Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

Placental and colostral transfer of antibodies reactive with enteropathogenic Escherichia coli intimins α, ß, or γ / Transferência placentária e colostral de anticorpos reativos a Escherichia coli enteropatogênica com expressão das intiminas α, ß, or γ

Abstract Objective: Intimins are protein adhesins of enteropathogenic Escherichia coli and enterohemorrhagic E. coli capable of inducing attachment and effacement lesions in enterocytes. Anti-intimin antibodies are important for the protection from enteropathogenic E. coli and enterohemorrhagic E. coli infections because these antibodies inhibit bacterial adhesion and impair the initial step of the pathogenesis. We studied the transfer of maternal anti-intimin antibodies from healthy Brazilian mothers to their newborns through the placenta and colostrum. Methods: Serum immunoglobulin G and secretory immunoglobulin A antibodies against conserved and variable regions of intimins α, β, and γ were analyzed using an enzyme linked-immunosorbent assay in the blood and colostrum from 45 healthy women as well as cord blood serum samples from their newborns. Results: The concentrations of antibodies reactive with α intimin were significantly lower than those of anti-γ and anti-conserved intimin antibodies in the colostrum samples. IgG serum antibodies reactive with all the subtypes of intimins were transferred to the newborns, but the concentrations of anti-conserved intimin serum antibodies were significantly higher in mothers and newborns than concentrations of antibodies against variable regions. The patterns of IgG transfer from mothers to newborns were similar for all anti-intimin antibodies. These values are similar to the percentage transference of total IgG. Conclusions: Anti-intimin antibodies are transferred from mothers to newborns through the placenta, and reinforce the protection provided by breastfeeding against diarrheagenic E. coli infections.

Resumo Objetivo: As intiminas são adesinas proteicas de Escherichia coli enteropatogênicas (EPEC) e enterro-hemorrágicas (EHEC) capazes de induzir as lesões attaching and effacing nos enterócitos. Anticorpos anti-intiminas são importantes para a proteção contra infecções por EPEC e EHEC porque esses anticorpos inibem a adesão bacteriana e impedem o passo inicial do mecanismo patogênico dessas bactérias. Nós estudamos a transferência de anticorpos maternos anti-intiminas de mães brasileiras saudáveis para os seus recém-nascidos através da placenta e do colostro. Métodos: Anticorpos séricos da classe IgG e secretórios da classe IgA (SIgA) reativos com as porções conservada (cons) e variáveis das intiminas α (vα), β (vβ) e γ (vγ) foram analisados pelo teste de ELISA no sangue e no colostro de 45 parturientes saudáveis e no sangue de cordão umbilical dos seus respectivos recém-nascidos. Resultados: As concentrações de anticorpos reativos com intimina vα foram significativamente mais baixas que as dos anticorpos anti-vγ e anti-cons nas amostras de colostro. Anticorpos IgG séricos reativos com todas as intiminas foram transferidos para os recém-nascidos, mas as concentrações de anti-cons foram significativamente mais altas tanto nas mães como nos recém-nascidos do que os anticorpos reativos com as regiões variáveis das intiminas. O padrão de transferência de IgG das mães para os recém-nascidos foi muito semelhante para todos os anticorpos anti-intiminas. Os valores de porcentagem de transferência foram semelhantes à transferência de IgG total. Conclusões: Anticorpos anti-intimina são transferidos das mães para os recém-nascidos pela placenta e corroboram a proteção contra infecções por Escherichia coli diarreiogênicas (DEC) conferida pelo aleitamento materno.

Placental and colostral transfer of antibodies reactive with enteropathogenic Escherichia coli intimins α, ß, or γ.

