Salmonella pathogenicity island 2-dependent macrophage death is mediated in part by the host cysteine protease caspase-1

Salmonella typhimurium invades host macrophages and can either induce a rapid cell death or establish an intracellular niche within the phagocytic vacuole. Rapid cell death requires the Salmonella pathogenicity island (SPI)1 and the host protein caspase-1, a member of the pro-apoptotic caspase family of proteases. Salmonella that do not cause this rapid cell death and instead reside in the phagocytic vacuole can trigger macrophage death at a later time point. We show here that the human pathogen Salmonella typhi also triggers both rapid, caspase-1-dependent and delayed cell death in human monocytes. The delayed cell death has previously been shown with S. typhimurium to be dependent on SPI2-encoded genes and ompR . Using caspase-1 ^–/– bone marrow-derived macrophages and isogenic S. typhimurium mutant strains, we show that a large portion of the delayed, SPI2-dependent death is mediated by caspase-1. The two known substrates of activated caspase-1 are the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18, which are cleaved to produce bioactive cytokines. We show here that IL-1β is released during both SPI1- and SPI2-dependent macrophage killing. Using IL-1β ^–/– bone marrow-derived macrophages and a neutralizing anti-IL-18 antibody, we show that neither IL-1β nor IL-18 is required for rapid or delayed macrophage death. Thus, both rapid, SPI1-mediated killing and delayed, SPI2-mediated killing require caspase-1 and result in the secretion of IL-1β, which promotes inflammation and may facilitate the spread of Salmonella beyond the gastrointestinal tract in systemic disease.     

Taking possession: biogenesis of the Salmonella-containing vacuole

The Gram-negative pathogen Salmonella enterica can survive and replicate within a variety of mammalian cells. Regardless of the cell type, internalized bacteria survive and replicate within the Salmonella -containing vacuole, the biogenesis of which is dependent on bacterially encoded virulence factors. In particular, Type III secretion systems translocate bacterial effector proteins into the eukaryotic cell where they can specifically interact with a variety of targets. Salmonella has two distinct Type III secretion systems that are believed to have completely different functions. The SPI2 system is induced intracellularly and is required for intracellular survival in macrophages; it plays no role in invasion but is categorized as being required for Salmonella -containing vacuole biogenesis. In contrast, the SPI1 Type III secretion system is induced extracellularly and is essential for invasion of nonphagocytic cells. Its role in post-invasion processes has not been well studied. Recent studies indicate that Salmonella -containing vacuole biogenesis may be more dependent on SPI1 than previously believed. Other non-SPI2 virulence factors and the host cell itself may play critical roles in determining the intracellular environment of this facultative intracellular pathogen. In this review we discuss the recent advances in determining the mechanisms by which Salmonella regulate Salmonella -containing vacuole biogenesis and the implications of these findings.     

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.     

Pyroptosis and host cell death responses during Salmonella infection

Salmonella enterica are facultatively intracellular pathogens causing diseases with markedly visible signs of inflammation. During infection, Salmonella interacts with various host cell types, often resulting in death of those cells. Salmonella induces intestinal epithelial cell death via apoptosis, a cell death programme with a notably non-inflammatory outcome. In contrast, macrophage infection triggers caspase-1-dependent proinflammatory programmed cell death, a recently recognized process termed pyroptosis, which is distinguished from other forms of cellular demise by its unique mechanism, features and inflammatory outcome. Rapid macrophage pyroptosis depends on the Salmonella pathogenicity island-1 type III secretion system (T3SS) and flagella. Salmonella dynamically modulates induction of macrophage pyroptosis, and regulation of T3SS systems permits bacterial replication in specialized intracellular niches within macrophages. However, these infected macrophages later undergo a delayed form of caspase-1-dependent pyroptosis. Caspase-1-deficient mice are more susceptible to a number of bacterial infections, including salmonellosis, and pyroptosis is therefore considered a generalized protective host response to infection. Thus, Salmonella-induced pyroptosis serves as a model to understand a broadly important pathway of proinflammatory programmed host cell death: examining this system affords insight into mechanisms of both beneficial and pathological cell death and strategies employed by pathogens to modulate host responses.     

