RESUMO
The minimal genetic requirements for microbes to survive within multiorganism communities, including host-pathogen interactions, remain poorly understood. Here, we combined targeted gene mutagenesis with phenotype-guided genetic reassembly to identify a cooperative network of SPI-2 T3SS effector genes that are sufficient for Salmonella Typhimurium (STm) to cause disease in a natural host organism. Five SPI-2 effector genes support pathogen survival within the host cell cytoplasm by coordinating bacterial replication with Salmonella-containing vacuole (SCV) division. Unexpectedly, this minimal genetic repertoire does not support STm systemic infection of mice. In vivo screening revealed a second effector-gene network, encoded by the spv operon, that expands the life cycle of STm from growth in cells to deep-tissue colonization in a murine model of typhoid fever. Comparison between Salmonella infection models suggests how cooperation between effector genes drives tissue tropism in a pathogen group.
Assuntos
Proteínas de Bactérias/genética , Redes Reguladoras de Genes , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Animais , Proteínas de Bactérias/metabolismo , Citoplasma/microbiologia , Feminino , Ilhas Genômicas , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Óperon , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Tropismo , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , VirulênciaRESUMO
The classical secretory pathway is essential for the transport of a host of proteins to the cell surface and/or extracellular matrix. While the pathway is well-established, many factors still remain to be elucidated. One of the most relevant biological processes that occur during transport involves the cleavage of pro-proteins by enzymes residing in the endoplasmic reticulum/Golgi/TransGolgi Network compartment. Teasing out the requirements involved in the classical secretory pathway and cleavage during transport would shed new light into mis-regulation leading to disease. Current methodologies fail to link transport and cleavage at the single cell level. Here, we describe a cell-based assay that relies on an engineered protein scaffold that can discriminate between transport to the cell surface, in the absence or presence of cleavage. Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively. Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle. Importantly, this assay can be easily coupled to existing siRNA-based screens to identify novel regulators and effectors involved in transport and/or cleavage of cell surface proteins. In addition, unlike other in vivo based assays, the assay described here can also be easily adapted to drug discovery purposes.