From microbial gene essentiality to novel antimicrobial drug targets

Background

Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials.

Result

Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept.

Conclusion

Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-958) contains supplementary material, which is available to authorized users.     

In vitro reconstitution of the Schizosaccharomyces pombe alternative excision repair pathway

Schizosaccharomyces pombe alternative excision repair has been shown genetically and biochemically to be involved in the repair of a wide variety of DNA lesions. AER is initiated by a damage-specific endonuclease (Uve1p) that recognizes UV-induced photoproducts, base mispairs, abasic sites, and platinum G-G diadducts and cleaves the DNA phosphodiester backbone 5' to a lesion. Several models exist that employ various mechanisms for damage removal based on the activities of Rad2p, a nuclease thought to be responsible for damage excision in AER. This study represents the first report of the biochemical reconstitution of the AER pathway. A base mispair-containing substrate is repaired in a reaction requiring S. pombe Uve1p, Rad2p, DNA polymerase delta, replication factor C, proliferating cell nuclear antigen, and T4 DNA ligase. Surprisingly, damage is removed exclusively by the 5' to 3' exonuclease activity of Rad2p and not its "flap endonuclease" activity and is absolutely dependent upon the presence of the 5'-phosphoryl moiety at the Uve1p cleavage site.     

A dynamic periplasmic electron transfer network enables respiratory flexibility beyond a thermodynamic regulatory regime

Microorganisms show an astonishing versatility in energy metabolism. They can use a variety of different catabolic electron acceptors, but they use them according to a thermodynamic hierarchy, which is determined by the redox potential of the available electron acceptors. This hierarchy is reflected by a regulatory machinery that leads to the production of respiratory chains in dependence of the availability of the corresponding electron acceptors. In this study, we showed that the γ-proteobacterium Shewanella oneidensis produces several functional electron transfer chains simultaneously. Furthermore, these chains are interconnected, most likely with the aid of c-type cytochromes. The cytochrome pool of a single S. oneidensis cell consists of ca. 700 000 hemes, which are reduced in the absence on an electron acceptor, but can be reoxidized in the presence of a variety of electron acceptors, irrespective of prior growth conditions. The small tetraheme cytochrome (STC) and the soluble heme and flavin containing fumarate reductase FccA have overlapping activity and appear to be important for this electron transfer network. Double deletion mutants showed either delayed growth or no growth with ferric iron, nitrate, dimethyl sulfoxide or fumarate as electron acceptor. We propose that an electron transfer machinery that is produced irrespective of a thermodynamic hierarchy not only enables the organism to quickly release catabolic electrons to a variety of environmental electron acceptors, but also offers a fitness benefit in redox-stratified environments.     

The effects of alkalinity and hypertonicity on the morphology and motility of Leptospira interrogans (biflexa) strain B16.

Effects of alkalinity and hypertonicity on the motile behaviour of Leptospira interrogans (biflexa) B16 were observed, quantified, and compared with effects previously shown by similar factors on the motility of eubacteria. Leptospira interrogans tolerated relatively high concentrations of hydroxide ions. Motility similar to that in controls was observed at pH values up to 9-8; but at pH 10-0 motility declined sharply with time of exposure, and there was structural alteration, visible as a blebbing of the cell envelope. Unlike the behaviour of eubacteria, immobilization of L. interrogans induced by hydroxide ions could not be reversed by lowering the pH. It is suggested that by restricting entry of hydroxide ions, the cell envelope protects its motility apparatus from adverse effects. Leptospira interrogans was completely immobilized in 0-5 M and 1-0 M-sucrose solutions. Unlike the eubacteria, leptospires were incapable of spontaneous reversion to motile forms and resumption of motility was dependent on both concentration and time of exposure to sucrose. Deuterium oxide did not affect movement, suggesting that even though leptospire endoflagella and the exoflagella of eubacteria are analogous, the motile behaviour of L. interrogans is significantly different from that of eubacteria.