Whole Genome Sequence Analysis Using JSpecies Tool Establishes Clonal Relationships between Listeria monocytogenes Strains from Epidemiologically Unrelated Listeriosis Outbreaks

In an effort to build a comprehensive genomic approach to food safety challenges, the FDA has implemented a whole genome sequencing effort, GenomeTrakr, which involves the sequencing and analysis of genomes of foodborne pathogens. As a part of this effort, we routinely sequence whole genomes of Listeria monocytogenes (Lm) isolates associated with human listeriosis outbreaks, as well as those isolated through other sources. To rapidly establish genetic relatedness of these genomes, we evaluated tetranucleotide frequency analysis via the JSpecies program to provide a cursory analysis of strain relatedness. The JSpecies tetranucleotide (tetra) analysis plots standardized (z-score) tetramer word frequencies of two strains against each other and uses linear regression analysis to determine similarity (r²). This tool was able to validate the close relationships between outbreak related strains from four different outbreaks. Included in this study was the analysis of Lm strains isolated during the recent caramel apple outbreak and stone fruit incident in 2014. We identified that many of the isolates from these two outbreaks shared a common 4b variant (4bV) serotype, also designated as IVb-v1, using a qPCR protocol developed in our laboratory. The 4bV serotype is characterized by the presence of a 6.3 Kb DNA segment normally found in serotype 1/2a, 3a, 1/2c and 3c strains but not in serotype 4b or 1/2b strains. We decided to compare these strains at a genomic level using the JSpecies Tetra tool. Specifically, we compared several 4bV and 4b isolates and identified a high level of similarity between the stone fruit and apple 4bV strains, but not the 4b strains co-identified in the caramel apple outbreak or other 4b or 4bV strains in our collection. This finding was further substantiated by a SNP-based analysis. Additionally, we were able to identify close relatedness between isolates from clinical cases from 1993–1994 and a single case from 2011 as well as links between two isolates from over 30 years ago. The identification of these potential links shows that JSpecies Tetra analysis can be a useful tool in rapidly assessing genetic relatedness of Lm isolates during outbreak investigations and for comparing historical isolates. Our analyses led to the identification of a highly related clonal group involved in two separate outbreaks, stone fruit and caramel apple, and suggests the possibility of a new genotype that may be better adapted for certain foods and/or environment.     

Microemulsion-Based Topical Hydrogels of Tenoxicam for Treatment of Arthritis

Tenoxicam (TNX) is a non-steroidal anti-inflammatory drug (NSAID) used for the treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, backache and pain. However, prolonged oral use of this drug is associated with gastrointestinal adverse events like peptic ulceration, thus necessitating its development as topical formulation that could obviate the adverse effects and improve patient compliance. The present study was aimed at development of microemulsion-based formulations of TNX for topical delivery at the affected site. The pseudoternary phase diagrams were developed and microemulsion formulations were prepared using Captex 300/oleic acid as oil, Tween 80 as surfactant and n-butanol/ethanol as co-surfactant. Optimized microemulsions were characterized for drug content, droplet size, viscosity, pH and zeta potential. The ex vivo permeation studies through Laca mice skin were performed using Franz diffusion cell assembly, and the permeation profile of the microemulsion formulation was compared with aqueous suspension of drug and drug incorporated in conventional cream. Microemulsion formulations of TNX showed significantly higher (p?<?0.001) mean cumulative percent permeation values in comparison to conventional cream and suspension of drug. In vivo anti-arthritic and anti-inflammatory activity of the developed TNX formulations was evaluated using various inflammatory models such as air pouch model, xylene-induced ear edema, cotton pellet granuloma and carrageenan-induced inflammation. Microemulsion formulations were found to be superior in controlling inflammation as compared to conventional topical dosage forms and showed efficacy equivalent to oral formulation. Results suggest that the developed microemulsion formulations may be used for effective topical delivery of TNX to treat various inflammatory conditions.     

SAR inspired by aldehyde oxidase (AO) metabolism: Discovery of novel,CNS penetrant tricyclic M4 PAMs

This letter describes progress towards an M₄ PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M₄ PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.     

