Salmonella Pathogenicity Island 1 T3SS Research Articles

SALMONELLA INFECTION: INTERPLAY BETWEEN THE T3SSS EFFECTORS AND NF-ΚB SIGNALING PATHWAY

Salmonella is an important foodborne pathogen that can evade host immune defense by evolving unique mechanisms. Salmonella manipulate host cell various signaling pathways by delivering specific effectors into target cells to establish infection. The nuclear factor-κB (NF-κB) is an important nuclear transcription factor that regulates the host immune system in Salmonella infection. The Salmonella pathogenicity island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2) encode type III secretion systems (T3SSs), effectors that are associated with the NF-κB signaling pathway through regulate host inflammation response. SPI-1 effectors SipA, SopE, SopE2, and SopB all can activate NF-κB signaling pathway to facilitate Salmonella invasion and intracellular carriage. Studies have shown that T3SS1 and/or T3SS2 effectors such as GtgA, GogA and PipA contain two histidine residues and have metalloprotease activity to control Salmonella replication. These zinc metalloproteases redundantly target the NF-κB subunits p65, RelB, and c-Rel, whereas GogA and GtgA only inhibit NF-κB-dependent gene transcription. The T3SS2 effectors SseK1, SseK2, and SseK3 are death domain-containing proteins with N-linked glycosyltransferase characteristics that can inhibit NF-κB activity by inhibiting IκBα phosphorylation in TNF- α-treated 293ET cells. Among them, SseK1 and SseK3 also suppress Salmonella-induced NF-κB activity in macrophages. SseK3-mediated inhibition of the NF-B signaling pathway is not required for protein 32 containing a tripartite E3-ubiquitin ligase motif. In addition, the SPI-2 T3SS effector SpvD inhibits NF-κB activity by preventing nuclear translocation of p65 through interaction with Exportin-2, but this does not affect IκBα degradation, which ultimately leads to systemic Salmonella growth. However, other effectors SptP, AvrA, IpaJ, SspH1, GtgA, GogA, and SPI-2 encoded SseL, SpvB, SseK1, and GogB all can effectively inhibit NF-κB signaling pathway, and contribute to Salmonella intracellular replication and virulence. In this mini-review, we summarize the special mechanism how NF-κB signaling pathway is regulated by Salmonella T3SSs effectors in the persistent infection of Salmonella, which will further elucidate the pathogenesis of Salmonella.

Construction of a constitutively active type III secretion system for heterologous protein secretion.

Proteins comprise a multibillion-dollar industry in enzymes and therapeutics, but bacterial protein production can be costly and inefficient. Proteins of interest (POIs) must be extracted from lysed cells and inclusion bodies, purified, and resolubilized, which adds significant time and cost to the protein-manufacturing process. The Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS) has been engineered to address these problems by secreting soluble, active proteins directly into the culture media, reducing the number of purification steps. However, the current best practices method of T3SS pathway activation is not ideal for industrial scaleup. Previously, the T3SS was activated by plasmid-based overexpression of the T3SS transcriptional regulator, hilA, which requires the addition of a small molecule inducer (IPTG) to the culture media. IPTG adds significant cost to production and plasmid-based expression is subject to instability in large-scale fermentation. Here, we modulate the upstream transcriptional regulator, hilD, to activate the T3SS via three distinct methods. In doing so, we develop a toolbox of T3SS activation methods and construct constitutively active T3SS strains capable of secreting a range of heterologous proteins at titers comparable to plasmid-based hilA overexpression. We also explore how each activation method in our toolbox impacts the SPI-1 regulatory cascade and discover an epistatic relationship between T3SS regulators, hilE and the hilD 3' untranslated region (hilD 3'UTR). Together, these findings further our goal of making an industrially competitive protein production strain that reduces the challenges associated with plasmid induction and maintenance. KEY POINTS: • Characterized 3 new type III secretion system (T3SS) activation methods for heterologous protein secretion, including 2 constitutive activation methods. • Eliminated the need for a second plasmid and a small molecule inducer to activate the system, making it more suitable for industrial production. • Discovered new regulatory insights into the SPI-1 T3SS, including an epistatic relationship between regulators hilE and the hilD 3' untranslated region.

Envelope Stress and Regulation of the Salmonella Pathogenicity Island 1 Type III Secretion System.

