Salmonella typhimurium strains carrying independent mutations display similar virulence phenotypes yet are controlled by distinct host defense mechanisms

The outcome of Salmonella infection in the mammalian host favors whoever succeeds best in disturbing the equilibrium between coordinate expression of bacterial (virulence) genes and host defense mechanisms. Intracellular persistence in host cells is critical for pathogenesis and disease, because Salmonella typhimurium strains defective in this property are avirulent. We examined whether similar host defense mechanisms are required for growth control of two S. typhimurium mutant strains. Salmonella pathogenicity island 2 (SPI2) and virulence plasmid-cured Salmonella mutants display similar virulence phenotypes in immunocompetent mice, yet their gene loci participate in independent virulence strategies. We determined the role of TNF-alpha and IFN-gamma as well as different T cell populations in infection with these Salmonella strains. After systemic infection, IFN-gamma was essential for growth restriction of plasmid-cured S. typhimurium, while SPI2 mutant infections were controlled in the absence of IFN-gamma. TNFRp55-deficiency restored systemic virulence to both Salmonella mutants. After oral inoculation, control of plasmid-cured bacteria substantially relied on both IFN-gamma and TNF-alpha signaling while control of SPI2 mutants did not. However, for both mutants, ultimate clearance of bacteria from infected mice depended on alphabeta T cells.     

Eat, kill or die: when amoeba meets bacteria

The core function of the innate immune response, phagocytosis, did not evolve first in metazoans but rather in primitive unicellular eukaryotes. Thus, though amoebae separated from the tree leading to metazoan shortly after the divergence of plants, they share many specific functions with mammalian phagocytic cells. Dictyostelium discoideum is by far the most studied amoeba, and it is proving useful to analyze phagocytosis and intracellular killing of bacteria. Since the basic mechanisms involved appear extremely conserved, Dictyostelium provides novel insights into the function of many new gene products. Bacterial pathogenicity was certainly largely developed to resist predatory amoebae in the environment, and this accounts for the fact that a large number of bacterial virulence traits can be studied using Dictyostelium as a host. This provides a particularly powerful system to analyze the complex interactions between pathogenic bacteria and host cells, where both the Dictyostelium host and the bacteria can be manipulated genetically with relative ease.     

蒙脱石对细菌黏附Caco-2细胞的影响

采用Caco-2细胞培养模型,观察两歧双歧杆菌、嗜酸乳杆菌、嗜水气单胞菌、副溶血弧菌、大肠杆菌、鼠伤寒沙门菌的黏附率,并在培养液中加入蒙脱石,计算蒙脱石对细菌黏附的阻断率,探讨蒙脱石对上述细菌黏附作用的影响。结果表明:所试菌与Caco-2细胞均有不同程度的黏附作用;蒙脱石对细菌黏附Caco-2细胞均有不同程度的阻断作用,对病原菌黏附Caco-2细胞的阻断作用要明显大于其对益生菌的阻断效果,其中对大肠杆菌、鼠伤寒沙门菌、嗜水气单胞菌、副溶血弧菌黏附的阻断率分别为54.22%、48.41%、60.53%、50.64%,而对两歧双歧杆菌、嗜酸乳杆菌黏附的阻断率分别为25.64%和21.49%。结果提示蒙脱石可有效阻断病原菌黏附,从而防治肠道细菌感染和细菌移位。     

Salmonella-containing vacuoles: directing traffic and nesting to grow

Salmonella enterica serovar Typhimurium (S. typhimurium) is a gram-negative facultative intracellular pathogen that can infect a broad range of mammalian hosts. Following invasion of host cells, the majority of S. typhimurium are known to reside in a membrane-bound compartment known as the Salmonella-containing vacuole (SCV). S. typhimurium actively remodels this compartment using bacterial virulence proteins, called effectors, to establish a protected niche where it can replicate. S. typhimurium delivers more than 30 effectors into the host cell cytosol by bacterial type three secretion systems, encoded by Salmonella pathogenicity island 1 or 2 (SPI-1 or SPI-2). Recent studies have revealed a critical role for the SPI-1 effector SopB in 'directing traffic' at early stages of infection, allowing the bacteria to control SCV maturation by modulating its interaction with the endocytic system. At later stages of infection, the SCV establishes a 'nest' near the Golgi where optimal bacterial growth takes place. In this study, we highlight these recent developments in our understanding of SCV trafficking.     

Requirement for N-ethylmaleimide-sensitive factor activity at different stages of bacterial invasion and phagocytosis

Bacterial invasion, like the process of phagocytosis, involves extensive and localized protrusion of the host cell plasma membrane. To examine the molecular mechanisms of the membrane remodeling that accompanies bacterial invasion, soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic was studied in cultured cells during infection by Salmonella typhimurium. A green fluorescent protein-tagged chimera of VAMP3, a SNARE characteristic of recycling endosomes, was found to accumulate at sites of Salmonella invasion. To analyze the possible role of SNARE-mediated membrane traffic in bacterial infection, invasion was measured in cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (NSF), an essential regulator of membrane fusion. Inhibition of NSF activity did not affect cellular invasion by S. typhimurium nor the associated membrane remodeling. By contrast, Fcgamma receptor-mediated phagocytosis was greatly reduced in the presence of the mutant NSF. Most important, dominant-negative NSF significantly impaired the fusion of Salmonella-containing vacuoles with endomembranes. These observations indicate that the membrane protrusions elicited by Salmonella invasion, unlike those involved in phagocytosis, occur via an NSF-independent mechanism, whereas maturation of Salmonella-containing vacuoles is NSF-dependent.     