Archaeoglobus fulgidus CopB is a thermophilic Cu2+-ATPase: functional role of its histidine-rich-N-terminal metal binding domain

P1B-type ATPases transport heavy metal ions across cellular membranes. Archaeoglobus fulgidus CopB is a member of this subfamily. We have cloned, expressed in Escherichia coli, and functionally characterized this enzyme. CopB and its homologs are distinguished by a metal binding sequence Cys-Pro-His in their sixth transmembrane segment (H6) and a His-rich N-terminal metal binding domain (His-N-MBD). CopB is a thermophilic protein active at 75 degrees C and high ionic strength. It is activated by Cu2+ with high apparent affinity (K1/2 = 0.28 microm) and partially by Cu+ and Ag+ (22 and 55%, respectively). The higher turnover was associated with a faster phosphorylation rate in the presence of Cu2+. A truncated CopB lacking the first 54 amino acids was constructed to characterize the His-N-MBD. This enzyme showed reduced ATPase activity (50% of wild type) but no changes in metal selectivity, ATP dependence, or phosphorylation levels. However, a slower rate of dephosphorylation of the E2P(Cu2+) form was observed for truncated CopB. The data suggest that the presence of the His residue in the putative transmembrane metal binding site of CopB determines a selectivity for this enzyme that is different for that observed in Cu+/Ag+-ATPases carrying a Cys-Pro-Cys sequence. The His-NMBD appears to have a regulatory role affecting the metal transport rate by controlling the metal release/dephosphorylation rates.     

Functional roles of metal binding domains of the Archaeoglobus fulgidus Cu(+)-ATPase CopA

CopA, a thermophilic membrane ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu(+) or Ag(+) [Mandal et al. (2002) J. Biol. Chem. 277, 7201-7208]. This, as other P(IB)-ATPases, is characterized by a putative metal binding sequence (C(380)PC(382)) in its sixth transmembrane fragment and cytoplasmic metal binding sequences in its NH(2)- and COOH-terminal ends (C(27)AMC(30) and C(751)HHC(754)). Using isolated CopA, we have studied the functional role of these three putative metal binding domains. Replacement of transmembrane Cys residues by Ala results in nonfunctional enzymes that are unable to hydrolyze ATP. However, the CPC --> APA substituted enzyme binds ATP, indicating its correct folding and suggesting that enzyme turnover is prevented by the lack of metal binding to the transmembrane site. Replacement of C-terminal Cys by Ala (C(751,754)A) has no significant effect on ATPase activity, enzyme phosphorylation, apparent binding affinities of ligands, or E1-E2 equilibrium. In contrast, replacement of Cys in the N-terminal metal binding domain (N-MBD) (C(27,30)A) leads to 40% reduction in enzyme turnover. The C(27,30)A enzyme binds Cu(+), Ag(+), and ATP with the same high apparent affinities as the wild-type CopA. Evidence that N-MBD disruption has no effect on the E1-E2 equilibrium is provided by the normal interaction of ATP acting with low affinity and the unaffected IC(50) for vanadate inhibition observed in the C(27,30)A-substituted enzyme. However, replacement C(27,30)A slowed the dephosphorylation of the E2P(metal) form of the enzyme, suggesting a reduction in the rate of metal release. Other investigators have shown the Cu-dependent interaction of isolated N-MBDs from the Wilson disease Cu-ATPase with the ATP binding cytoplasmic domain [Tsivkovskii et al. (2001) J. Biol. Chem. 276, 2234-2242]. Therefore, the data suggest a regulatory mechanism in which the Cu-dependent N-MBD/ATP binding domain interaction would accelerate cation release, the enzyme rate-limiting step, and consequently Cu(+) transport.     

Catalytic phosphorylation of Na,K-ATPase drives the outward movement of its cation-binding H5-H6 hairpin

The Na,K-ATPase undergoes conformational transitions during its catalytic cycle that mediate energy transduction between the phosphorylation and cation-binding sites. Structure-function studies have shown that transmembrane segments H5 and H6 in the alpha subunit of the enzyme participate in cation binding and transport. The Ca-ATPase crystal structure indicates that the H5 helix extends into the cytoplasmic ATP binding domain, finishing 4-5 A from the phosphorylation site. Here, we test whether the phosphorylation of the Na,K-ATPase leads to conformational changes in the cation-binding H5-H6 hairpin. Using as background an enzyme where all wild-type Cys in the transmembrane region were replaced, Cys were introduced in the joining loop and extracellular ends of H5 and H6. Mutated proteins were expressed in COS cells and probed with Hg(2+), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and biotin-maleimide, applied to the extracellular media while placing the cells in two different media (K-medium and Na-medium). We assumed that under these treatment conditions most of the enzyme would be in one of two predominant conformations: E1 (K-medium) and E2P (Na-medium). The extent of enzyme inactivation by Hg(2+) or MTSET treatment was dependent on the targeted position; i.e., proteins carrying Cys in the outermost positions were more affected by treatment. Moreover, in the case of proteins carrying Cys at positions 785, 787, and 797, driving the enzyme to phosphorylated conformations (Na-media) led to a larger inactivation. Similarly, biotinylation of introduced Cys was also influenced by the enzyme conformation, with a larger extent of modification after treatment of cells in the Na-medium (E2P form). These results can be explained by the enzyme phosphorylation driving the outward movement of the H5 helix. Thus, they provide experimental evidence for a structure-function mechanism where, via H5, enzyme phosphorylation leads to a conformational change at the cation-binding site and the consequent cation translocation.     

