##These authors contributed equally to this work
To allow the fast and simultaneous detection of novel bacterial virulence genes, we developed PCR screening-based targeted mutagenesis. We show that the careful design of gene-specific primers allows the direct relative quantification of mixed mutants in infected mouse organs by direct PCR screening of pooled bacterial mutants. This approach provides a sensitive measure of virulence attenuation that requires less time and fewer animals than alteranative procedures, and does not require rigorous DNA purification, complicated PCR set-up, or special equipment.
Understanding the strategies used by pathogens to infect, survive, and proliferate in their hosts requires the identification of virulence factors. We developed PCR-based screening of targeted mutants to facilitate quick, simultaneous detection of multiple novel bacterial virulence genes. Based on direct PCR screening of pooled targeted mutants, this approach provides a fast and sensitive measure of virulence attenuation while significantly reducing the number of animals and time required. We demonstrate that the careful design of gene-specific primers allows the direct relative quantification of mixed mutants in infected mouse organs. Indeed, we show that the band intensity of the PCR product is directly related to the quantity of the corresponding strain in a pool of mutants. We applied the PCR-based screening of targeted mutants to the murine model of listeriosis and revealed new genes required for full pathogenicity of Listeria monocytogenes, a facultative human intracellular pathogen. PCR-based screening is a simple, useful, and fast technique to test pools of targeted bacterial mutants in vivo, without the requirements for a rigorous purification step, complicated PCR set-up, or special equipment. This approach can be adapted to other bacterial systems, constituting a significant advance in the field of infection biology.
Virulence genes encode products necessary for the entry, survival, or persistence of pathogens within their hosts (1). The identification of virulence determinants is thus required to understand the mechanisms used by bacterial pathogens to establish infection. A number of different strategies have been applied to identify virulence-related genes. The construction of gene disruption mutants and their individual in vivo phenotype analysis is a common approach for the functional characterization of targeted genes (2, 3). However, when applied to a large number of genes, this approach is time and animal consuming. Random mutagenesis strategies like signature-tagged mutagenesis (STM) (4) are useful approaches for large-scale screenings of mutants in vivo, but cannot be used to target predefined genes. A multiple competitive index assay was previously developed (5), but it only allows the simultaneous analysis of a maximum of four strains and requires the additional integration of site-specific vectors that prevents future mutant complementation by integrative vectors. Other techniques were also recently applied to follow the in vivo behavior of diverse pathogens (6, 7). Although these strategies allowed important advances in the understanding of infectious processes, all are complex and involve an important development step. PCR is a useful technique that can be directly applied to different templates such as bacteria. This approach was previously adapted to screen pools of STM mutants (8) and then successively used for other STM screens (9, 10). However, PCR-based screening has never been applied to pools of targeted mutants and was previously used only to discriminate between the presence and absence of a mutant in a pool without relative quantification.
Our aim was to develop a PCR-based screening of targeted mutants to (i) achieve a fast and simple characterization of predefined target genes, while reducing and refining the use of animals; (ii) allow the simultaneous analysis of selected bacterial mutants in a single group of mice by direct PCR on bacteria; and (iii) correlate the PCR product to the quantity of the corresponding strain in a pool. In addition, our purpose was to design the simplest, most useful, and fastest technique to test pools of targeted bacterial mutants in vivo, without rigorous purification steps, complicated PCR set-up, or special equipment.
Our general approach (Figure 1) consists of individually culturing bacterial mutants for selected genes. Individual cultures were then mixed in equal amounts to constitute the input pool, which was inoculated to a single group of mice. Afterward, bacteria were recovered from infected mouse organs (output pool). The relative abundance of each mutant in the output pool was calculated and compared with the input pool by direct conventional PCR using bacterial cells as the DNA template. The design of sequence-specific primers is critical for this approach. Indeed, the primers should have similar compositions and melting temperatures to simplify mutant comparison in a single PCR run. Primers were designed following these simple rules: (a) avoid cross homology with other sequences in the genome; (b) the primer length should be between 19 and 22 nucleotides; (c) GC content should be around 50%; (d) the melting temperature should be around 60°C; (e) a GC clamp should be introduced at the 3′ end of primers but avoiding runs of more than three G/C; and (f) avoid complementary sequences within and between primers and avoid repeats.
Using the Gram-positive facultative intracellular pathogen Listeria monocytogenes (Lm) as an infection model, we showed that the careful design of gene-specific primers allowed the direct relative quantification of mixed selected mutants in infected mouse organs. In addition, we demonstrated that PCR-based screening of targeted mutants could be used not only with tagged insertion mutants, but also with deletion mutants. This approach provides a fast and sensitive measure of virulence attenuation while significantly reducing the time and number of animals required.Materials and methods Bacterial strains and media
Lm EGDe (ATCC BAA-679) was used. Listeria were grown in BHI and Escherichia coli in LB medium at 37°C with shaking. When required, the following antibiotics were used: Chloramphenicol 7µg/mL for Listeria and 20µg/mL for E. coli; Erythromycin 5µg/mL for Listeria and 150µg/mL for E. coli; Ampicilin 100µg/mL for E. coli.