The Rhl Quorum-Sensing System Is at the Top of the Regulatory Hierarchy under Phosphate-Limiting Conditions in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is a major nosocomial pathogen that presents high-level resistance to antibiotics. Its ability to cause infections relies on the production of multiple virulence factors. Quorum sensing (QS) regulates the expression of many of these virulence factors through three QS systems: Las, Rhl, and PQS. The Las system positively regulates the other two systems, so it is at the top of a hierarchized regulation. Nevertheless, clinical and environmental strains that lack a functional Las system have been isolated, and, surprisingly, some of them still have the ability to produce virulence factors and infect animal models, so it has been suggested that the hierarchy is flexible under some conditions or with atypical strains. Here, we analyze the PAO1 type strain and its ΔlasR-derived mutant and report, for the first time, a growth condition (phosphate limitation) where LasR absence has no effect either on virulence factor production or on the gene expression profile, in contrast to a condition of phosphate repletion where the LasR hierarchy is maintained. This work provides evidence on how the QS hierarchy can change from being a strictly LasR-dependent to a LasR-independent RhlR-based hierarchy under phosphate limitation even in the PAO1 type strain.IMPORTANCEPseudomonas aeruginosa is an important pathogen, considered a priority for the development of new therapeutic strategies. An important approach to fight its infections relies on blocking quorum sensing. The Las system is the main regulator of the quorum-sensing response, so many research efforts aim to block this system to suppress the entire response. In this work, we show that LasR is dispensable in a phosphate-limited environment in the PAO1 type strain, which has been used to define the quorum-sensing response hierarchy, and that under this condition RhlR is at the top of the regulation hierarchy. These results are highly significant, since phosphate limitation represents a similar environment to the one that P. aeruginosa faces when establishing infections.

Overexpression of the celA1 gene in BCG modifies surface pellicle, glucosamine content in biofilms, and affects in vivo replication.

Biofilm formed in vitro by mycobacteria has been associated with increased antibiotic tolerance as compared with planktonic cells. Cellulose has been identified as a component of DTT-exposed biofilms formed by M. tuberculosis. The celA1 gene of M. tuberculosis encodes a cellulase, which could affect the formation of biofilm by slow-growing mycobacteria. In this work, the celA1 gene of M. tuberculosis was cloned into the integrative pMV361 plasmid and then transformed into M. bovis BCG Pasteur to produce BCG:celA1, to have celA1 expressed from the strong promoter hsp60. We compared planktonic and biofilm growth, possible presence of CelA1 in whole protein extracts, quantitated biofilm, presence of monosaccharides, and bacillary burden in lungs after aerosol infection in BALB/c mice. Differences in the appearance of the surface pellicle and of the biofilm attached to the substrate were observed. In biofilms, we observed a significant decrease of glucosamine in BCG:celA1 compared with BCG:pMV361. Finally, BCG:celA1 had lower viable bacteria than the BCG:pMV361 strain after 24 h and 3 weeks post-infection, but no difference was found at 9 weeks post-infection.

A new serine protease family with elastase activity is produced by Streptomyces bacteria.

We found an elastolytic activity in the culture supernatant of Streptomyces sp. P-3, and the corresponding enzyme (streptomycetes elastase, SEL) was purified to apparent homogeneity from the culture supernatant. The molecular mass of purified SEL was approximately 18 kDa as judged by SDS-PAGE analysis and gel-filtration chromatography. Utilizing information from N-terminal amino acid sequencing of SEL and mass spectrometry of SEL tryptic fragments, we succeeded in cloning the gene-encoding SEL. The cloned SEL gene contains a 726 bp ORF, which encodes a 241 amino acid polypeptide containing a putative signal peptide for secretion (28 amino acid) and pro-sequence (14 amino acid). Although the deduced primary structure of SEL has sequence similarity to proteins in the S1 protease family, the amino acid sequence shares low identity (< 31.5 %) with any known elastase. SEL efficiently hydrolyses synthetic peptides having Ala or Val in the P1 position such as N-succinyl-Ala-Ala-(Pro or Val)-Ala-p-nitroanilide (pNA), whereas reported proteases by streptomycetes having elastolytic activity prefer large residues, such as Phe and Leu. Compared of kcat/Km ratios for Suc-Ala-Ala-Val-Ala-pNA and Suc-Ala-Ala-Pro-Ala-pNA with subtilisin YaB, which has high elastolytic activity, Streptomyces sp. P-3 SEL exhibits 12- and 121-fold higher, respectively. Phylogenetic analyses indicate that the predicted SEL protein, together with predicted proteins in streptomycetes, constitutes a novel group within the S1 serine protease family. These characteristics suggest that SEL-like proteins are new members of the S1 serine protease family, which display elastolytic activity.

