In Macrophages, Caspase-1 Activation by SopE and the Type III Secretion System-1 of S. Typhimurium Can Proceed in the Absence of Flagellin

The innate immune system is of vital importance for protection against infectious pathogens. Inflammasome mediated caspase-1 activation and subsequent release of pro-inflammatory cytokines like IL-1β and IL-18 is an important arm of the innate immune system. Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium, SL1344) is an enteropathogenic bacterium causing diarrheal diseases. Different reports have shown that in macrophages, S. Typhimurium may activate caspase-1 by at least three different types of stimuli: flagellin, the type III secretion system 1 (T1) and the T1 effector protein SopE. However, the relative importance and interdependence of the different factors in caspase-1 activation is still a matter of debate. Here, we have analyzed their relative contributions to caspase-1 activation in LPS-pretreated RAW264.7 macrophages. Using flagellar mutants (fliGHI, flgK) and centrifugation to mediate pathogen-host cell contact, we show that flagellins account for a small part of the caspase-1 activation in RAW264.7 cells. In addition, functional flagella are of key importance for motility and host cell attachment which is a prerequisite for mediating caspase-1 activation via these three stimuli. Using site directed mutants lacking several T1 effector proteins and flagellin expression, we found that SopE elicits caspase-1 activation even when flagellins are absent. In contrast, disruption of essential genes of the T1 protein injection system (invG, sipB) completely abolished caspase-1 activation. However, a robust level of caspase-1 activation is retained by the T1 system (or unidentified T1 effectors) in the absence of flagellin and SopE. T1-mediated inflammasome activation is in line with recent work by others and suggests that the T1 system itself may represent the basic caspase-1 activating stimulus in RAW264.7 macrophages which is further enhanced independently by SopE and/or flagellin

Salmonella-Induced Mucosal Lectin RegIIIβ Kills Competing Gut Microbiota

Intestinal inflammation induces alterations of the gut microbiota and promotes overgrowth of the enteric pathogen Salmonella enterica by largely unknown mechanisms. Here, we identified a host factor involved in this process. Specifically, the C-type lectin RegIIIβ is strongly upregulated during mucosal infection and released into the gut lumen. In vitro, RegIIIβ kills diverse commensal gut bacteria but not Salmonella enterica subspecies I serovar Typhimurium (S. Typhimurium). Protection of the pathogen was attributable to its specific cell envelope structure. Co-infection experiments with an avirulent S. Typhimurium mutant and a RegIIIβ-sensitive commensal E. coli strain demonstrated that feeding of RegIIIβ was sufficient for suppressing commensals in the absence of all other changes inflicted by mucosal disease. These data suggest that RegIIIβ production by the host can promote S. Typhimurium infection by eliminating inhibitory gut microbiota

Like Will to Like: Abundances of Closely Related Species Can Predict Susceptibility to Intestinal Colonization by Pathogenic and Commensal Bacteria

The intestinal ecosystem is formed by a complex, yet highly characteristic microbial community. The parameters defining whether this community permits invasion of a new bacterial species are unclear. In particular, inhibition of enteropathogen infection by the gut microbiota ( = colonization resistance) is poorly understood. To analyze the mechanisms of microbiota-mediated protection from Salmonella enterica induced enterocolitis, we used a mouse infection model and large scale high-throughput pyrosequencing. In contrast to conventional mice (CON), mice with a gut microbiota of low complexity (LCM) were highly susceptible to S. enterica induced colonization and enterocolitis. Colonization resistance was partially restored in LCM-animals by co-housing with conventional mice for 21 days (LCMcon21). 16S rRNA sequence analysis comparing LCM, LCMcon21 and CON gut microbiota revealed that gut microbiota complexity increased upon conventionalization and correlated with increased resistance to S. enterica infection. Comparative microbiota analysis of mice with varying degrees of colonization resistance allowed us to identify intestinal ecosystem characteristics associated with susceptibility to S. enterica infection. Moreover, this system enabled us to gain further insights into the general principles of gut ecosystem invasion by non-pathogenic, commensal bacteria. Mice harboring high commensal E. coli densities were more susceptible to S. enterica induced gut inflammation. Similarly, mice with high titers of Lactobacilli were more efficiently colonized by a commensal Lactobacillus reuteri RR strain after oral inoculation. Upon examination of 16S rRNA sequence data from 9 CON mice we found that closely related phylotypes generally display significantly correlated abundances (co-occurrence), more so than distantly related phylotypes. Thus, in essence, the presence of closely related species can increase the chance of invasion of newly incoming species into the gut ecosystem. We provide evidence that this principle might be of general validity for invasion of bacteria in preformed gut ecosystems. This might be of relevance for human enteropathogen infections as well as therapeutic use of probiotic commensal bacteria

