Refolding and purification of the human secreted group IID phospholipase A(2) expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A(2) (sPLA(2)s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A(2) (hsPLA(2)-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA(2)-IID in Escherichia coli, the DNA-coding sequence for hsPLA(2)-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA(2)-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A(2). The refolded recombinant hsPLA(2)-IID demonstrated Ca(2+)-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA(2)-IID which will advance our understanding of the structure-function relationship and biological effects of the protein. (C) 2009 Elsevier Inc. All rights reserved.

Acquisition of Serum Antibodies Reactive With Enterohemorrhagic Escherichia coli Virulence-Associated Factors by Healthy Brazilian Children and Adults

Background: Patients with hemorrhagic colitis or hemolytic uremic syndrome due to enterohemorrhagic Escherichia coli (EHEC) develop serum IgM and IgG response to lipopolysaccharide (LPS) and to virulence factors such as intimin. The small numbers of cases of diarrhea associated with EHEC strains in Brazil suggests a pre-existing immunity probably due to previous contact with diarrheagenic E. coli. Our aim was to evaluate the development of the serum antibody repertoire to EHEC virulence factors in Brazilian children and adults. Methods: Serum IgM and IgG antibodies were determined by enzyme-linked immunosorbent assay with LPS O111, LPS O26, and LPS O157 in 101 children between 2 months and 10 years of age and in 100 adult sera, by immunoblotting with protein membrane extracts and purified beta intimin; the ability of adult sera to neutralize Shiga toxin2 was also investigated. Results: Children older than 24 months had IgM concentrations reactive with the 3 LPS equivalent to those seen in the adult group, and significantly higher than the group of younger children (P < 0.05). Anti-O26 and anti-O157 LPS IgG concentrations were equivalent between the 2 groups of children and were significantly different from the adult group (P < 0.05). The anti-O111 LPS IgG levels in older children were intermediate between the younger group, and adults (P < 0.05). Immunoblotting revealed strong protein reactivity, including the conserved and variable regions of beta intimin and more than 50% of the adult samples neutralized Shiga toxin 2. Conclusions: Our results demonstrate an increasing anti-LPS and antiprotein antibody response with age, which could provide protection against EHEC infections.

Atypical enteropathogenic Escherichia coli genomic background allows the acquisition of non-EPEC virulence factors

Atypical enteropathogenic Escherichia coli (aEPEC) has been associated with infantile diarrhea in many countries. The clonal structure of aEPEC is the object of active investigation but few works have dealt with its genetic relationship with other diarrheagenic E. coli (DEC). This study aimed to evaluate the genetic relationship of aEPEC with other DEC pathotypes. The phylogenetic relationships of DEC strains were evaluated by multilocus sequence typing. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE). The phylogram showed that aEPEC strains were distributed in four major phylogenetic groups (A, B1, B2 and D). Cluster I ( group B1) contains the majority of the strains and other pathotypes [enteroaggregative, enterotoxigenic and enterohemorrhagic E. coli ( EHEC)]; cluster II ( group A) also contains enteroaggregative and diffusely adherent E. coli; cluster III ( group B2) has atypical and typical EPEC possessing H6 or H34 antigen; and cluster IV ( group D) contains aEPEC O55:H7 strains and EHEC O157:H7 strains. PFGE analysis confirmed that these strains encompass a great genetic diversity. These results indicate that aEPEC clonal groups have a particular genomic background - especially the strains of phylogenetic group B1 that probably made possible the acquisition and expression of virulence factors derived from non-EPEC pathotypes.

Preterm and term neonates transplacentally acquire IgG antibodies specific to LPS from Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa

High incidences of Gram-negative bacteria are found in neonatal nosocomial infections. Our aim was to investigate placental transmission of immunoglobulin G (IgG) reactive with lipopolysaccharide from Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia colt O111, O6 and O26. The total and lipopolysaccharide-specific IgM and IgG were determined in 11 maternal/umbilical-cord sera aged <= 33 weeks (GI); 21 aged > 33 and < 37 weeks (GII); and 32 term newborns (GIII). The total and lipopolysaccharide-specific IgM concentrations were equivalent in maternal sera. The total IgG concentrations were equivalent in maternal and newborn sera, with the exception of GIII newborns as compared with their mothers (P < 0.0001) and with neonates from GI and GII (P < 0.05). Lipopolysaccharide-specific IgG concentrations were lower in GI neonates than in their mothers (P < 0.01) and lower in GII (P < 0.05). Lower lipopolysaccharide-specific IgG levels were observed among neonates only for O111 in GI (P < 0.05) and for 026 and Pseudomonas in GII, both as compared with GIII (P < 0.05). The anti-lipopolysaccharide IgG transfer ratios were lower in GI (except for 026) and in GII (except for Klebsiella and O111) as compared with GIII (P < 0.05). Our results suggest that the greater susceptibility to infections in preterm infants is influenced (besides the humoral response) by factors intrinsic and extrinsic to the condition of prematurity.