Elastase 3B mutation links to familial pancreatitis with diabetes and pancreatic adenocarcinoma.

While improvements in genetic analysis have greatly enhanced our understanding of the mechanisms behind pancreatitis, it continues to afflict many families for whom the hereditary factors remain unknown. Recent evaluation of a patient with a strong family history of pancreatitis sparked us to reexamine a large kindred originally reported over 50 years ago with an autosomal dominant inheritance pattern of chronic pancreatitis, diabetes and pancreatic adenocarcinoma. Whole exome sequencing analysis identified a rare missense mutation in the gene encoding pancreas-specific protease Elastase 3B (CELA3B) that cosegregates with disease. Studies of the mutant protein in vitro, in cell lines and in CRISPR-Cas9 engineered mice indicate that this mutation causes translational upregulation of CELA3B, which upon secretion and activation by trypsin leads to uncontrolled proteolysis and recurrent pancreatitis. Although lesions in several other pancreatitic proteases have been previously linked to hereditary pancreatitis, this is the first known instance of a mutation in CELA3B and a defect in translational control contributing to this disease.

Identification of the physiological substrates of PDIp, a pancreas-specific protein-disulfide isomerase family member.

About 20 members of the protein-disulfide isomerase (PDI) family are present in the endoplasmic reticulum of mammalian cells. They are thought to catalyze thiol-disulfide exchange reactions within secretory or membrane proteins to assist in their folding or to regulate their functions. PDIp is a PDI family member highly expressed in the pancreas and known to bind estrogen in vivo and in vitro However, the physiological functions of PDIp remained unclear. In this study, we set out to identify its physiological substrates. By combining acid quenching and thiol alkylation, we stabilized and purified the complexes formed between endogenous PDIp and its target proteins from the mouse pancreas. MS analysis of these complexes helped identify the disulfide-linked PDIp targets in vivo, revealing that PDIp interacts directly with a number of pancreatic digestive enzymes. Interestingly, when pancreatic elastase, one of the identified proteins, was expressed alone in cultured cells, its proenzyme formed disulfide-linked aggregates within cells. However, when pancreatic elastase was co-expressed with PDIp, the latter prevented the formation of these aggregates and enhanced the production and secretion of proelastase in a form that could be converted to an active enzyme upon trypsin treatment. These findings indicate that the main targets of PDIp are digestive enzymes and that PDIp plays an important role in the biosynthesis of a digestive enzyme by assisting with the proper folding of the proenzyme within cells.

Chemical Constituents from the Stems of Tinospora sinensis and Their Bioactivity.

Fifty-seven compounds were purified from the stems of Tinospora sinensis, including three new compounds characterized as a lignan (1), a pyrrole alkaloid (11), and a benzenoid (17), respectively. Their structures were elucidated and established by various spectroscopic and spectrometric analytical methods. Among the isolates, fifteen compounds were examined for their anti-inflammatory potential in vitro. The results showed that several compounds displayed moderate inhibition of N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release.

Quorum sensing inhibitory activity of sub-inhibitory concentrations of ß-lactams.