OBJECTIVE: Intimins are protein adhesins of enteropathogenic Escherichia coli and enterohemorrhagic E. coli capable of inducing attachment and effacement lesions in enterocytes. Anti-intimin antibodies are important for the protection from enteropathogenic E. coli and enterohemorrhagic E. coli infections because these antibodies inhibit bacterial adhesion and impair the initial step of the pathogenesis. We studied the transfer of maternal anti-intimin antibodies from healthy Brazilian mothers to their newborns through the placenta and colostrum. METHODS: Serum immunoglobulin G and secretory immunoglobulin A antibodies against conserved and variable regions of intimins α, ß, and γ were analyzed using an enzyme linked-immunosorbent assay in the blood and colostrum from 45 healthy women as well as cord blood serum samples from their newborns. RESULTS: The concentrations of antibodies reactive with α intimin were significantly lower than those of anti-γ and anti-conserved intimin antibodies in the colostrum samples. IgG serum antibodies reactive with all the subtypes of intimins were transferred to the newborns, but the concentrations of anti-conserved intimin serum antibodies were significantly higher in mothers and newborns than concentrations of antibodies against variable regions. The patterns of IgG transfer from mothers to newborns were similar for all anti-intimin antibodies. These values are similar to the percentage transference of total IgG. CONCLUSIONS: Anti-intimin antibodies are transferred from mothers to newborns through the placenta, and reinforce the protection provided by breastfeeding against diarrheagenic E. coli infections.

Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.

Alternatives to carbapenems in ESBL-producing Escherichia coli infections.

OBJECTIVES: The authors had for objective to assess the activity of a wide panel of antibiotics on extended-spectrum-ß-lactamase producing Escherichia coli isolates (ESBL-Ec), because of the sharp increase of their frequency, leading to an increased use of carbapenems. MATERIAL AND METHODS: We selected 100 ESBL-Ec in which ESBLs were identified by PCR and sequencing, between 2009 and 2010. We determined the MICs of amoxicillin-clavulanate, piperacillin-tazobactam, temocillin, mecillinam, cefoxitin, cefotaxime, ceftazidime, aztreonam, tigecycline, nitrofurantoin, and fosfomycin using reference methods. The susceptibility profiles were defined according to EUCAST 2012 recommendations. RESULTS: Fosfomycin, nitrofurantoin, and pivmecillinam were active against more than 90% of isolates and remain excellent choices for the oral treatment of urinary tract infections (UTIs). Temocillin and piperacillin-tazobactam are also good candidates for the treatment of pyelonephritis or bloodstream infections. Only 27, 23, and 8% of isolates were susceptible to ceftazidime, cefepime, and cefotaxime, respectively. CONCLUSION: Our study results prove that in many cases, there are non-carbapenem alternatives for the treatment of ESBL-Ec infections.

Bifidobacterium spp. influences the production of autoinducer-2 and biofilm formation by Escherichia coli O157:H7.

The effect of Bifidobacterium spp. on the production of quorum-sensing (QS) signals and biofilm formation by enterohemorrhagic Escherichia coli (EHEC) O157:H7 was investigated. In an AI-2 bioassay, cell extracts of Bifidobacterium longum ATCC 15707 resulted in a 98-fold reduction in AI-2 activity in EHEC O157:H7 as well as in the Vibrio harveyi reporter strain, even though they did not inhibit the growth of EHEC O157:H7. In addition, they resulted in a 36% reduction in biofilm formation by the organism. Consistently, the virulence of EHEC O157:H7 was significantly attenuated by the presence of cell extracts of B. longum ATCC 15707 in the Caenorhabditis elegans nematode in vivo model. By a proteome analysis using two dimensional electrophoresis (2-DE), we determined that seven proteins including formation of iron-sulfur protein (NifU), thiol:disulfide interchange protein (DsbA), and flagellar P-ring protein (FlgI) were differentially regulated in the EHEC O157:H7 when supplemented with cell extracts of B. longum ATCC 15707. Taken together, these findings propose a novel function of a dairy adjunct in repressing the virulence of EHEC O157:H7.

Two-strain, cell-selective protein labeling in mixed bacterial cultures.

