The overarching goal of this proposal is to assess the essentiality of L,L-diaminopimelate aminotransferase in pathogenic bacteria to facilitate the development of antibiotics. There is an urgent need for the development of novel antibiotics to combat the drastic rise in the number of antibiotic resistant bacteria. One of the bottlenecks that is impeding the development of antibiotics is the identification of novel enzymatic targets. The PI recently identified and elucidated a novel variant of the diaminopimelate/lysine biosynthesis pathway by identifying and characterizing the enzyme L,L-diaminopimelate aminotransferase. In pathogenic bacteria, such as Chlamydia trachomatis, diaminopimelate aminotransferase catalyzes a specific reaction in the diaminopimelate/lysine anabolic pathway that is necessary for both cell wall peptidoglycan and amino acid protein synthesis. The genomes of animals, particularly humans, do not contain the genetic machinery necessary to facilitate the synthesis of diaminopimelate/lysine. As such, the enzymes in this pathway are attractive targets for novel antibiotics. We hypothesize that inhibition of diaminopimelate aminotransferase in the pathogenic bacteria will cause a bactericidal effect through inhibition of peptidoglycan synthesis and protein synthesis. This is because 1) the intermediate meso-diaminopimelate/lysine serves a cross-linking amino acid in the peptidoglycan of bacteria and 2) lysine is one of the 20 common proteogenic amino acids. The proposed research is significant since we will assess the essentiality of the dapL gene in the Gram- negative bacterium Verrucomicrobium spinosum, the closest free living relative of Chlamydia, the causative bacterium in the sexually transmitted disease ?Chlamydia?. V. spinosum was chosen as a model because it employs the diaminopimelate aminotransferase pathway as the sole pathway for peptidoglycan and lysine biosynthesis. The organism is not pathogenic and it can be genetically manipulated. To test if diaminopimelate aminotransferase is a feasible target for the development of novel antibiotics we have delineated three aims. 1) We will assess the essentiality of diaminopimelate aminotransferase in eubacteria using the V. spinosum as a model using mutagenesis experiments employing transposon and/or gene replacement of the dapL gene. 2) Recent studies from PI?s lab have identified antagonistic lead compounds towards diaminopimelate aminotransferase using in vitro assays. As such, we will discern the specificity of these compounds using a in vivo system where we will use V. spinosum wild type and diaminopimelate aminotransferase mutants to assess if these identified compounds are specific for diaminopimelate aminotransferase and 3) The final aim of the project will identify the amino acids that are involved in the binding of antagonistic compounds by incubating the enzyme with these compounds followed by structural analyses facilitated by X-ray crystallography, which will underpin the development of second generation inhibitors.
The rise in the number of multidrug-resistant bacteria such as; Methicillin-resistant Staphylococcus aureus, Vancomycin inter-mediate and resistant Staphylococcus aureus, Pseudomonas aeruginosa (multidrug resistant strains), multi-drug resistant Mycobacterium tuberculosis among others has led to a significant increase in the morbidity and mortality of humans infected with pathogenic bacteria. We have identified the enzyme L,L-diaminopimelate aminotransferase (DapL) as a novel and attractive target for narrow-spectrum antibiotic development given, 1) the presence of the gene in approximately 13% of bacterial genomes, 2) its role in cell wall peptidoglycan biosynthesis; and 3) role in the biosynthesis of the amino acid lysine. This proposal will generate key data that underpins future projects aimed at the identification and or development of antibiotics that are specific for DapL in pathogenic bacteria, in addition to others that have yet to be identified that employ the DapL pathway for peptidoglycan and protein synthesis.
| Cala, Ali R; Nadeau, Maria T; Abendroth, Jan et al. (2016) The crystal structure of dihydrodipicolinate reductase from the human-pathogenic bacterium Bartonella henselae strain Houston-1 at 2.3?Å resolution. Acta Crystallogr F Struct Biol Commun 72:885-891 |
