Differential contribution of sodC1 and sodC2 to intracellular survival and pathogenicity of Salmonella enterica serovar Choleraesuis

Several of the most virulent Salmonella enterica strains possess two genes encoding periplasmic Cu,Zn superoxide dismutase, sodC1 and sodC2, located on a lambdoid prophage and on the chromosome, respectively. These genes contribute to Salmonella virulence by protecting bacteria from superoxide generated by the host's phagocytes. To investigate the respective contributions of sodC1 and sodC2 to the virulence of a clinical isolate of Salmonella enterica serovar Choleraesuis (S. choleraesuis), we have analyzed both the intracellular survival of wild type and sodC mutant strains within J774 macrophages and Caco-2 cells, and their ability to proliferate in intraperitoneally-infected mice in competition assays. In agreement with previous studies, mutant strains lacking one or both sodC genes were equally impaired in their ability to survive within activated macrophages. However, when macrophage killing experiments were carried out with non-opsonized bacteria, sodC2 contributed to intracellular survival more than sodC1, indicating that changes in the pathways of bacterial uptake can modify the relative role of the two sodC genes. More unexpectedly, we have found that the ability of S. choleraesuis to survive within Caco-2 cells was severely affected by inactivation of sodC genes, sodC2 being more important than sodC1. As Caco-2 cells actively produce superoxide, this suggests that oxygen radical production by colonic cells has a role in controlling proliferation of facultative intracellular bacteria. Mouse infection studies confirmed that, in the S. choleraesuis strain under investigation, both sodC genes are required to confer full virulence, sodC2 contributing slightly more than sodC1 to Salmonella pathogenesis. Our findings contrast with the results of other studies carried out in S. enterica serovar Typhimurium and suggest that the relative contributions of sodC1 and sodC2 to host-pathogen interactive biology may vary depending on the Salmonella serovar or strain.     

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.     

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.     

Bacterial copper- and zinc-cofactored superoxide dismutase contributes to the pathogenesis of systemic salmonellosis

Copper/zinc-cofactored superoxide dismutase ([Cu,Zn]-SOD) has been found in the periplasm of many bacterial species but its biological function is unknown. Here we report the cloning and characterization of sodC , encoding [Cu,Zn]-SOD, from Salmonella typhimurium . The predicted protein sequence shows only 58% identity to Escherichia coli SodC, and from this its chromosomal location and its immediate proximity to a phage gene, sodC , in Salmonella is speculated to have been acquired by bacteriophage-mediated horizontal transfer from an unknown donor. A sodC mutant of S . typhimurium was unimpaired on aerobic growth in rich medium but showed enhanced sensitivity in vitro to the microbicidal action of superoxide. S . typhimurium , S . choleraesuis and S . dublin sodC mutants showed reduced lethality in a mouse model of oral infection and persisted in significantly lower numbers in livers and spleens after intraperitoneal infection, suggesting that [Cu,Zn]-SOD plays a role in pathogenicity, protecting Salmonella against oxygen radical-mediated host defences. There was, however, no observable difference compared with wild type in the interaction of sodC mutants with porcine pleural, mouse peritoneal or J774 macrophages in vitro , perhaps reflecting the hierarchical capacity of different macrophage lines to kill Salmonella , the most efficient overwhelming the proposed protective effect of periplasmic SOD.     

Differential accumulation of Salmonella[Cu,Zn] superoxide dismutases SodCI and SodCII in intracellular bacteria: correlation with their relative contribution to pathogenicity

Most Salmonella enterica strains have two peri-plasmic [Cu, Zn] superoxide dismutases, SodCI and SodCII, encoded by prophage and chromosomal genes respectively. Both enzymes are thought to play a role in Salmonella pathogenicity by intercepting reactive oxygen species produced by the host's innate immune response. To examine the apparent redundancy, we have compared the levels of epitope-tagged SodCI and SodCII proteins in bacteria growing in vitro, as well as inside tissue culture cells and in mouse tissues. Concomitantly, we have measured the abilities of mutants of either or both sodC genes to proliferate in infected mice in competition assays. Our results show a striking variation in the relative abundance of the two proteins in different environments. In vitro, both proteins accumulate when bacteria enter stationary phase; however, the increase is much sharper and conspicuous for SodCII than for SodCI. In contrast, SodCI vastly predominates in intracellular bacteria where SodCII levels are negligible. In agreement with these findings, most, if not all, of the contribution of [Cu, Zn] superoxide dismutase activity to murine salmonellosis can be ascribed to the SodCI protein. Overall the results of this work suggest that the duplicate sodC genes of Salmonella have evolved to respond to different sets of conditions encountered by bacteria inside the host and in the environment.     