Salmonella enterica serovar Typhimurium uses a type three secretion system (T3SS) encoded on the Salmonella pathogenicity island 1 (SPI1) to invade intestinal epithelial cells and induce inflammatory diarrhea. The SPI1 T3SS is regulated by numerous environmental and physiological signals, integrated to either activate or repress invasion. Transcription of hilA, encoding the transcriptional activator of the SPI1 structural genes, is activated by three AraC-like regulators, HilD, HilC, and RtsA, that act in a complex feed-forward loop. Deletion of bamB, encoding a component of the β-barrel assembly machinery, causes a dramatic repression of SPI1, but the mechanism was unknown. Here, we show that partially defective β-barrel assembly activates the RcsCDB regulon, leading to decreased hilA transcription. This regulation is independent of RpoE activation. Though Rcs has been previously shown to repress SPI1 when disulfide bond formation is impaired, we show that activation of Rcs in a bamB background is dependent on the sensor protein RcsF, whereas disulfide bond status is sensed independently. Rcs decreases transcription of the flagellar regulon, including fliZ, the product of which indirectly activates HilD protein activity. Rcs also represses hilD, hilC, and rtsA promoters by an unknown mechanism. Both dsbA and bamB mutants have motility defects, though this is simply regulatory in a bamB background; motility is restored in the absence of Rcs. Effector secretion assays show that repression of SPI1 in a bamB background is also regulatory; if expressed, the SPI1 T3SS is functional in a bamB background. This emphasizes the sensitivity of SPI1 regulation to overall envelope homeostasis.IMPORTANCESalmonella causes worldwide foodborne illness, leading to massive disease burden and an estimated 600,000 deaths per year. Salmonella infects orally and invades intestinal epithelial cells using a type 3 secretion system that directly injects effector proteins into host cells. This first step in invasion is tightly regulated by a variety of inputs. In this work, we demonstrate that Salmonella senses the functionality of outer membrane assembly in determining regulation of invasion machinery, and we show that Salmonella uses distinct mechanisms to detect specific perturbations in envelope assembly.

SrcA is a chaperone for the Salmonella SPI-2 type three secretion system effector SteD.

Effector proteins of type three secretion systems (T3SS) often require cytosolic chaperones for their stabilization, to interact with the secretion machinery and to enable effector delivery into host cells. We found that deletion of srcA, previously shown to encode a chaperone for the Salmonella pathogenicity island 2 (SPI-2) T3SS effectors SseL and PipB2, prevented the reduction of mature Major Histocompatibility Complex class II (mMHCII) from the surface of antigen-presenting cells during Salmonella infection. This activity was shown previously to be caused by the SPI-2 T3SS effector SteD. Since srcA and steD are located in the same operon on the Salmonella chromosome, this suggested that the srcA phenotype might be due to an indirect effect on SteD. We found that SrcA is not translocated by the SPI-2 T3SS but interacts directly and forms a stable complex with SteD in bacteria with a 2 : 1 stoichiometry. We found that SrcA was not required for SPI-2 T3SS-dependent, neutral pH-induced secretion of either SseL or PipB2 but was essential for secretion of SteD. SrcA therefore functions as a chaperone for SteD, explaining its requirement for the reduction in surface levels of mMHCII.

The Type III Secretion System Effector SeoC of Salmonella enterica subsp. salamae and S. enterica subsp. arizonae ADP-Ribosylates Src and Inhibits Opsonophagocytosis.

Salmonella species utilize type III secretion systems (T3SSs) to translocate effectors into the cytosol of mammalian host cells, subverting cell signaling and facilitating the onset of gastroenteritis. In this study, we compared a draft genome assembly of Salmonella enterica subsp. salamae strain 3588/07 against the genomes of S. enterica subsp. enterica serovar Typhimurium strain LT2 and Salmonella bongori strain 12419. S. enterica subsp. salamae encodes the Salmonella pathogenicity island 1 (SPI-1), SPI-2, and the locus of enterocyte effacement (LEE) T3SSs. Though several key S. Typhimurium effector genes are missing (e.g., avrA, sopB, and sseL), S. enterica subsp. salamae invades HeLa cells and contains homologues of S. bongori sboK and sboC, which we named seoC. SboC and SeoC are homologues of EspJ from enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), which inhibit Src kinase-dependent phagocytosis by ADP-ribosylation. By screening 73 clinical and environmental Salmonella isolates, we identified EspJ homologues in S. bongori, S. enterica subsp. salamae, and Salmonella enterica subsp. arizonae. The β-lactamase TEM-1 reporter system showed that SeoC is translocated by the SPI-1 T3SS. All the Salmonella SeoC/SboC homologues ADP-ribosylate Src E310 in vitro. Ectopic expression of SeoC/SboC inhibited phagocytosis of IgG-opsonized beads into Cos-7 cells stably expressing green fluorescent protein (GFP)-FcγRIIa. Concurrently, S. enterica subsp. salamae infection of J774.A1 macrophages inhibited phagocytosis of beads, in a seoC-dependent manner. These results show that S. bongori, S. enterica subsp. salamae, and S. enterica subsp. arizonae share features of the infection strategy of extracellular pathogens EPEC and EHEC and shed light on the complexities of the T3SS effector repertoires of Enterobacteriaceae.

