Martin Karl Russel Burnham

A genomic analysis of two‐component signal transduction in Streptococcus pneumoniae

A genomics‐based approach was used to identify the entire gene complement of putative two‐component signal transduction systems (TCSTSs) in Streptococcus pneumoniae. A total of 14 open reading frames (ORFs) were identified as putative response regulators, 13 of which were adjacent to genes encoding probable histidine kinases. Both the histidine kinase and response regulator proteins were categorized into subfamilies on the basis of phylogeny. Through a systematic programme of mutagenesis, the importance of each novel TCSTS was determined with respect to viability and pathogenicity. One TCSTS was identified that was essential for the growth of S. pneumoniaeThis locus was highly homologous to the yycFG gene pair encoding the essential response regulator/histidine kinase proteins identified in Bacillus subtilis and Staphylococcus aureus. Separate deletions of eight other loci led in each case to a dramatic attenuation of growth in a mouse respiratory tract infection model, suggesting that these signal transduction systems are important for the in vivo adaptation and pathogenesis of S. pneumoniae. The identification of conserved TCSTSs important for both pathogenicity and viability in a Gram‐positive pathogen highlights the potential of two‐component signal transduction as a multicomponent target for antibacterial drug discovery.

A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function.

Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.

Biochemical Characterization of the First Essential Two-Component Signal Transduction System from Staphylococcus aureus and Streptococcus pneumoniae

The yycFG two-component signal transduction system (TCSTS) has been shown to be essential to the viability of several gram-positive bacteria. However, the function of the gene pair remains unknown. Interestingly, while both components are essential to Staphylococcus aureus and Bacillus subtilis, only the response regulator (yycF) is essential to Streptococcus pneumoniae. To study this essential TCSTS further, the S. pneumoniae and S. aureus truncated YycG histidine kinase and full-length YycF response regulator proteins were characterized at a biochemical level. The recombinant proteins from both organisms were expressed in Escherichia coli and purified. The YycG autophosphorylation activities were activated by ammonium. The apparent Km (ATP) of S. aureus YycG autophosphorylation was 130 µM and S. pneumoniae was 3.0 µM. Each had similar kcat values of 0.036 and 0.024 min–1, respectively. Cognate phosphotransfer was also investigated indicating different levels of the phosphorylated YycG intermediates during the reaction. The S. pneumoniae YycG phosphorylated intermediate was not detectable in the presence of its cognate YycF, while phosphorylated S. aureus YycG and YycF were detected concurrently. In addition, noncognate phosphotransfer was demonstrated between the two species. These studies thoroughly compare the essential YycFG TCSTS from the two species at the biochemical level and also establish methods for assaying the activities of these antibacterial targets.

The Contribution of Genomics to the Discovery of New Antibiotics

The emergence of common bacterial pathogens that are resistant to multiple antibiotics, coupled with the failure of traditional methods to yield new anti-infective agents, threatens current paradigms of therapeutic intervention (Omura, 1992; Shlaes et al., 1991; Tenover and Hughes, 1996). The current focus has been on improving existing antibiotic classes while little progress has been made in discovering chemically novel anti-infective agents. This unmet medical need may now be addressed if we can successfully exploit the new wealth of genomic sequence data to devise novel strategies for drug discovery (Moir et al., 1999). Whole genome comparative sequence analysis now allows the identification of genes/gene products that are common to many or all pathogenic bacteria of clinical importance. bioinformatics-based gene homology and motif analyses allow rapid phylogenetic comparisons to be made and, in addition, predict functional information critical to target selection. Putative targets can then be assessed rapidly using gene essentiality testing methods which allow analyses both in vitro and in models of the infection state. Sensitive and direct in vivo expression analyses confirm that the expression of the target gene is relevant to the establishment and maintenance of infection. Ultimately, the gene products must be screened against novel chemical libraries and natural product banks derived from a wide bio-diversity in order to identify lead compounds with potential antibiotic activities that will be developed to provide the next generation of therapeutic agents. Those companies which can adapt and implement these genomic-based technologies, derive significant competitive advantage in creating product portfolios that will address the unmet clinical need.