Avibactam and class C ß-lactamases: mechanism of inhibition, conservation of the binding pocket, and implications for resistance.

Avibactam is a novel non-ß-lactam ß-lactamase inhibitor that inhibits a wide range of ß-lactamases. These include class A, class C, and some class D enzymes, which erode the activity of ß-lactam drugs in multidrug-resistant pathogens like Pseudomonas aeruginosa and Enterobacteriaceae spp. Avibactam is currently in clinical development in combination with the ß-lactam antibiotics ceftazidime, ceftaroline fosamil, and aztreonam. Avibactam has the potential to be the first ß-lactamase inhibitor that might provide activity against class C-mediated resistance, which represents a growing concern in both hospital- and community-acquired infections. Avibactam has an unusual mechanism of action: it is a covalent inhibitor that acts via ring opening, but in contrast to other currently used ß-lactamase inhibitors, this reaction is reversible. Here, we present a high-resolution structure of avibactam bound to a class C ß-lactamase, AmpC, from P. aeruginosa that provided insight into the mechanism of both acylation and recyclization in this enzyme class and highlighted the differences observed between class A and class C inhibition. Furthermore, variants resistant to avibactam that identified the residues important for inhibition were isolated. Finally, the structural information was used to predict effective inhibition by sequence analysis and functional studies of class C ß-lactamases from a large and diverse set of contemporary clinical isolates (P. aeruginosa and several Enterobacteriaceae spp.) obtained from recent infections to understand any preexisting variability in the binding pocket that might affect inhibition by avibactam.

Development of EUCAST zone diameter breakpoints and quality control range for Staphylococcus aureus with ceftaroline 5-µg disk.

This ceftaroline MIC/disk comparison study for Staphylococcus aureus was performed for the purpose of establishing EUCAST zone diameter breakpoints. Ceftaroline susceptibility for a challenge set of 70 methicillin resistant- and 30 methicillin susceptible-S. aureus was determined by 5-µg disk diffusion and broth microdilution methods. Seventeen isolates were retested by disk and MIC, and the remaining 83 isolates were retested by MIC. Molecular testing was performed on 19 isolates with borderline susceptible ceftaroline MIC results to assess any differences in mecA and epidemiological correlation. An additional set of 101 consecutive clinical S. aureus isolates were tested using the 5-µg disk. S. aureus ATCC 29213 was tested by multiple sites and media for QC range determination. Replicate MIC results were within ±1 doubling dilution, with tendency for slightly lower repeat MICs, and there was minimal variation in replicate zone results. Based on susceptible breakpoints for MIC of ≤1 mcg/mL and for disk of >20 mm, there was 100 % categorical agreement for 30 MSSA and 92 % categorical agreement for 70 MRSA. There were no common MLST or PBP changes for strains with MICs of 1 and 2 mcg/mL. All ceftaroline disk results for the consecutively collected isolates were >20 mm. EUCAST selected the ceftaroline 5-µg disk breakpoint of Susceptible ≥20, Resistant <20 mm because it correlated best with the MIC breakpoint of Susceptible ≤1, Resistant >1 mg/L. A ceftaroline 5-µg disk QC range for S. aureus ATCC 29213 of 24-30 mm was also established by EUCAST.

Associations between autoantibodies against apolipoprotein B-100 peptides and vascular complications in patients with type 2 diabetes.

AIMS/HYPOTHESIS: Oxidation of LDL in the arterial extracellular matrix is a key event in the development of atherosclerosis and autoantibodies against oxidised LDL antigens reflect disease severity and the risk of developing acute cardiovascular events. Since type 2 diabetes is associated with increased oxidative stress, we tested the hypothesis that autoantibodies against oxidised LDL antigens are biomarkers for vascular complications in diabetes. METHODS: We studied 497 patients with type 2 diabetes without clinical signs of coronary heart disease. Oxidised LDL autoantibodies were determined by ELISA detecting IgG and IgM specific for native and malondialdehyde (MDA)-modified apolipoprotein B-100 peptides p45 and p210. The severity of coronary disease was assessed as the coronary artery calcium score. RESULTS: Patients affected by retinopathy had significantly higher levels of IgG against MDA-p45 and MDA-p210. In contrast, high levels of autoantibodies against the corresponding native peptides were associated with less coronary calcification and a lower risk of progression of coronary disease. CONCLUSIONS/INTERPRETATION: Our observations suggest that LDL oxidation is involved in the pathogenesis of diabetic retinopathy and that autoantibodies against apolipoprotein B peptides may act as biomarkers for both micro- and macrovascular complications in diabetes.