INTRODUCTION: The virulence factors of Pseudomonas aeruginosa are under the control of quorum sensing (QS) signals. Hence, interference with QS prevents its pathogenesis. OBJECTIVE: The aim of the present research is to assess the influence of some ß-lactam antibiotics on cell communication and the release of different virulence factors. METHODS: The minimal inhibitory concentrations of ceftazidime, cefepime and imipenem were evaluated by microbroth dilution method. The effect of sub-inhibitory concentration of the tested antibiotics on QS signals was investigated using reporter strain assay. In addition, different virulence factors (elastase, protease, pyocyanin and hemolysin) were estimated in the presence of their sub-inhibitory concentrations. RESULTS: Low concentrations of ceftazidime, cefepime and imipenem caused significant elimination of the QS signals 3OH-C12-HSL and C4-HSL up to 1/20 MIC. Furthermore, low concentrations of the tested antimicrobials suppressed virulence factors elastase and hemolysin. Moreover, 1/20 of their MICs reduced elastase, protease, pyocyanin and hemolysin. CONCLUSION: Utilization of ß-lactam antibiotics at low concentrations could be an effective approach for prevention and treatment of P. aeruginosa infection.

The Influences of Bacillus subtilis on the Virulence of Aeromonas hydrophila and Expression of luxS Gene of Both Bacteria Under Co-cultivation.

The aim of this study was to explore the influence of Bacillus subtilis CH9 on Aeromonas hydrophila SC2005. The transcription level of virulence genes of A. hydrophila SC2005 and its hemolysin activity as well as its cytotoxicity were analyzed when B. subtilis CH9 and A. hydrophila SC2005 were co-cultured. The results indicated that the transcription levels of four virulence genes of A. hydrophila, including aer, ahyB, hcp, and emp, decreased when A. hydrophila was cultured with B. subtilis CH9. Furthermore, the extracellular products of A. hydrophila showed attenuated hemolysin activity as well as cytotoxicity when A. hydrophila was cultured with B. subtilis CH9. Finally, the transcriptional levels of luxS genes of B. subtilis CH9 and A. hydrophila SC2005 were determined when these two species were co-cultured. RT-qPCR results suggested that the transcription level of A. hydrophila was down-regulated significantly. On the contrary, the transcription level of B. subtilis CH9 was up-regulated significantly. These results suggested that the probiotic role of B. subtilis CH9 is related to the inhibition of growth and virulence of A. hydrophila SC2005, and quorum sensing may be involved.

Penicillin V acylases from gram-negative bacteria degrade N-acylhomoserine lactones and attenuate virulence in Pseudomonas aeruginosa.

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation of AHLs leading to attenuation of virulence (quorum quenching) could pave the way for the development of new antibacterials. Penicillin V acylases (PVAs) belong to the Ntn hydrolase superfamily, together with AHL acylases. PVAs are exploited widely in the pharmaceutical industry, but their role in the natural physiology of their native microbes is not clearly understood. This report details the characterization of AHL degradation activity by homotetrameric PVAs from two gram-negative plant pathogenic bacteria, Pectobacterium atrosepticum (PaPVA) and Agrobacterium tumefaciens (AtPVA). Both the PVAs exhibited substrate specificity for degrading long-chain AHLs. Exogenous addition of these enzymes into Pseudomonas aeruginosa greatly diminished the production of elastase and pyocyanin and biofilm formation and increased the survival rate in an insect model of acute infection. Subtle structural differences in the PVA active site that regulate specificity for acyl chain length have been characterized, which could reflect the evolution of AHL-degrading acylases in relation to the environment of the bacteria that produce them and also provide strategies for enzyme engineering. The potential for using these enzymes as therapeutic agents in clinical applications and a few ideas about their possible significance in microbial physiology have also been discussed.

The ferrichrome receptor A as a new target for Pseudomonas aeruginosa virulence attenuation.

Pseudomonas aeruginosa is an opportunistic pathogen, known to develop robust biofilms. Its biofilm development increases when antibiotics are presented at subminimal inhibitory concentrations (MICs) for reasons that remain unclear. In order to identify genes that affect biofilm development under such a sublethal antibiotic stress condition, we screened a transposon (Tn) mutant library of PAO1, a prototype P. aeruginosa strain. Among ∼5000 mutants, a fiuA gene mutant was verified to form very defective biofilms in the presence of sub-MIC carbenicillin. The fiuA gene encodes ferrichrome receptor A, involved in the iron acquisition process. Of note, biofilm formation was not decreased in the ΔpchΔpvd mutant defective in the production of pyochelin and pyoverdine, two well-characterized P. aeruginosa siderophore molecules. Moreover, ΔfiuA, a non-polar fiuA deletion mutant, produced a significantly decreased level of elastase, a major virulence determinant. Mouse airway infection experiments revealed that the mutant expressed significantly less pathogenicity. Our results suggest that the fiuA gene has pleiotropic functions that affect P. aeruginosa biofilm development and virulence. The targeting of FiuA could enable the attenuation of P. aeruginosa virulence and may be suitable for the development of a drug that specifically controls the virulence of this important pathogen.

