Association between excreta management and incidence of extended-spectrum ß-lactamase-producing Enterobacteriaceae: role of healthcare workers' knowledge and practices.

BACKGROUND: The spread of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) in healthcare environments has become a major public health threat in recent years. AIM: To assess how healthcare workers (HCWs) manage excreta and the possible association with the incidence of ESBL-PE. METHODS: Eight hundred HCWs and 74 nurse-supervisors were questioned through two self-report questionnaires in order to assess their knowledge and practices, and to determine the equipment utilized for excreta management in 74 healthcare departments. Performance on equipment utilized, knowledge and practices were scored as good (score of 1), intermediate (score of 2) or poor (score of 3) on the basis of pre-established thresholds. Linear regression was performed to evaluate the association between HCWs' knowledge/practices and the incidence of ESBL-PE. FINDINGS: Six hundred and eighty-eight HCWs (86%) and all nurse-supervisors participated in the survey. The proportions of respondents scoring 1, 2 and 3 were: 14.8%, 71.6% and 17.6% for equipment; 30.1%, 40.6 % and 29.3% for knowledge; and 2.0%, 71.9% and 26.1% for practices, respectively. The single regression mathematic model highlighted that poor practices (score of 3) among HCWs was significantly associated with increased incidence of ESBL-PE (P = 0.002). CONCLUSIONS: A positive correlation was found between HCWs' practices for managing excreta and the incidence of ESBL-PE, especially in surgical units. There is an urgent need for development of public health efforts to enhance knowledge and practices of HCWs to better control the spread of multi-drug-resistant bacteria, and these should be integrated within infection control programmes.

[Hereditary optic atrophies]. / Les atrophies optiques héréditaires.

INTRODUCTION: Hereditary optic neuropathies, resulting from retinal ganglion cell degeneration, are a heterogeneous group of diseases ranging from asymptomatic forms to legal blindness. STATE OF KNOWLEDGE: Two most frequent phenotypes are Kjer's disease, an autosomal dominant optic atrophy caused by OPA1 gene mutations, and Leber's disease due to maternally inherited mitochondrial DNA mutations. PROSPECTS AND CONCLUSION: Both optic neuropathies usually isolated are sometimes associated with extraocular symptoms, especially neurological symptoms, thus justifying a systematic neurological evaluation and brain imaging.

Crystal structure of the CheA histidine phosphotransfer domain that mediates response regulator phosphorylation in bacterial chemotaxis.

The x-ray crystal structure of the P1 or H domain of the Salmonella CheA protein has been solved at 2.1-A resolution. The structure is composed of an up-down up-down four-helix bundle that is typical of histidine phosphotransfer or HPt domains such as Escherichia coli ArcB(C) and Saccharomyces cerevisiae Ypd1. Loop regions and additional structural features distinguish all three proteins. The CheA domain has an additional C-terminal helix that lies over the surface formed by the C and D helices. The phosphoaccepting His-48 is located at a solvent-exposed position in the middle of the B helix where it is surrounded by several residues that are characteristic of other HPt domains. Mutagenesis studies indicate that conserved glutamate and lysine residues that are part of a hydrogen-bond network with His-48 are essential for the ATP-dependent phosphorylation reaction but not for the phosphotransfer reaction with CheY. These results suggest that the CheA-P1 domain may serve as a good model for understanding the general function of HPt domains in complex two-component phosphorelay systems.

The molecular puzzle of two-component signaling cascades.

Two-component systems constitute prevalent signaling pathways in bacteria and mediate a large variety of adaptative cellular responses. Signaling proceeds through His-Asp phosphorelay cascades that involve two central partners, the histidine protein kinase and the response regulator protein. Structural studies have provided insights into some design principles and activation mechanisms of these multi-domain proteins implicated in the control of virulence gene expression in several pathogens.

Further insights into the mechanism of function of the response regulator CheY from crystallographic studies of the CheY--CheA(124--257) complex.

