Activation and Evasion of the Inflammasome by Yersinia

Multicellular organisms constantly encounter microbes, ranging from beneficial to pathogenic. In order to mount appropriate immune responses that allow the host to clear pathogens while maintaining a balance with nonpathogenic microbes, the innate immune system must discriminate between pathogens and commensals. Through the recognition of virulence structures and activities, innate immune cells can distinguish pathogens from commensals. One such virulence structure, the type III secretion system (T3SS), translocates effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. Over 25 species of pathogenic gram-negative bacteria depend upon T3SSs to cause productive infection. However, recognition of T3SS activity by cytosolic Pattern Recognition Receptors (PRRs) of the Nucleotide-Binding Domain Leucine Rich Repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Yersinia pseudotuberculosis (Yptb) is an ideal model for inflammasome recognition of the T3SS as Yptb expresses an archetypal T3SS and is a genetically tractable, natural rodent pathogen. Investigation of the interaction between the inflammasomes and the T3SS could reveal important mechanistic and cell biological information about the inflammasomes themselves as well as a potential target for treating T3SS expressing bacteria. Although effectors of Yptb has been shown to actively inhibit inflammasome activation, until this work very little was known about what inflammasome is actually activated by the T3SS, what activity of the T3SS is recognized, and how Yersinia’s YopK protein inhibits inflammasome activation. Therefore, we investigated the bacterial and host interactions required for inflammasome activation and the mechanism by which YopK inhibits inflammasome activation. To dissect the contribution of the different consequences of T3SSs, pore-formation and translocation, to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions. Our findings indicated that inflammasome activation required hyper-translocation of YopB/D. Using macrophages deficient in caspase-1, caspase-11, or certain guanylate binding proteins, we characterized the host pathways activated by hyper-translocation of YopD/B. Finally, using mutations in YopK, we characterized how YopK prevents inflammasome activation. Overall, our findings help define how bacterial virulence activities activate innate immune responses

Activation of Ca2+-activated Cl- current by depolarizing steps in rabbit urethral interstitial cells.

Interstitial cells were isolated from strips of rabbit urethra for study using the amphotericin B perforated-patch technique. Depolarizing steps to -30 mV or greater activated a Ca2+ current (ICa), followed by a Ca2+-activated Cl- current, and, on stepping back to -80 mV, large Cl- tail currents were observed. Both currents were abolished when the cells were superfused with Ca2+-free bath solution, suggesting that Ca2+ influx was necessary for activation of the Cl- current. The Cl- current was also abolished when Ba2+ was substituted for Ca2+ in the bath or the cell was dialyzed with EGTA (2 mM). The Cl- current was also reduced by cyclopiazonic acid, ryanodine, 2-aminoethoxydiphenyl borate (2-APB), and xestospongin C, suggesting that Ca2+-induced Ca2+ release (CICR) involving both ryanodine and inositol 1,4,5-trisphosphate receptors contributes to its activation

Caspase-11 Activation in Response to Bacterial Secretion Systems That Access the Host Cytosol

Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in inflammatory responses. The inflammasome activates caspase-1 to process and secrete IL-1β. However, the mechanisms governing IL-1α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1α and IL-1β. Caspase-11 activation in response to Gram-negative bacteria requires Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon production. Whether additional bacterial signals trigger caspase-11 activation is unknown. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, even in the absence of flagellin, these secretion systems induce inflammasome activation and the release of IL-1α and IL-1β, but the inflammasome pathways that mediate this response are unclear. We observe rapid IL-1α and IL-1β release and cell death in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis. Unlike IL-1β, IL-1α secretion does not require caspase-1. Instead, caspase-11 activation is required for both IL-1α secretion and cell death in response to the activity of these secretion systems. Interestingly, whereas caspase-11 promotes IL-1β release in response to the type IV secretion system through the NLRP3/ASC inflammasome, caspase-11-dependent release of IL-1α is independent of both the NAIP5/NLRC4 and NLRP3/ASC inflammasomes as well as TRIF and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila. Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response important for host defense

Engineering bacteria to solve the Burnt Pancake Problem

<p>Abstract</p> <p>Background</p> <p>We investigated the possibility of executing DNA-based computation in living cells by engineering <it>Escherichia coli </it>to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA.</p> <p>Results</p> <p>Inversions (or "flips") of the DNA fragment pancakes are driven by the <it>Salmonella typhimurium </it>Hin/<it>hix </it>DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in <it>E. coli</it>. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; <it>E. coli </it>cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of <it>in vivo </it>flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes.</p> <p>Conclusion</p> <p>The Hin/<it>hix </it>system is a proof-of-concept demonstration of <it>in vivo </it>computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/<it>hix </it>may provide a flexible new tool for manipulating transgenic DNA <it>in vivo</it>.</p