Intracellular Salmonella enterica redirect exocytic transport processes in a Salmonella pathogenicity island 2-dependent manner

During intracellular life, Salmonella enterica proliferate within a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and interfere with the microtubule cytoskeleton and cellular transport. To characterize the interaction of intracellular Salmonella with host cell transport processes, we utilized various model systems to follow microtubule-dependent transport. The vesicular stomatitis virus glycoprotein (VSVG) is a commonly used marker to follow protein transport from the Golgi to the plasma membrane. Using a VSVG-GFP fusion protein, we observed that virulent intracellular Salmonella alter exocytotic transport and recruit exocytotic transport vesicles to the SCV. This virulence function was dependent on the function of the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) and more specifically on a subset of SPI2 effector proteins. Furthermore, the Golgi to plasma membrane traffic of the shingolipid C(5)-ceramide was redirected to the SCV by virulent Salmonella. We propose that Salmonella modulates the biogenesis of the SCV by deviating this compartment from the default endocytic pathway to an organelle that interacts with the exocytic pathway. This observation might reveal a novel element of the intracellular survival and replication strategy of Salmonella.     

Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2

The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) is essential for virulence and intracellular proliferation of Salmonella enterica. We have previously identified SPI2-encoded proteins that are secreted and function as a translocon for the injection of effector proteins. Here, we describe the formation of a novel SPI2-dependent appendage structure in vitro as well as on the surface of bacteria that reside inside a vacuole of infected host cells. In contrast to the T3SS of other pathogens, the translocon encoded by SPI2 is only present singly or in few copies at one pole of the bacterial cell. Under in vitro conditions, appendages are composed of a filamentous needle-like structure with a diameter of 10 nm that was sheathed with secreted protein. The formation of the appendage in vitro is dependent on acidic media conditions. We analyzed SPI2-encoded appendages in infected cells and observed that acidic vacuolar pH was not required for induction of SPI2 gene expression, but was essential for the assembly of these structures and their function as translocon for delivery of effector proteins.     

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.     

Salmonella effectors within a single pathogenicity island are differentially expressed and translocated by separate type III secretion systems

Pathogenicity islands (PAIs) are large DNA segments in the genomes of bacterial pathogens that encode virulence factors. Five PAIs have been identified in the Gram-negative bacterium Salmonella enterica. Two of these PAIs, Salmonella pathogenicity island (SPI)-1 and SPI-2, encode type III secretion systems (TTSS), which are essential virulence determinants. These 'molecular syringes' inject effectors directly into the host cell, whereupon they manipulate host cell functions. These effectors are either encoded with their respective TTSS or scattered elsewhere on the Salmonella chromosome. Importantly, SPI-1 and SPI-2 are expressed under distinct environmental conditions: SPI-1 is induced upon initial contact with the host cell, whereas SPI-2 is induced intracellularly. Here, we demonstrate that a single PAI, in this case SPI-5, can encode effectors that are induced by distinct regulatory cues and targeted to different TTSS. SPI-5 encodes the SPI-1 TTSS translocated effector, SigD/SopB. In contrast, we report that the adjacently encoded effector PipB is part of the SPI-2 regulon. PipB is translocated by the SPI-2 TTSS to the Salmonella-containing vacuole and Salmonella-induced filaments. We also show that regions of SPI-5 are not conserved in all Salmonella spp. Although sigD/sopB is present in all Salmonella spp., pipB is not found in Salmonella bongori, which also lacks a functional SPI-2 TTSS. Thus, we demonstrate a functional and regulatory cross-talk between three chromosomal PAIs, SPI-1, SPI-2 and SPI-5, which has significant implications for the evolution and role of PAIs in bacterial pathogenesis.     

Host cell death induced by the egress of intracellular <Emphasis Type="Italic">Plasmodium</Emphasis> parasites

Intracellular pathogens are known to inhibit host cell apoptosis efficiently to ensure their own survival. However, following replication within a cell, they typically need to egress in order to infect new cells. For a long time it was assumed that this happens by simply disrupting the host cell and in some cases, such as for Plasmodium-infected erythrocytes, this seems indeed to be true. However, recently it has been shown that in Plasmodium-infected hepatocytes, an ordered form of cell death is initiated. This cell death is parasite-dependent and can clearly be distinguished from apoptosis and necrosis. The key event, and point of no return, appears to be the rupture of the parasitophorous vacuole membrane (PVM). PVM disruption and host cell death depend on the activation of cysteine proteases. Whether these are of parasite or host cell origin seems to rely on the life cycle stage of the Plasmodium parasite and the corresponding host cell.     