Inducible prophages contribute to Salmonella virulence in mice

We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy-1 and Gifsy-2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy-2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy-2 carries the sodC gene for a periplasmic [Cu,Zn]-superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy-1 prophage to virulence - undetectable in the presence of Gifsy-2 as prophage - becomes significant in cells that lack Gifsy-2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy-2-encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy-1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy-2 genome.     

Identification of a NIPSNAP homologue as host cell target for Salmonella virulence protein SpiC

Salmonella enterica uses a type III secretion system encoded by SPI-2 to target specific virulence factors into the host cytosol of macrophages to inhibit the phagosomal-lysosomal maturation pathway. This ensures survival of Salmonella inside its intracellular niche, the Salmonella -containing vacuole (SCV). One such virulence factor is SpiC, which was previously shown to interfere with intracellular vesicular trafficking. In this study we have used a yeast two-hybrid assay to identify a NIPSNAP homologue as host cell target for SpiC that we have termed TassC. In vitro and in vivo co-purification of SpiC and TassC confirm the specificity of this interaction. Suppression of TassC production compensates a SpiC production deficit and allows spiC ^– Salmonella to survive within macrophages at levels comparable to wild-type Salmonella . We hypothesize that TassC is a host cell factor that determines vesicular trafficking in macrophages and is inactivated by Salmonella SpiC.     

Salmonella redirects phagosomal maturation

Following invasion of host cells, Salmonella occupies an intracellular vacuole that superficially resembles a phagosome. However, unlike bona fide phagosomes that undergo a maturation process to become microbicidal phago-lysosomes, Salmonella avoid killing by arresting and diverting the maturation program of the invasion vacuole. As a result of this re-routing, the bacteria generate a protected niche where they survive and replicate.     

Remodelling of the actin cytoskeleton is essential for replication of intravacuolar Salmonella

Maturation and maintenance of the intracellular vacuole in which Salmonella replicates is controlled by virulence proteins including the type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2). Here, we show that, several hours after bacterial uptake into different host cell types, Salmonella induces the formation of an F-actin meshwork around bacterial vacuoles. This structure is assembled de novo from the cellular G-actin pool in close proximity to the Salmonella vacuolar membrane. We demonstrate that the phenomenon does not require the Inv/Spa type III secretion system or cognate effector proteins, which induce actin polymerization during bacterial invasion, but does require a functional SPI-2 type III secretion system, which plays an important role in intracellular replication and systemic infection in mice. Treatment with actin-depolymerizing agents significantly inhibited intramacrophage replication of wild-type Salmonella typhimurium . Furthermore, after this treatment, wild-type bacteria were released into the host cell cytoplasm, whereas SPI-2 mutant bacteria remained within vacuoles. We conclude that actin assembly plays an important role in the establishment of an intracellular niche that sustains bacterial growth.     

Salmonella – the ultimate insider. Salmonella virulence factors that modulate intracellular survival

Salmonella enterica serovar Typhimurium is a common facultative intracellular pathogen that causes food-borne gastroenteritis in millions of people worldwide. Intracellular survival and replication are important virulence determinants and the bacteria can be found in a variety of phagocytic and non-phagocytic cells in vivo . Invasion of host cells and intracellular survival are dependent on two type III secretion systems, T3SS1 and T3SS2, each of which translocates a distinct set of effector proteins. However, other virulence factors including ion transporters, superoxide dismutase, flagella and fimbriae are also involved in accessing and utilizing the intracellular niche.     

Salmonella-induced macrophage death: the role of caspase-1 in death and inflammation.