What makes Xanthomonas albilineans unique amongst xanthomonads?

Xanthomonas albilineans causes leaf scald, a lethal disease of sugarcane. Compared to other species of Xanthomonas, X. albilineans exhibits distinctive pathogenic mechanisms, ecology and taxonomy. Its genome, which has experienced significant erosion, has unique genomic features. It lacks two loci required for pathogenicity in other plant pathogenic species of Xanthomonas: the xanthan gum biosynthesis and the Hrp-T3SS (hypersensitive response and pathogenicity-type three secretion system) gene clusters. Instead, X. albilineans harbors in its genome an SPI-1 (Salmonella pathogenicity island-1) T3SS gene cluster usually found in animal pathogens. X. albilineans produces a potent DNA gyrase inhibitor called albicidin, which blocks chloroplast differentiation, resulting in the characteristic white foliar stripe symptoms. The antibacterial activity of albicidin also confers on X. albilineans a competitive advantage against rival bacteria during sugarcane colonization. Recent chemical studies have uncovered the unique structure of albicidin and allowed us to partially elucidate its fascinating biosynthesis apparatus, which involves an enigmatic hybrid PKS/NRPS (polyketide synthase/non-ribosomal peptide synthetase) machinery.

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.

Identification ofSalmonellaPathogenicity Island-2 Type III Secretion System Effectors Involved in Intramacrophage Replication of S. enterica Serovar Typhimurium: Implications for Rational Vaccine Design

ABSTRACTSalmonella enterica serovars cause severe diseases in humans, such as gastroenteritis and typhoid fever. The development of systemic disease is dependent on a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). Translocation of effector proteins across the Salmonella-containing vacuole, via the SPI-2 T3SS, enables bacterial replication within host cells, including macrophages. Here, we investigated the contribution of these effectors to intramacrophage replication of Salmonella enterica serovar Typhimurium using Fluorescence Dilution, a dual-fluorescence tool which allows direct measurement of bacterial replication. Of 32 strains, each carrying single mutations in genes encoding effectors, 10 (lacking sifA, sseJ, sopD2, sseG, sseF, srfH, sseL, spvD, cigR, or steD) were attenuated in replication in mouse bone marrow-derived macrophages. The replication profiles of strains combining deletions in effector genes were also investigated: a strain lacking the genes sseG, sopD2, and srfH showed an increased replication defect compared to single-mutation strains and was very similar to SPI-2 T3SS-deficient bacteria with respect to its replication defect. This strain was substantially attenuated in virulence in vivo and yet retained intracellular vacuole integrity and a functional SPI-2 T3SS. Moreover, this strain was capable of SPI-2 T3SS-mediated delivery of a model antigen for major histocompatibility complex (MHC) class I-dependent T-cell activation. This work establishes a basis for the use of a poly-effector mutant strain as an attenuated vaccine carrier for delivery of heterologous antigens directly into the cytoplasm of host cells.

Lack of Effect of the Salmonella Deubiquitinase SseL on the NF-κB Pathway

Intracellular replication of Salmonella enterica requires effector proteins translocated across the Salmonella-containing vacuolar membrane by Salmonella pathogenicity island-2 (SPI-2) encoded type III secretion system (T3SS). The SPI-2 T3SS effector SseL is a deubiquitinase that contributes to virulence in mice. Previous work has produced conflicting evidence as to the involvement of SseL in interference with the NF-κB pathway. To attempt to clarify these discrepancies, we compared mRNA levels in mouse primary bone marrow-derived macrophages infected with wild-type or sseL mutant strains using a genome-wide microarray. There was no detectable effect of loss of SseL on mRNA levels corresponding to any known NF-κB-regulated gene. In addition, there was no effect of SseL on (i) the activation or levels of both the canonical inhibitor of the NF-κB pathway (IκBα and phospho-IκBα), and the non-canonical NF-κB precursor p100/p52, (ii) the translocation of the NF-κB transcription factor p65 to the nucleus of infected macrophages and (iii) pro-inflammatory cytokines secretion. Furthermore, ectopic expression of SseL did not affect NF-κB activation in reporter cell lines. These results fail to support a role for SseL in the down-regulation of the host immune response and in particular the NF-κB pathway.

