66% at highest sub-MIC for C. violaceum, S. marcescens and P. aeruginosa, respectively. Observations on glass surfaces have shown remarkable reductions in biofilm formation, with less aggregation of bacteria and a reduced amount of extra polymeric materials being formed from the bacteria. Moreover, swimming motility and exopolysaccharides (EPS) was also found to reduce in the presence of AgNPs-LR. Therefore, these results clearly demonstrate that AgNPs-LR is highly effective in inhibiting the development of biofilms and the QS-mediated virulent traits of Gram-negative bacteria. In the future, AgNPs-LR may be used as an alternative to conventional antibiotics for the treatment of bacterial infections after careful evaluation in animal models, especially for the development of topical antimicrobial agents.

Antiquorum and Antibiofilm Activities of Piper bogotense C. DC. against Pseudomonas aeruginosa and Identification of Bioactive Compounds.

The present study describes the anti-biofilm and quorum sensing (QS) inhibitory potential of extracts and chemical constituents from Piper bogotense. Antibiofilm potential was determined through crystal violet assay against Pseudomonas aeruginosa, while QS inhibition efficacy was determined through violacein inhibition assay using Chromobacterium violaceum as a bacterial model. Additionally, this study reports the effects of the chemical constituents isolated in P. bogotense against various virulent factors associated with QS, such as the percentage decrease in pyocyanin, elastase, and protease production. The chemical study led to the isolation and identification of two prenylated benzoic acids (1 and 2) and a prenylated hydroquinone 3, of which compounds 1 and 2 are reported for the first time for P. bogotense. The ethanolic extract and the DCM fraction from P. bogotense stand out for reducing violacein production in C. violaceum, as well as the biofilm formation in P. aeruginosa. Compounds 2 and 3 stand out for having the lowest violacein production (43.8% and 68.3%), as well as the lowest production of virulence factors such as elastase (60.2% and 51.4%) and pyocyanin (39.7% and 33.2%). These results demonstrate the potential of P. bogotense components to be used as an alternative control against multidrug-resistant P. aeruginosa.

Effects of Pendrin Protein in Nasal Epithelial Cells on Mucin Production in the Context of Type 2 Inflammation.

Chronic rhinosinusitis (CRS) is a heterogeneous disease. The pathogenesis of chronic sinusitis is still unclear; however, the nasal cavity and paranasal sinuses are commonly affected by type 2 inflammation, which is caused by Th2 cytokines such as interleukin (IL)-5, IL-4, and IL-13. Previous studies have shown that pendrin promotes local infiltration of neutrophils through the production of human neutrophil elastase (HNE), which is essential for the secretion of mucin 5AC (MUC5AC) in chronic inflammatory diseases of the lower respiratory tract. This study investigated pendrin expression and its relationship to mucin in type 2 inflammation. A total of 40 patients (10 CRS patients with nasal polyps,10 CRS patients without nasal polyps, and 20 nasal septum deviation patients) were included in this study and were divided into the CRS group and the NC group. A normal nasal mucosa tissue culture model was established in vitro. IL-13 was used to stimulate primary cultures of human nasal epithelial cells (HNECs). Western blot (WB), enzyme-linked immunosorbent assay (ELISA), and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of pendrin, MUC5AC, and MUC5B. After transfecting HNECs with siRNA pendrin or negative control (NC), EGF receptor (EGFR), HNE, MUC5AC, and MUC5B expression were analyzed using qRT-PCR, WB, or ELISA in terms of their relationships with pendrin. Pendrin expression in the tissue was also analyzed. After IL-13 stimulation, pendrin, MUC5AC, and MUC5B expression levels were upregulated; the optimal concentration of IL-13 was 50 ng/mL. The expression levels of HNE, EGFR, MUC5AC, and MUC5B were downregulated after transfection with siRNA pendrin-1650. Pendrin expression in the NC group was lower than in the CRS group. IL-13 is implicated in the inflammation of nasal mucosa, and pendrin is closely related to the excessive secretion of mucin. The expression of mucin is downregulated after transfection with siRNA pendrin. There is a positive relationship between pendrin and EFGR/HNE. Moreover, pendrin plays an important role in type 2 inflammation.