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.     

Identification of Salmonella functions critical for bacterial cell division within eukaryotic cells

Salmonella typhimurium multiplication inside eukaryotic host cells is critical for virulence. Salmonella typhimurium strain SL1344 appears as filaments upon growth in macrophages and MelJuSo cells, a human melanoma cell line, indicating a specific blockage in the bacterial cell division process. Several studies have investigated the host cell response impairing bacterial division. However, none looked at the bacterial factors involved in inhibition of Salmonella division inside eukaryotic cells. We show here that blockage in the bacterial division process is sulA-independent and takes place after FtsZ-ring assembly. Salmonella typhimurium genes in which mutations lead to filamentous growth within host cells were identified by a large scale mutagenesis approach on strain 12023, revealing bacterial functions crucial for cell division within eukaryotic cells. We finally demonstrate that SL1344 filamentation is a result of hisG mutation, requires the activity of an enzyme of the histidine biosynthetic pathway HisFH and is specific for the vacuolar environment.     

No effect of Wolbachia on resistance to intracellular infection by pathogenic bacteria in Drosophila melanogaster

Multiple studies have shown that infection with the endosymbiotic bacterium Wolbachia pipientis confers Drosophila melanogaster and other insects with resistance to infection by RNA viruses. Studies investigating whether Wolbachia infection induces the immune system or confers protection against secondary bacterial infection have not shown any effect. These studies, however, have emphasized resistance against extracellular pathogens. Since Wolbachia lives inside the host cell, we hypothesized that Wolbachia might confer resistance to pathogens that establish infection by invading host cells. We therefore tested whether Wolbachia-infected D. melanogaster are protected against infection by the intracellular pathogenic bacteria Listeria monocytogenes and Salmonella typhimurium, as well as the extracellular pathogenic bacterium Providencia rettgeri. We evaluated the ability of flies infected with Wolbachia to suppress secondary infection by pathogenic bacteria relative to genetically matched controls that had been cured of Wolbachia by treatment with tetracycline. We found no evidence that Wolbachia alters host ability to suppress proliferation of any of the three pathogenic bacteria. Our results indicate that Wolbachia-induced antiviral protection does not result from a generalized response to intracellular pathogens.     

TLR signaling is required for Salmonella typhimurium virulence

Toll-like receptors (TLRs) contribute to host resistance to microbial pathogens and can drive the evolution of virulence mechanisms. We have examined the relationship between host resistance and pathogen virulence using mice with a functional allele of the nramp-1 gene and lacking combinations of TLRs. Mice deficient in both TLR2 and TLR4 were highly susceptible to the intracellular bacterial pathogen Salmonella typhimurium, consistent with reduced innate immune function. However, mice lacking additional TLRs involved in S. typhimurium recognition were less susceptible to infection. In these TLR-deficient cells, bacteria failed to upregulate Salmonella pathogenicity island 2 (SPI-2) genes and did not form a replicative compartment. We demonstrate that TLR signaling enhances the rate of acidification of the Salmonella-containing phagosome, and inhibition of this acidification prevents SPI-2 induction. Our results indicate that S. typhimurium requires cues from the innate immune system to regulate virulence genes necessary for intracellular survival, growth, and systemic infection.     

Antagonistic activity against pathogenic bacteria by human vaginal lactobacilli

This study attempted to isolate lactobacilli strains from healthy vaginal ecosystem to search for a new effective antibacterial probiotic strain. The strains were identified and characterized for their probiotic properties including bile salt and acid tolerance, growth at acidic pH, their ability to utilize protein, starch, and lipid, the production of hydrogen peroxide and bacteriocin as well as their antibiotic resistance patterns. The antibacterial activity of the culture supernatant of these strains were tested against a wide range of Gram-positive and Gram-negative pathogenic bacteria including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae. Salmonella typhi, and Salmonella typhimurium. None of the strains inhibited the growth of Gram-negative bacteria. Contrastly, the culture supernatant of strain L 22, identified as Lactobacillus reuteri, significantly inhibited all of the clinical isolates of methicillin-resistant S. aureus (MRSA). The antibacterial effect of the selected strain L 22 was further investigated. In the presence of L 22, the bacterial growth was assessed in vitro by viable bacterial counting. The numbers of viable cells were significantly lower in L 22-containing broth than those in the control by 6h. This finding clearly demonstrates that strain L 22 can produce an anti-MRSA effect. The antibacterial ability of the strain L 22 was fundamentally attributed to their bacteriocin production which can cause both cell inhibition and cell death.     

