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Chapter 2 : Methods and Strategies for the Detection of Bacterial Virulence Factors Associated with Pathogenicity Islands, Plasmids, and Bacteriophages

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Methods and Strategies for the Detection of Bacterial Virulence Factors Associated with Pathogenicity Islands, Plasmids, and Bacteriophages, Page 1 of 2

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Abstract:

As many techniques to study individual genes (e.g., by knockout mutations, such as insertion, inframe, point, and deletion mutations) become available, more complex genetic structures like plasmids, phage genomes, and pathogenicity islands (PAIs) can also be addressed. This chapter provides a summary and overview of recently developed strategies to detect virulence genes in PAI and other genetic elements. It specifically addresses the techniques of differential display (DD) and representational difference analysis (RDA) applicable to prokaryotes. A combination of genomic subtraction, pulsed-field gel electrophoresis (PFGE), and Southern hybridization techniques was used to compare two C strains, one isolate from a cystic fibrosis patient and one aquatic isolate; this study revealed that the overall gene order remained relatively constant although insertions and deletions of large blocks of DNA in defined regions of the chromosome were observed. Furthermore, new developments of powerful integrative methods such as DNA chip technology which allows screening for differential gene expression on microfabricated chips as published recently for , , and , and fast and reliable detection techniques like strand displacement amplification are just two examples. In the future, sophisticated genetic approaches in combination with genomics- and proteomics-derived information and methods will allow one to efficiently investigate aspects of bacterial pathogenesis, addressing the evolution of pathogenic bacteria and their molecular analysis, diagnosis, prevention, and therapy.

Citation: Reidl J. 1999. Methods and Strategies for the Detection of Bacterial Virulence Factors Associated with Pathogenicity Islands, Plasmids, and Bacteriophages, p 13-32. In Kaper J, Hacker J (ed), Pathogenicity Islands and Other Mobile Virulence Elements. ASM Press, Washington, DC. doi: 10.1128/9781555818173.ch2

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Figure 1

IVET ( ). A plasmid library containing putative promoter fragments cloned in front of on pIVET1 was constructed. These plasmids were then conjugated to and integrated into the chromosome, generating a merodiploid state ( and ). Pooled transconjugants were then used to infect mice. After incubation for 3 days, S. typhimurium cells were recovered from the spleens and plated onto MacConkey lactose plates. In vivo LacZ colonies were then recognized under ex vivo conditions as LacZ colonies and further identified as containing genes.

Citation: Reidl J. 1999. Methods and Strategies for the Detection of Bacterial Virulence Factors Associated with Pathogenicity Islands, Plasmids, and Bacteriophages, p 13-32. In Kaper J, Hacker J (ed), Pathogenicity Islands and Other Mobile Virulence Elements. ASM Press, Washington, DC. doi: 10.1128/9781555818173.ch2
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Figure 2

DD ( ) and DD-PCR ( ) techniques. Different mRNA expression patterns of one species will be compared; e.g., mRNA extracted from broth-cultured bacteria is compared to mRNA extracted from bacteria cultured in macrophages. In a first step, cDNA is synthesized from mRNA. (A) After primer-adapter ligation, these cDNA fragments were subcloned and cDNA derived from step a was amplified and biotin labeled. cDNA derived from steps a and b was denatured, mixed, and hybridized. By magnetic separation, the biotin-labeled and mixed hybridized fragments were removed. By using macrophage-derived adapter-specific oligonucleotides in PCR amplification, the remaining fragments were amplified, isolated, and further characterized as macrophage-induced genes. (B) For DD-PCR, after production of cDNA as a template, two sets of random primers were used to amplify and simultaneously label the cDNA-derived DNA fragments with S-dATP. Subsequent separation by gel electrophoresis allowed the identification of differently positioned DNA fragments, which were eluted and amplified for further subcloning and identification.

Citation: Reidl J. 1999. Methods and Strategies for the Detection of Bacterial Virulence Factors Associated with Pathogenicity Islands, Plasmids, and Bacteriophages, p 13-32. In Kaper J, Hacker J (ed), Pathogenicity Islands and Other Mobile Virulence Elements. ASM Press, Washington, DC. doi: 10.1128/9781555818173.ch2
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Figure 3

RDA ( ). This analysis allows the detection of genomic differences of related species. Starting material was genomic DNA of organism 1 (tester) and organism 2 (driver). After the generation of restriction fragments, ligation of adapters (consisting of long oligonucleotide adapters and nonligated short oligonucleotide) to tester DNA led to tester-specific amplicons. Tester and driver fragments were then denatured, mixed, and hybridized to form tester-tester, tester-driver, and driver-driver fragments. Subsequent exponential amplification of tester-specific fragments led to organism (tester)-specific DNA.

Citation: Reidl J. 1999. Methods and Strategies for the Detection of Bacterial Virulence Factors Associated with Pathogenicity Islands, Plasmids, and Bacteriophages, p 13-32. In Kaper J, Hacker J (ed), Pathogenicity Islands and Other Mobile Virulence Elements. ASM Press, Washington, DC. doi: 10.1128/9781555818173.ch2
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Figure 4

STM ( ). (A) The design of a tagged transposon system is shown by the Km mini-Tn transposon containing the kanamycin resistance gene and the tag region. This region contained a restriction site (R), which could be used to isolate the variable region, which was used as hybridization probe DNA. (B) An input pool of tagged insertion mutants was generated in and used to infect mice, after which the spleens were removed and bacterial cells were isolated. Probe DNA of the variable regions of the recovered pool was produced and hybridized with the input pool. Nonhybridizing representatives were observed, and such clones were further identified to contain transposon insertions in genes essential for surviving in the in vivo model.

Citation: Reidl J. 1999. Methods and Strategies for the Detection of Bacterial Virulence Factors Associated with Pathogenicity Islands, Plasmids, and Bacteriophages, p 13-32. In Kaper J, Hacker J (ed), Pathogenicity Islands and Other Mobile Virulence Elements. ASM Press, Washington, DC. doi: 10.1128/9781555818173.ch2
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Figure 5

Example of an in vivo transposon mutagenesis ( ). ( ) A lysogenic cell harboring an integrated bacteriophage (represented by a black bar, Φ) was mutagenized with , and randomized insertion was selected by ampicillin resistance. (II) Phage lysate production was induced to obtain a heterogeneous lysate, containing the insertion on the phage genome. (III) The phage lysate was then used to infect a reference strain, and ampicillin-resistant transductants were selected. Subsequent isolation of such colonies and further phage purification steps resulted in the isolation of lysogenic expressed and phage-encoded -lactamase hybrid proteins, indicating secreted or membrane-derived phage products to be further identified as potential virulence factors.

Citation: Reidl J. 1999. Methods and Strategies for the Detection of Bacterial Virulence Factors Associated with Pathogenicity Islands, Plasmids, and Bacteriophages, p 13-32. In Kaper J, Hacker J (ed), Pathogenicity Islands and Other Mobile Virulence Elements. ASM Press, Washington, DC. doi: 10.1128/9781555818173.ch2
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Tables

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Table 1

Methods developed to detect virulence genes by differential analysis

Citation: Reidl J. 1999. Methods and Strategies for the Detection of Bacterial Virulence Factors Associated with Pathogenicity Islands, Plasmids, and Bacteriophages, p 13-32. In Kaper J, Hacker J (ed), Pathogenicity Islands and Other Mobile Virulence Elements. ASM Press, Washington, DC. doi: 10.1128/9781555818173.ch2

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