Cell-selective metabolic labeling of proteins with noncanonical amino acids enables the study of proteomic changes in specified subpopulations of complex multicellular systems. For example, azidonorleucine (Anl) and 2-aminooctynoic acid, both of which are activated by an engineered methionyl-tRNA synthetase (designated NLL-MetRS), are excluded from proteins made in wild-type cells but incorporated readily into proteins made in cells that carry NLL-MetRS. To expand the set of tools available for cell-selective metabolic labeling, we sought a MetRS variant capable of activating propargylglycine (Pra). Pra was chosen as the target amino acid because its alkynyl side chain can be selectively and efficiently conjugated to azide-functionalized fluorescence probes and affinity tags. Directed evolution, using active-site randomization and error-prone PCR, yielded a MetRS variant (designated PraRS) capable of incorporating Pra at near-quantitative levels into proteins made in a Met-auxotrophic strain of Escherichia coli cultured in Met-depleted media. Proteins made in E. coli strains expressing PraRS were labeled with Pra in Met-supplemented media as shown by in-gel fluorescence after conjugation to Cy5-azide. The combined use of NLL-MetRS and PraRS enabled differential, cell-selective labeling of marker proteins derived from two bacterial strains cocultured in media supplemented with Met, Anl, and Pra. Treatment of the mixed marker proteins by sequential strain-promoted and copper(I)-catalyzed cycloadditions allowed straightforward identification of the cellular origin of each protein.

Expression of the cholera toxin B subunit (CT-B) in maize seeds and a combined mucosal treatment against cholera and traveler's diarrhea.

The non-toxic B subunit (CT-B) of cholera toxin from Vibrio cholerae is a strong immunogen and amplifies the immune reaction to conjugated antigens. In this work, a synthetic gene encoding for CT-B was expressed under control of a γ-zein promoter in maize seeds. Levels of CT-B in maize plants were determined via ganglioside dependent ELISA. The highest expression level recorded in T(1) generation seeds was 0.0014% of total aqueous soluble protein (TASP). Expression level of the same event in the T(2) generation was significantly increased to 0.0197% of TASP. Immunogenicity of maize derived CT-B was evaluated in mice with an oral immunization trial. Anti-CTB IgG and anti-CTB IgA were detected in the sera and fecal samples of the orally immunized mice, respectively. The mice were protected against holotoxin challenge with CT. An additional group of mice was administrated with an equal amount (5 µg per dose each) of mixed maize-derived CT-B and LT-B (B subunit of E. coli heat labile toxin). In the sera and fecal samples obtained from this group, the specific antibody levels were enhanced compared to either the same or a higher amount of CT-B alone. These results suggest that a synergistic action may be achieved using a CT-B and LT-B mixture that can lead to a more efficacious combined vaccine to target diarrhea induced by both cholera and enterotoxigenic strains of Escherichia coli.

Extending the usability of the phasing power of diselenide bonds: SeCys SAD phasing of CsgC using a non-auxotrophic strain.

The CsgC protein is a component of the curli system in Escherichia coli. Reported here is the successful incorporation of selenocysteine (SeCys) and selenomethionine (SeMet) into recombinant CsgC, yielding derivatized crystals suitable for structural determination. Unlike in previous reports, a standard autotrophic expression strain was used and only single-wavelength anomalous dispersion (SAD) data were required for successful phasing. The level of SeCys/SeMet incorporation was estimated by mass spectrometry to be about 80%. The native protein crystallized in two different crystal forms (form 1 belonging to space group C222(1) and form 2 belonging to space group C2), which diffracted to 2.4 and 2.0 Šresolution, respectively, whilst Se-derivatized protein crystallized in space group C2 and diffracted to 1.7 Šresolution. The Se-derivatized crystals are suitable for SAD structure determination using only the anomalous signal derived from the SeCys residues. These results extend the usability of SeCys labelling to more general and less favourable cases, rendering it a suitable alternative to traditional phasing approaches.

Antipathogenic properties of green tea polyphenol epigallocatechin gallate at concentrations below the MIC against enterohemorrhagic Escherichia coli O157:H7.

