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

Pseudomonas aeruginosa quorum sensing inhibition by clinical isolate Delftia tsuruhatensis 11304: involvement of N-octadecanoylhomoserine lactones

Pseudomonas aeruginosa is one of the most common opportunistic pathogens that use quorum sensing (QS) system to regulate virulence factors expression and biofilm development. Delftia sp. 11304 was selected among 663 Gram-negative clinical isolates based on its QS inhibitory activity against P. aeruginosa MMA83 clinical isolate. Whole genome sequencing identified this isolate as D. tsuruhatensis and revealed genetic armamentarium of virulence factors and antibiotic resistance determinants. Ethyl acetate extract of D. tsuruhatensis 11304 culture supernatant (QSI extract) prevented biofilm formation of P. aeruginosa MMA83, but was unable to cause biofilm decomposition. QSI extract showed a synergistic effect in combination with meropenem and gentamycin, against P. aeruginosa MMA83. A dose-dependent reduction of the virulence factors: elastase, rhamnolipid and pyocyanin production by P. aeruginosa MMA83 and significant downregulation of lasI, lasR, rhlI, rhlR, pqs and mvfR expression were observed. Matrix-assisted Laser Desorption Ionization (MALDI) mass spectrometry of D. tsuruhatensis 11304 QSI extract revealed the presence of N-acyl homoserine lactones (AHL) with chain lengths of C12 to C18. The main ion peak was identified as N-octadecanoylhomoserine lactone (C18-HSL). Commercial C18-HSL (20 µM) reduced pyocyanin production as well as mRNA level of the lasI gene. A novel AHL species, dihydroxy-N-octadecanoylhomoserine lactone, was also described

Amide containing NBTI antibacterials with reduced hERG inhibition, retained antimicrobial activity against gram-positive bacteria and in vivo efficacy

Novel bacterial topoisomerase inhibitors (NBTIs) are new promising antimicrobials for the treatment of multidrug-resistant bacterial infections. In recent years, many new NBTIs have been discovered, however most of them struggle with the same issue - the balance between antibacterial activity and hERG-related toxicity. We started a new campaign by optimizing the previous series of NBTIs, followed by the design and synthesis of a new, amide-containing focused NBTI library to reduce hERG inhibition and maintain antibacterial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). This optimization strategy yielded the lead compound 12 that exhibits potent antibacterial activity against Gram-positive bacteria, reduced hERG inhibition, no cardiotoxicity in zebrafish model, and a favorable in vivo efficacy in a neutropenic murine thigh infection model of MRSA infection

Glycosylation and pH stability of penicillin G acylase from providencia rettgeri produced in Pichia pastoris

Penicilin G acilaza (PAC) je jedan od najšire korišćenih enzima u industrijskoj sintezi polusintetskih antibiotika. U ovom radu dobijeni nivo ekspresije PAC gena iz Providencia rettgeri u ekspresionom sistemu Pichia pastoris iznosio je 2.7 U/ml. Rekombinantni enzim je prečišćen i određen je njegov glikozilacioni status. Nađeno je da osim što su obe subjedinice enzima (α i β) N-glikozilovane, β subjedinica sadrži još i O-glikane. Takođe je ustanovljeno da je rekombinantna PACP. rett. stabilna u širokom pH opsegu što ju je, zajedno sa predhodno ustanovljenom visokom termostabilnošću, učinilo izuzetno privlačnim biokatalizatorom sa industrijske tačke gledišta.Penicillin G acylase (PAC) is one of the most widely used enzymes in industrial synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (α and β) of the enzyme were N-glycosylated, while the β-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view

The critical role of macrophage migration inhibitory factor in insulin activity

Macrophage migration inhibitory factor (MIF) is a molecule with plethora of functions such as regulation of immune response, hormone-like, enzymatic and chaperone-like activity. Further, MIF is a major participant in glucose homeostasis since it is an autocrine stimulator of insulin secretion. MIF absence in male knockout mice (MW-MO) results in development of glucose intolerance, while sensitivity to insulin is fully preserved. Since our results confirm that beta cells from MIF-KO mice express, produce and secrete insulin similarly to beta cells of their wild type (WT) counterparts C57BL/6 mice, we hypothesize that MIF-KO-derived insulin is less active. Indeed, insulin from MIF-KO islets is unable to significantly induce glucose uptake into hepatocytes and to efficiently promote insulin-triggered Akt phosphorylation determined by immunoblot. However, MIF's tautomerase function is not crucial for insulin biosynthesis since MIF inhibitors had no impact on WT insulin activity. Importantly, MIF recognition by anti-MIF anti-body (ELISA) after in vitro co-incubation with purified insulin was significantly lower suggesting that insulin covers MIF immunodominant epitope. In addition, MIF binds insulin within beta cell as confirmed by co-immunoprecipitation. WT and MIF-KO-derived insulin exhibited different cleavage patterns suggesting different protein conformations. Finally, pre-incubation of recombinant MIF with insulin promotes formation of insulin hexamers. These results imply that MIF probably enables proper insulin folding what results in insulin full activity. This newly discovered feature of the cytokine MIF could be potentially important for commercially produced insulin, for increasing its stability and/or bioavailability. (C) 2014 Elsevier Ltd. All rights reserved.Ministry of Education, Science and Technological Development, Republic of Serbia {[}173013, 173048

