Analysis of Cells Targeted by Salmonella Type III Secretion In Vivo

The type III secretion systems (TTSS) encoded in Salmonella pathogenicity island-1 and -2 (SPI-1 and -2) are virulence factors required for specific phases of Salmonella infection in animal hosts. However, the host cell types targeted by the TTSS have not been determined. To investigate this, we have constructed translational fusions between the ß-lactamase reporter and a broad array of TTSS effectors secreted via SPI-1, SPI-2, or both. Secretion of the fusion protein to a host cell was determined by cleavage of a specific fluorescent substrate. In cultured cells, secretion of all six effectors could be observed. However, two to four days following i.p. infection of mice, only effectors secreted by SPI-2 were detected in spleen cells. The cells targeted were identified via staining with nine different cell surface markers followed by FACS analysis as well as by conventional cytological methods. The targeted cells include B and T lymphocytes, neutrophils, monocytes, and dendritic cells, but not mature macrophages. To further investigate replication in these various cell types, Salmonella derivatives were constructed that express a red fluorescent protein. Bacteria could be seen in each of the cell types above; however, most viable bacteria were present in neutrophils. We find that Salmonella is capable of targeting most phagocytic and non-phagocytic cells in the spleen but has a surprisingly high preference for neutrophils. These findings suggest that Salmonella specifically target splenic neutrophils presumably to attenuate their microbicidal functions, thereby promoting intracellular survival and replication in the mouse

Identification of New Secreted Effectors in Salmonella enterica Serovar Typhimurium

A common theme in bacterial pathogenesis is the secretion of bacterial products that modify cellular functions to overcome host defenses. Gram-negative bacterial pathogens use type III secretion systems (TTSSs) to inject effector proteins into host cells. The genes encoding the structural components of the type III secretion apparatus are conserved among bacterial species and can be identified by sequence homology. In contrast, the sequences of secreted effector proteins are less conserved and are therefore difficult to identify. A strategy was developed to identify virulence factors secreted by Salmonella enterica serovar Typhimurium into the host cell cytoplasm. We constructed a transposon, which we refer to as mini-Tn5-cycler, to generate translational fusions between Salmonella chromosomal genes and a fragment of the calmodulin-dependent adenylate cyclase gene derived from Bordetella pertussis (cyaA′). In-frame fusions to bacterial proteins that are secreted into the eukaryotic cell cytoplasm were identified by high levels of cyclic AMP in infected cells. The assay was sufficiently sensitive that a single secreted fusion could be identified among several hundred that were not secreted. This approach identified three new effectors as well as seven that have been previously characterized. A deletion of one of the new effectors, steA (Salmonella translocated effector A), attenuated virulence. In addition, SteA localizes to the trans-Golgi network in both transfected and infected cells. This approach has identified new secreted effector proteins in Salmonella and will likely be useful for other organisms, even those in which genetic manipulation is more difficult

Nod2 activates NF-kB in CD4+ T cells but its expression is dispensable for T cell-induced colitis.

Although the etiology of Crohn's disease (CD) remains elusive this disease is characterized by T cell activation that leads to chronic inflammation and mucosal damage. A potential role for maladaptation between the intestinal microbiota and the mucosal immune response is suggested by the fact that mutations in the pattern recognition receptor Nod2 are associated with higher risks for developing CD. Although Nod2 deletion in CD4(+) T cells has been shown to impair the induction of colitis in the murine T cell transfer model, the analysis of T cell intrinsic Nod2 function in T cell differentiation and T cell-mediated immunity is inconsistent between several studies. In addition, the role of T cell intrinsic Nod2 in regulatory T cell (Treg) development and function during colitis remain to be analyzed. In this study, we show that Nod2 expression is higher in activated/memory CD4(+) T cells and its expression was inducible after T cell receptor (TCR) ligation. Nod2 stimulation with muramyl dipeptide (MDP) led to a nuclear accumulation of c-Rel NF-kB subunit. Although functionally active in CD4(+) T cells, the deletion of Nod2 did not impair the induction and the prevention of colitis in the T cell transfer model. Moreover, Nod2 deletion did not affect the development of Foxp3(+) Treg cells in the spleen of recipient mice and Nod2 deficient CD4 T cells expressing the OVA specific transgenic TCR were able to differentiate in Foxp3(+) Treg cells after OVA feeding. In vitro, CD25(+) Nod2 deficient T cells suppressed T cell proliferation as well as wild type counter parts and T cell stimulation with MDP did not affect the proliferation and the cytokine secretion of T cells. In conclusion, our data indicate that Nod2 is functional in murine CD4(+) T cells but its expression is dispensable for the T cell regulation of colitis

Nod2 is expressed in CD4⁺ T cells and inducible after TCR ligation.