The inhibitory effects of green tea polyphenol epigallocatechin gallate (EGCG) on virulence phenotypes and gene expression regulated by quorum sensing (QS) in Escherichia coli O157:H7 were demonstrated at concentrations of 1 to 100 microg/ml, which are lower than the MIC (539 +/- 22 microg/ml). At 25 microg/ml, the growth rate was not affected, but autoinducer 2 concentration, biofilm formation, and swarm motility decreased to 13.2, 11.8, and 50%, respectively. Survival at 5 days of nematodes (Caenorhabditis elegans) that were fed the pathogen without and with EGCG were 47.1 and 76%, respectively. Real-time PCR data indicated decreased transcriptional level in many quorum sensing-regulated virulence genes at 25 microg/ml. Our results suggest that EGCG at concentrations below itsMIC has significant antipathogenic effects against E. coli O157:H7.

An improved method for a rapid determination of phytase activity in animal feed.

The current direct colorimetric assay for phytase activity in feeds has interference from high P background and other factors. Our objective was to develop a rapid and reliable spin column method to accurately determine phytase activity in feed ingredients or complete diets. After the feed sample was extracted by stirring in 0.2 M citrate buffer, pH 5.5, for 30 min at room temperature, the oily layer of the supernatant fraction was removed by passing through an acrodisc syringe filter (0.45-microm HT Tuffryn membrane, Gelman Laboratory, Ann Arbor, MI). The filtrate was then loaded onto a spin column (MW cutoff 30,000, Millipore, Bedford, MA) to remove free phosphate before the phytase activity assay. Compared with the direct assay, this new procedure improved both accuracy and reproducibility. When diets contained phytase at 0 to 1,500 U/kg (as fed), the CV for multiple assays of the same samples (n = 6) by the new method ranged from 1 to 6% compared with 28 to 39% by the direct method. A linear relationship was found between the added phytase activity in practical diets and the analyzed activity by the new method (r2 = 0.99; P < 0.01). In conclusion, the spin column method is an improved assay for phytase activity in animal feed, and may be used for quality control of phytase supplementation.

Optimization of an Escherichia coli system for cell-free synthesis of selectively N-labelled proteins for rapid analysis by NMR spectroscopy.

Cell-free protein synthesis offers rapid access to proteins that are selectively labelled with [15N]amino acids and suitable for analysis by NMR spectroscopy without chromatographic purification. A system based on an Escherichia coli cell extract was optimized with regard to protein yield and minimal usage of 15N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to 1.8 mg of human cyclophilin A per mL of reaction medium were obtained by expression of a synthetic gene. Equivalent yields were obtained using transcription directed by either T7 or tandem phage lambdapR and pL promoters, when the reactions were supplemented with purified phage T7 or E. coli RNA polymerase. Nineteen samples, each selectively labelled with a different 15N-enriched amino acid, were produced and analysed directly by NMR spectroscopy after ultracentrifugation. Cross-peaks from metabolic by-products were evident in the 15N-HSQC spectra of 13 of the samples. All metabolites were found to be small molecules that could be separated readily from the labelled proteins by dialysis. No significant transamination activity was observed except for [15N]Asp, where an enzyme in the cell extract efficiently converted Asp-->Asn. This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate. In addition, the activity of peptide deformylase appeared to be generally reduced in the cell-free expression system.

Off-line monitoring of bacterial stress response during recombinant protein production using an optical biosensor.

A surface plasmon resonance (SPR) biosensor was used to monitor the profiles of the heat-shock protein (DnaK) and the expression of a heterologous protein to map the dynamics of the cellular stress response in Escherichia coli. As expression system was used an E. coli strain overproducing human recombinant superoxide dismutase (rhSOD). Expression of DnaK showed complex patterns differing with strength of induction. The strong up-regulation of DnaK expression was observed in all cultivations which over-produced of rhSOD. Similar patterns were not observed in non-induced reference cultures. Differences in DnaK concentration profiles were correlated with induction strength. Presented data, carried out in shake flask and glucose limited fed-batch cultivation, show a good consistency with previously published transcriptional profiling results and provide complementary information to understand stress response related to overproduction of recombinant protein. The study also demonstrates the feasibility of using the SPR as a two channel protein array for monitoring of intracellular components.