N-benzyl derivatives of long-chained 4-Amino-7-chloro-quionolines as inhibitors of pyocyanin production in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a leading cause of nosocomial infections that are becoming increasingly difficult to treat due to the occurrence of antibiotic resistant strains. Since P. aeruginosa virulence is controlled through quorum sensing, small molecule treatments inhibiting quorum sensing signaling pathways provide a promising therapeutic option. Consequently, we synthesized a series of N-octaneamino-4-aminoquinoline derivatives to optimize this chemotype’s antivirulence activity against P. aeruginosa via inhibition of pyocyanin production. The most potent derivative, which possesses a benzofuran substituent, provided effective inhibition of pyocyanin production (IC50 = 12 μM), biofilm formation (BFIC50 = 50 μM), and motility. Experimentally, the compound’s activity is achieved through competitive inhibition of PqsR, and structure–activity data were rationalized using molecular docking studies.The peer-reviewed version: [http://cer.ihtm.bg.ac.rs/handle/123456789/3341

Anti-quorum sensing activity, toxicity in zebrafish (Danio rerio) embryos and phytochemical characterization of Trapa natans leaf extracts

© 2018 Ethnopharmacological relevance: Trapa natans L. (water chestnut or water caltrop) is a widespread aquatic plant, which has been cultivated for food and traditional medicine since ancient times. Pharmacological studies showed that water chestnut exhibits the wide range of biological activities, such as antimicrobial, antioxidative, analgesic, anti-inflammatory, as well as antiulcer. Aim of the study: Evaluation of anti-virulence potential and toxicity of T. natans methanol (TnM), acetone (TnA) and ethyl acetate (TnEA) leaf extracts. Materials and methods: The anti-quorum sensing activity of Tn extracts was addressed by measuring their effects on biofilm formation, swarming motility and pyocyanin and elastase production in Pseudomonas aeruginosa. Specific P. aeruginosa biosensors were used to identify which of the signaling pathways were affected. The lethal and developmental toxicity of extracts were addressed in vivo using the zebrafish (Danio rerio) model system. The phenolic composition of T. natans leafs extracts was analyzed by a linear ion trap-OrbiTrap hybrid mass spectrometer (LTQ OrbiTrapMS) and UHPLC system configured with a diode array detector (DAD) hyphenated with the triple quadrupole mass spectrometer. Results: Subinhibitory concentrations of Tn leaf extracts (0.2 MIC) inhibited pyocyanin and elastase production up to 50% and 60%, respectively, and reduced swarming zones, comparing to non-treated P. aeruginosa. TnA inhibited biofilm formation by 15%, TnM showed a stimulatory effect on biofilm formation up to 20%, while TnEA showed no effect. The bioactive concentrations of TnM and TnA were not toxic in the zebrafish model system. Twenty-two phenolic compounds were tentatively identified in TnM, where thirteen of them were identified in T. natans for the first time. Tn extracts, as well as their major components, ellagic and ferulic acids, demonstrated the ability to interfere with P. aeruginosa Las and PQS signaling pathways. Conclusions: This study demonstrates anti-virulence potential of Tn leaf extracts against medically important pathogen P. aeruginosa and confirms the ethnopharmacological application of this plant against microbial infections

Undecylprodigiosin conjugated monodisperse gold nanoparticles efficiently cause apoptosis in colon cancer cells in vitro

Bacterial pigment undecylprodigiosin (UP) was produced using Streptomyces sp. JS520 and conjugated to monodisperse gold nanoparticles (UP-Au). Both UP and UP-Au showed cytocidal activity towards melanoma (A375), lung carcinoma (A549), breast cancer (MCF-7) and colon cancer (HCT-116) cells, inducing apoptosis with IC50 values ranging from 0.4 to 4 mu g ml(-1). Unconjugated UP had a tendency to lose its activity over time and to change biophysical characteristics over pH. The loss of the pigment potency was overcome by conjugation with gold nanoparticles. UP-Au exhibited high stability over pH 3.8 to 7.4 and its activity remained unaffected in time. Nano-packing changed the mechanism of UP toxicity by converting the intracellular signals from a mitochondrial dependent to a mitochondrial independent apoptotic process. The availability of nonpyrogenic UP in high amounts, together with specific anticancer activity and improved stability in the complex with gold nanoparticles, presents a novel platform for further development of UP-Au complexes as an anticancer drug suitable for clinical applications.Ministry of Education, Science and Technological Development of Serbia {[}173048, 173013]; U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences {[}DE-AC02-06CH11357