Salmonella infections are a leading cause of bacterial foodborne illness in the United States and the European Union. Antimicrobial therapy is often administered to treat the infection but increasing isolates are being detected that demonstrate resistance to multiple antibiotics. Salmonella enterica contains two virulence related type-III secretion systems (T3SS): one promotes invasion of the intestine and the other one mediates systemic disease. Both of them secrete the SlrP protein acting as E3 ubiquitin ligase in human host cells where it targets thioredoxin-1 (Trx1). SlrP belongs to the NEL family of bacterial E3 ubiquitin ligases that have been observed in two distinct autoinhibitory conformations. We solved the 3D structure of the SlrP/Trx1 complex and determined the Trx1 ubiquitination site. The description of the substrate-binding mode sheds light on the first step of the activation mechanism of SlrP. Comparison with the available structural data of other NEL effectors allowed us to gain new insights into their autoinhibitory mechanism. We propose a molecular mechanism for the regulation of SlrP in which structural constraints sequestrating the NEL domain would be sequentially released. This work thus constitutes a new milestone in the understanding of how these T3SS effectors influence pathogen virulence. It also provides the fundamental basis for future development of new antimicrobials.

Virulence-related type III secretion systems are present in many Gram-negative bacterial pathogens. These complex devices translocate proteins, called effectors, from the bacterium into the eukaryotic host cell. Here, we identify the product of srfJ, a Salmonella enterica serovar Typhimurium gene regulated by SsrB, as a new substrate of the type III secretion system encoded by Salmonella pathogenicity island 2. The N-terminal 20-amino-acid segment of SrfJ was recognized as a functional secretion and translocation signal specific for this system. Transcription of srfJ was positively regulated by the PhoP/PhoQ system in an SsrBdependent manner and was negatively regulated by the Rcs system in an SsrB-independent manner. A screen for regulators of an srfJ-lacZ transcriptional fusion using the T-POP transposon identified IolR, the regulator of genes involved in myo-inositol utilization, as an srfJ repressor. Our results suggest that SrfJ is synthesized both inside the host, in response to intracellular conditions, and outside the host, in myo-inositol-rich environments.T ype III secretion systems (T3SS) are complex devices that are present in many Gram-negative bacteria, including the animal pathogens Yersinia spp., enteropathogenic Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri, Chlamydia spp., and Salmonella enterica, and the plant pathogens or symbionts Pseudomonas syringae, Ralstonia solanacearum, Erwinia spp., Rhizobium spp., and Xanthomonas campestris (29). T3SS allows secretion and translocation of effector proteins from bacteria to eukaryotic cells. The injected proteins are often able to interfere with host signal transduction pathways to enable bacterial invasion and survival in subcellular niches.Salmonella enterica is a facultative intracellular bacterium responsible for gastroenteritis and systemic infections in many animals, including humans (45). S. enterica virulence depends on two distinct T3SS, T3SS1 and T3SS2. T3SS1 translocates proteins through the plasma membrane of the host cell and is necessary for invasion, whereas T3SS2 is induced intracellularly, injects effector through the membrane of the Salmonella-containing vacuole (SCV), and is essential for survival and proliferation inside host cells. Both systems, however, depend on each other for efficient functioning (1). The genes encoding the structural components, many effectors, and some regulators of T3SS1 and T3SS2 are located in two Salmonella pathogenicity islands, SPI1 and SPI2, respectively (16,17,21,41,44,54). Some effector proteins are encoded by genes outside SPI1 and SPI2.SsrA (also named SpiR) and SsrB are the sensor and the response regulator, respectively, of a two-component regulatory system encoded by SPI2 that is necessary for the expression of T3SS2 (9, 28, 44). A genetic screen identified 12 genes (named srfA to srfM, for SsrB-regulated factors) outside SPI2 that were regulated by SsrB (62). These genes were located in horizontally acquired regions and, because of their pattern of expression, were suggested as c...

SlrP is an E3 ubiquitin ligase that can be translocated into eukaryotic host cells by the two type III secretion systems that are expressed by Salmonella enterica serovar Typhimurium and are encoded in Salmonella pathogenicity islands 1 (SPI1) and 2 (SPI2). Expression of slrP and translocation of its product were examined using lac, 3؋FLAG, and cyaA= translational fusions. Although slrP was expressed in different media, optimal expression was found under conditions that imitate the intravacuolar environment and promote synthesis of the SPI2-encoded type III secretion system. Translocation into mammalian cells took place through the SPI1-or the SPI2-encoded type III secretion system, depending on specific host cell type and timing. A search for genetic factors involved in controlling the expression of slrP unveiled LeuO, Lon, and the two-component system PhoQ/PhoP as novel regulators of slrP. Our experiments suggest that LeuO and Lon act through HilD under SPI1-inducing conditions, whereas PhoP directly interacts with the slrP promoter to activate transcription under SPI2 inducing conditions.

The virulence of the human and animal pathogen Salmonella enterica serovar Typhimurium is dependent on two type III secretion systems. These systems translocate proteins called effectors into eukaryotic host cells. SlrP is a Salmonella type III secretion effector with ubiquitin ligase activity. Here, we used two complementary proteomic approaches, two-dimensional gel electrophoresis and iTRAQ (isobaric tags for relative and absolute quantification) to study the consequences of the presence of SlrP in human epithelial cells. We identified 37 proteins that were differentially expressed in HeLa cells expressing slrP compared to control cells. Microarray analysis revealed that more than a half of differentially expressed proteins did not show changes in the transcriptome, suggesting post-transcriptional regulation. A gene ontology overrepresentation test carried out on the differentially expressed proteins revealed enrichment of ontology terms related to several types of junctions mediating adhesion in epithelial cells. Consistently, slrP-transfected cells showed defects in migration and adhesion. Our results suggest that the modification of cell-cell interaction ability of the host could be one of the final consequences of the action of SlrP during an infection.

CyaA from Bordetella pertussis is a calmodulin-dependent adenylate cyclase. Fusions to the catalytic domain of CyaA (CyaA') are useful tools to detect translocation of type III secretion system effectors from gram-negative pathogens like Salmonella enterica. These fusions are usually generated using plasmids with strong promoters. Here, we describe a protocol to insert the CyaA'-encoding sequence in a specific site in the bacterial chromosome in order to get a monocopy fusion whose expression is driven by the native promoter. We also describe the procedure to detect translocation of a CyaA' fusion into mammalian cells.