High variability in quorum quenching and growth inhibition by furanone C-30 in Pseudomonas aeruginosa clinical isolates from cystic fibrosis patients.

Pseudomonas aeruginosa colonizes the lungs of cystic fibrosis patients causing severe damage. This bacterium is intrinsically resistant to antibiotics and shows resistance against new antimicrobials and its virulence is controlled by the quorum-sensing response. Thus, attenuating its virulence by quorum quenching instead of inhibiting its growth has been proposed to minimize resistance; however, resistance against the canonical quorum quencher furanone C-30 can be achieved by mutations leading to increased efflux. In the present work, the effect of C-30 in the attenuation of the QS-controlled virulence factors elastase and pyocyanin was investigated in 50 isolates from cystic fibrosis patients. The results demonstrate that there is a high variability in the expression of both elastase and pyocyanin and that there are many naturally resistant C-30 strains. We report that the main mechanism of C-30 resistance in these strains was not due to enhanced efflux but a lack of permeability. Moreover, C-30 strongly inhibited the growth of several of the isolates studied, thus imposing high selective pressure for the generation of resistance.

A novel regulator PA5022 (aefA) is involved in swimming motility, biofilm formation and elastase activity of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen contributing to a range of nosocomial infections. To identify new genes involved in P. aeruginosa swimming motility, an important mechanism of pathogenesis, mutants with altered swimming motility patterns from Mu transposon mutagenesis library of the P. aeruginosa clinical strain PA68 were isolated and characterized. We identified a mutant with transposon inactivation of PA5022 has completely abolished its swimming motility while still possesses a normal terminal flagellum according to electronic microscopy analysis. Microscopic examination revealed that the PA5022 mutant forms thicker biofilms compared to the PA68 wild-type strain and is impaired in its elastase activity. To exclude the possibility of genetic diversity in affecting gene functions among different strains, we constructed a PA5022 knock out mutant based on the PAK lab strain. The PAKΔPA5022 has similar phenotypes to the PA5022 (PA5022::Mu) mutant of PA68 strain. Furthermore, transcriptional fusion assays were carried out to investigate the regulatory mechanism of PA5022 by using the PlasI-lacZ, PrhlI-lacZ, PrpoN-lacZ, PrpoS-lacZ, PqscR-lacZ, PvqsR-lacZ fusions. ß-Galactosidase activity assays indicated that the expression of the vqsR, lasI and rhlI promotors was reduced in the PA5022 mutant compared to the PA68 wild-type. Our study showed that PA5022 links swimming motility and quorum sensing, which might be an important regulator for the pathogenesis of P. aeruginosa.

Production of IL-8, VEGF and Elastase by Circulating and Intraplaque Neutrophils in Patients with Carotid Atherosclerosis.

OBJECTIVES: Polymorphonuclear neutrophils (PMN) in atherosclerotic plaques have been identified only recently, and their contribution to plaque development is not yet fully understood. In this study, production of elastase, interleukin (IL)-8 and vascular endothelial growth factor (VEGF) by PMN was investigated in subjects with carotid stenosis undergoing carotid endarterectomy (CEA). METHODS: The study enrolled 50 patients (Pts) and 10 healthy subjects (HS). Circulating PMN (cPMN) isolated from venous blood (in both Pts and HS) and from plaques (pPMN, in Pts) were cultured, alone or with 0.1 µM fMLP. Elastase, IL-8 and VEGF mRNA were analyzed by real-time PCR. In CEA specimens, PMN were localized by immunohistochemistry. RESULTS: In both Pts cPMN and pPMN, IL-8 mRNA was higher at rest but lower after fMLP (P<0.01 vs HS), and VEGF mRNA was higher both at rest and after fMLP (P<0.01 vs HS), while elastase mRNA was not significantly different. On the contrary, protein production was always higher in cPMN of HS with respect to values measured in cells of Pts. In CEA specimens, CD66b+ cells localized to areas with massive plaque formation close to neovessels. Pts with soft and mix plaques, as defined by computed tomography, did not differ in cPMN or pPMN IL-8, VEGF or elastase mRNA, or in intraplaque CD66b+ cell density. However, Pts with soft plaques had higher white blood cell count due to increased PMN. CONCLUSIONS: In Pts with carotid plaques, both circulating and intraplaque PMN produce IL-8, VEGF and elastase, which are crucial for plaque development and progression. These findings suggest mechanistic explanations to the reported correlation between PMN count and cardiovascular mortality in carotid ATH.