New crystallographic structures of the response regulator CheY in association with CheA(124--257), its binding domain in the kinase CheA, have been determined. In all crystal forms, the molecular interactions at the heterodimer interface are identical. Soaking experiments have been performed on the crystals using acetyl phosphate as phosphodonor to CheY. No phosphoryl group attached to Asp57 of CheY is visible from the electron density, but the response regulator in the CheY-CheA(124--257) complex may have undergone a phosphorylation-dephosphorylation process. The distribution of water molecules and the geometry of the active site have changed and are now similar to those of isolated CheY. In a second soaking experiment, imido-diphosphate, an inhibitor of the phosphorylation reaction, was used. This compound binds in the vicinity of the active site, close to the N-terminal part of the first alpha-helix. Together, these results suggest that the binding of CheY to CheA(124--257) generates a geometry of the active site that favours phosphorylation and that imido-diphosphate interferes with phosphorylation by precluding structural changes in this region.

The high resolution crystal structure for class A beta-lactamase PER-1 reveals the bases for its increase in breadth of activity.

The treatment of infectious diseases by beta-lactam antibiotics is continuously challenged by the emergence and dissemination of new beta-lactamases. In most cases, the cephalosporinase activity of class A enzymes results from a few mutations in the TEM and SHV penicillinases. The PER-1 beta-lactamase was characterized as a class A enzyme displaying a cephalosporinase activity. This activity was, however, insensitive to the mutations of residues known to be critical for providing extended substrate profiles to TEM and SHV. The x-ray structure of the protein, solved at 1.9-A resolution, reveals that two of the most conserved features in class A beta-lactamases are not present in this enzyme: the fold of the Omega-loop and the cis conformation of the peptide bond between residues 166 and 167. The new fold of the Omega-loop and the insertion of four residues at the edge of strand S3 generate a broad cavity that may easily accommodate the bulky substituents of cephalosporin substrates. The trans conformation of the 166-167 bond is related to the presence of an aspartic acid at position 136. Selection of class A enzymes based on the occurrence of both Asp(136) and Asn(179) identifies a subgroup of enzymes with high sequence homology.

Structural transitions in the FixJ receiver domain.

BACKGROUND: Two-component signal transduction pathways are sophisticated phosphorelay cascades widespread in prokaryotes and also found in fungi, molds and plants. FixL/FixJ is a prototypical system responsible for the regulation of nitrogen fixation in the symbiotic bacterium Sinorhizobium meliloti. In microaerobic conditions the membrane-bound kinase FixL uses ATP to transphosphorylate a histidine residue, and the response regulator FixJ transfers the phosphoryl group from the phosphohistidine to one of its own aspartate residues in a Mg(2+)-dependent mechanism. RESULTS: Seven X-ray structures of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) have been solved from two crystal forms soaked in different conditions. Three conformations of the protein were found. In the first case, the protein fold impairs metal binding in the active site and the structure reveals a receiver domain that is self-inhibited for catalysis. In the second conformation, the canonical geometry of the active site is attained, and subsequent metal binding to the protein induces minimal conformational changes. The third conformation illustrates a non-catalytic form of the protein where unwinding of the N terminus of helix alpha 1 has occurred. Interconversion of the canonical and self-inhibited conformations requires a large conformational change of the beta 3-alpha 3 loop region. CONCLUSIONS: These unphosphorylated structures of FixJN stress the importance of flexible peptide segments that delineate the active site. Their movements may act as molecular switches that define the functional status of the protein. Such observations are in line with structural and biochemical results obtained on other response regulator proteins and may illustrate general features that account for the specificity of protein-protein interactions.

Electrostatic analysis of TEM1 beta-lactamase: effect of substrate binding, steep potential gradients and consequences of site-directed mutations.

