Target Identification and Validation

Comparative genome analysis allows identification of the proteins that are conserved across the medically important pathogens. However, broad conservation of genes does not necessarily mean that each protein carries out an essential function for bacterial growth. Genetic analyses in different species have revealed that the same gene may encode an essential function in one organism but not in another. Such a function can be made dispensable by the presence of biochemical bypasses or additional analogous enzymes, which are structurally unrelated but catalyze the same reaction. Genetic diversity needs to be considered in target evaluation even for different strains of the same species. Differences in S. pneumoniae strains relating to the essentiality of the broadly conserved methionyl-tRNA synthetase were detected by researchers of GlaxoSmithKline [46]. They discovered and optimized an antibacterial compound class active against this target. However, 40% of the clinical isolates of S. pneumoniae were resistant against this compound class, since these isolates harbor an additional insensitive methionyl-tRNA synthetase. This finding demonstrates that we have not reached the end of the genomic sequencing era. An essential prerequisite for the study of species-immanent variations is the continuation of sequencing numerous isolates from important pathogens.

A gene is regarded as being essential if the bacterium cannot tolerate its genetic inactivation. For this reason, genome-wide gene inactivation studies represent the starting point for target-based drug discovery. In many studies, genes are disrupted by insertions of transposons. Such mobile genetic elements are randomly inserted into genomes mediated via intracellularly expressed transposases or via delivery of transposon-transposase complexes ("transposomes") to the cell [4750]. Alternatively, such insertions are achieved by in vitro transposon insertion into purified chromosomes [51] or defined chromosomal areas [52, 53], followed by cellular transformation and genomic recombination. Genetic footprinting using diverse hybridization and polymerase chain reaction (PCR) techniques enables mapping of transposon insertion sites in the genomes. Only in vitro transposition into defined chromosomal fragments, which are usually generated by PCR, reduces this mapping effort [52, 53]. Other genome-wide gene inactivation studies are either based on cloning a portion of a targeted gene into a plasmid suicide vector, which is integrated into the genome by single recombination [54, 55], or marker sequences are inserted into PCR-amplified sequences using crossover PCR techniques before integration into the bacterial genome by double recombination [35]. The most precise way of gene inactivation is base pair position-specific gene deletion without leaving long marker sequences (such as resistance cassettes) in the genome. Methods for this, generally based on PCR techniques, are more elaborate and are mainly reported from the best studied model bacterium, E. coli [56, 57]. In any case, the fact that a gene cannot be inactivated is not final proof of its essentiality; there may be experimental reasons why gene insertion or deletion is impaired.

Only conditional mutants, such as temperature-sensitive (ts) mutations and controlled gene expression systems, allow the essential role of a gene to be demonstrated. Today, genes of interest are generally put under the control of regulable promoter systems. In most cases inducible promoters are used, regulated by ara-binose, rhamnose, tetracycline, or lactose (IPTG) in gram-negative species, and by tetracycline, lactose (IPTG), xylose, fucose, or acetamide in gram-positives including mycobacteria (for a review see Ref. [15]). Instead of directly controlling the gene of interest, gene-specific antisense DNA can be cloned together with induci-ble promoters enabling genome-wide approaches of conditional gene expression. Forsyth et al. and Ji et al. applied the naturally occurring RNA antisense principle for gene silencing by conditional expression of random genomic fragments and then screening for fragments whose expression blocks growth of S. aureus [58, 59]. Recently, a tetracycline-dependent repression system has been developed [60]. Unfortunately, an example of the effect of repression of essential bacterial genes has not yet been published. The genome-wide conditional and nonconditional gene inactivation studies make it possible to estimate how many genes are essential for growth in the different bacterial species (Table 23.2). The number of genes encoding putative broad-spectrum targets is below 200 for major gram-positive/gramnegative or exclusively gram-positive pathogenic bacterial species (Table23.3). The tests are generally performed in nutrient-rich medium, since one assumes that most of the essential genes identified in such a medium are also required for growth in the host. Only a few genes shown to be essential in vitro have already been checked for their importance for bacterial growth in animal models, such as in the study performed by Ji et al., who used inducible antisense constructs [59]. Whatever growth condition for conditional mutants is chosen (in vitro or in vivo), one has to thoroughly check how the mutant has been generated and whether any polar or other side effect can be excluded that might affect another essential gene besides the one of interest.

23.3 Contributions of Genomic Technologies to Antibacterial Research | 515 Table 23.2 Results of genome-wide gene inactivation studies.

Organism

Gene inactivation method

Total number of genes

Number of (potentially) essential genes[a]

Bacillus subtilis

Plasmid-insertion mutagenesis; conditional mutants; estimations derived from literature study [102]

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