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.

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.

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.

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.

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.

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.

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.

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.

Characterization of the hlyB gene and its role in the production of the El Tor haemolysin of Vibrio cholerae O1.

El Tor strains of Vibrio cholerae are capable of producing a haemolysin which they actively secrete into the growth medium. This requires translation to produce the protein at the surface of the cytoplasmic membrane and translocation across this membrane, the periplasmic space and the outer membrane. The mechanism by which this occurs is poorly understood. In addition to the structural gene for the haemolysin (hlyA), we have cloned a second adjacent gene, hlyB. By site-directed mutagenesis, specific hlyB mutants have been constructed. These mutants are defective in the secretion of HlyA in the early to mid-exponential phase of growth and the haemolysin becomes trapped within the cell and is only released in stationary phase. Nucleotide sequence analysis and cell fractionations reveal HlyB to be a 60.3 kD putative outer membrane-associated protein.

Identification of a gene, pilV, required for type 4 fimbrial biogenesis in Pseudomonas aeruginosa, whose product possesses a pre-pilin-like leader sequence.

Type 4 fimbriae are important colonization factors in Pseudomonas aeruginosa and other pathogens that mediate attachment to epithelial cells of the host. They are also responsible for a form of translocation termed 'twitching motility' and are implicated in the susceptibility to fimbrial-specific bacteriophage. Analysis of a transposon mutant which lacks functional fimbriae has identified a new gene which is required for fimbrial biogenesis. This gene, termed pilV, is located on chromosomal SpeI fragment E, 2 kb downstream of the previously characterized pilSR genes involved in transcriptional activation of the fimbrial subunit gene. The pilV gene encodes a 20 kDa membrane-located protein with considerable amino-terminal homology to the type 4 consensus pre-pilin leader sequence, suggesting that it is processed by a leader peptidase. Site-directed mutagenesis has shown that PilV requires such cleavage to be functional. PilV also exhibits close similarity to a group of proteins involved in extracellular protein secretion from a number of Gram-negative bacteria, suggesting that the biogenesis of type 4 fimbriae may have a similar basis.

Identification of two genes with prepilin-like leader sequences involved in type 4 fimbrial biogenesis in Pseudomonas aeruginosa.

Type 4 fimbriae are surface filaments produced by a range of bacterial pathogens for colonization of host epithelial surfaces. In Pseudomonas aeruginosa, they are involved in adhesion as well as in a form of surface translocation called twitching motility, and sensitivity to infection by fimbria-specific bacteriophage. Analysis of the 2.5-kb intergenic region between the previously defined pilR and pilV genes on P. aeruginosa genomic SpeI fragment E has identified three new genes, fimT, fimU, and dadA*. The predicted 18.5-kDa products of the fimT and fimU genes contain prepilin-like leader sequences, whereas the third gene, dadA*, encodes a protein similar to the D-amino acid dehydrogenase of Escherichia coli. Isogenic mutants constructed by allelic exchange demonstrated that the fimU gene was required for fimbrial biogenesis and twitching motility, whereas the fimT and dada* mutants retained wild-type phenotypes. However, overexpression of the fimT gene was found to be able to functionally replace the lack of a fimU gene product, suggesting a subtle role in fimbrial biogenesis. The identification of these proteins increases the similarity between type 4 fimbrial biogenesis and the supersystems involved in macromolecular traffic, such as extracellular protein secretion and DNA uptake, all of which now possess multiple protein species that possess prepilin-like leader sequences.

Biotype-specific probe for Vibrio cholerae serogroup O1.

The O1 serogroup of Vibrio cholerae can be divided into two biotypes, El Tor and Classical. Current tests to distinguish between these biotypes are often difficult to interpret. On the basis of the difference in sequence of the hlyA gene in these biotypes, we have developed a simple probe that can easily and reliably differentiate between the two biotypes.

Extracellular proteins of Vibrio cholerae: nucleotide sequence of the structural gene (hlyA) for the haemolysin of the haemolytic El Tor strain 017 and characterization of the hlyA mutation in the non-haemolytic classical strain 569B.

The EI T or haemolysin, product of hlyA, is exported from Vibrio cholerae as a Mr 80,000 protein which can be subsequently cleaved to give two proteins of Mr 65,000 and 15,000. Nucleotide sequence analysis has demonstrated that hlyA encodes a protein of Mr 82,250 with a potential 18-amino-acid signal sequence. The non-haemolytic classical strain 569B has been shown to have a structural gene defect rather than a defect in secretion. By non-reciprocal recombination it was possible to transfer this defect onto a plasmid and show that a truncated hlyA product of Mr 27,000 is made in Escherichia coli K-12 minicells. Nucleotide sequence analysis demonstrates an 11-base-pair deletion which would result in a Mr 26,940 protein probably loosely associated with the membrane.