Enhanced expression of recombinant elastase in Pichia pastoris through the substitution of Thr for Ser in Asn-Xaa-Ser sequons.

N-glycosylation usually occurs at the Asn-Xaa-Ser/Thr sequon of glycoproteins in Pichia pastoris, exerting great effects on expression efficiency; however, Asn-Xaa-Thr is more efficiently glycosylated than Asn-Xaa-Ser. In this study, the role of the two sequons in the expression of recombinant elastase (rPAE) was investigated. At N43, N212, and N280 of rPAE, Asn-Xaa-Thr was substituted for the native Asn-Xaa-Ser sequon through site-directed mutagenesis, and the two sequon forms were introduced into rPAE at N36 and N264. As expected, substitution at N36, N43, N212, and N280 enhanced the degree of N-glycosylation. At N212 or N280, substitution increased rPAE production effectively by 43 and 25 %, respectively. In comparison, at N36, N43, and N264, the change inhibited rPAE expression to varying extents; specifically, substitution at N36 resulted in a 31 % decrease, while substitution at N43 or N264 resulted in a decrease of less than 9 %. It is suggested that the effect of the substitution of Asn-Xaa-Thr for Asn-Xaa-Ser on rPAE expression is roughly related to the role of the original Asn-Xaa-Ser sequon. As the conversion of Ser to Thr at N-glycosylation sites through site-directed mutagenesis is easily achieved, it is a feasible means of improving the expression of recombinant proteins in P. pastoris.

Seventy years of pancreatic physiology: take a look back.

This review article has 4 major objectives to follow pancreatic physiology development more than close to 70 years of intensive and productive basic research. At first, the review will focus on secretion of the pancreatic enzymes with (1) the controls involved, (2) the interrelations existing between secretion and synthesis of these enzymes, (3) the enzymes' adaptation to the constituents of the diet, and (4) whether secretion of the different enzymes is parallel or nonparallel. Second, growth and regeneration of the pancreatic gland will be looked at in relation to the factors involved and the target cells implicated.

Sodium ascorbate as a quorum sensing inhibitor of Pseudomonas aeruginosa.

AIMS: Quorum sensing circuits regulate virulence factors in Pseudomonas aeruginosa and coordinate bacterial pathogenicity. We are interested in exploring available medications for their antiquorum sensing activity. METHODS AND RESULTS: First, we determined the MIC of ascorbate against Ps. aeruginosa strain PAO1, and all further experiments used concentrations below the MIC so that results could not be caused by reduced viability. Tests of subinhibitory concentrations of sodium ascorbate on cell signals were performed using a reporter strain assay. Sub-MICs of sodium ascorbate resulted in significant reduction of the signalling molecules C4-HSL and 3-oxo-C12-HSL (P < 0·01). The influence of sub-MIC of sodium ascorbate on virulence factors was also determined and ascorbate treatment led to significant depression of elastase, protease and haemolysin activities. In addition, inhibition of pyocyanin production, attenuation of biofilm formation and alteration of Pseudomonas motility was observed. Analysis by RT-PCR tested the effect of ascorbate on the expression of QS regulatory genes. Expression of QS regulatory genes, lasI, lasR, rhlI, rhlR, pqsR and pqsA, was repressed compared to untreated Ps. aeruginosa PAO1, confirming that ascorbate QS inhibition works on gene expression at the molecular level. CONCLUSION: Sodium ascorbate, even at low concentrations, inhibited QS and related virulence factors of Ps. aeruginosa PAO1. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that sodium ascorbate could function as signal modulator and virulence inhibitor in Ps. aeruginosa.