Salmonella typhimurium invades host macrophages and can induce either an almost immediate cell death or establish an intracellular niche within the phagocytic vacuole. Rapid cell death depends on the Salmonella pathogenicity island SPI1 and the host protein caspase-1, a member of the pro-apoptotic caspase family of proteases. Caspase-1-dependent cell death leads to the activation of the potent pro-inflammatory cytokines interleukin (IL)-1beta and IL-18 to produce bioactive cytokines. Animal studies indicate that the activation of these cytokines is necessary for efficient colonization of the mouse gastrointestinal tract. Salmonella that reside in the phagocytic vacuole do not cause this early cell death and can trigger a macrophage death at a much later time point. This late-phase cell death is dependent on SPI2-encoded genes and ompR.     

The Salmonella effector AvrA mediates bacterial intracellular survival during infection in vivo

The enteric pathogen Salmonella typhimurium secretes the preformed AvrA effector protein into host cells. This acetyltransferase has been shown to modulate mammalian intestinal immune and survival responses by inhibition of JNK MAPK. To study the role of this effector in natural enteric infection, we used a mouse model to compare wild-type S. typhimurium to an isogenic AvrA null Salmonella mutant. Salmonella lacking AvrA induced increased intestinal inflammation, more intense systemic cytokine responses, and increased apoptosis in epithelial cells. Increased apoptosis was also observed in extra epithelial macrophages. AvrA null-infected mice consistently showed higher bacterial burden within mucosal lymphoid tissues, spleen and liver by 5 days post infection, which indicated a more severe clinical course. To study the molecular mechanisms involved, recombinant adenoviruses expressing AvrA or mutant AvrA proteins were constructed, which showed appropriate expression and mediated the expected inhibition of JNK signalling. Cultured epithelial cells and macrophages transduced with AvrA expressing adenovirus were protected from apoptosis induced by exogenous stimuli. In conclusion, the results demonstrated that Salmonella AvrA modulates survival of infected macrophages likely via JNK suppression, and prevents macrophage death and rapid bacterial dissemination. AvrA suppression of apoptosis in infected macrophages may allow for establishment of a stable intracellular niche typical of intracellular pathogens.     

Growth control in the Salmonella-containing vacuole

Salmonella enterica is an intracellular bacterial pathogen that inhabits membrane-bound vacuoles of eukaryotic cells. Coined as the 'Salmonella-containing vacuole' (SCV), this compartment has been studied for two decades as a replicative niche. Recent findings reveal, however, marked differences in the lifestyle of bacteria enclosed in the SCV of varied host cell types. In fibroblasts, the emerging view supports a model of bacteria facing in the SCV a 'to grow' or 'not to grow' dilemma, which is solved by entering in a dormancy-like state. Fine-tuning of host cell defense/survival routes, drastic metabolic shift down, adaptation to hypoxia conditions, and attenuation of own virulence systems emerge as strategies used by Salmonella to intentionally reduce the growth rate inside the SCV.     

布鲁氏菌胞内生存机制研究进展

布鲁氏菌是一种胞内寄生菌,可以在专业和非专业吞噬细胞内生存和复制。当布鲁氏菌与细胞接触时,细菌可以通过受体分子进入细胞。布鲁氏菌在细胞内首先定位于早期吞噬体,然后,在胞内改变其运输方向,最终抵达其胞内复制部位内质网,开始大量复制。这种复制既不影响细胞的基本功能,也不诱导细胞的损伤。主要综述了布鲁氏菌对细胞的侵袭、胞内运输和复制的相关研究进展。     

Salmonella-induced tubular networks

Salmonella virulence relies on its capacity to replicate inside various cell types in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). A unique feature of Salmonella-infected cells is the presence of tubular structures originating from and connected to the SCV, which often extend throughout the cell cytoplasm. These tubules include the well-studied Salmonella-induced filaments (SIFs), enriched in lysosomal membrane proteins. However, recent studies revealed that the Salmonella-induced tubular network is more extensive than previously thought and includes three types of tubules distinct from SIFs: sorting nexin tubules, Salmonella-induced secretory carrier membrane protein 3 (SCAMP3) tubules and lysosome-associated membrane protein 1 (LAMP1)-negative tubules. In this review, we examine the molecular mechanisms involved in the formation of Salmonella-induced tubular networks and discuss the importance of the tubules for Salmonella virulence and establishment of a Salmonella intracellular replicative niche.     

