Structural characterization of the DNA-binding mechanism underlying the copper(II)-sensing MarR transcriptional regulator.

Multiple antibiotic resistance regulator (MarR) family proteins are widely conserved transcription factors that control bacterial resistance to antibiotics, environmental stresses, as well as the regulation of virulence determinants. Escherichia coli MarR, the prototype member of this family, has recently been shown to undergo copper(II)-catalyzed inter-dimer disulfide bond formation via a unique cysteine residue (Cys80) residing in its DNA-binding domain. However, despite extensive structural characterization of the MarR family proteins, the structural mechanism for DNA binding of this copper(II)-sensing MarR factor remains elusive. Here, we report the crystal structures of DNA-bound forms of MarR, which revealed a unique, concerted generation of two new helix-loop-helix motifs that facilitated MarR's DNA binding. Structural analysis and electrophoretic mobility shift assays (EMSA) show that the flexibility of Gly116 in the center of helix α5 and the extensive hydrogen-bonding interactions at the N-terminus of helix α1 together assist the reorientation of the wHTH domains and stabilize MarR's DNA-bound conformation.

The multiple antibiotic resistance regulator MarR is a copper sensor in Escherichia coli.

The widely conserved multiple antibiotic resistance regulator (MarR) family of transcription factors modulates bacterial detoxification in response to diverse antibiotics, toxic chemicals or both. The natural inducer for Escherichia coli MarR, the prototypical transcription repressor within this family, remains unknown. Here we show that copper signaling potentiates MarR derepression in E. coli. Copper(II) oxidizes a cysteine residue (Cys80) on MarR to generate disulfide bonds between two MarR dimers, thereby inducing tetramer formation and the dissociation of MarR from its cognate promoter DNA. We further discovered that salicylate, a putative MarR inducer, and the clinically important bactericidal antibiotics norfloxacin and ampicillin all stimulate intracellular copper elevation, most likely through oxidative impairment of copper-dependent envelope proteins, including NADH dehydrogenase-2. This membrane-associated copper oxidation and liberation process derepresses MarR, causing increased bacterial antibiotic resistance. Our study reveals that this bacterial transcription regulator senses copper(II) as a natural signal to cope with stress caused by antibiotics or the environment.

Crystallization and preliminary diffraction analysis of Wzi, a member of the capsule export and assembly pathway in Escherichia coli.

External polysaccharide capsules provide a physical barrier that is employed by many species of bacteria for the purposes of host evasion and persistence. Wzi is a 53 kDa outer membrane ß-barrel protein that is thought to play a role in the attachment of group 1 capsular polysaccharides to the cell surface. The purification and crystallization of an Escherichia coli homologue of Wzi is reported and diffraction data from native and selenomethionine-incorporated protein crystals are presented. Crystals of C-terminally His6-tagged Wzi diffracted to 2.8 Šresolution. Data processing showed that the crystals belonged to the orthorhombic space group C222, with unit-cell parameters a=128.8, b=152.8, c=94.4 Å, α=ß=γ=90°. A His-tagged selenomethionine-containing variant of Wzi has also been crystallized in the same space group and diffraction data have been recorded to 3.8 Šresolution. Data processing shows that the variant crystal has similar unit-cell parameters to the native crystal.

Improved diagnostic precision of urine cytology by implementation of The Paris System and the use of cell blocks.