The Salmonella Deubiquitinase SseL Inhibits Selective Autophagy of Cytosolic Aggregates

Cell stress and infection promote the formation of ubiquitinated aggregates in both non-immune and immune cells. These structures are recognised by the autophagy receptor p62/sequestosome 1 and are substrates for selective autophagy. The intracellular growth of Salmonella enterica occurs in a membranous compartment, the Salmonella-containing vacuole (SCV), and is dependent on effectors translocated to the host cytoplasm by the Salmonella pathogenicity island-2 (SPI-2) encoded type III secretion system (T3SS). Here, we show that bacterial replication is accompanied by the formation of ubiquitinated structures in infected cells. Analysis of bacterial strains carrying mutations in genes encoding SPI-2 T3SS effectors revealed that in epithelial cells, formation of these ubiquitinated structures is dependent on SPI-2 T3SS effector translocation, but is counteracted by the SPI-2 T3SS deubiquitinase SseL. In macrophages, both SPI-2 T3SS-dependent aggregates and aggresome-like induced structures (ALIS) are deubiquitinated by SseL. In the absence of SseL activity, ubiquitinated structures are recognized by the autophagy receptor p62, which recruits LC3 and targets them for autophagic degradation. We found that SseL activity lowers autophagic flux and favours intracellular Salmonella replication. Our data therefore show that there is a host selective autophagy response to intracellular Salmonella infection, which is counteracted by the deubiquitinase SseL.

Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors

Salmonella enterica serovars cause severe disease in humans, such as gastroenteritis and typhoid fever. The bacteria are able to invade and replicate within host cells, including epithelial cells and macrophages. Pathogenesis of Salmonella is facilitated by a type III secretion system (T3SS) encoded by genes of Salmonella pathogenicity island 2 (SPI-2). Intracellular replication occurs in a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and depends on translocation of approximately 30 effector proteins via the SPI-2 T3SS into the host endomembrane system and cytoplasm. In this review we discuss the many different functions of these effectors, which range from maintaining the integrity of the SCV and its juxtanuclear location, to interference with the host cytoskeleton and immune signalling.

Effect of the Salmonella Pathogenicity Island 2 Type III Secretion System on Salmonella Survival in Activated Chicken Macrophage-Like HD11 Cells

In order to better identify the role of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (T3SS) in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a wild-type Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) strain, a SPI-2 mutant S. Typhimurium strain, a wild-type Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-uptake (PU) by the HD11 cells, up to 24 h PU, while the E. coli strain was no longer recoverable by 3 h PU. We can conclude from these observations that the SPI-2 T3SS of S. Typhimurium and S. Enteritidis is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment, as E. coli is effectively eliminated.

Tandem Translation Generates a Chaperone for the Salmonella Type III Secretion System Protein SsaQ

Type III secretion systems (T3SSs) of bacterial pathogens involve the assembly of a surface-localized needle complex, through which translocon proteins are secreted to form a pore in the eukaryotic cell membrane. This enables the transfer of effector proteins from the bacterial cytoplasm to the host cell. A structure known as the C-ring is thought to have a crucial role in secretion by acting as a cytoplasmic sorting platform at the base of the T3SS. Here, we studied SsaQ, an FliN-like putative C-ring protein of the Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS. ssaQ produces two proteins by tandem translation: a long form (SsaQ(L)) composed of 322 amino acids and a shorter protein (SsaQ(S)) comprising the C-terminal 106 residues of SsaQ(L). SsaQ(L) is essential for SPI-2 T3SS function. Loss of SsaQ(S) impairs the function of the T3SS both ex vivo and in vivo. SsaQ(S) binds to its corresponding region within SsaQ(L) and stabilizes the larger protein. Therefore, SsaQ(L) function is optimized by a novel chaperone-like protein, produced by tandem translation from its own mRNA species.

Correction: The role of coupled positive feedback in the expression of the SPI1 type three secretion system in Salmonella.