Inflammasome activation in response to the Yersinia type III secretion system requires hyperinjection of translocon proteins YopB and YopD

Type III secretion systems (T3SS) translocate effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. However, recognition of T3SS activity by cytosolic pattern recognition receptors (PRRs) of the nucleotide-binding domain leucine rich repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Macrophages infected with Yersinia pseudotuberculosis strains lacking all known effectors or lacking the translocation regulator YopK induce rapid activation of both the canonical NLRP3 and noncanonical caspase-11 inflammasomes. While this inflammasome activation requires a functional T3SS, the precise signal that triggers inflammasome activation in response to Yersinia T3SS activity remains unclear. Effectorless strains of Yersinia as well as ΔyopK strains translocate elevated levels of T3SS substrates into infected cells. To dissect the contribution of pore formation and translocation to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions of the T3SS. Notably, YopD variants that abrogated translocation but not pore-forming activity failed to induce inflammasome activation. Furthermore, analysis of individual infected cells revealed that inflammasome activation at the single-cell level correlated with translocated levels of YopB and YopD themselves. Intriguingly, LcrH mutants that are fully competent for effector translocation but produce and translocate lower levels of YopB and YopD also fail to trigger inflammasome activation. Our findings therefore suggest that hypertranslocation of YopD and YopB is linked to inflammasome activation in response to the Yersinia T3SS

IL-1α and IL-1β control bacterial burden and neutrophil recruitment <i>in vivo</i>.

<p>(<b>A</b>) 8–12 week old B6 or <i>Il1r1^−/−</i> mice were infected with 1×10⁶ Δ<i>flaA L. pneumophila</i> intranasally (IN). Lungs were plated to quantify CFU per gram. Graph shows the mean ± SEM of three or four infected mice per group. Dashed line represents the limit of detection. (<b>B</b> and <b>C</b>) B6 or <i>Il1r1^−/−</i> mice were infected with 1×10⁶ Δ<i>flaA</i> Lp IN. 24 hours post-infection, bronchoalveolar lavage fluid (BALF) was collected and the percentage of neutrophils in the BALF was quantified by flow cytometry. Percentages are reported as the frequency of live cells in the BALF. (B) Representative flow cytometry plots showing the percentage of Gr-1⁺Ly6G⁺ neutrophils. (C) Graph showing the percentage of neutrophils. Each point represents an individual mouse and lines indicate the mean of 4 mice per group. (<b>D, E</b>, and <b>F</b>) B6 mice were injected intraperitoneally (IP) with either PBS, 100 µg isotype control antibody (iso), 100 µg anti-IL-1α antibody, 100 µg anti-IL-1β antibody, or 100 µg each of anti-IL-1α and anti-IL-1β (anti-IL-1α/β) 16 hours before infection. The mice were then intranasally infected with either 1×10⁶ Δ<i>flaA</i> Lp or mock infected with PBS. (D and E) 24 hours post-infection, BALF was collected and flow cytometry was performed to quantify the percentage of neutrophils. (D) Representative flow cytometry plots showing the percentage of Gr-1⁺Ly6G⁺ neutrophils. (E) Graph showing the percentage of neutrophils. Each point represents an individual mouse, lines indicate the mean of 8 mice per group, and error bars represent SEM. Shown are the pooled results of two independent experiments. (F) 72 hours post-infection, the lungs were plated to quantify CFU per gram. Each point represents an individual mouse. Line indicates the mean of 4 infected mice per group with error bars representing SEM. *** is p<0.001 by one-way ANOVA with Tukey post-test or unpaired t-test (C). **is p<0.01 and *is p<0.05 by unpaired t-test. NS is not significant.</p

Activity of Uncleaved Caspase-8 Controls Anti-bacterial Immune Defense and TLR-Induced Cytokine Production Independent of Cell Death.

Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense

Caspase-11 mediates inflammasome activation in response to a functional <i>Yersinia</i> type III secretion system.

<p>BMDMs from B6, <i>Casp1^−/−Casp11^−/−</i>, <i>Casp1^−/−</i>, or <i>Casp11^−/−</i> mice were primed with 0.05 µg/mL LPS for 4 hours and infected with type III secretion system-deficient <i>Y. pseudotuberculosis</i> (Δ<i>yopB</i> Yp), effectorless <i>Y. pseudotuberculosis</i> ΔHOJMEK (Δ6 Yp), or PBS (mock infection) or treated with 2.5 mm ATP for 4 hours. (<b>A</b>) Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. (<b>B</b>) Cell death (% cytotoxicity) was measured by lactate dehydrogenase (LDH) release relative to Triton X-100-lysed cells. Graphs show the mean ± SEM of triplicate wells. Data are representative of two independent experiments. *** is p<0.001 and ** is p<0.01 by two-way ANOVA with Bonferroni post-test. NS is not significant.</p