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

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

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

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

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

A Genome-Wide siRNA Screen Implicates Spire1/2 in SipA-Driven <i>Salmonella</i> Typhimurium Host Cell Invasion

<div><p><i>Salmonella</i> Typhimurium (<i>S</i>. Tm) is a leading cause of diarrhea. The disease is triggered by pathogen invasion into the gut epithelium. Invasion is attributed to the SPI-1 type 3 secretion system (T1). T1 injects effector proteins into epithelial cells and thereby elicits rearrangements of the host cellular actin cytoskeleton and pathogen invasion. The T1 effector proteins SopE, SopB, SopE2 and SipA are contributing to this. However, the host cell factors contributing to invasion are still not completely understood. To address this question comprehensively, we used Hela tissue culture cells, a genome-wide siRNA library, a modified gentamicin protection assay and <i>S</i>. Tm^SipA, a <i>sopBsopE2sopE</i> mutant which strongly relies on the T1 effector protein SipA to invade host cells. We found that <i>S</i>. Tm^SipA invasion does not elicit membrane ruffles, nor promote the entry of non-invasive bacteria "in trans". However, SipA-mediated infection involved the SPIRE family of actin nucleators, besides well-established host cell factors (WRC, ARP2/3, RhoGTPases, COPI). Stage-specific follow-up assays and knockout fibroblasts indicated that SPIRE1 and SPIRE2 are involved in different steps of the <i>S</i>. Tm infection process. Whereas SPIRE1 interferes with bacterial binding, SPIRE2 influences intracellular replication of <i>S</i>. Tm. Hence, these two proteins might fulfill non-redundant functions in the pathogen-host interaction. The lack of co-localization hints to a short, direct interaction between <i>S</i>. Tm and SPIRE proteins or to an indirect effect.</p></div

Infection step assays using iMEF cell lines.

<p>(A) Binding assay using <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- iMEFs normalized to the data from the wild type iMEF control cell line. <i>Spire1</i>^gt/gt show reduced <i>Salmonella</i> binding. (B) Effector injection assay using <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- iMEFs normalized to the wild type iMEF control cell line. <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- cells show attenuation and increase in effector injection, respectively. (C) Modified gentamicin protection assay using <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- iMEFs normalized to the wild type iMEF control cell line. <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- cell lines show decrease in invasion after 4h. Invasion is not dependent on <i>Salmonella</i> effectors or invasion type. (D) Intracellular replication in <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- iMEFs normalized to the wild type iMEF control cell line measure by plating assay. <i>Spire2</i>^-/- cell line shows decrease of intracellular bacterial replication. (E) Absolute number of CFUs corresponding to D. A-C show data from 3 independent experiments with 2 replicates each in HeLa Kyoto cells. D and E show data from 2 independent experiments with 3 replicates each. Asteriscs indicate significant differences. *: p<0.05.</p

Bacterial strains.

<p>Bacterial strains.</p