Salmonella enterica serovar Typhimurium is a common food-borne pathogen that induces inflammatory diarrhea and invades intestinal epithelial cells using a type three secretion system (T3SS) encoded within Salmonella pathogenicity island 1 (SPI1). The genes encoding the SPI1 T3SS are tightly regulated by a network of interacting transcriptional regulators involving three coupled positive feedback loops. While the core architecture of the SPI1 gene circuit has been determined, the relative roles of these interacting regulators and associated feedback loops are still unknown. To determine the function of this circuit, we measured gene expression dynamics at both population and single-cell resolution in a number of SPI1 regulatory mutants. Using these data, we constructed a mathematical model of the SPI1 gene circuit. Analysis of the model predicted that the circuit serves two functions. The first is to place a threshold on SPI1 activation, ensuring that the genes encoding the T3SS are expressed only in response to the appropriate combination of environmental and cellular cues. The second is to amplify SPI1 gene expression. To experimentally test these predictions, we rewired the SPI1 genetic circuit by changing its regulatory architecture. This enabled us to directly test our predictions regarding the function of the circuit by varying the strength and dynamics of the activating signal. Collectively, our experimental and computational results enable us to deconstruct this complex circuit and determine the role of its individual components in regulating SPI1 gene expression dynamics.

The role of coupled positive feedback in the expression of the SPI1 type three secretion system in Salmonella.

Salmonella enterica serovar Typhimurium is a common food-borne pathogen that induces inflammatory diarrhea and invades intestinal epithelial cells using a type three secretion system (T3SS) encoded within Salmonella pathogenicity island 1 (SPI1). The genes encoding the SPI1 T3SS are tightly regulated by a network of interacting transcriptional regulators involving three coupled positive feedback loops. While the core architecture of the SPI1 gene circuit has been determined, the relative roles of these interacting regulators and associated feedback loops are still unknown. To determine the function of this circuit, we measured gene expression dynamics at both population and single-cell resolution in a number of SPI1 regulatory mutants. Using these data, we constructed a mathematical model of the SPI1 gene circuit. Analysis of the model predicted that the circuit serves two functions. The first is to place a threshold on SPI1 activation, ensuring that the genes encoding the T3SS are expressed only in response to the appropriate combination of environmental and cellular cues. The second is to amplify SPI1 gene expression. To experimentally test these predictions, we rewired the SPI1 genetic circuit by changing its regulatory architecture. This enabled us to directly test our predictions regarding the function of the circuit by varying the strength and dynamics of the activating signal. Collectively, our experimental and computational results enable us to deconstruct this complex circuit and determine the role of its individual components in regulating SPI1 gene expression dynamics.

Protection of epithelial cells from Salmonella enterica serovar Enteritidis invasion by antibodies against the SPI-1 type III secretion system

Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) is one of the major causes of bacterial food-borne illness in humans. During the course of infection, Salmonella Enteritidis uses 2 type III secretion systems (T3SS), one of which is encoded on Salmonella pathogenicity island 1 (SPI-1). SPI-1 plays a major role in the invasion process. In the present study, we evaluated the effect of sera against the SPI-1 T3SS components on invasion in vitro using polarized human intestinal epithelial cells (Caco-2). Antisera to SipD protected Caco-2 cells against entry of wild-type Salmonella Enteritidis. On the other hand, sera against InvG, PrgI, SipA, SipC, SopB, SopE, and SopE2 did not affect Salmonella Enteritidis entry. To illustrate the specificity of anti-SipD mediated inhibition, SipD-specific antibodies were depleted from the serum. Antiserum depleted of SipD-specific antibodies lost its capacity to inhibit Salmonella Enteritidis entry. Thus, we demonstrate for the first time that antibodies against the SPI-1 needle tip protein (SipD) inhibit Salmonella Enteritidis invasion and that the SipD protein may be an important target in blocking SPI-1 mediated virulence of Salmonella Enteritidis.

Differential expression of Salmonella type III secretion system factors InvJ, PrgJ, SipC, SipD, SopA and SopB in cultures and in mice