BACKGROUND: Escherichia coli TEM1 is a penicillinase and belongs to class A beta-lactamases. Its naturally occurring mutants are responsible for bacterial resistance to beta-lactamin-based antibiotics. X-ray structure determinations show that all class A beta-lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated. We address the questions of what the molecular contexts during the acylation and deacylation steps are and how they contribute to the efficiency of these penicillinases. RESULTS: Electrostatic analysis of the 1.8 A resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl-enzyme complexes, showed that substrate binding induces an upward shift in the pKa of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote acylation. In the acyl-enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the estercarbonyl carbon of the acyl-enzyme complex, in an electrostatic potential gradient amounting to 30 kTe-1 A-1. Other residues, not directly involved in catalysis, also contribute to the formation of this gradient. The deacylation rate is related to the magnitude of the gradient. The kinetic behavior of site-directed mutants that affect the protonation state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism. CONCLUSIONS: In the wild-type enzyme, the very high rates of acylation and deacylation of class A beta-lactamases arise from an optimal chemical setup in which the acylation reaction seems triggered by substrate binding that changes the general base property of Lys73. In site-directed mutants where Lys73 is protonated, acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.

Crystallization and preliminary X-ray investigation of barstar, the intracellular inhibitor of barnase.

Crystals of barstar, the intracellular inhibitor of the extracellular ribonuclease produced by Bacillus amyloliquefaciens (barnase), were obtained through vapor phase equilibration using the hanging drop technique. Three crystal forms have been characterized. Forms I and II, crystallized either in potassium phosphate or sodium citrate, are tetragonal; they exhibit a superstructure along the c-axis. Form III crystals, suitable for a high resolution structure determination, were grown from 55-65% ammonium sulfate. This crystal form is hexagonal and diffracts to at least 2 A resolution at a synchrotron radiation source. It belongs to the hexagonal space group P6, with unit cell dimensions a = b = 143.6 A, c = 35.6 A. There are four molecules of barstar in the asymmetric unit. X-ray data have been collected to 2.2 A Bragg spacing. The structure determination is underway in order to analyze conformational changes of barstar upon complexation with barnase.

Recognition between a bacterial ribonuclease, barnase, and its natural inhibitor, barstar.

BACKGROUND: Protein-protein recognition is fundamental to most biological processes. The information we have so far on the interfaces between proteins comes largely from several protease-inhibitor and antigen-antibody complexes. Barnase, a bacterial ribonuclease, and barstar, its natural inhibitor, form a tight complex which provides a good model for the study and design of protein-protein non-covalent interactions. RESULTS: Here we report the structure of a complex between barnase and a fully functional mutant of barstar determined by X-ray analysis. Barstar is composed of three parallel alpha-helices stacked against a three-stranded parallel, beta-sheet, and sterically blocks the active site of the enzyme with an alpha-helix and adjacent loop. The buried surface in the interface between the two molecules totals 1630 A2. The barnase-barstar complex is predominantly stabilized by charge interactions involving positive charges in the active site of the enzyme. Asp39 of barstar binds to the phosphate-binding site of barnase, mimicking enzyme-substrate interactions. CONCLUSION: The phosphate-binding site of the enzyme is the anchor point for inhibitor binding. We propose that this is also likely to be the case for other ribonuclease inhibitors.

Three-dimensional structure of a barnase-3'GMP complex at 2.2A resolution.

Barnase has been co-crystallized at neutral pH with its natural product the 3'-guanylic acid. The X-ray structure was solved by molecular replacement methods and refined to a final R-factor of 18.7%. The protein folding is essentially the same as that of the native form. The base recognition site is almost identical to that of the homologous binase-3'GMP complex, but the nucleotide is bound in a productive binding mode for a substrate with a syn glycosyl torsion angle allowing the general base Glu73 to hydrogen bond with the 2'O of the nucleotide as is assumed in the classical catalytic mechanism. The two molecules of the asymmetric unit form a dimer and the positions of the two nucleotides partially mimic the interaction of the RNA with the enzyme, one of the inhibitors being located in a secondary subsite.