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Background: The innate immune response of urinary tract is critically important in the defense to microbial attack. Toll-like receptor 4 (TLR4) controls initial mucosal response to uropathogenic Escherichia coli (UPEC). However, excessive and dysfunctional TLR signaling may result in severe inflammation and inappropriate tissue damage. Previous studies have demonstrated that single immunoglobulin IL-1R-related receptor/Toll IL-1 receptor 8 (SIGIRR/TIR8) is a member of the toll-interleukin-1 receptor (TIR) family that can negatively modulate TLR4 mediated signaling, but its role in the innate immunity of urinary tract infection remains incompletely defined. In this study, we investigated its cellular distribution and mechanisms involved within the human bladder epithelial cells after LPS stimulation. Results: Immunostaining, reverse transcription PCR and Western blot results showed that SIGIRR was constitutively expressed in the human bladder epithelial cell lines and was downregulated after LPS stimulation. To further define the role of SIGIRR, cells were transiently transfected with SIGIRR siRNA and stimulated with LPS. SIGIRR gene silencing augmented chemokine expression in response to LPS, as indicated by increased levels of IL-6 and IL-8 secretions in the supernatants compared with negative control siRNA. Furthermore, LPS tolerance, a protective mechanism against second LPS stimulation, was significantly reduced in SIGIRR siRNA transfected cells. Moreover, transient gene silencing augmented LPS-induced NF-?B and MAPK activation. Conclusions: In conclusion, our results suggest that SIGIRR plays an important role in the negative regulation of LPS response and tolerance in human bladder epithelial cells, possibly through its impact on TLR-mediated signaling. [ABSTRACT FROM AUTHOR]

The Rhesus (Rh) proteins are a family of integral membrane proteins found throughout the animal kingdom that also occur in a number of lower eukaryotes. The significance of Rh proteins derives from their presence in the human red blood cell membrane, where they constitute the second most important group of antigens used in transfusion medicine after the ABO group. Rh proteins are related to the ammonium transport (Amt) protein family and there is considerable evidence that, like Amt proteins, they function as ammonia channels. We have now solved the structure of a rare bacterial homologue (from Nitrosomonas europaea) of human Rh50 proteins at a resolution of 1.3 Å. The protein is a trimer, and analysis of its subunit interface strongly argues that all Rh proteins are likely to be homotrimers and that the human erythrocyte proteins RhAG and RhCE/D are unlikely to form heterooligomers as previously proposed. When compared with structures of bacterial Amt proteins, NeRh50 shows several distinctive features of the substrate conduction pathway that support the concept that Rh proteins have much lower ammonium affinities than Amt proteins and might potentially function bidirectionally. [ABSTRACT FROM AUTHOR]

As a substrate of extracellular signal-related kinase (ERK), the p90 ribosome S6 kinase 1 (RSK1) is at the terminus of the Ras/ERK pathway. Residues 411–735 of human RSK1, covering the C-terminal serine/threonine kinase catalytic domain and the functionally important tail, were cloned into an Escherichia coli expression vector. The protein was expressed, purified and crystallized. The crystals diffracted to 2.7 Å and belonged to space group P21, with unit-cell parameters a = 39.8, b = 143.8, c = 59.9 Å, β = 95.7°. [ABSTRACT FROM AUTHOR]

Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2'-5' RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21- DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°. [ABSTRACT FROM AUTHOR]

The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEPpyruvate- AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in Escherichia coli and Edwardsiella tarda. These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism. [ABSTRACT FROM AUTHOR]

The article presents a study that investigates the structure of the O-antigen of Salmonella O66 and the differences and similarities between the O-antigens of Salmonella O66 and Escherichia coli O166. It states that the O-antigen of Salmonella O66 differs from those of Escherichia coli O166 in one linkage and O-acetylation. The study has found out that the O-antigen gene clusters of Escherichia coli O166 and Salmonella O66 have similar organizations, except in their wzy genes.

Background Emergence of multidrug resistance in Enterobacteriaceae limits the selection of antimicrobials for treatment of infectious diseases. Identification of NDM-1 makes more difficulty in treating multidrug-resistant Enterobacteriaceae infections. Carbapenem-resistant Escherichia coli clinical isolates from a tertiary hospital in Wenzhou, east China, were investigated for NDM-1 production. Results The two tested isolates were negative for modified Hodge test, but positive for a double-disc synergy test used for detecting metallo-β-lactamase production. E. coli WZ33 and WZ51 exhibited discrepant-level resistance to most clinically frequent used antimicrobials, but still susceptible to trimethoprim/sulfamethoxazole, amikacin, fosfomycin, tigecycline and polymyxin B. E. coli WZ33 and WZ51 were positive for blaNDM-1 determined by PCR and DNA sequencing. Other than blaNDM-1, E. coli WZ33 also harbored blaCTX-M-14 and blaCMY-42, while E. coli WZ51 simultaneously harbored blaSHV-12, blaCTX-M-14 and blaCMY-42. Carbapenem resistance for E. coli WZ51 and WZ33 could not be transferred to E. coli recipients through conjugation, but could be transferred to E. coli recipients by chemical transformation. The EcoR1-digested DNA pattern of plasmids from the transformant of E. coli WZ51 was different from that of E. coli WZ51. MLST showed that E. coli WZ33 and WZ51 belonged to an animal-associated clone (ST167). Conclusion The present study is the first report of blaNDM-1 carriage in E. coli ST167 isolates and coexistence of blaNDM-1 and blaCMY-42 in same isolate. Systemic surveillance should focus on the dissemination of blaNDM-1 among Enterobacteriaceae, especially E. coli ST167 clone associated with animal infection. [ABSTRACT FROM AUTHOR]

