Gene Expression In Vivo IVETand STM

Growing a pathogenic organism in vitro and studying its gene expression pattern may not tell us which genes are important for causing pathogenicity in a living organism. Genes involved in these processes can only be determined in vivo. A genetic system termed IVET (in vivo expression technology) was developed to identify bacterial genes that are induced when a pathogen infects its host [20]. A subset of these induced genes should include those that encode virulence factors - products specifically required for the infection process. The system is based on complementation of an attenuating auxotrophic mutation by gene fusion, and it is designed to be used in a wide variety of pathogenic organisms. However, alternative systems have been proposed based on reporter genes encoding resistance to antibiotics [21], or encoding the green fluorescent protein (GFP).

The IVET system has several applications in the area of vaccine and antimicrobial drug development. The technique was designed for the identification of virulence factors and thus may lead to the discovery of new antigens useful as vaccine components. The IVET system facilitates the isolation of mutations in genes involved in virulence and therefore should aid in the construction of live-attenuated vaccines. In addition, the identification of promoters that are optimally expressed in animal tissues provides a means of establishing in vivo regulated expression of heterologous antigens in live vaccines, an area that has been previously problematic. Finally, this methodology may also be used to uncover many biosynthetic, catabolic, and regulatory genes that are required for growth of microbes in animal tissues. The elucidation of these gene products should provide new targets for antimicrobial drug development.

Recently, a new version of the IVET method has been proposed, called RIVET (recombination-based in vivo expression technology), which allows the detection of genes that are transiently turned on during adaptation to a new environment [22].

The principle of in vivo induced antigen technology (IVIAT) is vice versa that of identifying those gene products targeted by the host immune system [23].

Although IVET and IVIAT have been both developed and applied to the identification of virulence genes independently of genomics, the genome sequence of the microorganism under study can facilitate their use, by assisting the design of gene libraries intended for the experimental screenings as well as the rapid identification of genes by short fragment sequencing.

Another approach in vaccine design, which is also facilitated by genome sequencing, is signature-tagged mutagenesis (STM), developed by David Holden and coworkers [24]. A bacterial pathogen is subjected to random transposon-medi-ated mutagenesis to identify genes required for in vivo survival. Each mutant incorporates a specific DNA sequence tag and can be recovered and recognized after infection of the animal. Mutants that fail to be recovered after the infection are likely to be attenuated and therefore altered in virulence or essential genes. The advantage of using this approach is that the technique allows for the identification of attenuated mutants that may be used as live vaccines. Moreover, proteins identified as being essential for infection or disease are likely to be good candidates for inclusion in subunit vaccines.

STM has been successfully used to discover virulence genes from a variety of bacterial species including M. tuberculosis [25], S. aureus [26, 27], Salmonella typhi-murium [24], Vibrio cholerae [28], Yersinia enterocolitica [29], Streptococcus pneumoniae [30], and N. meningitidis [31]. In the case of N. meningitidis Sun and coworkers [31], combining the use of STM technology with two available genome sequences, identified in an infant rat model 73 genes that are essential for bacteremia, many of them of unknown function. The majority were novel genes, and in particular 16 surface-exposed antigens are currently under investigation as potential vaccine candidates.

Other transposon-based approaches for the identification of essential genes required for bacterial growth include genome analysis and mapping by in vitro transposition (GAMBIT) and transposon site hybridization (TraSH). The first approach uses high-density mutagenesis of restricted regions of the genome. Using this technique, Mekalanos and collaborators identified the complete set of genes required by H. influenzae for growth and viability in vitro [32]. Genes essential for growth in Mycoplasma were defined using the same approach [33, 34]. The combined use of transposon mutagenesis with microarray hybridization resulted in the development of TraSH, a method suitable for the identification of bacterial genes that are required for growth under specific conditions [35]. TraSH has been applied to identify conditionally essential genes of Mycobacterium bovis BCG, which represent promising targets for rational attenuation.

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