Salmonella acquires lysosome-associated membrane protein 1 (LAMP1) on phagosomes from Golgi via SipC protein-mediated recruitment of host Syntaxin6

Several intracellular pathogens have developed diverse strategies to avoid targeting to lysosomes. However, they universally recruit lysosome-associated membrane protein 1 (LAMP1); the mechanism of LAMP1 recruitment remains unclear. Here, we report that a Salmonella effector protein, SipC, specifically binds with host Syntaxin6 through its C terminus and thereby recruits Syntaxin6 and other accessory molecules like VAMP2, Rab6, and Rab8 on Salmonella-containing phagosomes (SCP) and acquires LAMP1 by fusing with LAMP1-containing Golgi-derived vesicles. In contrast, sipC knock-out:SCP (sipC(-):SCP) or sipC(M398K):SCP fails to obtain significant amounts of Syntaxin6 and is unable to acquire LAMP1. Moreover, phagosomes containing respective knock-out Salmonella like sipA(-), sipB(-), sipD(-), sopB(-), or sopE(-) recruit LAMP1, demonstrating the specificity of SipC in this process. In addition, depletion of Syntaxin6 by shRNA in macrophages significantly inhibits LAMP1 recruitment on SCP. Additionally, survival of sipC(-):Salmonella in mice is found to be significantly inhibited in comparison with WT:Salmonella. Our results reveal a novel mechanism showing how Salmonella acquires LAMP1 through a SipC-Syntaxin6-mediated interaction probably to stabilize their niche in macrophages and also suggest that similar modalities might be used by other intracellular pathogens to recruit LAMP1.     

The Salmonella-containing vacuole is a major site of intracellular cholesterol accumulation and recruits the GPI-anchored protein CD55

Intracellular, pathogenic Salmonella typhimurium avoids phago-lysosome fusion, and exists within a unique vacuolar niche that resembles a late endosome. This model has emerged from studying the trafficking of host proteins to the Salmonella-containing vacuole (SCV). Very little is known about the role of major host lipids during infection. Here, we show using biochemical analyses as well as fluorescence microscopy, that intracellular infection perturbs the host sterol biosynthetic pathway and induces cholesterol accumulation in the SCV. Cholesterol accumulation is seen in both macrophages and epithelial cells: at the terminal stages of infection, as much as 30% of the total cellular cholesterol resides in the SCV. We find that accumulation of cholesterol in the SCV is linked to intracellular bacterial replication and may be dependent on Salmonella pathogenicity island 2 (SPI-2). Furthermore, the construction of a three-dimensional space-filling model yields novel insights into the structure of the SCV: bacteria embedded in cholesterol-rich membranes. Finally, we show that the glycosylphosphatidylinositol (GPI)-anchored protein CD55 is recruited to the SCV. These data suggest that, in contrast to prevailing models, the SCV accumulates components of cholesterol-rich early endocytic pathways during intracellular bacterial replication.     

Role of lipid-mediated signal transduction in bacterial internalization

Receptor-mediated phagocytosis normally represents an important first line of immune defence. Invading microbes are internalized into phagosomes and are typically killed by exposure to a battery of microbicidal agents. To some intracellular pathogens, however, receptor-mediated phagocytosis represents an opportunity to access a protected niche within the host cell. Another type of intracellular pathogen, including Salmonella enterica serovar Typhimurium and Shigella flexneri, invade host cells in a more direct manner. These pathogens deliver effectors into the host cell via a type III secretion apparatus, initiating a ruffling response that leads to their uptake into intracellular vacuoles. Recent studies have demonstrated the importance of lipid signal transduction events in the uptake of pathogenic bacteria by both receptor-mediated phagocytosis and type III secretion-mediated invasion. In this review we highlight some of these discoveries, with a focus on phospholipid-dependent signalling events.     

Identification of a single-nucleotide insertion in the promoter region affecting the sodC promoter activity in Brucella neotomae

Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.