title: PCR-based screening of targeted mutants for the fast and simultaneous identification of bacterial virulence factors.
abstract: 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.
authors: IBMC - Instituto de Biologia Molecular e Celular, Group of Molecular Microbiology, Universidade do Porto, Porto, Portugal.
authors: Ana
authors: A
authors: Henriques
authors: IBMC - Instituto de Biologia Molecular e Celular, Group of Molecular Microbiology, Universidade do Porto, Porto, Portugal.
authors: Filipe
authors: F
authors: Carvalho
authors: IBMC - Instituto de Biologia Molecular e Celular, Group of Molecular Microbiology, Universidade do Porto, Porto, Portugal.
authors: Rita
authors: R
authors: Pombinho
authors: IBMC - Instituto de Biologia Molecular e Celular, Group of Molecular Microbiology, Universidade do Porto, Porto, Portugal.
authors: Olga
authors: O
authors: Reis
authors: IBMC - Instituto de Biologia Molecular e Celular, Group of Molecular Microbiology, Universidade do Porto, Porto, Portugal.
authors: Sandra
authors: S
authors: Sousa
authors: IBMC - Instituto de Biologia Molecular e Celular, Group of Molecular Microbiology, Universidade do Porto, Porto, Portugal.
authors: Didier
authors: D
authors: Cabanes
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D005798:Genes, Bacterial / Q000235:genetics*; D054884:Host-Pathogen Interactions / Q000235:genetics; D008089:Listeria monocytogenes / Q000235:genetics* / Q000472:pathogenicity*; D008088:Listeriosis / Q000382:microbiology; D051379:Mice; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014774:Virulence / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D037521:Virulence Factors
keywords: Listeria; PCR, infection biology; mutagenesis screening; pathogen; virulence-gene