Salmonella type III effector SpvC, a phosphothreonine lyase, contributes to reduction in inflammatory response during intestinal phase of infection

Salmonella phosphothreonine lyase SpvC inactivates the dual-phosphorylated host mitogen-activated protein kinases (MAPK) through β-elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)-1 and SPI-2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI-2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI-1 and SPI-2 T3SSs. Dephosphorylation of the extracellular signal-regulated protein kinases (ERK) was detected in an SPI-1 T3SS-dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild-type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro-inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an anti-inflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.     

Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence plasmid are essential for Salmonella-induced apoptosis in intestinal epithelial cells

Intestinal epithelial cells are an important site of the host's interaction with enteroinvasive bacteria. Genes in the chromosomally encoded Salmonella pathogenicity island 2 (SPI 2) that encodes a type III secretion system and genes on the virulence plasmid pSDL2 of Salmonella enteritica serovar Dublin (spv genes) are thought to be important for Salmonella dublin survival in host cells. We hypothesized that genes in those loci may be important also for prolonged Salmonella growth and the induction of apoptosis induced by Salmonella in human intestinal epithelial cells. HT-29 human intestinal epithelial cells were infected with wild-type S. dublin or isogenic mutants deficient in the expression of spv genes or with SPI 2 locus mutations. Neither the spv nor the SPI 2 mutations affected bacterial entry into epithelial cells or intracellular proliferation of Salmonella during the initial 8 h after infection. However, at later periods, bacteria with mutations in the SPI 2 locus or in the spv locus compared to wild-type bacteria, manifested a marked decrease in intracellular proliferation and a different distribution pattern of bacteria within infected cells. Epithelial cell apoptosis was markedly increased in response to infection with wild-type, but not the mutant Salmonella. However, apoptosis of epithelial cells infected with wild-type S. dublin was delayed for approximately 28 h after bacterial entry. Apoptosis was preceded by caspase 3 activation, which was also delayed for approximately 24 h after infection. Despite its late onset, the cellular commitment to apoptosis was determined in the early period after infection as inhibition of bacterial protein synthesis during the first 6 h after epithelial cell infection with wild-type S. dublin, but not at later times, inhibited the induction of apoptosis. These studies indicate that genes in the SPI 2 and the spv loci are crucial for prolonged bacterial growth in intestinal epithelial cells. In addition to their influence on intracellular proliferation of Salmonella, genes in those loci determine the ultimate fate of infected epithelial cells with respect to caspase 3 activation and undergoing death by apoptosis.     

Vacuolar proteases livening up programmed cell death

The molecular identity of the key executioners involved in controlling plant programmed cell death (PCD) has been elusive. In a recent paper published in Science, Hatsugai and coworkers reported that a well-characterized protease called VPE from the plant cell vacuole can cleave caspase-specific substrates and is required for cell death activation by tobacco mosaic virus. This work provides clear evidence for the importance of the vacuole in plant PCD and a novel regulatory function for this organelle as well as for VPE proteases.     

Role of the ESCRT‐III complex in controlling integrity of the Salmonella‐containing vacuole

Intracellular pathogens need to establish specialised niches for survival and proliferation in host cells. The enteropathogen Salmonella enterica accomplishes this by extensive reorganisation of the host endosomal system deploying the SPI2‐encoded type III secretion system (SPI2‐T3SS). Fusion events of endosomal compartments with the Salmonella‐containing vacuole (SCV) form elaborate membrane networks within host cells enabling intracellular nutrition. However, which host compartments exactly are involved in this process and how the integrity of Salmonella‐modified membranes is accomplished are not fully resolved. An RNA interference knockdown screen of host factors involved in cellular logistics identified the ESCRT (endosomal sorting complex required for transport) system as important for proper formation and integrity of the SCV in infected epithelial cells. We demonstrate that subunits of the ESCRT‐III complex are specifically recruited to the SCV and membrane network. To investigate the role of ESCRT‐III for the intracellular lifestyle of Salmonella, a CHMP3 knockout cell line was generated. Infected CHMP3 knockout cells formed amorphous, bulky SCV. Salmonella within these amorphous SCV were in contact with host cell cytosol, and the attenuation of an SPI2‐T3SS‐deficient mutant strain was partially abrogated. ESCRT‐dependent endolysosomal repair mechanisms have recently been described for other intracellular pathogens, and we hypothesise that minor damages of the SCV during bacterial proliferation are repaired by the action of ESCRT‐III recruitment in Salmonella‐infected host cells.     