Peroral ciprofloxacin therapy impairs the generation of a protective immune response in a mouse model for Salmonella enterica serovar Typhimurium diarrhea, while parenteral ceftriaxone therapy does not

Nontyphoidal Salmonella (NTS) species cause self-limiting diarrhea and sometimes severe disease. Antibiotic treatment is considered only in severe cases and immune-compromised patients. The beneficial effects of antibiotic therapy and the consequences for adaptive immune responses are not well understood. We used a mouse model for Salmonella diarrhea to assess the effects of per os treatment with ciprofloxacin (15 mg/kg of body weight intragastrically 2 times/day, 5 days) or parenteral ceftriaxone (50 mg/kg intraperitoneally, 5 days), two common drugs used in human patients. The therapeutic and adverse effects were assessed with respect to generation of a protective adaptive immune response, fecal pathogen excretion, and the emergence of nonsymptomatic excreters. In the mouse model, both therapies reduced disease severity and reduced the level of fecal shedding. In line with clinical data, in most animals, a rebound of pathogen gut colonization/fecal shedding was observed 2 to 12 days after the end of the treatment. Yet, levels of pathogen shedding and frequency of appearance of nonsymptomatic excreters did not differ from those for untreated controls. Moreover, mice treated intraperitoneally with ceftriaxone developed an adaptive immunity protecting the mice from enteropathy in wild-type Salmonella enterica serovar Typhimurium challenge infections. In contrast, the mice treated intragastrically with ciprofloxacin were not protected. Thus, antibiotic treatment regimens can disrupt the adaptive immune response, but treatment regimens may be optimized in order to preserve the generation of protective immunity. It might be of interest to determine whether this also pertains to human patients. In this case, the mouse model might be a tool for further mechanistic studies

Accelerated Type III Secretion System 2-Dependent Enteropathogenesis by a Salmonella enterica Serovar Enteritidis PT4/6 Strain▿

Salmonella enterica subsp. I serovars Typhimurium and Enteritidis are major causes of enteric disease. The pathomechanism of enteric infection by serovar Typhimurium has been studied in detail. Serovar Typhimurium employs two pathways in parallel for triggering disease, i.e., the “classical” pathway, triggered by type III secretion system 1 (TTSS-1), and the “alternative” pathway, mediated by TTSS-2. It had remained unclear whether these two pathways would also explain the enteropathogenesis of strains from other serovars. We chose the isolate P125109 of the epidemic serovar Enteritidis PT4/6, generated isogenic mutants, and studied their virulence. Using in vitro and in vivo infection experiments, a dendritic cell depletion strategy, and MyD88−/− knockout mice, we found that P125109 employs both the “classical” and “alternative” pathways for triggering mucosal inflammation. The “classical” pathway was phenotypically similar in serovar Typhimurium strain SL1344 and in P125109. However, the kinetics of the “alternative” pathway differed significantly. Via TTSS-2, P125109 colonized the gut tissue more efficiently and triggered mucosal inflammation approximately 1 day faster than SL1344 did. In conclusion, our data demonstrate that different Salmonella spp. can differ in their capacity to trigger mucosal inflammation via the “alternative” pathway in vivo

SopE and an intact T1 system contribute to flagellin-independent caspase-1 activation.

<p><b>A</b>) SopE is the main effector protein mediating caspase-1 activation in the absence of flagellin. LDH release induced by strains expressing SopE and SopE2 (Δ<i>sipA</i> Δ<i>sopB;</i> SopE/E2, SopE/E2^{<b>M−F−</b>}, and SopE/E2^{<b>M−F+</b>}) is equivalent to LDH release induced by strains additionally lacking SopE2 (Δ<i>sipA</i> Δ<i>sopB</i> Δ<i>sopE2;</i> SopE/E2, SopE/E2^{<b>M−F−</b>}, and SopE/E2^{<b>M−F+</b>}). Note that data shown in A) and C) were obtained from the same experiments. The value for WT in A) was replotted in C) for better comparison. <b>B</b>). The catalytic activity of SopE (infection with SopE^M45 strain) is required for full LDH release. A strain with a catalytically inactive SopE mutant (SopE^M45G168V; Δ<i>sipA</i> Δ<i>sopB</i> Δ<i>sopE2</i>) induces the same level of LDH release as a mutant lacking four effector proteins including SopE (Δ4; Δ<i>sipA</i> Δ<i>sopB</i> Δ<i>sopE</i> Δ<i>sopE2</i>). <b>C</b>) Mutants lacking four (Δ4; Δ<i>sipA</i> Δ<i>sopB</i> Δ<i>sopE</i> Δ<i>sopE2</i>) or eight (Δ8; Δ<i>sipA</i> Δ<i>sopB</i> Δ<i>sopE</i> Δ<i>sopE2</i> Δ<i>sopA</i> Δ<i>sptP</i> Δ<i>spvB</i> Δ<i>spvC</i>) virulence proteins induce LDH release with (Δ4^{<b>M−F+</b>}, Δ8^{<b>M−F+</b>}) or without flagellin (Δ4^{<b>M−F−</b>}, Δ8^{<b>M−F−</b>}), whereas a <i>sipB</i> mutant that lacks the ability for translocon insertion does not. <b>D</b>) IL-1 maturation induced by Δ4, Δ8, Δ4^{<b>M−F+</b>}, Δ8^{<b>M−F+</b>}, Δ4^{<b>M−F−</b>}, and Δ8^{<b>M−F−</b>}. n.d.: not detected. Mean +/− standard deviation of triplicates from at least 2 independent experiments. n.s.: not significant; *: p-value ≤0.05 (paired t-test in panel B; Mann-Whitney U test in panel C). Data shown in D) are representative of 3 independent experiments.</p

