The Vitamin B1 Metabolism of Staphylococcus aureus Is Controlled at Enzymatic and Transcriptional Levels

Vitamin B1 is in its active form thiamine pyrophosphate (TPP), an essential cofactor for several key enzymes in the carbohydrate metabolism. Mammals must salvage this crucial nutrient from their diet in order to complement the deficiency of de novo synthesis. In the human pathogenic bacterium Staphylococcus aureus, two operons were identified which are involved in vitamin B1 metabolism. The first operon encodes for the thiaminase type II (TenA), 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (ThiD), 5-(2-hydroxyethyl)-4-methylthiazole kinase (ThiM) and thiamine phosphate synthase (ThiE). The second operon encodes a phosphatase, an epimerase and the thiamine pyrophosphokinase (TPK). The open reading frames of the individual operons were cloned, their corresponding proteins were recombinantly expressed and biochemically analysed. The kinetic properties of the enzymes as well as the binding of TPP to the in vitro transcribed RNA of the proposed operons suggest that the vitamin B1 homeostasis in S. aureus is strongly regulated at transcriptional as well as enzymatic levels

Identification of Inhibitors against Mycobacterium tuberculosis Thiamin Phosphate Synthase, an Important Target for the Development of Anti-TB Drugs

Tuberculosis (TB) continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the non-compliance to 6–9 months long chemotherapeutic regimens often results in the emergence of multidrug resistant strains of Mycobacterium tuberculosis adding to the precariousness of the situation. This has necessitated the development of more effective drugs. Thiamin biosynthesis, an important metabolic pathway of M.tuberculosis, is shown to be essential for the intracellular growth of this pathogen and hence, it is believed that inhibition of this pathway would severely affect the growth of M.tuberculosis. In this study, a comparative homology model of M.tuberculosis thiamin phosphate synthase (MtTPS) was generated and employed for virtual screening of NCI diversity set II to select potential inhibitors. The best 39 compounds based on the docking results were evaluated for their potential to inhibit the MtTPS activity. Seven compounds inhibited MtTPS activity with IC50 values ranging from 20 – 100 µg/ml and two of these exhibited weak inhibition of M.tuberculosis growth with MIC99 values being 125 µg/ml and 162.5 µg/ml while one compound was identified as a very potent inhibitor of M.tuberculosis growth with an MIC99 value of 6 µg/ml. This study establishes MtTPS as a novel drug target against M.tuberculosis leading to the identification of new lead molecules for the development of antitubercular drugs. Further optimization of these lead compounds could result in more potent therapeutic molecules against Tuberculosis

Genome-Scale Identification Method Applied to Find Cryptic Aminoglycoside Resistance Genes in Pseudomonas aeruginosa

BACKGROUND:The ability of bacteria to rapidly evolve resistance to antibiotics is a critical public health problem. Resistance leads to increased disease severity and death rates, as well as imposes pressure towards the discovery and development of new antibiotic therapies. Improving understanding of the evolution and genetic basis of resistance is a fundamental goal in the field of microbiology. RESULTS:We have applied a new genomic method, Scalar Analysis of Library Enrichments (SCALEs), to identify genomic regions that, given increased copy number, may lead to aminoglycoside resistance in Pseudomonas aeruginosa at the genome scale. We report the result of selections on highly representative genomic libraries for three different aminoglycoside antibiotics (amikacin, gentamicin, and tobramycin). At the genome-scale, we show significant (p<0.05) overlap in genes identified for each aminoglycoside evaluated. Among the genomic segments identified, we confirmed increased resistance associated with an increased copy number of several genomic regions, including the ORF of PA5471, recently implicated in MexXY efflux pump related aminoglycoside resistance, PA4943-PA4946 (encoding a probable GTP-binding protein, a predicted host factor I protein, a delta 2-isopentenylpyrophosphate transferase, and DNA mismatch repair protein mutL), PA0960-PA0963 (encoding hypothetical proteins, a probable cold shock protein, a probable DNA-binding stress protein, and aspartyl-tRNA synthetase), a segment of PA4967 (encoding a topoisomerase IV subunit B), as well as a chimeric clone containing two inserts including the ORFs PA0547 and PA2326 (encoding a probable transcriptional regulator and a probable hypothetical protein, respectively). CONCLUSIONS:The studies reported here demonstrate the application of new a genomic method, SCALEs, which can be used to improve understanding of the evolution of antibiotic resistance in P. aeruginosa. In our demonstration studies, we identified a significant number of genomic regions that increased resistance to multiple aminoglycosides. We identified genetic regions that include open reading frames that encode for products from many functional categories, including genes related to O-antigen synthesis, DNA repair, and transcriptional and translational processes

Bt-maize event MON 88017 expressing Cry3Bb1 does not cause harm to non-target organisms

This review paper explores whether the cultivation of the genetically modified Bt-maize transformation event MON 88017, expressing the insecticidal Cry3Bb1 protein against corn rootworms (Coleoptera: Chrysomelidae), causes adverse effects to non-target organisms (NTOs) and the ecological and anthropocentric functions they provide. Available data do not reveal adverse effects of Cry3Bb1 on various NTOs that are representative of potentially exposed taxonomic and functional groups, confirming that the insecticidal activity of the Cry3Bb1 protein is limited to species belonging to the coleopteran family of Chrysomelidae. The potential risk to non-target chrysomelid larvae ingesting maize MON 88017 pollen deposited on host plants is minimal, as their abundance in maize fields and the likelihood of encountering harmful amounts of pollen in and around maize MON 88017 fields are low. Non-target adult chrysomelids, which may occasionally feed on maize MON 88017 plants, are not expected to be affected due to the low activity of the Cry3Bb1 protein on adults. Impacts on NTOs caused by potential unintended changes in maize MON 88017 are not expected to occur, as no differences in composition, phenotypic characteristics and plant-NTO interactions were observed between maize MON 88017 and its near-isogenic line