Living in the danger zone: innate immunity to Salmonella

Phagocytic cells, including macrophages, neutrophils and dendritic cells, are critical components of the innate immune response to bacterial pathogens such as Salmonella typhimurium. These cells can have several roles during the early stage of an infection including controlling bacterial replication and producing cytokines and chemokines that activate and recruit additional cells. Macrophages, neutrophils and dendritic cells increase in number early after oral Salmonella infection and produce cytokines important in host survival such as tumor necrosis factor alpha (TNF-alpha). All three phagocytic cell types also harbor bacteria during infection. Natural killer cells, natural killer T cells and T cell receptor alpha beta T cells also respond rapidly to infection and are early sources of interferon-gamma during infection with Salmonella. Studies using infection models with Salmonella are providing a picture of the innate response to bacteria and insight into the role of defined cell types and cytokines important in the transition from innate to adaptive immunity.     

Delineation of upstream signaling events in the salmonella pathogenicity island 2 transcriptional activation pathway

Survival and replication in the intracellular environment are critical components of the ability of Salmonella enterica serovar Typhimurium to establish systemic infection in the murine host. Intracellular survival is mediated by a number of genetic loci, including Salmonella pathogenicity island 2 (SPI2). SPI2 is a 40-kb locus encoding a type III secretion system that secretes effector molecules, which permits bacterial survival and replication in the intracellular environment of host cells. A two-component regulatory system, ssrAB, is also encoded in SPI2 and controls expression of the secretion system and effectors. While the environmental signals to which SPI2 responds in vivo are not known, activation of expression is dependent on OmpR and can be stimulated in vitro by chelation of cations or by a shift from rich to acidic minimal medium. In this work, we demonstrated that SPI2 activation is associated with OmpR in the phosphorylated form (OmpR-P). Mutations in envZ and ackA-pta, which disrupted two distinct sources of OmpR phosphorylation, indicated that SPI2 activation by chelators or a shift from rich to acidic minimal medium is largely dependent on functional EnvZ. In contrast, the PhoPQ pathway is not required for SPI2 activation in the presence of OmpR-P. As in the case of in vitro stimulation, SPI2 expression in macrophages correlates with the presence of OmpR-P. Additionally, EnvZ, but not acetyl phosphate, is required for maximal expression of SPI2 in the intracellular environment, suggesting that the in vitro SPI2 activation pathway is the same as that used in vivo.     

Functional dissection of translocon proteins of the <Emphasis Type="Italic">Salmonella</Emphasis> Pathogenicity Island 2-encoded type III secretion system

Background  

Type III secretion systems (T3SS) are essential virulence factors of most Gram-negative bacterial pathogens. T3SS deliver effector proteins directly into the cytoplasm of eukaryotic target cells and for this function, the insertion of a subset of T3SS proteins into the target cell membrane is important. These proteins form hetero-oligomeric pores acting as translocon for the delivery of effector proteins. Salmonella enterica is a facultative intracellular pathogen that uses the Salmonella Pathogenicity Island 2 (SPI2)-encoded T3SS to manipulate host cells in order to survive and proliferate within the Salmonella-containing vacuole of host cells. Previous work showed that SPI2-encoded SseB, SseC and SseD act to form the translocon of the SPI2-T3SS.     

Presence of SopE and mode of infection result in increased Salmonella‐containing vacuole damage and cytosolic release during host cell infection by Salmonella enterica

Salmonella enterica serovar Typhimurium (STM) is an invasive, facultative intracellular pathogen that has evolved sophisticated molecular mechanisms to establish an intracellular niche within a specialised vesicular compartment, the Salmonella‐containing vacuole (SCV). The loss of the SCV and release of STM into the cytosol of infected host cells was observed, and a bimodal intracellular lifestyle of STM in the SCV versus life in the cytosol is currently discussed. We set out to investigate the parameters affecting SCV integrity and cytosolic release. A fluorescent protein‐based cytosolic reporter approach was established to quantify, time‐resolved, and on a single cell level, the release of STM into the cytosol of host cells. We observed that the extent of SCV damage and cytosolic release is highly dependent on experimental conditions such as multiplicity of infection, type of host cell line, and STM strain background. Trigger invasion mediated by the Salmonella Pathogenicity Island 1‐encoded type III secretion system (SPI1‐T3SS) and its effector proteins promoted cytosolic release, whereas cytosolic bacteria were rarely observed if entry was mediated by zipper invasion. Presence of SPI1‐T3SS effector SopE was identified as major factor for damage of the SCV in the early phase after STM invasion and sopE‐expressing strains showed higher levels of cytosolic release.