IL-1 maturation and LDH release induced by flagellin-deficient <i>S</i>. Typhimurium.

<p><b>A</b>) Western Blot analysis of <i>Salmonella</i> flagellins (FliC and FljB) and the T1 effector SopE in lysates (P) and supernatants (SN) of flagella wildtype strains and Δ<i>fliGHI</i> (M−F−). WT: wildtype, T1⁻: no T3SS-1, SopE/E2: Δ<i>sipA</i> Δ<i>sopB</i>; *:unspecific band as loading control. <b>B</b>) Flagellin-deficient <i>S</i>. Typhimurium induce LDH release from LPS-pretreated RAW264.7 macrophages. Infection was performed with the indicated <i>S</i>. Typhimurium strains (MOI 150) either without (black bars) or with centrifugation (grey bars) of cell plates. <b>C</b>) Release of mature IL-1 after infection of LPS-pretreated RAW264.7 macrophages with flagellin-deficient <i>S</i>. Typhimurium (Δ<i>fliGHI,</i> M−F−) following centrifugation. Experiments were performed in triplicate; mean +/− SD. n.s.: not significant; *: p-value ≤0.05 (Mann-Whitney U test).</p

Motility defect but not lack of flagellin leads to failure in caspase-1 induction.

<p><b>A</b>–<b>E</b>) LPS-primed RAW264.7 macrophages were infected with or without centrifugation with different strains of <i>S</i>. Typhimurium (MOI 150) that have <i>sopE</i> substituted by <i>sopE^m45-tem-1</i>. WT_TEM or T1⁻_TEM either have normal flagella (wildtype flagella), lack flagellin expression (M−F−), or express monomeric flagellin but do not assemble flagella (M−F+). <b>A</b>) SopE^M45-TEM-1 effector translocation into RAW264.7 macrophages was detected by measuring conversion of the TEM-1 beta-lactamase fluorescent substrate CCF2-AM. Values were normalized to the WT_TEM strain. Centrifugation restores effector translocation by WT_TEM^{<b>M−F−</b>} and WT_TEM^{<b>M−F+</b>}. <b>B</b>) Infection was performed with WT_TEM^{<b>M−F−</b>} (left side) or WT_TEM^{<b>M−F+</b>} (right side), respectively, where after cells were washed extensively, fixed and stained with DAPI (blue), phalloidin-TRITC (red), and anti-Salmonella LPS antibody (green) to visualize attachment of bacteria. Cells with attached WT_TEM^{<b>M−F−</b>} or WT_TEM^{<b>M−F+</b>} without (upper panels) or with centrifugation (lower panels), or with WT_TEM, were quantified as shown in C). Scale bar: 50 µm. <b>C</b>) Black circles: not centrifuged; grey circles: with centrifugation. Data shown from two independent experiments performed in duplicate. Black bar: mean of four data points. <b>D</b>) LDH release and <b>E</b>) IL-1 maturation after infection without (black bars) or with centrifugation (grey bars). Experiments were performed in triplicate; mean +/− SD.; n.s.: not significant; *: p-value ≤0.05.</p

Strains used in this study.

<p>a. M<b>⁻</b>F<b>⁻</b>: no expression of flagellin, no flagella (amotile).</p><p>b. M<b>⁻</b>F<b>⁺</b>: expression of flagellins (FliC and FljB), no assembly of flagella (amotile).</p

Effector- and T1-induced caspase-1 activation in the absence of flagellin is dose-dependent.

<p><b>A</b>) SopE^M45-TEM-1 translocation by strains SopE/E2_TEM (no centrifugation: black circles; centrifugation: open triangles), SopE/E2_TEM^{<b>M−F−</b>} (no centrifugation: open circles; centrifugation: black squares), and T1⁻_TEM (no centrifugation: black triangles; centrifugation: open squares) at different MOI. <b>B</b>) LDH release induced by the same strains as in A) correlates with SopE^M45-TEM-1 translocation in a dose-dependent manner. Data are representative of 3 independent experiments.</p