Salmonella AvrA Coordinates Suppression of Host Immune and Apoptotic Defenses via JNK Pathway Blockade

Salmonellae are bacterial pathogens that have evolved sophisticated strategies to evade host immune defenses. These strategies include the secretion of effector proteins into mammalian cells so as to subvert innate immune and apoptotic signaling pathways, thereby allowing Salmonella to avoid elimination. Here, we show that the secreted Salmonella typhimurium effector protein AvrA possesses acetyltransferase activity toward specific mitogen-activated protein kinase kinases (MAPKKs) and potently inhibits c-Jun N-terminal kinase (JNK) and NF-kappaB signaling pathways in both transgenic Drosophila and murine models. Furthermore, we show that AvrA dampens the proapoptotic innate immune response to Salmonella at the mouse intestinal mucosa. This activity is consistent with the natural history of Salmonella in mammalian hosts, where the bacteria elicit transient inflammation but do not destroy epithelial cells. Our findings suggest that targeting JNK signaling to dampen apoptosis may be a conserved strategy for intracellular pathogens.     

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.     

Salmonella type III effector AvrA stabilizes cell tight junctions to inhibit inflammation in intestinal epithelial cells

Salmonella Typhimurium is a major cause of human gastroenteritis. The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junction (TJ) structure and function in intestinal epithelial cells. AvrA is a newly described bacterial effector found in Salmonella. We report here that AvrA expression stabilizes cell permeability and tight junctions in intestinal epithelial cells. Cells colonized with an AvrA-deficient bacterial strain (AvrA-) displayed decreased cell permeability, disruption of TJs, and an increased inflammatory response. Western blot data showed that TJ proteins, such as ZO-1, claudin-1, decreased after AvrA- colonization for only 1 hour. In contrast, cells colonized with AvrA-sufficient bacteria maintained cell permeability with stabilized TJ structure. This difference was confirmed in vivo. Fluorescent tracer studies showed increased fluorescence in the blood of mice infected with AvrA- compared to those infected with the AvrA-sufficient strains. AvrA- disrupted TJ structure and function and increased inflammation in vivo, compared to the AvrA- sufficient strain. Additionally, AvrA overexpression increased TJ protein expression when transfected into colonic epithelial cells. An intriguing aspect of this study is that AvrA stabilized TJs, even though the other TTSS proteins, SopB, SopE, and SopE2, are known to disrupt TJs. AvrA may play a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. Our findings indicate an important role for the bacterial effector AvrA in regulation of intestinal epithelial cell TJs during inflammation. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampen the inflammatory response.     

Selective Inhibition of Type III Secretion Activated Signaling by the Salmonella Effector AvrA

Salmonella enterica utilizes a type III secretion system (TTSS) encoded in its pathogenicity island 1 to mediate its initial interactions with intestinal epithelial cells, which are characterized by the stimulation of actin cytoskeleton reorganization and a profound reprogramming of gene expression. These responses result from the stimulation of Rho-family GTPases and downstream signaling pathways by specific effector proteins delivered by this TTSS. We show here that AvrA, an effector protein of this TTSS, specifically inhibits the Salmonella-induced activation of the JNK pathway through its interaction with MKK7, although it does not interfere with the bacterial infection-induced NF-κB activation. We also show that AvrA is phosphorylated at evolutionary conserved residues by a TTSS-effector-activated ERK pathway. This interplay between effector proteins delivered by the same TTSS highlights the remarkable complexity of these systems.     

