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.     

The Salmonella SPI1 effector SopB stimulates nitric oxide production long after invasion

The ability of Salmonella enterica to invade and replicate within host cells depends on two type III secretion systems (TTSSs) encoded on pathogenicity islands 1 and 2 (SPI1 and SPI2). The current paradigm holds that these systems translocate two classes of effectors that operate sequentially and independently. In essence, the SPI1 TTSS mediates early events (i.e. invasion) whereas the SPI2 TTSS mediates post-invasion processes (i.e. replication, vacuole maturation). Contrary to this model, we have found in infected macrophages that a SPI1 effector, SopB/SigD, increased inducible nitric oxide synthase levels and nitric oxide production, host cell process previously known only to be a target of the SPI2 TTSS. Furthermore, SopB protein and message persist many hours after invasion. Our findings reveal an unanticipated potential for dialogue between the SPI1 and SPI2 TTSS and the host cell response.     

Global Impact of Salmonella Pathogenicity Island 2-secreted Effectors on the Host Phosphoproteome

During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated.Type III secretion systems (T3SSs)¹ are specialized virulence factors in Gram-negative pathogens that play an important role in delivering effector proteins to host cells. Salmonella enterica employs two distinct T3SSs encoded in Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2), with numerous effectors encoded around the genome, including a small number in SPI1 and SPI2 (1). SPI1 T3SS effectors are required for the bacterial internalization by intestinal epithelial cells at early stages of infection after oral ingestion. Although Salmonella is subsequently taken up by intestinal macrophages via phagocytosis, SPI2 T3SS effectors function to promote intracellular replication. Part of the role of SPI2 effectors is to control the maturation of the membrane-enclosed, Salmonella-containing vacuole (SCV) where Salmonella survives and replicates, eventually leading to a systemic infection known as typhoid fever (2, 3).Approximately 30 effectors are known to be translocated by the SPI2 T3SS but the actions and targets of most of these effectors are largely unknown (1, 3, 4). A recent systematic study using a single mutant collection of SPI2 genes showed particular virulence factors (e.g. SpvB, SifA, and SteC) play a dominant role in replication within macrophages (5). It is known that SpvB induces cytotoxicity through its ADP-ribosyltransferase activity (6), and SifA is required for maturation of the SCV and the formation of Salmonella-induced filaments (7). SteC has been identified as the sole serine/threonine protein kinase encoded in the Salmonella genome (8), but the target substrates of this kinase within the host are not fully understood, although it has been demonstrated that SteC partially targets the MAP kinase MEK (9). Interestingly, SteC is capable of promoting assembly of an F-actin meshwork around the SCV; this is dependent on its kinase activity but does not require activation of signaling pathways through Rho-associated protein kinase (8), Cdc42, Rac, N-WASP, Scar/WAVE, and Arp2/3 (10). These host signaling proteins are the main targets of T3SS-secreted effectors from many pathogens, including the SPI1 system in Salmonella (11) and Shigella (12). Therefore, SteC is thought to manipulate actin in a unique way through phosphorylation of host protein target(s).Recent advances in high throughput measurements allow us to characterize host gene expression profiles (13) and host phosphoproteme dynamics (14) dependent on the presence of SPI1 effectors in an unbiased, comprehensive manner. However, although it is clear that SPI2 T3SS is a major virulence factor contributing to systemic infection, our knowledge of its effects on host responses is limited. In this study, we used a mass spectrometry (MS)-based quantitative proteomics approach and measured global host phosphorylation changes as well as proteome abundance altered by SPI2 effectors. Furthermore, we explore a molecular target of SPI2 effector kinase SteC by integrating the phosphoproteomics data and other quantitative proteomics screens.     