Although over 50 complete Escherichia coli/Shigella genome sequences are available, it is only for closely related strains, for example the O55:H7 and O157:H7 clones of E. coli, that we can assign differences to individual evolutionary events along specific lineages. Here we sequence the genomes of 14 isolates of a uropathogenic E. coli clone that persisted for 3 years within a household, including a dog, causing a urinary tract infection (UTI) in the dog after 2 years. The 20 mutations observed fit a single tree that allows us to estimate the mutation rate to be about 1.1 per genome per year, with minimal evidence for adaptive change, including in relation to the UTI episode. The host data also imply at least 6 host transfer events over the 3 years, with 2 lineages present over much of that period. To our knowledge, these are the first direct measurements for a clone in a well-defined host community that includes rates of mutation and host transmission. There is a concentration of non-synonymous mutations associated with 2 transfers to the dog, suggesting some selection pressure from the change of host. However, there are no changes to which we can attribute the UTI event in the dog, which suggests that this occurrence after 2 years of the clone being in the household may have been due to chance, or some unknown change in the host or environment. The ability of a UTI strain to persist for 2 years and also to transfer readily within a household has implications for epidemiology, diagnosis, and clinical intervention. [ABSTRACT FROM AUTHOR]

The occurrence of unilateral flagellar phase variation was previously demonstrated in Escherichia coli strains carrying the non-fliC flagellin-specifying locus flk. In this study, we investigated the mechanism involved in this process. By using sequencing and sequence analysis, the flk region between the chromosomal genes yhaC and rnpB was characterized in all described flk-positive E. coli strains, including the H35 strain identified in this study (the other strains used are H3, H36, H47, and H53 strains), and this region was found to contain a putative integrase gene and flanking direct repeats in addition to the flk flagellin-specifying gene flkA and a fliC repressor gene, flkB, indicating that there is a typical genomic islet (GI), which was designated the flk GI. The horizontal transfer potential of the flk GI was indicated by detection of the excised extrachromosomal circular form of the flk GI. By generating fliC-expressing variants of H3 and H47 strains, unilateral flagellar phase variation in flk-positive strains was shown to be mediated by excision of the flk GI. The function of the proposed integrase gene was confirmed by deletion and a complementation test. The potential integration sites of the flk GI were identified. A general model for flagellar phase variation in flk-positive E. coli strains can be expressed as fliCoff + flkAon fliCon + flkAnone. This is the first time that a molecular mechanism for flagellar phase variation has been reported for E. coli. [ABSTRACT FROM AUTHOR]

Amt proteins are ubiquitous channels for the conduction of ammonia in archaea, eubacteria, fungi, and plants. In Escherichia coli, previous studies have indicated that binding of the PII signal transduction protein GInK to the ammonia channel AmtB regulates the channel thereby controlling ammonium influx in response to the intracellular nitrogen status. Here, we describe the crystal structure of the complex between AmtB and GInK at a resolution of 2.5 Å. This structure of PII in a complex with one of its targets reveals physiologically relevant conformations of both AmtB and GInK. GInK interacts with AmtB almost exclusively via a long surface loop containing Y51 (T-loop), the tip of which inserts deeply into the cytoplasmic pore exit, blocking ammonia conduction. Y51 of GlnK is also buried in the pore exit, explaining why uridylylation of this residue prevents complex formation. [ABSTRACT FROM AUTHOR]

Amt proteins constitute a class of ubiquitous integral membrane proteins that mediate movement of ammonium across cell membranes. They are homotrimers, in which each subunit contains a narrow pore through which substrate transport occurs. Two conserved histidine residues in the pore have been proposed to be necessary for ammonia conductance. By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance. Crystal structures of variants confirm that substitution of the histidine residues does not affect AmtB structure. In a subgroup of Amt proteins, found only in fungi, one of the histidines is replaced by glutamate. The equivalent substitution in E. coil AmtB is partially active, and the structure of this variant suggests that the glutamate side chain can make similar interactions to those made by histidine. [ABSTRACT FROM AUTHOR]

Ammonium is one of the most important nitrogen sources for bacteria, fungi, and plants, but it is toxic to animals. The ammonium transport proteins (methylamine permeases/ammonium transporters/rhesus) are present in all domains of life; however. functional studies with members of this family have yielded controversial results with respect to the chemical identity (NH4+ or NH3) of the transported species. We have solved the structure of wild-type AmtB from Escherichla coil in two crystal forms at 1.8- and 2.1-Å resolution, respectively. Substrate transport occurs through a narrow mainly hydrophobic pore located at the center of each monomer of the trimeric AmtB. At the periplasmic entry, a binding site for NH4+ is observed. Two phenylalanine side chains (F107 and F215) block access into the pore from the periplasmic side. Further into the pore, the side chains of two highly conserved histidine residues (H168 and H318) bridged by a H-bond lie adjacent with their edges pointing into the cavity. These histidine residues may facilitate the deprotonation of an ammonium ion entering the pore. Adiabatic free energy calculations support the hypothesis that an electrostatic barrier between H168 and H318 hinders the permeation of cations but not that of the uncharged NH3. The structural data and energetic considerations strongly indicate that the methylamine permeases/ammonium transport- ers/rhesus proteins are ammonia gas channels. Interestingly, at the cytoplasmic exit of the pore, two different conformational states are observed that might be related to the inactivation mechanism by its regulatory partner. [ABSTRACT FROM AUTHOR]