Immune responses against fibronectin modified by lipoprotein oxidation and their association with cardiovascular disease.

OBJECTIVES: Accumulation and subsequent oxidation of LDL in the arterial wall are considered as key events in the development of atherosclerosis. We have investigated the possibility that LDL oxidation results in release of aldehydes that modify surrounding matrix proteins and that this may target immune responses against the plaque extracellular matrix and modulate the disease progression. RESULTS: Using custom-made ELISAs we demonstrate that human plasma contains autoantibodies against aldehyde-modified fibronectin (FN) and to a lesser extent also other extracellular matrix proteins including collagen type I, type III, and tenascin-C. Immunohistochemistry and western blot analysis showed that aldehyde-modified FN is present in human atherosclerotic plaques and that aldehydes generated by oxidation of LDL formed adducts with FN in vitro. We also demonstrate that aldehyde-modification of FN results in a loss of its ability to promote basal secretion of cytokines and growth factors from cultured macrophages without affecting the ability of the cells to respond to stimulation with LPS. A prospective clinical study demonstrated that subjects that subsequently developed acute myocardial infarction or sudden cardiac death had lower baseline levels of autoantibodies against aldehyde-modified FN than matched controls. CONCLUSIONS: These observations demonstrate that oxidation of LDL in the arterial wall may lead to aldehyde-modification of surrounding extracellular matrix proteins and that these modifications may affect macrophage function and activate autoimmune responses of pathophysiological importance for the development of atherosclerosis.

Molecular regulation of GLUT-4 targeting in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and adipose tissue by triggering the movement of the glucose transporter GLUT-4 from an intracellular compartment to the cell surface. Fundamental to this process is the intracellular sequestration of GLUT-4 in nonstimulated cells. Two distinct targeting motifs in the amino and carboxy termini of GLUT-4 have been previously identified by expressing chimeras comprised of portions of GLUT-4 and GLUT-1, a transporter isoform that is constitutively targeted to the cell surface, in heterologous cells. These motifs-FQQI in the NH2 terminus and LL in the COOH terminus-resemble endocytic signals that have been described in other proteins. In the present study we have investigated the roles of these motifs in GLUT-4 targeting in insulin-sensitive cells. Epitope-tagged GLUT-4 constructs engineered to differentiate between endogenous and transfected GLUT-4 were stably expressed in 3T3-L1 adipocytes. Targeting was assessed in cells incubated in the presence or absence of insulin by subcellular fractionation. The targeting of epitope-tagged GLUT-4 was indistinguishable from endogenous GLUT-4. Mutation of the FQQI motif (F5 to A5) caused GLUT-4 to constitutively accumulate at the cell surface regardless of expression level. Mutation of the dileucine motif (L489L490 to A489A490) caused an increase in cell surface distribution only at higher levels of expression, but the overall cells surface distribution of this mutant was less than that of the amino-terminal mutants. Both NH2- and COOH-terminal mutants retained insulin-dependent movement from an intracellular to a cell surface locale, suggesting that neither of these motifs is involved in the insulin-dependent redistribution of GLUT-4. We conclude that the phenylalanine-based NH2-terminal and the dileucine-based COOH-terminal motifs play important and distinct roles in GLUT-4 targeting in 3T3-L1 adipocytes.

Helicobacter pylori physiology predicted from genomic comparison of two strains.

Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outcome of H. pylori-related disease.

Organ cultures of human fetal hepatocytes in the study of extra-and intracellular alpha1-antitrypsin.

The rate of synthesis of alpha 1-antitrypsin has been studied in organ cultures of fetal human liver. By de novo synthesis, alpha 1-antitrypsin of the same electrophoretic mobility and molecular size as plasma alpha 1-antitrypsin was produced. Synthetic rate was comparable to in vivo conditions and was suppressed by cycloheximide, colchicine and neuraminidase. By increasing alpha 1-antitrypsin levels in cultre medium, suppression of alpha 1-antitrypsin release from the intra-to the extracellular site was achieved, i.e., synthesis does not proceed autonomously. This suppression was preceded by a temporary enhancement of synthesis. Both effects were found to be independent of degree of sialylation of add-d alpha 1-antitrypsin. In contrast to alpha 1-antitrypsin released in tissue culture, the intracellular protein, as analyzed by crossed immunoelectrophoresis of Triton X-100 extracts from fetal liver, was found to occur partly as slowly moving peaks. Whether these peaks represent proforms or incompletely glycosylated precursors of export alpha 1-antitrypsin or complexes with proteases remains unsettled. A variety of other plasma proteins are released in organ cultures making the system suitable for study of factors regulating plasma protein synthesis.

