Assessment of susceptibility to metronidazole and vancomycin of Clostridium difficile strains isolated between 1998-2002

The drugs of choice used to treat C. diffcile associated diarrhoea (CDAD) are metronidazole and vancomycin. C. difficile strains isolated in most laboratories are susceptible to metronidazole and vancomycin. Communication about emergence of antimicrobial resistance among C. difficile strains in some countries to metronidazole and intermediate resistance to vancomycin are alarming. This study was performed to determine the susceptibility to metronidazole and vancomycin of 140 C. difficile strains isolated from patients with CDAD hospitalised in academic hospital between 1999-2002. Resistance to metronidazole and vancomycin was not observed.     

Comparison of AP-PCR methods,ribotyping (PCR-ribotyping) and pulsed-field electrophoresis (PFGE) for strains of Clostridium difficile producing toxin B and not producing toxin A

In this study were used AP-PCR, PCR-ribotyping and pulsed-field elecrophoresis (PFGE) for comparative study of toxin A-negative/toxin B-posi-tive Clostridium difficile strains with deletion in toxin A gen. We investigated nine unrelated clinical strains, isolated from different units and different time from patients suffering to antibiotic associated diarrhea (AAD). We found that toxin A-negative/toxin B-positive C. difficile strains isolated in Poland belonging to a single genotype A, are being similar to the Japanese strains.     

Crystal structure of a trimeric form of dephosphocoenzyme A kinase from Escherichia coli

Coenzyme A (CoA) is an essential cofactor used in a wide variety of biochemical pathways. The final step in the biosynthesis of CoA is catalyzed by dephosphocoenzyme A kinase (DPCK, E.C. 2.7.1.24). Here we report the crystal structure of DPCK from Escherichia coli at 1.8 A resolution. This enzyme forms a tightly packed trimer in its crystal state, in contrast to its observed monomeric structure in solution and to the monomeric, homologous DPCK structure from Haemophilus influenzae. We have confirmed the existence of the trimeric form of the enzyme in solution using gel filtration chromatography measurements. Dephospho-CoA kinase is structurally similar to many nucleoside kinases and other P-loop-containing nucleotide triphosphate hydrolases, despite having negligible sequence similarity to these enzymes. Each monomer consists of five parallel beta-strands flanked by alpha-helices, with an ATP-binding site formed by a P-loop motif. Orthologs of the E. coli DPCK sequence exist in a wide range of organisms, including humans. Multiple alignment of orthologous DPCK sequences reveals a set of highly conserved residues in the vicinity of the nucleotide/CoA binding site.     

Crystal structure of a dodecameric FMN-dependent UbiX-like decarboxylase (Pad1) from Escherichia coli O157: H7

The crystal structure of the flavoprotein Pad1 from Escherichia coli O157:H7 complexed with the cofactor FMN has been determined by the multiple anomalous diffraction method and refined at 2.0 A resolution. This protein is a paralog of UbiX (3-octaprenyl-4-hydroxybenzoate carboxylyase, 51% sequence identity) that catalyzes the third step in ubiquinone biosynthesis and to Saccharomyces cerevisiae Pad1 (54% identity), an enzyme that confers resistance to the antimicrobial compounds phenylacrylic acids through decarboxylation of these compounds. Each Pad1 monomer consists of a typical Rossmann fold containing a non-covalently bound molecule of FMN. The fold of Pad1 is similar to MrsD, an enzyme associated with lantibiotic synthesis; EpiD, a peptidyl-cysteine decarboxylase; and AtHAL3a, the enzyme, which decarboxylates 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine during coenzyme A biosynthesis, all with a similar location of the FMN binding site at the interface between two monomers, yet each having little sequence similarity to one another. All of these proteins associate into oligomers, with a trimer forming the common structural unit in each case. In MrsD and EpiD, which belong to the homo-dodecameric flavin-containing cysteine decarboxylase (HFCD) family, these trimers associate further into dodecamers. Pad1 also forms dodecamers, although the association of the trimers is completely different, resulting in exposure of a different side of the trimer unit to the solvent. This exposure affects the location of the substrate binding site and, specifically, its access to the FMN cofactor. Therefore, Pad1 forms a separate family, distinguishable from the HFCD family.     