The SdiA-Regulated Gene srgE Encodes a Type III Secreted Effector

Salmonella enterica serovar Typhimurium is a food-borne pathogen that causes severe gastroenteritis. The ability of Salmonella to cause disease depends on two type III secretion systems (T3SSs) encoded in two distinct Salmonella pathogenicity islands, 1 and 2 (SPI1 and SPI2, respectively). S. Typhimurium encodes a solo LuxR homolog, SdiA, which can detect the acyl-homoserine lactones (AHLs) produced by other bacteria and upregulate the rck operon and the srgE gene. SrgE is predicted to encode a protein of 488 residues with a coiled-coil domain between residues 345 and 382. In silico studies have provided conflicting predictions as to whether SrgE is a T3SS substrate. Therefore, in this work, we tested the hypothesis that SrgE is a T3SS effector by two methods, a β-lactamase activity assay and a split green fluorescent protein (GFP) complementation assay. SrgE with β-lactamase fused to residue 40, 100, 150, or 300 was indeed expressed and translocated into host cells, but SrgE with β-lactamase fused to residue 400 or 488 was not expressed, suggesting interference by the coiled-coil domain. Similarly, SrgE with GFP S11 fused to residue 300, but not to residue 488, was expressed and translocated into host cells. With both systems, translocation into host cells was dependent upon SPI2. A phylogenetic analysis indicated that srgE is found only within Salmonella enterica subspecies. It is found sporadically within both typhoidal and nontyphoidal serovars, although the SrgE protein sequences found within typhoidal serovars tend to cluster separately from those found in nontyphoidal serovars, suggesting functional diversification.     

The Salmonella SPI2 Effector SseI Mediates Long-Term Systemic Infection by Modulating Host Cell Migration

Host-adapted strains of Salmonella enterica cause systemic infections and have the ability to persist systemically for long periods of time despite the presence of a robust immune response. Chronically infected hosts are asymptomatic and transmit disease to naïve hosts via fecal shedding of bacteria, thereby serving as a critical reservoir for disease. We show that the bacterial effector protein SseI (also called SrfH), which is translocated into host cells by the Salmonella Pathogenicity Island 2 (SPI2) type III secretion system (T3SS), is required for Salmonella typhimurium to maintain a long-term chronic systemic infection in mice. SseI inhibits normal cell migration of primary macrophages and dendritic cells (DC) in vitro, and such inhibition requires the host factor IQ motif containing GTPase activating protein 1 (IQGAP1), an important regulator of cell migration. SseI binds directly to IQGAP1 and co-localizes with this factor at the cell periphery. The C-terminal domain of SseI is similar to PMT/ToxA, a bacterial toxin that contains a cysteine residue (C1165) that is critical for activity. Mutation of the corresponding residue in SseI (C178A) eliminates SseI function in vitro and in vivo, but not binding to IQGAP1. In addition, infection with wild-type (WT) S. typhimurium suppressed DC migration to the spleen in vivo in an SseI-dependent manner. Correspondingly, examination of spleens from mice infected with WT S. typhimurium revealed fewer DC and CD4⁺ T lymphocytes compared to mice infected with ΔsseI S. typhimurium. Taken together, our results demonstrate that SseI inhibits normal host cell migration, which ultimately counteracts the ability of the host to clear systemic bacteria.     

Vaccination of Chickens with SPI1-lon and SPI1-lon-fliC Mutant of Salmonella enterica Serovar Enteritidis

The prevalence of Salmonella enterica serovar Enteritidis is gradually decreasing in poultry flocks in the EU, which may result in the demand for a vaccine that allows for the differentiation of vaccinated flocks from those infected by wild-type S. Enteritidis. In this study, we therefore constructed a (Salmonella Pathogenicity Island 1) SPI1-lon mutant with or without fliC encoding for S. Enteritidis flagellin. The combination of SPI1-lon mutations resulted in attenuated but immunogenic mutant suitable for oral vaccination of poultry. In addition, the vaccination of chickens with the SPI1-lon-fliC mutant enabled the serological differentiation of vaccinated and infected chickens. The absence of fliC therefore did not affect the immunogenicity of the vaccine strain and allowed for serological differentiation of the vaccinated chickens. The SPI1-lon-fliC mutant is therefore a suitable marker vaccine strain for oral vaccination of poultry.