Salmonella infection of bone marrow-derived macrophages and dendritic cells: influence on antigen presentation and initiating an immune response

Traditionally macrophages (MPhi) have been considered to be the key type of antigen presenting cells (APC) to combat bacterial infections by phagocytosing and destroying bacteria and presenting bacteria-derived antigens to T cells. However, data in recent years have demonstrated that dendritic cells (DC), at their immature stage of differentiation, are capable of phagocytosing particulate antigens including bacteria. Thus, DC may also be important APC for initiating an immune response to bacterial infections. Our studies focus on studying how DC and MPhi process antigens derived from bacteria with no known mechanism of phagosomal escape (i.e. Salmonella typhimurium) for T cell stimulation as well as what role these APC types have in Salmonella infection in vivo. Using an in vitro antigen processing and presentation assay with bone marrow-derived (BM) APC showed that, in addition to peritoneal elicited MPhi and BMMPhi, BMDC can phagocytose and process Escherichia coli and S. typhimurium for peptide presentation on major histocompatibility complex (MHC) class I (MHC-I) and class II MHC-II. These studies showed that both elicited peritoneal MPhi and BMMPhi use an alternate MHC-I presentation pathway that does not require the transporter associated with antigen processing (TAP) or the proteasome and involves peptide loading onto a preformed pool of post-Golgi MHC-I molecules. In contrast, DC process E. coli and S. typhimurium for peptide presentation on MHC-I using the cytosolic MHC-I presentation pathway that requires TAP, the proteasome and uses newly synthesized MHC-I molecules. We further investigated the interaction of Salmonella with BMDC and BMMPhi by analyzing surface molecule expression and cytokine secretion following S. typhimurium infection of BMDC and BMMPhi. These data reveal that Salmonella co-incubation with BMDC as well as BMMPhi results in upregulation of MHC-I and MHC-II as well as several co-stimulatory molecules including CD80 and CD86. Salmonella infection of BMDC or BMMPhi also results in secretion of cytokines including IL-6 and IL-12. Finally, injecting mice with BMDC that have been loaded in vitro with S. typhimurium primes na?ve CD4(+) and CD8(+) T cells to Salmonella-encoded antigens. Taken together, our data suggest that DC may be an important type of APC that contributes to the immune response to Salmonella.     

Genetic control of the innate resistance of mice to Salmonella typhimurium: expression of the Ity gene in peritoneal and splenic macrophages isolated in vitro

The mouse Chromosome 1 locus Ity regulates the extent to which Salmonella typhimurium replicates within the reticuloendothelial cell system (RES) during the first days of infection. If animals are homozygous for the Itys susceptibility allele, the Gram-negative bacterium undergoes rapid net multiplication, and mice die of a typhoid fever-like disease by day 10 of infection. Animals that are homozygous or heterozygous for the resistance allele, Ityr, control net bacterial replication and survive the first phase of salmonellosis. Indirect studies have implicated the resident macrophage as the effector cell for regulation of early in vivo salmonellae growth. To verify this supposition and to evaluate the phenotypic expression of Ity, we developed an in vitro assay to compare kinetics of S. typhimurium growth within Ityr and Itys macrophages. Resident peritoneal and splenic macrophages were used from inbred Ityr and Itys mice and from Ity congeneic mice. With these mice and through the use of radiolabeled S. typhimurium and an avirulent temperature-sensitive mutant of the bacterium, we found that: phagocytosis of S. typhimurium by Ityr and by Itys macrophages was the same; S. typhimurium grew to a greater extent in Itys peritoneal and splenic macrophages than in Ityr cells; Ityr macrophages killed intracellular salmonellae more efficiently than did Itys macrophages. Thus, we have demonstrated directly that Ity is expressed by the macrophage and have shown for the first time with Ity congeneic mice that the basis for differential net growth of virulent S. typhimurium in Ityr and Itys macrophages is a variation in the degree of bacterial kill.     

Polymorphonuclear leukocyte migration across model intestinal epithelia enhances Salmonella typhimurium killing via the epithelial derived cytokine,IL-6

The host response to Salmonella typhimurium involves movement of polymorphonuclear leukocytes (PMN) across the epithelium and into the intestinal lumen. Following their arrival in the lumen, the PMN attempt to combat bacterial infection by activating antimicrobial defenses such as granule release, oxidative burst, phagocytosis, and cell signaling. We sought to examine PMN-S. typhimurium interaction following PMN arrival in the lumenal compartment. Here, for the first time, we demonstrate that PMN that have transmigrated across model intestinal epithelia have an enhanced ability to kill S. typhimurium. Our data provide evidence to indicate that the extracellular release of the primary and secondary granules of PMN, myeloperoxidase and lactoferrin, respectively, is correlated with enhanced bacterial killing. Furthermore, epithelial cells, during PMN transmigration, release the cytokine IL-6. IL-6 is known to increase intracellular stores of Ca(2+), and we have determined that this epithelial released cytokine is not only responsible for priming the PMN to release their granules, but also stimulating the PMN to kill S. typhimurium. These results substantiate the pathway in which PMN transmigration activates the epithelial release of IL-6, which in turn increases intracellular Ca(2+) storage. Our results, herein, extend this pathway to include an enhanced PMN granule release and an enhanced killing of S. typhimurium.