Salmonella typhimurium induces apoptosis in human monocyte-derived macrophages

Salmonella species represent a leading cause of gastroenteritis worldwide. More recently, they have been proposed as putative vaccine delivery vehicles in humans. Oral infection with Salmonella leads to invasion of the intestinal epithelial barrier and subsequent interaction with mucosal macrophages. In this study, we investigated the fate of Salmonella typhimurium-infected human macrophages differentiated from blood monocytes by GM-CSF. Wild type S. typhimurium strain SL1344 induced macrophage surface blebbing and caused the release of host cytoplasmic lactate dehydrogenase beginning 30 min post-infection. Three hours later more than 80% of the macrophages in the culture were killed. In contrast, during the same period, macrophages infected with the non-invasive S. typhimurium strain BJ66 remained viable. Chromatin fragmentation is a hallmark of cells undergoing apoptosis. Using TUNEL analysis, we observed chromatin fragmentation in macrophages infected with SL1344 but not in BJ66 infected cells. Consistent with this observation, we found that pretreatment of human macrophages with an inhibitor of caspase-3, a member of the pro-apoptotic enzyme family shown to be involved in S. typhimurium-induced killing of mouse macrophages, reduced SL1344-mediated cytotoxicity by 40%. Our study provides the first evidence that invasive S. typhimurium induces apoptosis in human macrophages that were differentiated from blood monocytes by GM-CSF, and that cell death is a caspase-dependent phenomenon.     

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.     

The Salmonella effector protein SopB protects epithelial cells from apoptosis by sustained activation of Akt

Invasion of epithelial cells by Salmonella enterica is mediated by bacterial "effector" proteins that are delivered into the host cell by a type III secretion system. Although primarily known for their roles in actin rearrangements and membrane ruffling, translocated effectors also affect host cell processes that are not directly associated with invasion. Here, we show that SopB/SigD, an effector with phosphoinositide phosphatase activity, has anti-apoptotic activity in Salmonella-infected epithelial cells. Salmonella induced the sustained activation of Akt/protein kinase B, a pro-survival kinase, in a SopB-dependent manner. Failure to activate Akt resulted in increased levels of apoptosis after infection with a sopB deletion mutant (DeltasopB). Furthermore, cells infected with wild type bacteria, but not the DeltasopB strain, were protected from camptothecin-induced cleavage of caspase-3 and subsequent apoptosis. The anti-apoptotic activity of SopB was dependent on its phosphatase activity, because a catalytically inactive mutant was unable to protect cells from the effects of camptothecin. Finally, small interfering RNA was used to demonstrate the essential role of Akt in SopB-mediated protection against apoptosis. These results provide new insights into the mechanisms of apoptosis and highlight how bacterial effectors can intercept signaling pathways to manipulate host responses.     

The inflammation-associated Salmonella SopA is a HECT-like E3 ubiquitin ligase

Salmonella translocate a group of type III effectors into the host cells to induce entry, promote survival and cause intestinal inflammation. Although the biochemical and cellular mechanisms of how bacterial effectors function inside host cells remain largely unknown, studies have indicated that a likely strategy is to exploit host cellular pathways through functional mimicry. We report here that SopA, a Salmonella type III effector, mimics the mammalian HECT E3 ubiquitin ligase. SopA preferentially uses the host UbcH5a, UbcH5c and UbcH7 as E2s, which are involved in inflammation. Both the wild-type SopA and the mutant SopAC753S were expressed and translocated at similar levels during the infection of HeLa cells. A Salmonella strain expressing a catalytically incompetent SopAC753S mutant had reduced Salmonella-induced polymorphonuclear leukocytes transepithelial migration. We speculate that SopA ubiquitinate bacterial/host proteins involved in Salmonella-induced intestinal inflammation.     

Esterification of cholesterol by a type III secretion effector during intracellular Salmonella infection

Survival of Salmonella typhimurium within a vacuole in host cells depends on secreted virulence factors encoded by the Salmonella pathogenicity island 2 (SPI-2). High levels of cholesterol are detected at the Salmonella -containing vacuole (SCV). Here we show that the SPI-2 effector SseJ esterifies cholesterol in vitro , in cells and during infection. Intracellular infections with wild-type as compared with Δ sseJ bacteria led to higher levels of cholesterol ester production in HeLa cells and RAW macrophages and were shown to increase levels of lipid droplets (structures enriched in cholesterol esters). Ectopic expression of SseJ reduced cholesterol levels in cellular membranes and antagonized a major membrane activity of a second bacterial effector known to be important to the stability of the SCV. Previous studies in mouse models of infection have established a virulence defect in Δ sseJ bacteria and have suggested a role for SseJ in regulating SCV dynamics. Our data indicating the molecular activity of SseJ suggest that cholesterol and its esterification at the SCV are functionally important for intracellular bacterial survival.     

