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

Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens

Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended “on-target” mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by “off-target” effects resulting from partial complementarity of siRNAs to multiple mRNAs via the “seed” region (i.e., nucleotides 2–8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended on-target effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying “seed reagents” for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens

Abstracts from the 20th International Symposium on Signal Transduction at the Blood-Brain Barriers

https://deepblue.lib.umich.edu/bitstream/2027.42/138963/1/12987_2017_Article_71.pd

Regulation of Caffeate Respiration in the Acetogenic Bacterium Acetobacterium woodii▿

The anaerobic acetogenic bacterium Acetobacterium woodii can conserve energy by oxidation of various substrates coupled to either carbonate or caffeate respiration. We used a cell suspension system to study the regulation and kinetics of induction of caffeate respiration. After addition of caffeate to suspensions of fructose-grown cells, there was a lag phase of about 90 min before caffeate reduction commenced. However, in the presence of tetracycline caffeate was not reduced, indicating that de novo protein synthesis is required for the ability to respire caffeate. Induction also took place in the presence of CO2, and once a culture was induced, caffeate and CO2 were used simultaneously as electron acceptors. Induction of caffeate reduction was also observed with H2 plus CO2 as the substrate, but the lag phase was much longer. Again, caffeate and CO2 were used simultaneously as electron acceptors. In contrast, during oxidation of methyl groups derived from methanol or betaine, acetogenesis was the preferred energy-conserving pathway, and caffeate reduction started only after acetogenesis was completed. The differential flow of reductants was also observed with suspensions of resting cells in which caffeate reduction was induced prior to harvest of the cells. These cell suspensions utilized caffeate and CO2 simultaneously with fructose or hydrogen as electron donors, but CO2 was preferred over caffeate during methyl group oxidation. Caffeate-induced resting cells could reduce caffeate and also p-coumarate or ferulate with hydrogen as the electron donor. p-Coumarate or ferulate also served as an inducer for caffeate reduction. Interestingly, caffeate-induced cells reduced ferulate in the absence of an external reductant, indicating that caffeate also induces the enzymes required for oxidation of the methyl group of ferulate

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

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

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