Lipopolysaccharide from Escherichia coli prevents indomethacin-induced gastric damage in rats: role of non-protein sulfhydryl groups and leukocyte adherence

To investigate the role of non-protein sulfhydryl groups (NP-SH) and leukocyte adhesion in the protective effect of lipopolysaccharide (LPS) from Escherichia coli against indomethacin-induced gastropathy. Male Wistar rats were divided into four groups: saline, LPS, saline + indomethacin and LPS + indomethacin, with six rats in each group. Rats were pretreated with LPS (300 mu g/kg, by intravenous) or saline. After 6 h, indomethacin was administered (20 mg/kg, by gavage). Three hours after treatments, rats were killed. Macroscopic gastric damage, gastric NP-SH concentration, myeloperoxidase (MPO) activity and mesenteric leukocyte adhesion (intravital microscopy) were assessed. Statistical analysis was performed using one-way analysis of variance followed by the Newman-Keuls test. Statistical significance was set at P < 0.05. LPS reduced the gastric damage, gastric MPO activity and increased gastric NP-SH concentration in indomethacin-induced gastropathy. LPS alone increased gastric NP-SH when compared to saline. Indomethacin increased leukocyte adhesion when compared to the saline, and LPS reduced indomethacin-induced leukocyte adhesion. In addition, LPS alone did not change leukocyte adhesion, when compared to the saline. LPS protective effect against indomethacin-induced gastropathy is mediated by an increase in the NP-SH and a decrease in leukocyte-endothelial adhesion.

Molecular characteristics of extended-spectrum beta-lactamase-producing Escherichia coli from Rio de Janeiro, Brazil

Twenty-five extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates from Rio de Janeiro, Brazil were characterized by isoelectric focusing, PCR and sequencing of bla(ESBL) genes, plasmid-mediated quinolone resistance determinants, phylogenetic groups, replicon typing, pulsed-field electrophoresis, and multilocus sequencing typing. Twenty-three (92%) ESBL-producing E. coli isolates were positive for bla(CTX-M) genes, aac(6`)-lb-cr, and qnrB. Genetic relatedness of ESBL producers clustered seven (28%) CTX-M-15-producing isolates as sequence type (ST) 410, clonal complex (CC) 23, and two (8%) as clone O25-ST131. Our results illustrate the predominance of phylo-group A (52%), ST410 (CC 23) and CTX-M-15 among ESBL-producing E. coli isolates from hospitals in Rio de Janeiro.

Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher`s disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

Two Atypical Enteropathogenic Escherichia coli Strains Induce the Production of Secreted and Membrane-Bound Mucins To Benefit Their Own Growth at the Apical Surface of Human Mucin-Secreting Intestinal HT29-MTX Cells

In rabbit ligated ileal loops, two atypical enteropathogenic Escherichia coli (aEPEC) strains, 3991-1 and 0421-1, intimately associated with the cell membrane, forming the characteristic EPEC attachment and effacement lesion of the brush border, induced a mucous hypersecretion, whereas typical EPEC (tEPEC) strain E2348/69 did not. Using cultured human mucin-secreting intestinal HT29-MTX cells, we demonstrate that apically aEPEC infection is followed by increased production of secreted MUC2 and MUC5AC mucins and membrane-bound MUC3 and MUC4 mucins. The transcription of the MUC5AC and MUC4 genes was transiently upregulated after aEPEC infection. We provide evidence that the apically adhering aEPEC cells exploit the mucins` increased production since they grew in the presence of membrane-bound mucins, whereas tEPEC did not. The data described herein report a putative new virulence phenomenon in aEPEC.

Escherichia coli strains of serotype O51 : H40 comprise typical and atypical enteropathogenic E. coli strains and are potentially diarrheagenic

Escherichia coli strains of serotype O51:H40 were studied with regard to the presence of several virulence properties and their genetic diversity and enteropathogenicity in rabbit ileal loops. This serotype encompasses potential enteropathogenic strains mostly classified as being atypical enteropathogenic E. coli (EPEC) strains, which are genetically closer to enterohemorrhagic E. coli than to typical EPEC strains.

Beef Carcass Contamination by Shiga Toxin-Producing Escherichia coli Strains in an Abattoir in Brazil: Characterization and Resistance to Antimicrobial Drugs

A survey was performed to estimate the frequency of Escherichia coli and Shiga toxin-producing E. coli (STEC) in carcasses obtained from an abattoir in Brazil between February 2006 and June 2007. A total of 216 beef carcasses were sampled at three stages of the slaughter process-preevisceration, postevisceration, and postprocessing-during the rain and dry seasons, respectively. Of the carcasses sampled, 58%, were preevisceration E. coli positive, 38% were postevisceration positive, and 32% postprocessing positive. At the postprocessing stage, the isolation of E. coli was twice as high in the rain season. E. coli was isolated from 85 carcasses of which only 3 (1.4%) were positive for stx-encoding genes. No E. coli O157 serogroup isolates were detected. No antimicrobial resistance was found in nine of the isolates (10% of the total). The most frequent resistances were seen against cephalothin (78%), streptomycin (38%), nalidixic acid (36%), and tetracycline (30%). Multidrug resistance (MDR) to three or more antimicrobial agents was determined in 28 (33%) E. coli isolates. The presence of STEC and MDR strains among the isolates in the beef carcasses emphasizes the importance of proper handling to prevent carcass contamination.