Intracellular replication of Salmonella typhimurium strains in specific subsets of splenic macrophages in vivo

We used flow cytometry and confocal immunofluorescence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice. Animals were inoculated intragastrically or intraperitoneally with S. typhimurium strains, constitutively expressing green fluorescent protein. Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation. Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S. typhimurium bacteria reside within macrophages. Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population. We have demonstrated that the Salmonella SPI-2 type III secretion system is required for replication within splenic macrophages, and that sifA ⁻ mutant bacteria are found within the cytosol of these cells. These results confirm that SifA and SPI-2 are involved in maintenance of the vacuolar membrane and intracellular replication in vivo .     

ATP scavenging by the intracellular pathogen Porphyromonas gingivalis inhibits P2X7-mediated host-cell apoptosis

The purinergic receptor P2X₇ is involved in cell death, inhibition of intracellular infection and secretion of inflammatory cytokines. The role of the P2X₇ receptor in bacterial infection has been primarily established in macrophages. Here we show that primary gingival epithelial cells, an important component of the oral innate immune response, also express functional P2X₇ and are sensitive to ATP-induced apoptosis. Porphyromonas gingivalis, an intracellular bacterium and successful colonizer of oral tissues, can inhibit gingival epithelial cell apoptosis induced by ATP ligation of P2X₇ receptors. A P. gingivalis homologue of nucleoside diphosphate kinase (NDK), an ATP-consuming enzyme, is secreted extracellularly and is required for maximal suppression of apoptosis. An ndk -deficient mutant was unable to prevent ATP-induced host-cell death nor plasma membrane permeabilization in the epithelial cells. Treatment with purified recombinant NDK inhibited ATP-mediated host-cell plasma membrane permeabilization in a dose-dependent manner. Therefore, NDK promotes survival of host cells by hydrolysing extracellular ATP and preventing apoptosis-mediated through P2X₇.     

Applications for green fluorescent protein (GFP) in the study of hostpathogen interactions

The green fluorescent protein (GFP) from Aequorea victoria is a novel fluorescent marker that has potential use in the study of bacterial pathogenicity. To explore some of the potential applications of GFP to the study of host-parasite interactions, we constructed two GFP expression vectors suitable for different facultative intracellular bacterial pathogens. The first expression vector was tested in the enteric pathogens, Salmonella typhimurium and Yersinia pseudotuberculosis, and the second vector tested in Mycobacterium marinum (Mm). Both expression vectors were found to be stable and to direct high levels of GFP synthesis. Standard epifluorescence microscopy was used to detect all three bacterial pathogenic species during the early and late stages of infection of live mammalian cells. Mm expressing gfp was also visualized in infected animal tissues, gfp expression did not adversely affect bacterial survival, nor did it compromise entry into mammalian cells or their survival within macrophages. In addition, all three gfp-expressing bacterial pathogens could be detected and sorted in a flow cytometer, either alone or in association with epithelial cells or macrophages. Therefore, GFP not only provides a convenient tool to image pathogenic bacteria, but allows the quantitative measurement of bacterial association with mammalian cells.     

Salmonella secreted factor L deubiquitinase of Salmonella typhimurium inhibits NF-kappaB, suppresses IkappaBalpha ubiquitination and modulates innate immune responses

Salmonella enterica translocates virulent factors into host cells using type III secretion systems to promote host colonization, intracellular bacterial replication and survival, and disease pathogenesis. Among many effectors, the type III secretion system encoded in Salmonella pathogenicity island 2 translocates a Salmonella-specific protein, designated Salmonella secreted factor L (SseL), a putative virulence factor possessing deubiquitinase activity. In this study, we attempt to elucidate the mechanism and the function of SseL in vitro, in primary host macrophages and in vivo in infected mice. Expression of SseL in mammalian cells suppresses NF-kappaB activation downstream of IkappaBalpha kinases and impairs IkappaBalpha ubiquitination and degradation, but not IkappaBalpha phosphorylation. Disruption of the gene encoding SseL in S. enterica serovar typhimurium increases IkappaBalpha degradation and ubiquitination, as well as NF-kappaB activation in infected macrophages, compared with wild-type bacteria. Mice infected with SseL-deficient bacteria mount stronger inflammatory responses, associated with increased production of NF-kappaB-dependent cytokines. Thus, SseL represents one of the first bacterial deubiquitinases demonstrated to modulate the host inflammatory response in vivo.     

The Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms

Salmonella typhimurium can colonize the gut, invade intestinal tissues, and cause enterocolitis. In vitro studies suggest different mechanisms leading to mucosal inflammation, including 1) direct modulation of proinflammatory signaling by bacterial type III effector proteins and 2) disruption or penetration of the intestinal epithelium so that penetrating bacteria or bacterial products can trigger innate immunity (i.e., TLR signaling). We studied these mechanisms in vivo using streptomycin-pretreated wild-type and knockout mice including MyD88(-/-) animals lacking an adaptor molecule required for signaling via most TLRs. The Salmonella SPI-1 and the SPI-2 type III secretion systems (TTSS) contributed to inflammation. Mutants that retain only a functional SPI-1 (M556; sseD::aphT) or a SPI-2 TTSS (SB161; DeltainvG) caused attenuated colitis, which reflected distinct aspects of the colitis caused by wild-type S. typhimurium: M556 caused diffuse cecal inflammation that did not require MyD88 signaling. In contrast, SB161 induced focal mucosal inflammation requiring MyD88. M556 but not SB161 was found in intestinal epithelial cells. In the lamina propria, M556 and SB161 appeared to reside in different leukocyte cell populations as indicated by differential CD11c staining. Only the SPI-2-dependent inflammatory pathway required aroA-dependent intracellular growth. Thus, S. typhimurium can use two independent mechanisms to elicit colitis in vivo: SPI-1-dependent and MyD88-independent signaling to epithelial cells and SPI-2-dependent intracellular proliferation in the lamina propria triggering MyD88-dependent innate immune responses.     

Eukaryotic signaling pathways targeted by <Emphasis Type="Italic">Salmonella</Emphasis> effector protein AvrA in intestinal infection <Emphasis Type="Italic">in vivo</Emphasis>

Background  

The Salmonella AvrA gene is present in 80% of Salmonella enterica serovar strains. AvrA protein mimics the activities of some eukaryotic proteins and uses these activities to the pathogen's advantage by debilitating the target cells, such as intestinal epithelial cells. Therefore, it is important to understand how AvrA works in targeting eukaryotic signaling pathways in intestinal infection in vivo. In this study, we hypothesized that AvrA interacts with multiple stress pathways in eukaryotic cells to manipulate the host defense system. A whole genome approach combined with bioinformatics assays was used to investigate the in vivo genetic responses of the mouse colon to Salmonella with or without AvrA protein expression in the early stage (8 hours) and late stage (4 days). Specifically, we examined the gene expression profiles in mouse colon as it responded to pathogenic Salmonella stain SL1344 (with AvrA expression) or SB1117 (without AvrA expression).     

SseA is required for translocation of Salmonella pathogenicity island-2 effectors into host cells

The Salmonella pathogenicity island-2 (SPI2) is a virulence locus on the bacterial chromosome required for intracellular proliferation and systemic infection in mice. Cell culture models and a murine model of systemic infection were used to address the role of an uncharacterized SPI2 open reading frame, designated as sseA, in Salmonella virulence. A Salmonella strain with an unmarked internal deletion of sseA displayed a phenotype that was similar to an SPI2-encoded type III secretion system apparatus mutant. Moreover, SseA was required for survival and replication within epithelial cells and macrophages. Murine infection studies confirmed that the DeltasseA strain was severely attenuated for virulence. Using immunofluorescence microscopy, the virulence defect in the DeltasseA strain was attributed to an inability to translocate SPI2 effector proteins into host cells. These data demonstrate that SseA is essential for SPI2-mediated translocation of effector proteins.