Insertion mutants are faster to obtain than deletion mutants but can have disadvantages (such as polar effects). Therefore, we tested the feasibility of our approach using targeted deletion mutants and pairs of gene-specific primers designed to have similar melting temperatures. The pool of deletion mutants included a negative control strain (DM-) carrying a deletion mutation for a major Lm virulence factor, and a positive control strain (DM+) carrying a deletion of a gene not required for virulence. The input pool, containing deletion mutants for three additional genes with unknown function in virulence (DM1-DM3), was produced and verified as described above. The relative presence of each strain in the liver and spleen of infected mice was monitored 72h after oral infection. The proportion of each mutant was estimated in the output pool compared with the input pool after PCR screening and calculation of relative band intensities. DM- was, as expected, undetectable in both organs (Figure 3B). For DM1, we also observed a dramatic reduction in band intensity in all conditions, indicating that DM1 is also highly attenuated (Figure 3B). In addition, DM2 showed a relative decrease of more than 30% in both organs (Figure 3B). Together, these results demonstrate the feasibility and potential of the PCR-based screening of targeted mutants using deletion mutants for the identification of genes implicated in virulence.Confirmation of virulence defects by individual mutant testing
To validate our approach, virulence-attenuated mutants identified by PCR-based screening were individually tested in vivo. BALB/c mice were infected with Lm WT, DM+, TM2, DM1, and DM2. The number of bacteria in mouse organs was monitored 72h after individual oral inoculation. TM2, DM1, and DM2 showed virulence attenuation in mouse organs (Figure 3C) that largely corroborate virulence reductions observed by PCR-based screening (Figure 3A-3B). Results obtained by individual testing and by PCR-based screening are comparable not only in terms of quality (attenuated or not in mice), but also regarding the attenuation level. The individual characterization of mutants confirmed their virulence phenotype, validating the PCR-based screening of targeted mutants as a powerful method for the rapid identification of genes required for full pathogenesis.
As a proof of concept, we used pools of up to six strains. Theoretically, the number of mutants is only limited by the ability to produce the expected and specific PCR fragment. However, oligonucleotides should be carefully designed to avoid cross-amplification, which could be difficult for highly complex templates. Besides PCR-screening associated limitations, other issues need to be considered. The animal model might limit the complexity of the inoculum and increasing pool complexity will reduce the respective quantity of each mutant. Thus, in a complex pool with several attenuated mutants, the inoculum could be insufficiently virulent, resulting in the elimination of all of the bacteria by the host. Moreover, the use of highly complex pools may exacerbate technical limitations. For example, a secreted molecule may complement a virulence defect of another strain in the pool or the route of infection may be hijacked by a defective competing strain.
The possibility of using a small number of animals to screen large numbers of mutants is important economically and ethically (15). The individual analysis of the virulence potential of five mutants currently requires the use of at least 30 mice. To test the same number of mutants, our approach only involves five mice, representing a reduction of more than 80% in animals, time, and financial resources. In addition, intra-animal experiments minimize inherent inter-animal variations and improve the identification of mutants with reduced competitive fitness within the host.
The advantage of the PCR-based screening of targeted mutants over conventional random mutagenesis techniques (4) is the ability to target specific genes. In addition, we showed that this technique could be used not only with insertion but also with deletion mutants, usually preferred in order to avoid insertional mutagenesis pitfalls. Even if it was not the original purpose of this study, it would be interesting to apply the method to insertion and deletion mutants for the same gene and analyze results in separate assays to rule out any differences between the two approaches. This would also support the validity of the assay in reproducibly identifying attenuations. Nonetheless, we are aware of the technical limitations of our approach. In particular, the use of purified bacterial genomic DNA instead of direct PCR on bacteria, as well as analysis by quantitative PCR, would improve the accuracy and sensibility of the method. However, our purpose was to design the simplest technique to test pools of bacterial mutants in vivo. We demonstrated here that PCR-based screening, without a rigorous purification step, complicated PCR set-up, or special equipment, is able to allow an easy and simultaneous detection of mutants impaired in virulence.
We used mutants for a major Listeria virulence factor to validate the efficiency of the in vivo selection and showed that highly attenuated mutants are easily detected by PCR-based screening. PCR-based screening allowed the detection of mutants with only one log decrease in virulence, which corresponds to the minimal decrease generally considered as a virulence attenuation (14). In addition, we observed a good correlation between the level of virulence attenuation measured by PCR-based screening and individual in vivo infection, thus definitively establishing the value of our strategy.
We chose to recover mouse organs 72h post infection at the peak of the infection. Since virulence factors can be expressed and active at different time points of the infection, in different organs, and following different routes of inoculation (16), the technique described here can be applied to bacterial mixtures extracted from various mouse organs, over the time course of the infection, and following diverse routes of inoculation.