RESUMEN
The facultative intracellular pathogen Salmonella enterica serovar Typhimurium relies on its Salmonella pathogenicity island 2 (SPI2) type III secretion system (T3SS) for intracellular replication and virulence. We report that the oxidoreductase thioredoxin 1 (TrxA) and SPI2 are coinduced for expression under in vitro conditions that mimic an intravacuolar environment, that TrxA is needed for proper SPI2 activity under these conditions, and that TrxA is indispensable for SPI2 activity in both phagocytic and epithelial cells. Infection experiments in mice demonstrated that SPI2 strongly contributed to virulence in a TrxA-proficient background whereas SPI2 did not affect virulence in a trxA mutant. Complementation analyses using wild-type trxA or a genetically engineered trxA coding for noncatalytic TrxA showed that the catalytic activity of TrxA is essential for SPI2 activity in phagocytic cells whereas a noncatalytic variant of TrxA partially sustained SPI2 activity in epithelial cells and virulence in mice. These results show that TrxA is needed for the intracellular induction of SPI2 and provide new insights into the functional integration between catalytic and noncatalytic activities of TrxA and a bacterial T3SS in different settings of intracellular infections.
Asunto(s)
Proteínas Bacterianas/fisiología , Islas Genómicas/fisiología , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad , Tiorredoxinas/fisiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Perros , Femenino , Citometría de Flujo , Regulación Bacteriana de la Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Islas Genómicas/genética , Immunoblotting , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Salmonelosis Animal/microbiología , Salmonella typhimurium/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Virulencia/genéticaRESUMEN
A collection of nine salicylidene acylhydrazide compounds were tested for their ability to inhibit the activity of virulence-associated type III secretion systems (T3SSs) in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island 1 (SPI1) T3SS-mediated invasion of epithelial cells and in vitro secretion of SPI1 invasion-associated effector proteins. The use of a SPI1 effector beta-lactamase fusion protein implicated intracellular entrapment of the protein construct upon application of a salicylidene acylhydrazide, whereas the use of chromosomal transcriptional gene fusions revealed a compound-mediated transcriptional silencing of SPI1. Salicylidene acylhydrazides also affected intracellular bacterial replication in murine macrophage-like cells and blocked the transport of an epitope-tagged SPI2 effector protein. Two of the compounds significantly inhibited bacterial motility and expression of extracellular flagellin. We conclude that salicylidene acylhydrazides affect bacterial T3SS activity in S. enterica and hence could be used as lead substances when designing specific inhibitors of bacterial T3SSs in order to pharmaceutically intervene with bacterial virulence.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Salmonella typhimurium/efectos de los fármacos , Animales , Antibacterianos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Perros , Células Epiteliales/microbiología , Flagelos/fisiología , Regulación Bacteriana de la Expresión Génica , Humanos , Hidrazinas/química , Hidrazinas/farmacología , Macrófagos/microbiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Salmonella typhimurium/patogenicidad , Salmonella typhimurium/fisiología , VirulenciaRESUMEN
Mutational inactivation of the cold-shock-associated exoribonuclease polynucleotide phosphorylase (PNPase; encoded by the pnp gene) in Salmonella enterica serovar Typhimurium was previously shown to enable the bacteria to cause chronic infection and to affect the bacterial replication in BALB/c mice (M. O. Clements et al., Proc. Natl. Acad. Sci. USA 99:8784-8789, 2002). Here, we report that PNPase deficiency results in increased expression of Salmonella plasmid virulence (spv) genes under in vitro growth conditions that allow induction of spv expression. Furthermore, whole-genome microarray-based transcriptome analyses of bacteria growing inside murine macrophage-like J774.A.1 cells revealed six genes as being significantly up-regulated in the PNPase-deficient background, which included spvABC, rtcB, entC, and STM2236. Mutational inactivation of the spvR regulator diminished the increased expression of spv observed in the pnp mutant background, implying that PNPase acts upstream of or at the level of SpvR. Finally, competition experiments revealed that the growth advantage of the pnp mutant in BALB/c mice was dependent on spvR as well. Combined, our results support the idea that in S. enterica PNPase, apart from being a regulator of the cold shock response, also functions in tuning the expression of virulence genes and bacterial fitness during infection.