The microheterogeneity of desialylated alpha 1-antichymotrypsin: the occurrence of two amino-terminal isoforms, one lacking a His-Pro dipeptide.

ACT (alpha 1-antichymotrypsin), a serine antiproteinase with specificity against neutrophil cathepsin G, is homologous with alpha 1-antitrypsin, plasminogen activator inhibitor and angiotensinogen, all with known amino-terminal microheterogeneity. Here we report that the two predominant isoforms of desialylated ACT obtained on isoelectric focusing correspond to a microheterogeneity at the amino terminus of ACT: one isoform (His-Pro-Asn-Ser-Pro-) and a two residues shorter isoform (Asn-Ser-Pro-). The relative occurrence of the two isoforms was comparable both in normal plasma, acute-phase plasma and plasma from subjects with heterozygous familial ACT deficiency. When desialylated ACT, isolated by affinity chromatography from ACT-deficient, normal or acute-phase plasma, was compared with regard to mass and charge microheterogeneity, we found no significant differences in either respect. Nor was the isoform pattern of desialylated plasma from patients with rheumatoid arthritis different. Although the occurrence of heterozygous familial ACT deficiency implies genotypic variation, isolated ACT from patients with the trait was not found to exhibit any phenotypic variation detectable by standard electrophoretic methods.

Significance of duplicated flagellin genes in Campylobacter.

The complex flagellum of Campylobacter coli VC167 contains two highly related (98%) flagellin subunit proteins which are produced from two 92% homologous, tandemly orientated genes, flaA and flaB. Mutants expressing only flaA form a full-length flagellar filament that confers slightly less than wild-type motility to the bacterium. However, flagellin mutants expressing only flaB produce extremely short, truncated filaments, and are only slightly motile. We have shown that the presence of two essentially identical genes is advantageous, in that flaAflaB+ mutants become highly motile upon passage by an event which allows the production of a full length simple flagellar filament containing a single FlaA-FlaB chimeric flagellin protein. Furthermore, we have demonstrated that the reassortment of DNA that results in this chimeric protein can occur by two mechanisms: intragenomic recombination and transformation-mediated intergenomic recombination.

Analysis of the genetic diversity of Helicobacter pylori: the tale of two genomes.

Infection with Helicobacter pylori has been linked to numerous severe gastroduodenal diseases including peptic ulcer and gastric cancer. Several techniques have been used to measure the genetic heterogeneity of H. pylori at several different levels and to determine whether there is any correlation with severity of disease. The availability of two completed genome sequences from unrelated strains (J99 and 26,695) has allowed an analysis of the level of diversity from a large-scale yet detailed perspective. Although the two chromosomes are organized differently in a limited number of discrete regions, the genome size and gene order of these two "high-virulence" (cagA+ and vacA+) H. pylori isolates was found to be highly similar. The regions of organizational difference are associated with insertion sequences, DNA restriction/modification genes, repeat sequences, or a combination of the above. A significant level of variation at the nucleotide level is seen across the genome, providing an explanation for why the nucleotide-based typing techniques have such high discriminatory power among independent H. pylori isolates. This nucleotide variation together with the organizational rearrangements appears to have provided an over-estimation of the gene order diversity of H. pylori as assessed by pulse-field gel electrophoresis. Functional assignments are assigned to approximately only 60% of the gene products in each strain, with one-half of the remaining gene products of unknown function having homologues in other bacteria, while the remainder appear to be H. pylori-specific. Between 6% and 7% of the coding capacity of each strain are genes that are absent from the other strain, with almost one-half of these strain-specific genes located in a single hypervariable region called the plasticity zone. The majority of the strain-specific genes in each strain are also H. pylori-specific, with no homologues being identified in the public databases. Significantly, over one-half of the functionally assigned strain-specific genes in both H. pylori J99 and 26695 encode DNA restriction/modification enzymes. Analysis of the level of conservation between orthologues from the two strains indicates that the H. pylori specific genes have a lower level of conservation than those orthologues to which a putative function can be assigned. The plasticity zone represents one of several regions across each genome that is comprised of lower (G+C)% content DNA, some of which has been detected in self-replicating plasmids, suggesting that both horizontal transfer from other species and plasmid integration are responsible for the strain-specific diversity at this locus. These analyses have yielded results with important implications for understanding the genetic diversity of H. pylori and its associated diseases, and imply a need to reassess the respective roles of bacterial and host factors in H. pylori associated diseases.

Genes involved in the biogenesis and function of type-4 fimbriae in Pseudomonas aeruginosa.