BACKGROUND: The Paris System for Reporting Urinary Cytology (TPS) published in 2016 provides a standardized approach to evaluating urine cytology. In the authors' practice, a TPS-like approach was adopted in 2012 using similarly defined cytologic criteria and correlating cystoscopic findings, and they also began incorporating the use of cell block (CB) material. The objective of the current study was to assess whether this TPS-like approach with the use of CB, as well as direct implementation of TPS, improved the diagnostic value of urine cytology. METHODS: In total, 188 consecutive urine cytology specimens from 2010 through 2016 that had concurrent or subsequent histologic specimens available were retrospectively analyzed for diagnostic correlation. Urine cytology performance was compared between the periods 2010 to 2012 (pre-TPS-like), 2013 to 2016 (TPS-like), and after TPS reclassification, including blind review by a cytopathologist. The contribution of available CB material to final diagnoses also was assessed. RESULTS: Both the TPS-like approach and TPS reclassification resulted in significantly lowering the rate of atypical urothelial cells (AUC) diagnosis from 48%, to 21%, to 9% (from pre-TPS, to TPS-like, to after TPS reclassification, respectively; P < 0.01) while increasing the positive predictive value of an AUC diagnosis for high-grade urothelial carcinoma from 21%, to 43%, to 83%, respectively. The use of CBs contributed to a definitive final diagnosis in 24 of 36 cases (66.7%). CONCLUSIONS: In support of the new TPS recommendations, the application of stringent "TPS-like" and TPS criteria improves the value of an AUC diagnosis for clinicians and patients by lowering the AUC rate and increasing the positive predictive value of AUC for high-grade urothelial carcinoma. CBs can be used to help resolve problematic cases for more definitive diagnostic categorization.

Protein and Chemical Determinants of BL-1249 Action and Selectivity for K2P Channels.

K2P potassium channels generate leak currents that stabilize the resting membrane potential of excitable cells. Various K2P channels are implicated in pain, ischemia, depression, migraine, and anesthetic responses, making this family an attractive target for small molecule modulator development efforts. BL-1249, a compound from the fenamate class of nonsteroidal anti-inflammatory drugs is known to activate K2P2.1(TREK-1), the founding member of the thermo- and mechanosensitive TREK subfamily; however, its mechanism of action and effects on other K2P channels are not well-defined. Here, we demonstrate that BL-1249 extracellular application activates all TREK subfamily members but has no effect on other K2P subfamilies. Patch clamp experiments demonstrate that, similar to the diverse range of other chemical and physical TREK subfamily gating cues, BL-1249 stimulates the selectivity filter "C-type" gate that controls K2P function. BL-1249 displays selectivity among the TREK subfamily, activating K2P2.1(TREK-1) and K2P10.1(TREK-2) ∼10-fold more potently than K2P4.1(TRAAK). Investigation of mutants and K2P2.1(TREK-1)/K2P4.1(TRAAK) chimeras highlight the key roles of the C-terminal tail in BL-1249 action and identify the M2/M3 transmembrane helix interface as a key site of BL-1249 selectivity. Synthesis and characterization of a set of BL-1249 analogs demonstrates that both the tetrazole and opposing tetralin moieties are critical for function, whereas the conformational mobility between the two ring systems impacts selectivity. Together, our findings underscore the landscape of modes by which small molecules can affect K2P channels and provide crucial information for the development of better and more selective K2P modulators of the TREK subfamily.

Wzi is an outer membrane lectin that underpins group 1 capsule assembly in Escherichia coli.

Many pathogenic bacteria encase themselves in a polysaccharide capsule that provides a barrier to the physical and immunological challenges of the host. The mechanism by which the capsule assembles around the bacterial cell is unknown. Wzi, an integral outer-membrane protein from Escherichia coli, has been implicated in the formation of group 1 capsules. The 2.6 Å resolution structure of Wzi reveals an 18-stranded ß-barrel fold with a novel arrangement of long extracellular loops that blocks the extracellular entrance and a helical bundle that plugs the periplasmic end. Mutagenesis shows that specific extracellular loops are required for in vivo capsule assembly. The data show that Wzi binds the K30 carbohydrate polymer and, crucially, that mutants functionally deficient in vivo show no binding to K30 polymer in vitro. We conclude that Wzi is a novel outer-membrane lectin that assists in the formation of the bacterial capsule via direct interaction with capsular polysaccharides.

Altered antibiotic transport in OmpC mutants isolated from a series of clinical strains of multi-drug resistant E. coli.

Antibiotic-resistant bacteria, particularly gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane.