Transfer of the Salmonella type III effector sopE between unrelated phage families.

Salmonella spp. are pathogenic enterobacteria that employ type III secretion systems to translocate effector proteins and modulate responses of host cells. The repertoire of translocated effector proteins is thought to define host specificity and epidemic virulence, and varies even between closely related Salmonella strains. Therefore, horizontal transfer of effector protein genes between Salmonella strains plays a key role in shaping the Salmonella-host interaction. Several effector protein genes are located in temperate phages. The P2-like phage SopE Phi encodes SopE and the lambda-like GIFSY phages encode several effector proteins of the YopM/IpaH-family. Lysogenic conversion with these phages is responsible for much of the diversity of the effector protein repertoires observed among Salmonella spp. However, free exchange of effector proteins by lysogenic conversion can be restricted by superinfection immunity. To identify genetic mechanisms that may further enhance horizontal transfer of effector genes, we have analyzed sopE loci from Salmonella spp. that do not harbor P2-like sequences of SopE Phi. In two novel sopE loci that were identified, the 723 nt sopE gene is located in a conserved 1.2 kb cassette present also in SopE Phi. Most strikingly, in Salmonella enterica subspecies I serovars Gallinarum, Enteritidis, Hadar and Dublin, the sopE-cassette is located in a cryptic lambda-like prophage with similarity to the GIFSY phages. This provides the first evidence for transfer of virulence genes between different phage families. We show that such a mechanism can circumvent restrictions to phage-mediated gene transfer and thereby enhances reassortment of the effector protein repertoires in Salmonella spp.

Salmonella host cell invasion emerged by acquisition of a mosaic of separate genetic elements, including Salmonella pathogenicity island 1 (SPI1), SPI5, and sopE2.

Salmonella spp. possess a conserved type III secretion system encoded within the pathogenicity island 1 (SPI1; centisome 63), which mediates translocation of effector proteins into the host cell cytosol to trigger responses such as bacterial internalization. Several translocated effector proteins are encoded in other regions of the Salmonella chromosome. It remains unclear how this complex chromosomal arrangement of genes for the type III apparatus and the effector proteins emerged and how the different effector proteins cooperate to mediate virulence. By Southern blotting, PCR, and phylogenetic analyses of highly diverse Salmonella spp., we show here that effector protein genes located in the core of SPI1 are present in all Salmonella lineages. Surprisingly, the same holds true for several effector protein genes located in distant regions of the Salmonella chromosome, namely, sopB (SPI5, centisome 20), sopD (centisome 64), and sopE2 (centisomes 40 to 42). Our data demonstrate that sopB, sopD, and sopE2, along with SPI1, were already present in the last common ancestor of all contemporary Salmonella spp. Analysis of Salmonella mutants revealed that host cell invasion is mediated by SopB, SopE2, and, in the case of Salmonella enterica serovar Typhimurium SL1344, by SopE: a sopB sopE sopE2-deficient triple mutant was incapable of inducing membrane ruffling and was >100-fold attenuated in host cell invasion. We conclude that host cell invasion emerged early during evolution by acquisition of a mosaic of genetic elements (SPI1 itself, SPI5 [sopB], and sopE2) and that the last common ancestor of all contemporary Salmonella spp. was probably already invasive.

Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for Cdc42 of the host cell.

Salmonella typhimurium translocates effector proteins into host cells via the SPI1 type III secretion system to induce responses such as membrane ruffling and internalization by non-phagocytic cells. Activation of the host cellular RhoGTPase Cdc42 is thought to be a key event during internalization. The translocated Salmonella protein SopE is an activator for Cdc42. Because SopE is absent from most S. typhimurium strains it remains unclear whether all S. typhimurium strains rely on activation of Cdc42 to invade host cells. We have identified SopE2, a translocated effector protein common to all S. typhimurium strains. SopE2 is a guanine nucleotide exchange factor for Cdc42 and shows 69% sequence similarity to SopE. Analysis of S. typhimurium mutants demonstrated that SopE2 plays a role in recruitment of the actin-nucleating Arp2/3 complex to the membrane ruffles and in efficient host cell invasion. Transfection experiments showed that SopE2 is sufficient to activate host cellular Cdc42, to recruit the actin-nucleating Arp2/3 complex and to induce actin cytoskeletal rearrangements and internalization. In conclusion, as a result of SopE2 all S. typhimurium strains tested have the capacity to activate Cdc42 signalling inside host cells which is important to ensure efficient entry.

Prevalence and polymorphism of genes encoding translocated effector proteins among clinical isolates of Salmonella enterica.

Pathogenic Salmonella enterica strains are capable of causing local and/or systemic infections. They employ two type III secretion systems to translocate an array of virulence-associated proteins (effector proteins) directly into the cytosol of target cells of the host. Earlier data had shown that changes in the repertoire of translocated effector proteins may contribute to the adaptation of Salmonella strains to new hosts and to the emergence of epidemic strains. Using PCR and Southern blot techniques the presence of and the polymorphism among the genes encoding the translocated effector proteins SopB, SopD, SopE, SopE2, SipA, SipB, SipC, AvrA, and SptP was studied in 71 phylogenetically well characterised S. enterica subspecies I (subspecies enterica) strains of the SARB collection and in 209 clinical and epidemic isolates of S. enterica subspecies I belonging to various serovars, phage types, and genotypes. All these Salmonella strains harbour all these respective genes with the exception of sopE and avrA which have been identified in only some of them. Several of the studied genes display genetic polymorphisms (RFLP). These RFLP patterns did not show a strict correlation with the genetic distance, the grouping genes in order to understand their role in the evolution of Salmonella as a pathogen.

Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for RhoGTPases.

RhoGTPases are key regulators of eukaryotic cell physiology. The bacterial enteropathogen Salmonella typhimurium modulates host cell physiology by translocating specific toxins into the cytoplasm of host cells that induce responses such as apoptotic cell death in macrophages, the production of proinflammatory cytokines, the rearrangement of the host cell actin cytoskeleton (membrane ruffling), and bacterial entry into host cells. One of the translocated toxins is SopE, which has been shown to bind to RhoGTPases of the host cell and to activate RhoGTPase signaling. SopE is sufficient to induce profuse membrane ruffling in Cos cells and to facilitate efficient bacterial internalization. We show here that SopE belongs to a novel class of bacterial toxins that modulate RhoGTPase function by transient interaction. Surface plasmon resonance measurements revealed that the kinetics of formation and dissociation of the SopE.CDC42 complex are in the same order of magnitude as those described for complex formation of GTPases of the Ras superfamily with their cognate guanine nucleotide exchange factors (GEFs). In the presence of excess GDP, dissociation of the SopE.CDC42 complex was accelerated more than 1000-fold. SopE-mediated guanine nucleotide exchange was very efficient (e.g. exchange rates almost 10(5)-fold above the level of the uncatalyzed reaction; substrate affinity), and the kinetic constants were similar to those described for guanine nucleotide exchange mediated by CDC25 or RCC1. Far-UV CD spectroscopy revealed that SopE has a high content of alpha-helical structure, a feature also found in Dbl homology domains, Sec7-like domains, and the Ras-GEF domain of Sos. Despite the lack of any obvious sequence similarity, our data suggest that SopE may closely mimic eukaryotic GEFs.

Isolation of a temperate bacteriophage encoding the type III effector protein SopE from an epidemic Salmonella typhimurium strain.

Salmonella typhimurium employs the specialized type III secretion system encoded in pathogenicity island 1 (SPI1) to translocate effector proteins into host cells and to modulate host cell signal transduction. The SPI1 type III system and the effector proteins are conserved among all salmonellae and are thought to be acquired by horizontal gene transfer. The genetic mechanisms mediating this horizontal transfer are unknown. Here, we describe that SopE, a SPI1-dependent translocated effector protein, is present in relatively few S. typhimurium isolates. We have isolated a temperate phage that encodes SopE. Phage morphology and DNA hybridization, as well as partial sequence information, suggest that this phage (SopEPhi) is a new member of the P2 family of bacteriophages. By lysogenic conversion this phage can horizontally transfer genes between different S. typhimurium strains. Strikingly, most of the isolates harboring SopEPhi belong to the small group of epidemic strains of S. typhimurium that have been responsible for a large percentage of human and animal salmonellosis and have persisted for a long period of time. Our data suggest that horizontal transfer of type III dependent effector proteins by lysogenic infection with bacteriophages (lysogenic conversion) may provide an efficient mechanism for fine-tuning the interaction of Salmonella spp. with their hosts.