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) is important for the invasion of epithelial cells during development of Salmonella-associated enterocolitis. It has been suggested that the level and timing of the expression of the SPI-1 T3SS proteins and effectors dictate the consequences of bacterial infection and pathogenesis. However, the expression of these proteins has not been extensively studied in vivo, especially during the later stages of salmonellosis when the infection is established. We have constructed recombinant Salmonella strains that contain a FLAG epitope inserted in-frame to genes invJ, prgJ, sipC, sipD, sopA and sopB, and investigated the expression of the tagged proteins both in vitro and in vivo during murine salmonellosis. Mice were inoculated intraperitoneally or intragastrically with the tagged Salmonella strains. At different time points post-infection, bacteria were recovered from various organs, and the expression of the tagged proteins was determined. Our results provide direct evidence that PrgJ and SipD are expressed in Salmonella colonizing the liver and ileum of infected animals at both the early and late stages of infection. Furthermore, our study has shown that the InvJ protein is expressed preferentially in Salmonella colonizing the ileum but not the liver, while SipC is expressed preferentially in Salmonella colonizing the liver but not the ileum. Thus, Salmonella appears to express different SPI-1 proteins and effectors when colonizing specific tissues. Our results suggest that differential expression of these proteins may be important for tissue-specific aspects of bacterial pathogenesis such as gastroenterititis in the ileum and systemic infection in the liver.

Mutations in theSalmonella entericaserovar Choleraesuis cAMP-receptor protein gene lead to functional defects in the SPI-1 Type III secretion system

Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis) causes a lethal systemic infection (salmonellosis) in swine. Live attenuated Salmonella Choleraesuis vaccines are effective in preventing the disease, and isolates of Salmonella Choleraesuis with mutations in the cAMP-receptor protein (CRP) gene (Salmonella Choleraesuis ∆crp) are the most widely used, although the basis of the attenuation remains unclear. The objective of this study was to determine if the attenuated phenotype of Salmonella Choleraesuis ∆crp was due to alterations in susceptibility to gastrointestinal factors such as pH and bile salts, ability to colonize or invade the intestine, or cytotoxicity for macrophages. Compared with the parental strain, the survival rate of Salmonella Choleraesuis ∆crp at low pH or in the presence of bile salts was higher, while the ability of the mutant to invade intestinal epithelia was significantly decreased. In examining the role of CRP on the secretory function of the Salmonella pathogenicity island 1 (SPI-1) encoded type III secretion system (T3SS), it was shown that Salmonella Choleraesuis ∆crp was unable to secrete the SPI-1 T3SS effector proteins, SopB and SipB, which play a role in Salmonella intestinal invasiveness and macrophage cytotoxicity, respectively. In addition, caspase-1 dependent cytotoxicity for macrophages was significantly reduced in Salmonella Choleraesuis ∆crp. Collectively, this study demonstrates that the CRP affects the secretory function of SPI-1 T3SS and the resulting ability to invade the host intestinal epithelium, which is a critical element in the pathogenesis of Salmonella Choleraesuis.

Analysis of functional domains present in the N-terminus of the SipB protein

SipB (593 aa), one of the Salmonella invasion proteins (Sips), is secreted via the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS). Here, we report the delineation of several functional regions present in the SipB protein. Our data show that residues 3-8 of the SipB protein are essential for its secretion from the bacterial cell and that the SicA chaperone, which is important to ensure stability of SipB and SipC in the bacterial cytosol, binds to SipB somewhere between amino acids 80 and100 of the SipB N-terminal region. Interestingly, the N-terminal region (residues 1-160) of SipB (SipB160) cannot be secreted via the SPI-1 T3SS, but fusion of the C-terminal amphipathic region (residues 300-593) to SipB160 can restore secretion via this system.

SteC is a Salmonella kinase required for SPI-2-dependent F-actin remodelling

Salmonella enterica serovar Typhimurium (S. Typhimurium) replicates inside mammalian cells within membrane-bound compartments called Salmonella-containing vacuoles. Intracellular replication is dependent on the activities of several effector proteins translocated across the vacuolar membrane by the Salmonella pathogenicity island 2 (SPI-2)-type III secretion system (T3SS). This is accompanied by the formation in the vicinity of bacterial vacuoles of an F-actin meshwork, thought to be involved in maintaining the integrity of vacuolar membranes. In this study, we investigated the function of the SPI-2 T3SS effector SteC. An steC mutant strain was not defective for intracellular replication or attenuated for virulence in mice. However, the steC mutant was defective for SPI-2-dependent F-actin meshwork formation in host cells, although the vacuolar membranes surrounding mutant bacteria appeared to be normal. Expression of SteC in fibroblast cells following transfection caused extensive rearrangements of the F-actin cytoskeleton. Sequence analysis identified amino acid similarity between SteC and the human kinase Raf-1. A His-tagged SteC fusion protein had kinase activity in vitro and a point mutant lacking kinase activity was unable to induce F-actin rearrangements in vivo. We conclude that SPI-2-dependent F-actin meshwork formation depends on the kinase activity of SteC, which resembles more closely eukaryotic than prokaryotic kinases.