Pancreatic exocrine enzyme-producing cell differentiation via embryoid bodies from human embryonic stem cells.

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all-trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.

The flavanone naringenin reduces the production of quorum sensing-controlled virulence factors in Pseudomonas aeruginosa PAO1.

Preliminary screening of the Malagasy plant Combretum albiflorum for compounds attenuating the production of quorum sensing (QS)-controlled virulence factors in bacteria led to the identification of active fractions containing flavonoids. In the present study, several flavonoids belonging to the flavone, flavanone, flavonol and chalcone structural groups were screened for their capacity to reduce the production of QS-controlled factors in the opportunistic pathogen Pseudomonas aeruginosa (strain PAO1). Flavanones (i.e. naringenin, eriodictyol and taxifolin) significantly reduced the production of pyocyanin and elastase in P. aeruginosa without affecting bacterial growth. Consistently, naringenin and taxifolin reduced the expression of several QS-controlled genes (i.e. lasI, lasR, rhlI, rhlR, lasA, lasB, phzA1 and rhlA) in P. aeruginosa PAO1. Naringenin also dramatically reduced the production of the acylhomoserine lactones N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), which is driven by the lasI and rhlI gene products, respectively. In addition, using mutant strains deficient for autoinduction (ΔlasI and ΔrhlI) and LasR- and RhlR-based biosensors, it was shown that QS inhibition by naringenin not only is the consequence of a reduced production of autoinduction compounds but also results from a defect in the proper functioning of the RlhR-C4-HSL complex. Widely distributed in the plant kingdom, flavonoids are known for their numerous and determinant roles in plant physiology, plant development and in the success of plant-rhizobia interactions, but, as shown here, some of them also have a role as inhibitors of the virulence of pathogenic bacteria by interfering with QS mechanisms.

Myeloperoxidase and elastase are only expressed by neutrophils in normal and in inflamed liver.

Myeloperoxidase (MPO) is involved in acute and chronic inflammatory diseases. The source of MPO in acute liver diseases is still a matter of debate. Therefore, we analysed MPO-gene expression on sections from normal and acutely damaged [carbon tetrachloride-(CCl(4)) or whole liver γ-Irradiation] rat liver by immunohistochemistry, real time PCR and Western blot analysis of total RNA and protein. Also total RNA and protein from isolated Kupffer cells, hepatic stellate cells, Hepatocytes, endothelial cells and neutrophil granulocytes (NG) was analysed by real time PCR and Western blot, respectively. Sections of acutely injured human liver were prepared for MPO and CD68 immunofluorescence double staining. In normal rat liver MPO was detected immunohistochemically and by immunofluorescence double staining only in single NG. No MPO was detected in isolated parenchymal and non-parenchymal cell populations of the normal rat liver. In acutely damaged rat liver mRNA of MPO increased 2.8-fold at 24 h after administration of CCl(4) and 3.3-fold at 3 h after γ-Irradiation and MPO was detected by immunofluorescence double staining only in elastase (NE) positive NGs but not in macrophages (ED1 or CD68 positive cells). Our results demonstrate that, increased expression of MPO in damaged rat and human liver is due to recruited elastase positive NGs.

Characterization of a phosphotriesterase-like lactonase from Sulfolobus solfataricus and its immobilization for disruption of quorum sensing.

SsoPox, a bifunctional enzyme with organophosphate hydrolase and N-acyl homoserine lactonase activities from the hyperthermophilic archaeon Sulfolobus solfataricus, was overexpressed and purified from recombinant Pseudomonas putida KT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference for N-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lower K(m) values for these substrates. The highest specificity constant obtained was for N-3-oxo-decanoyl homoserine lactone (k(cat)/K(m) = 5.5 × 10(3) M(-1)·s(-1)), but SsoPox can also degrade N-butyryl homoserine lactone (C(4)-HSL) and N-oxo-dodecanoyl homoserine lactone (oxo-C(12)-HSL), which are important for quorum sensing in our Pseudomonas aeruginosa model system. When P. aeruginosa PAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C(4)-HSL- and oxo-C(12)-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.