Clonal Relationship among Atypical Enteropathogenic Escherichia coli Strains Isolated from Different Animal Species and Humans

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.

Phylogenetic group distribution among Escherichia coli isolated from rivers in Sao Paulo State, Brazil

The phylogenetic group distribution of Escherichia coli strains isolated from the Sorocaba and Jaguari Rivers located in the State of Sao Paulo, Brazil, is described. E. coli strains from group D were found in both rivers while one strain from group B2 was isolated from the Sorocaba river. These two groups often include strains that can cause extraintestinal diseases. Most of the strains analyzed were allocated into the phylogenetic groups A and B1, supporting the hypothesis that strains from these phylogenetic groups are more abundant in tropical areas. Though both rivers are located in urbanized and industrialized areas where the main source of water pollution is considered to derive from domestic sewage, our results suggest that the major sources of contamination in the sampling sites of both rivers might have originated from animals and not humans.

Strain variation in ppGpp concentration and RpoS levels in laboratory strains of Escherichia coli K-12

Laboratory strains and natural isolates of Escherichia coli differ in their level of stress resistance due to strain variation in the level of the sigma factor sigma(S) (or RpoS), the transcriptional master controller of the general stress response. We found that the high level of RpoS in one laboratory strain (MC4100) was partially dependent on an elevated basal level of ppGpp, an alarmone responding to stress and starvation. The elevated ppGpp was caused by two mutations in spoT, a gene associated with ppGpp synthesis and degradation. The nature of the spoT allele influenced the level of ppGpp in both MC4100 and another commonly used K-12 strain, MG1655. Introduction of the spoT mutation into MG1655 also resulted in an increased level of RpoS, but the amount of RpoS was lower in MG1655 than in MC4100 with either the wild-type or mutant spoT allele. In both MC4100 and MG1655, high ppGpp concentration increased RpoS levels, which in turn reduced growth with poor carbon sources like acetate. The growth inhibition resulting from elevated ppGpp was relieved by rpoS mutations. The extent of the growth inhibition by ppGpp, as well as the magnitude of the relief by rpoS mutations, differed between MG1655 and MC4100. These results together suggest that spoT mutations represent one of several polymorphisms influencing the strain variation of RpoS levels. Stress resistance was higher in strains with the spoT mutation, which is consistent with the conclusion that microevolution affecting either or both ppGpp and RpoS can reset the balance between self-protection and nutritional capability, the SPANC balance, in individual strains of E coli.

Boosting systemic and secreted antibody responses in mice orally immunized with recombinant Bacillus subtilis strains following parenteral priming with a DNA vaccine encoding the enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae B subunit

Recombinant Bacillus subtilis strains, either spores or vegetative cells, may be employed as safe and low cost orally delivered live vaccine vehicles. In this study, we report the use of an orally delivered B. subtilis vaccine strain to boost systemic and secreted antibody responses in mice i.m. primed with a DNA vaccine encoding the structural subunit (CfaB) of the CFA/I fimbriae encoded by enterotoxigenic Escherichia coli (ETEC), an important etiological agent of diarrhea among travelers and children living in endemic regions. DBA/2 female mice submitted to the prime-boost immunization regimen developed synergic serum (IgG) and mucosal (IgA) antibody responses to the target CfaB antigen. Moreover, in contrast to mice immunized only with one vaccine formulation, sera harvested from prime-boosted vaccinated individuals inhibited adhesion of ETEC cells to human red blood cells. Additionally, vaccinated dams conferred full passive protection to suckling newborn mice challenged with a virulent ETEC strain. Taken together the present results further demonstrate the potential use of recombinant B. subtilis strains as an alternative live vaccine vehicle. (C) 2008 Elsevier Ltd. All rights reserved.

The pst operon of enteropathogenic Escherichia coli enhances bacterial adherence to epithelial cells

Enteropathogenic Escherichia coli (EPEC) adheres in vivo and in vitro to epithelial cells. Two main adhesins, the bundle-forming pilus and intimin, encoded by the Up operon and eae, respectively, are responsible for the localized and the intimate adherence phenotypes. Deletion of the pst operon of EPEC abolishes the transport of inorganic phosphate through the phosphate-specific transport system and causes the constitutive expression of the PHO regulon genes. In the absence of pst there is a decrease in the expression of the main EPEC adhesins and a reduction in bacterial adherence to epithelial cells in vitro. This effect is not related to PHO constitutivity, because a Delta pst phoB double mutant that is defective in the transcription of the PHO genes also displayed low levels of adherence and expression of adhesins. Likewise, a PHO-constitutive phoR mutation did not affect bacterial adherence. The expression of the per operon, which encodes the Up and ler regulators PerA and PerC, is also negatively affected by the pst deletion. Overall, the data presented here demonstrate that the pst operon of EPEC plays a positive role in the bacterial adherence mechanism by increasing the expression of perA and perC and consequently the transcription of bfp and eae.