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20 New Tools for Virulence Gene Discovery

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20 New Tools for Virulence Gene Discovery, Page 1 of 2

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Abstract:

Early forays into identifying virulence genes used the basic tools of bacterial genetics: mutation and complementation. The fundamental biology of some host-pathogen interactions can limit the ability to apply signature-tagged mutagenesis (STM) and related approaches. One of the most versatile tools to probe host-pathogen interactions is the green fluorescent protein (GFP). Differential fluorescence induction (DFI)-based screens have yielded a high proportion of virulence genes relative to housekeeping genes. Part of this success results not from any particular advantage of the DFI technique per se but from a principle that is important when designing any screen based on analysis of differential expression, be it DNA microarrays or in vivo expression technology (IVET). is one of the prevalent model systems used to study the development of multicellular organisms. The availability of whole genomic sequences presents another method of virulence gene discovery: bioinformatics. This chapter outlines a variety of approaches used to identify virulence genes. The genome sequence of every major pathogen (and most minor ones) is or will soon be available. These genomic sequences, augmented by the gene discovery methods described in the chapter, should permit the discovery of novel virulence genes at an unprecedented pace.

Citation: McDaniel T, Valdivia R. 2004. 20 New Tools for Virulence Gene Discovery, p 473-488. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch20
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Figures

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Figure 20.1

Signature-tagged mutagenesis. This diagram shows the procedure for a pool of three mutants. In practice, pools of up to 96 mutants have been used to infect a single animal.

Citation: McDaniel T, Valdivia R. 2004. 20 New Tools for Virulence Gene Discovery, p 473-488. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch20
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Image of Figure 20.3
Figure 20.3

In vivo expression technology with and .

Citation: McDaniel T, Valdivia R. 2004. 20 New Tools for Virulence Gene Discovery, p 473-488. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch20
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Image of Figure 20.4
Figure 20.4

Resolvase as an IVET reporter. Promoter fragments are inserted before a promoterless gene, which encodes a resolvase enzyme. If the gene is fused to an active promoter, the resolvase is produced and deletes an antibiotic resistance gene that has been inserted into the bacterial chromosome flanked by two resolvase recognition sites. Loss of antibiotic resistance therefore permanently records that the promoter was activated.

Citation: McDaniel T, Valdivia R. 2004. 20 New Tools for Virulence Gene Discovery, p 473-488. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch20
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Figure 20.5

Isolation of host cell-induced microbial genes by differential fluorescence induction.

Citation: McDaniel T, Valdivia R. 2004. 20 New Tools for Virulence Gene Discovery, p 473-488. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch20
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Image of Figure 20.7
Figure 20.7

Categorization of serovar Typhimurium genes based on comparative genomic studies of serovar Typhimurium strain LT2 to five other strains of and three other enteric bacterial strains. Note the small fraction of genes unique to serovar Typhimurium. The precise number of genes in any given category is not static since the numbers shown depend on the limited set of strains examined.

Citation: McDaniel T, Valdivia R. 2004. 20 New Tools for Virulence Gene Discovery, p 473-488. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch20
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References

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1. Camilli, A.,, and J. J. Mekalanos. 1995. Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection. Mol. Microbiol. 18:671683.
2. Falkow, S. 1988. Molecular Koch’s postulates applied to microbial pathogenicity. Rev. Infect. Dis. 10:S274S276.
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4. Guttman, D. S.,, B. A Vinatzer,, S. F. Sarkar,, M. V. Ranall,, G. Kettler,, and J. T. Greenberg. 2002. A functional screen for the type III (Hrp) secretome of the plant pathogen Pseudomonas syringae. Science 295:17221726. A clever approach to identify targets of the TTSS system.
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6. Mahan, M. J.,, J. M. Slauch,, and J. J. Mekalanos. 1993. Selection of bacterial virulence genes that are specifically induced in host tissues. Science 259:686688. The “original” in vivo expression technology (IVET) paper. This work introduced the concept that bacterial genes required for survival in host tissues are most likely preferentially expressed in host tissues.
7. McClelland, M.,, K. E. Sanderson,, J. Spieth,, S. W. Clifton,, P. Latreille,, L. Courtney,, S. Porwollik,, J. Ali,, M. Dante,, F. Du,, S. Hou,, D. Layman,, S. Leonard,, C. Nguyen,, K. Scott,, A. Holmes,, N. Grewal,, E. Mulvaney,, E. Ryan,, H. Sun,, L. Florea,, W. Miller,, T. Stoneking,, M. Nhan,, R. Waterston,, and R. K. Wilson. 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852856.
8. Valdivia, R. H.,, and S. Falkow. 1997. Fluorescence-based isolation of bacterial genes expressed within host cells. Science 277:20072011. This paper describes the use of gfp to identify bacterial genes expressed during infection. It also details the advantages of monitoring bacterial gene expression with single-cell resolution in complex host environments.

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