title: An in vivo gene deletion system for determining temporal requirement of bacterial virulence factors.
abstract: Analysis of phenotypes associated with specific mutants has been instrumental in determining the roles of a bacterial gene in a biological process. However, this technique does not allow one to address whether a specific gene or gene set is necessary to maintain such a process once it has been established. In the study of microbial pathogenesis, it is important but difficult to determine the temporal requirement of essential pathogenic determinants in the entire infection cycle. Here we report a Cre/loxP-based genetic system that allowed inducible deletion of specific bacterial genes after the pathogen had been phagocytosed by host cells. Using this system, we have examined the temporal requirement of the Dot/Icm type IV protein transporter of Legionella pneumophila during infection. We found that deletion of single essential dot/icm genes did not prevent the internalized bacteria from completing one cycle of intracellular replication. Further analyses indicate that the observed phenotypes were due to the high stability of the examined Dot/Icm protein. However, postinfection deletion within 8 h of the gene coding for the Dot/Icm substrate, SdhA, abolishes intracellular bacterial growth. This result indicates that the Dot/Icm transporter is important for intracellular bacterial growth after the initial biogenesis of the vacuole. Our study has provided a technical concept for analyzing the temporal requirement of specific bacterial proteins or protein complexes in infection or development.
authors.affiliation: Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, IN 47907, USA.
authors.forename: Yancheng
authors.initials: Y
authors.lastname: Liu
authors.forename: Ping
authors.initials: P
authors.lastname: Gao
authors.forename: Simran
authors.initials: S
authors.lastname: Banga
authors.forename: Zhao-Qing
authors.initials: ZQ
authors.lastname: Luo
mesh_terms: D001424:Bacterial Infections / Q000382:microbiology; D001426:Bacterial Proteins / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002876:Chromosomes, Bacterial / Q000378:metabolism; D017353:Gene Deletion; D005798:Genes, Bacterial; D042541:Intracellular Space / Q000187:drug effects / Q000382:microbiology; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D016952:Legionella pneumophila / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D008264:Macrophages / Q000187:drug effects / Q000382:microbiology; D026901:Membrane Transport Proteins / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects; D013997:Time Factors; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; C432955:ICMQ protein, Legionella pneumophila; D026901:Membrane Transport Proteins; D037521:Virulence Factors; D007544:Isopropyl Thiogalactoside