Type-4 fimbriae are filamentous polar organelles which are found in a wide variety of pathogenic bacteria. Their biogenesis and function is proving to be extremely complex, involving the expression and coordinate regulation of a large number of genes. Type-4 fimbriae mediate attachment to host epithelial tissues and a form of surface translocation called twitching motility. In Pseudomonas aeruginosa they also appear to function as receptors for fimbrial-dependent bacteriophages. Analysis of mutants defective in fimbrial function has allowed the identification of many of the genes involved in the biogenesis of these organelles. Thus far over 30 genes have been characterized, which fall into two broad categories: those encoding regulatory networks that control the production and function of these fimbriae (and other virulence determinants such as alginate) in response to alterations in environmental conditions; and those encoding proteins involved in export and assembly of these organelles, many of which are similar to proteins involved in protein secretion and DNA uptake. These systems all appear to be closely related and to function in the assembly of surface-associated protein complexes that have been adapted to different biological functions.

Construction of new Campylobacter cloning vectors and a new mutational cat cassette.

We have developed new Campylobacter shuttle vectors which are 6.5-6.8-kb plasmids carrying Campylobacter and Escherichia coli replicons, a multiple cloning site (MCS), the lacZ alpha gene, oriT and either a kanamycin or chloramphenicol resistance-encoding gene (KmR or CmR) from Campylobacter which functions in both hosts. These vectors can be mobilized efficiently from E. coli into C. jejuni or C. coli, and stably maintained in these hosts. Plasmids pRY107 and pRY108 carry a KmR marker and 17 unique cloning sites in two different orientations in lacZ alpha, allowing easy blue/white color selection. Plasmids pRY111 and pRY112 contain a CmR gene and 17 unique sites in both orientations. In addition, MCS are flanked by T7 and T3 late promoters and M13 forward and reverse primer sites, facilitating expression in T7 or T3 expression systems and sequence analysis. A Campylobacter CmR gene cartridge, bracketed by six restriction sites, has been developed for use in site-specific mutagenesis of Campylobacter genes.

Effect of heat denaturation of target DNA on the PCR amplification.

The polymerase chain reaction (PCR) and amplification of specific regions of DNA in vitro is a widely used and powerful technique, and the optimization of conditions used to maximize PCR product yield has received much attention. We have shown that lengthy denaturation times of template DNA ranging from 1 to 7 min at pH 7.0-8.0, that are often employed prior to the start of a PCR reaction, result in marked degradation of the template. This can result in a significant reduction in the yield of PCR products larger than 500 bp, by up to 99%. This effect was demonstrated for both complex genomic template DNA, and also for a 2691-bp linear piece of template DNA using both a rapid hot-air thermocycler and a conventional block thermocycler. This decrease in product yield is likely due to the increased degradation of the template or target DNA as a result of pre-amplification denaturation (PAD). We therefore recommend that when amplifying larger pieces of DNA, the template DNA should not be exposed to PAD prior to a PCR reaction, irrespective of the starting pH of the template solution.

Sequencing and expression of the aroA gene from Dichelobacter nodosus.

The aroA locus of the Gram- pathogen Dichelobacter nodosus, which encodes 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, has been sequenced and expressed in Escherichia coli. The gene is located on a 1.48-kb DraI-HindIII fragment located directly upstream and in opposite transcriptional orientation to the gene encoding the fimbrial structural subunit. The deduced open reading frame is 1329 nucleotides in length, which encodes a protein of 443 amino acids (aa) with a calculated M(r) of 47,413, which was visualized in E. coli minicells, under the control of its native promoter. This derived aa sequence displays significant similarities with the sequences of the aroA gene products from a variety of microorganisms.

Construction of improved vectors for protein production in Pseudomonas aeruginosa.

We report the construction of two cloning vectors that are based on the Pseudomonas-Escherichia shuttle vector, pUCP19. The new vectors, pUCPKS and pUCPSK, contain a significantly expanded multiple cloning site (MCS) with an adjacent T7 promoter sequence. In conjunction with specifically engineered host strains encoding an inducible T7 RNA polymerase, these vectors allow the controlled production of plasmid-encoded proteins in both Escherichia coli and Pseudomonas aeruginosa to analyse the spectrum of products encoded by cloned segments of DNA. The usefulness of these vectors was demonstrated by expressing the chloramphenicol acetyltransferase (CAT)-encoding gene.

Functional expression of heterologous type 4 fimbriae in Pseudomonas aeruginosa.

