RESUMEN
Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA, a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Código Genético , Salmonella typhimurium/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Factores de Virulencia/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Microscopía Confocal , Microscopía Fluorescente , Ingeniería de Proteínas , Salmonella typhimurium/genética , Sistemas de Secreción Tipo III/genética , Factores de Virulencia/genéticaRESUMEN
After Salmonella is phagocytosed, it resides in an acidic vacuole. Its cytoplasm acidifies to pH 5.6; acidification activates pathogenicity island 2 (SPI-2). SPI-2 encodes a type three secretion system whose effectors modify the vacuole, driving endosomal tubulation. Using super-resolution imaging in single bacterial cells, we show that low pH induces expression of the SPI-2 SsrA/B signaling system. Single particle tracking, atomic force microscopy, and single molecule unzipping assays identified pH-dependent stimulation of DNA binding by SsrB. A so-called phosphomimetic form (D56E) was unable to bind to DNA in live cells. Acid-dependent DNA binding was not intrinsic to regulators, as PhoP and OmpR binding was not pH-sensitive. The low level of SPI-2 injectisomes observed in single cells is not due to fluctuating SsrB levels. This work highlights the surprising role that acid pH plays in virulence and intracellular lifestyles of Salmonella; modifying acid survival pathways represents a target for inhibiting Salmonella.
Asunto(s)
Ácidos/farmacología , Proteínas Bacterianas/metabolismo , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/metabolismo , Conformación Molecular/efectos de los fármacos , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/efectos de los fármacos , Citoplasma , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Histidina Quinasa/metabolismo , Proteínas de la Membrana/efectos de los fármacos , Regiones Promotoras Genéticas , Salmonella typhimurium/citología , Salmonella typhimurium/genética , Transactivadores/metabolismo , Factores de Transcripción/efectos de los fármacos , Vacuolas/metabolismo , VirulenciaRESUMEN
The bglA gene of Escherichia coli encodes phospho-ß-glucosidase A capable of hydrolyzing the plant-derived aromatic ß-glucoside arbutin. We report that the sequential accumulation of mutations in bglA can confer the ability to hydrolyze the related aromatic ß-glucosides esculin and salicin in two steps. In the first step, esculin hydrolysis is achieved through the acquisition of a four-nucleotide insertion within the promoter of the bglA gene, resulting in enhanced steady-state levels of the bglA transcript. In the second step, hydrolysis of salicin is achieved through the acquisition of a point mutation within the bglA structural gene close to the active site without the loss of the original catabolic activity against arbutin. These studies underscore the ability of microorganisms to evolve additional metabolic capabilities by mutational modification of preexisting genetic systems under selection pressure, thereby expanding their repertoire of utilizable substrates.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Evolución Molecular , Glucósidos/metabolismo , Mutación Puntual , beta-Glucosidasa/genética , Secuencia de Bases , Alcoholes Bencílicos/química , Alcoholes Bencílicos/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glucósidos/química , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , beta-Glucosidasa/química , beta-Glucosidasa/metabolismoRESUMEN
We report that the bgl operon of Escherichia coli, encoding the functions necessary for the uptake and metabolism of aryl-ß-glucosides, is involved in the regulation of oligopeptide transport during stationary phase. Global analysis of intracellular proteins from Bgl-positive (Bgl(+)) and Bgl-negative (Bgl(-)) strains revealed that the operon exerts regulation on at least 12 downstream target genes. Of these, oppA, which encodes an oligopeptide transporter, was confirmed to be upregulated in the Bgl(+) strain. Loss of oppA function results in a partial loss of the growth advantage in stationary-phase (GASP) phenotype of Bgl(+) cells. The regulatory effect of the bgl operon on oppA expression is indirect and is mediated via gcvA, the activator of the glycine cleavage system, and gcvB, which regulates oppA at the posttranscriptional level. We show that BglG destabilizes the gcvA mRNA in vivo, leading to reduced expression of gcvA in the stationary phase. Deletion of gcvA results in the downregulation of gcvB and upregulation of oppA and can partially rescue the loss of the GASP phenotype seen in ΔbglG strains. A possible mechanism by which oppA confers a competitive advantage to Bgl(+) cells relative to Bgl(-) cells is discussed.