Crystal structure of Escherichia coli PdxA,an enzyme involved in the pyridoxal phosphate biosynthesis pathway

Pyridoxal 5'-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-l-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP+ or NAD+ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn2+. The protein forms tightly bound dimers. Each monomer has an alpha/beta/alpha-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn2+ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp247 and Asp267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP.     

Myocardial function defined by strain rate and strain during alterations in inotropic states and heart rate

For porcine myocardium, ultrasonic regional deformation parameters, systolic strain (epsilon(sys)) and peak systolic strain rate (SR(sys)), were compared with stroke volume (SV) and contractility [contractility index (CI)] measured as the ratio of end-systolic strain to end-systolic wall stress. Heart rate (HR) and contractility were varied by atrial pacing (AP = 120-180 beats/min, n = 7), incremental dobutamine infusion (DI = 2.5-20 microg. kg(-1). min(-1), n = 7), or continuous esmolol infusion (0.5 mg. kg(-1). min(-1)) + subsequent pacing (120-180 beats/min) (EI group, n = 6). Baseline SR(sys) and epsilon(sys) averaged 5.0 +/- 0.4 s(-1) and 60 +/- 4%. SR(sys) and CI increased linearly with DI (20 microg. kg(-1). min(-1); SR(sys) = 9.9 +/- 0.7 s(-1), P < 0.0001) and decreased with EI (SR(sys) = 3.4 +/- 0.1 s(-1), P < 0.01). During pacing, SR(sys) and CI remained unchanged in the AP and EI groups. During DI, epsilon(sys) and SV initially increased (5 microg. kg(-1). min(-1); epsilon(sys) = 77 +/- 6%, P < 0.01) and then progressively returned to baseline. During EI, SV and epsilon(sys) decreased (epsilon(sys) = 38 +/- 2%, P < 0.001). Pacing also decreased SV and epsilon(sys) in the AP (180 beats/min; epsilon(sys) = 36 +/- 2%, P < 0.001) and EI groups (180 beats/min; epsilon(sys) = 25 +/- 3%, P < 0.001). Thus, for normal myocardium, SR(sys) reflects regional contractile function (being relatively independent of HR), whereas epsilon(sys) reflects changes in SV.     

Structural basis for specificity of papain-like cysteine protease proregions toward their cognate enzymes

Synthetic peptides corresponding to the proregions of papain-like cysteine proteases have been shown to be good and selective inhibitors of their parental enzymes. The molecular basis for their selectivity, quite remarkable in some cases, is not fully understood. The recent determination of the crystal structures of three distinct papain-like cysteine protease zymogens allows detailed structural comparisons to be made. The reasons for the specificity shown by each proregion toward its cognate enzyme are explained in terms of the three-dimensional structure of the proregion and the interface between the mature enzyme and the proregion. These comparisons reveal that insertion and substitution of amino acids within the proregion cause major rearrangement of sidechains on the enzyme/proregion interface, allowing detailed surface and charge recognition. Proteins 32:504–514, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of the outer domain of the gp120 glycoprotein from human immunodeficiency virus type 1

The core of the gp120 glycoprotein from human immunodeficiency virus type 1 (HIV-1) is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain is exposed on the HIV-1 envelope glycoprotein trimer and contains binding surfaces for neutralizing antibodies such as 2G12, immunoglobulin G1b12, and anti-V3 antibodies. We expressed the outer domain of HIV-1(YU2) gp120 as an independent protein, termed OD1. OD1 efficiently bound 2G12 and a large number of anti-V3 antibodies, indicating its structural integrity. Immunochemical studies with OD1 indicated that antibody responses against the outer domain of the HIV-1 gp120 envelope glycoprotein are rare in HIV-1-infected human sera that potently neutralize the virus. Surprisingly, such outer-domain-directed antibody responses are commonly elicited by immunization with recombinant monomeric gp120. Immunization with soluble, stabilized HIV-1 envelope glycoprotein trimers elicited antibody responses that more closely resembled those in the sera of HIV-1-infected individuals. These results underscore the qualitatively different humoral immune responses elicited during natural infection and after gp120 vaccination and help to explain the failure of gp120 as an effective vaccine.     

Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases

Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 A resolution, respectively. YdiF is organized into tetramers, with each monomer having an open alpha/beta structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent gamma-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.