Type 4 fimbriae are surface organelles produced by a wide range of bacterial pathogens. In Pseudomonas aeruginosa they are associated with a form of surface translocation known as twitching motility and have also been implicated as the receptor for a number of fimbrial-specific bacteriophages. The infrastructural machinery required for type 4 fimbrial biogenesis appears to be conserved as heterologous subunits from other species can be expressed in P. aeruginosa. All of these studies have, until now, been performed in non-functional Pseudomonas host strains which lack twitching motility. We have constructed isogenic mutants of two commonly studied wild-type P. aeruginosa strains, PAK and PAO1, by replacing the entire pilA gene which encodes the fimbrial subunit. Fimbrial expression and twitching motility were restored by complementation in trans with either the homologous or heterologous subunits from these strains, as well as that from another type 4 fimbriate species, Dichelobacter nodosus. The expression of different subunits allowed us to investigate the precise role that the individual subunit proteins contribute to bacteriophage infection by several fimbrial-specific bacteriophages. Sensitivity to bacteriophages B3cts and D3112cts was restored by the expression of any fimbrial subunit in both PAO1 and PAK cells, indicating that infection by these bacteriophages is fimbrial dependent but not fimbrial specific. In contrast, while sensitivity to the PAK-specific bacteriophage PO4 was restored by the expression of any fimbrial subunit in PAK cells, this did not occur in PAO1 cells except when expressing the PAK subunit. In all cases, the presence of fimbriae was absolutely required to allow a productive bacteriophage infection to occur.

The molecular genetics of type-4 fimbriae in Pseudomonas aeruginosa--a review.

Type-4 fimbriae (or pili) are filaments found at the poles of a wide range of bacterial pathogens, including Neisseria gonorrhoeae, Moraxella bovis, Dichelobacter nodosus and Pseudomonas aeruginosa. They are composed of a small subunit which is highly conserved among different species and appear to mediate adhesion and translocation across epithelial surfaces via a phenomenon termed "twitching motility'. These fimbriae are key host colonisation factors and important protective antigens. We have analysed the genetics and biosynthesis of type-4 fimbriae in P. aeruginosa, which is an opportunistic pathogen of compromised individuals, including those suffering cystic fibrosis, AIDS or burns. A library of P. aeruginosa transposon mutants was constructed which exhibited loss of twitching motility, as determined by altered colony morphology. Analysis of these mutants, and of similar collections by other groups, have revealed that there are at least 22 genes involved in type-4 fimbrial assembly and function. A large number (pilA, B, C, D, E, M, N, O, P, Q, T, U, V and Z) appear to be involved in the biogenesis of the fimbriae and to represent a subset of a supersystem involved in the assembly of surface-associated protein complexes. Homologs of at least some of these genes have subsequently been identified in other type-4 fimbriate bacteria. In P. aeruginosa, the system is also regulated via two signal transduction pathways-a classic sensor-regulator system (encoded by pilS, pilR and rpoN) which controls transcription of the fimbrial subunit, presumably in response to host cues, and a chemotactic system (encoded by pilG, H, I, J, K and L) which may be involved in the directional or rate control of twitching motility in response to local environmental variables.

Identification of a gene, pilF, required for type 4 fimbrial biogenesis and twitching motility in Pseudomonas aeruginosa.

Many bacterial pathogens produce a class of surface structures called type 4 fimbriae. In Pseudomonas aeruginosa these fimbriae are responsible for adhesion and translocation across host epithelial surfaces. We have identified a novel gene involved in the complex process of type 4 fimbrial biogenesis. This gene, termed pilF, is located on SpeI fragment S at 30 min on the P. aeruginosa genomic map, which is the sixth region on the chromosome shown to contain a fimbrial-associated gene. The PilF protein has a predicted M(r) of 22402, and together with a highly homologous upstream ORF shares a chromosomal arrangement similar to that found in Haemophilus influenzae. A pilF mutant is blocked in the export/assembly of the fimbrial subunit PilA, and accumulates this protein in the membrane fraction. Complementation studies indicate that the cloned pilF gene is able to restore the expression of surface fimbriae, twitching motility and susceptibility to fimbrial-specific bacteriophage.

Biosynthesis of abnormally glycosylated hepatoma secretory proteins in cell cultures.

We studied, by electrophoretic techniques, the physiochemical properties of 4 glycoproteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein and transferrin synthesized by three different human hepatoma cell lines. A common feature was the export of glycoproteins with retarded electrophoretic mobility, indicating incomplete sialylation, and a predominance of atypical, highly branched carbohydrate chains. The abnormal glycosylation pattern may be specific for malignant transformation of hepatocytes and possibly related to the intracellular accumulation of some of these proteins in malignant cells.