Genomic Characterization of Listeria monocytogenes Strains Involved in a Multistate Listeriosis Outbreak Associated with Cantaloupe in US

A multistate listeriosis outbreak associated with cantaloupe consumption was reported in the United States in September, 2011. The outbreak investigation recorded a total of 146 invasive illnesses, 30 deaths and one miscarriage. Subtyping of the outbreak associated clinical, food and environmental isolates revealed two serotypes (1/2a and 1/2b) and four pulsed-field gel electrophoresis two-enzyme pattern combinations I, II, III, and IV, including one rarely seen before this outbreak. A DNA-microarray, Listeria GeneChip®, developed by FDA from 24 Listeria monocytogenes genome sequences, was used to further characterize a representative sample of the outbreak isolates. The microarray data (in the form of present or absent calls of specific DNA sequences) separated the isolates into two distinct groups as per their serotypes. The gene content of the outbreak-associated isolates was distinct from that of the previously-reported outbreak strains belonging to the same serotypes. Although the 1/2b outbreak associated isolates are closely related to each other, the 1/2a isolates could be further divided into two distinct genomic groups, one represented by pattern combination I strains and the other represented by highly similar pattern combinations III and IV strains. Gene content analysis of these groups revealed unique genomic sequences associated with these two 1/2a genovars. This work underscores the utility of multiple approaches, such as serotyping, PFGE and DNA microarray analysis to characterize the composition of complex polyclonal listeriosis outbreaks.     

Identification of Novel Regulatory Small RNAs in Acinetobacter baumannii

Small RNA (sRNA) molecules are non-coding RNAs that have been implicated in regulation of various cellular processes in living systems, allowing them to adapt to changing environmental conditions. Till date, sRNAs have not been reported in Acinetobacter baumannii (A. baumannii), which has emerged as a significant multiple drug resistant nosocomial pathogen. In the present study, a combination of bioinformatic and experimental approach was used for identification of novel sRNAs. A total of 31 putative sRNAs were predicted by a combination of two algorithms, sRNAPredict and QRNA. Initially 10 sRNAs were chosen on the basis of lower E- value and three sRNAs (designated as AbsR11, 25 and 28) showed positive signal on Northern blot. These sRNAs are novel in nature as they do not have homologous sequences in other bacterial species. Expression of the three sRNAs was examined in various phases of bacterial growth. Further, the effect of various stress conditions on sRNA gene expression was determined. A detailed investigation revealed differential expression profile of AbsR25 in presence of varying amounts of ethidium bromide (EtBr), suggesting that its expression is influenced by environmental or internal signals such as stress response. A decrease in expression of AbsR25 and concomitant increase in the expression of bioinformatically predicted targets in presence of high EtBr was reverberated by the decrease in target gene expression when AbsR25 was overexpressed. This hints at the negative regulation of target genes by AbsR25. Interestingly, the putative targets include transporter genes and the degree of variation in expression of one of them (A1S_1331) suggests that AbsR25 is involved in regulation of a transporter. This study provides a perspective for future studies of sRNAs and their possible involvement in regulation of antibiotic resistance in bacteria specifically in cryptic A. baumannii.     

Detection of chemicals that stimulate Tn9 transposition in Escherichia coli K12

Summary A spot test has been developed for detecting substances that enhance the transposition of Tn9 in Escherichia coli. Phage :: Tn9-infected cells were plated on chloramphenicol media and a drop of the test substance was placed at the center of the plate. Following incubation, chloramphenicol-resistant colonies appeared due to the transposition of Tn9 to the bacterial chromosome. By comparing the test plate and a control plate with respect to the number and distribution of colonies, the effect of the test compound can be evaluated.Out of over 100 compounds tested, acetate, two detergents (Brij 58 and Nonidet P40) and dimethylsulfoxide were found to enhance transposition 3–20 fold. Acetate was also found to enhance the transposition of Tn5 and Tn10. The stimulating effect of Brij 58 was lost when palmitic acid was added with the Brij 58. The nature of these substances, which we refer to as transposagens, suggests an involvement of lipid or membrane in the transposition process.Abbreviations AMP-R, CAM-R, KAN-R, SPC-R, TET-R resistance to ampicillin, chloramphenicol, kanamycin, spectinomycin and tetracycline, respectively - DMSO dimethylsulfoxide A preliminary report of this work was presented at the Fifth Mid-Atlantic Extrachromosomal Genetic Elements Meeting, 1981 (Datta, Randolph and Rosner, Plasmid 7:99, 1982)