<p>(A) Splenic CD4⁺ T cells isolated from C57BL/6 or NOD2^-/- mice were cultured alone or in presence of anti-CD3 (1-10µg/mL) and anti-CD28 (2µg/mL) monoclonal antibodies for 24 hours. Nod2 expression was assessed by western blot. (B) Naïve CD4⁺CD45RB^high, effector CD4⁺CD45RB^lowFoxp3^- and regulatory CD4⁺CD45RB^lowFoxp3⁺ splenic T cell subsets were isolated from <i>Foxp3-GFP</i> mice and Nod2 mRNA level was assessed by RT-qPCR. (C) <i>Lamina propria</i> lymphocytes were isolated from the cecum of NOD2-GFP mice and analyzed by flow cytometry. The histograms show the level of GFP expression. Cells were gated on total viable TCRβ⁺CD4⁺ cells (left panel), on  TCRβ⁺CD4⁺CD44^- cells (middle panel), or TCRβ⁺CD4⁺CD44⁺ cells (right panel). </p

Transfer of naive and effector <i>NOD2</i>^<i>-/-</i> T cells effectively induced and prevented colitis.

<p><i>Rag1</i>^<i>-/-</i> mice were transferred with 3x10⁵ wild type or <i>NOD2</i>^<i>-/-</i> naïve CD4⁺CD45Rb^high T cells in presence or absence of CD4⁺CD45Rb^low T cells and (A) percentage of initial body weight change, (B) colon pathology scores and (C) colon weight to length ratio were measured. (D) Percentage of Foxp3⁺CD4⁺ T cells in the spleen of T cell transferred <i>Rag1</i>^<i>-/-</i> mice. Results show the mean ± SEM and are representative of two independent experiments, n=6-9 mice per group. Colon pathology scores were assessed in one experiment, n=3-5 mice per group. </p

Nod1 and Nod2 Regulation of Inflammation in the Salmonella Colitis Model▿ †

The pattern recognition molecules Nod1 and Nod2 play important roles in intestinal homeostasis; however, how these proteins impact on the development of inflammation during bacterial colitis has not been examined. In the streptomycin-treated mouse model of Salmonella colitis, we found that mice deficient for both Nod1 and Nod2 had attenuated inflammatory pathology, reduced levels of inflammatory cytokines, and increased colonization of the mucosal tissue. Nod1 and Nod2 from both hematopoietic and nonhematopoietic sources contributed to the pathology, and all phenotypes were recapitulated in mice deficient for the signaling adaptor protein Rip2. However, the influence of Rip2 was strictly dependent on infection conditions that favored expression of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS), as Rip2 was dispensable for inflammation when mice were infected with bacteria grown under conditions that promoted expression of the SPI-1 TTSS. Thus, Nod1 and Nod2 can modulate inflammation and mediate efficient clearance of bacteria from the mucosal tissue during Salmonella colitis, but their role is dependent on the expression of the SPI-2 TTSS

Role of Nod1 in Mucosal Dendritic Cells during Salmonella Pathogenicity Island 1-Independent Salmonella enterica Serovar Typhimurium Infection ▿

Recent advances in immunology have highlighted the critical function of pattern-recognition molecules (PRMs) in generating the innate immune response to effectively target pathogens. Nod1 and Nod2 are intracellular PRMs that detect peptidoglycan motifs from the cell walls of bacteria once they gain access to the cytosol. Salmonella enterica serovar Typhimurium is an enteric intracellular pathogen that causes a severe disease in the mouse model. This pathogen resides within vacuoles inside the cell, but the question of whether cytosolic PRMs such as Nod1 and Nod2 could have an impact on the course of S. Typhimurium infection in vivo has not been addressed. Here, we show that deficiency in the PRM Nod1, but not Nod2, resulted in increased susceptibility toward a mutant strain of S. Typhimurium that targets directly lamina propria dendritic cells (DCs) for its entry into the host. Using this bacterium and bone marrow chimeras, we uncovered a surprising role for Nod1 in myeloid cells controlling bacterial infection at the level of the intestinal lamina propria. Indeed, DCs deficient for Nod1 exhibited impaired clearance of the bacteria, both in vitro and in vivo, leading to increased organ colonization and decreased host survival after oral infection. Taken together, these findings demonstrate a key role for Nod1 in the host response to an enteric bacterial pathogen through the modulation of intestinal lamina propria DCs

Increased early influx of neutrophils and macrophages in <i>Nod2^−/−</i> mice.

<p>Streptomycin-pretreated C57Bl/6 and <i>Nod2</i>^−/− mice were orally infected <i>with S.</i> Typhimurium Δ<i>msbB</i> for 2 days and cecum sections were stained to visualize neutrophils and macrophages, respectively. (A) Cecum sections were stained for DAPI (blue), E-cadherin (epithelial cells, red) and MPO (neutrophils, green). (B) Cecum sections were stained for DAPI (blue) and CD68 (macrophages, red). Original magnification: 400×, scale bars = 50 µm. (C) Quantification of MPO+ and (D) CD68+ cells. HPF = high power field. Statistical analysis: Student's <i>t</i> test. *** <i>p</i><0.001.</p

<i>S.</i> Typhimurium Δ<i>msbB</i> infection induces pro-inflammatory gene expression in C57Bl/6 and <i>Nod2^−/−</i> mice.

<p>Gene expression of <i>mcp1</i> and <i>tnfα</i>, were measured by quantitative real-time PCR. Data were normalized to <i>gapdh</i> expression levels; Statistical analysis: 1way ANOVA with Tukey's multiple comparison post-test. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001; ns: not significant.</p