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. 2013 Nov 12;110(46):E4345-54.
doi: 10.1073/pnas.1303641110. Epub 2013 Oct 28.

Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation

Ida Paciello  1 , Alba Silipo, Luigi Lembo-Fazio, Laura Curcurù, Anna Zumsteg, Gaëlle Noël, Valeria Ciancarella, Luisa Sturiale, Antonio Molinaro, Maria Lina Bernardini
Affiliations

Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation

Ida Paciello et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2013 Dec 17;110(51):20843

Abstract

LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection.

Keywords: PAMPs/PRRs; enteric pathogen; immune evasion; innate immunity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Acylation and glycosylation pattern of iLPS and aLPS of S. flexneri. (A) Structure of the Lipid A (Right) and of the core region (Left) produced by iLPS of S. flexneri. The lipid A is constituted by a mixture of species differing for the acylation pattern; red acyl chains are not stoichiometric. The core is formed by an undecasaccharide carrying a phosphate group on the first heptose residue. (B) Comparison between negative ion MALDI-TOF spectra from iLPS (Left) and aLPS (Right) shows change in the distribution of lipid A species. The relative intensity of the peaks related to the lipid A clearly shows the abundance of species with a low acylation pattern in the iLPS and the presence of a single heptaacylated species in the aLPS and a minor pyrophosphorylated form. In the MALDI TOF mass spectrum of the iLPS, the ion peaks from the core oligosaccharide are also indicated.
Fig. 2.
Fig. 2.
TLR4 engagement by iLPS and aLPS. Activation of NF-κB, iL-8 mRNA expression, and IL-8 production in HEK 293 hTLR4/MD2-CD14 stimulated with iLPS, aLPS, and E. coli LPS (A) and TLR4-mediated competition of iLPS and aLPS on E. coli LPS (B). (A) Fold of NF-κB activation upon stimulation of HEK 293 hTLR4/MD2-CD14 with 1 and 10 ng/mL of LPS derived from intracellular shigellae (iLPS) and shigellae grown in culture medium (aLPS) for 4 h. Commercial hexaacylated E. coli LPS was used as a control; qPCR of il-8 mRNA induction after stimulation with the three LPS forms for 4 h; and IL-8 secretion after stimulation for 18 h as described earlier. (B) Fold of NF-κB activation upon stimulation of HEK 293 hTLR4/MD2-CD14 for 1 h with 1, 10, and 100 ng/mL of iLPS or aLPS before exposure to E. coli LPS 100 ng/mL for 4 h. For activation of NF-κB and IL-8 production, mean values (±SEM) of three representative experiments are shown (*P < 0.05, **P < 0.01, and ***P < 0.001). For qPCR, results are normalized to the internal gapdh gene control and are presented as the ratio of gene expression between stimulated and unstimulated HEK 293 cells.
Fig. 3.
Fig. 3.
Modulation of cytokine release in BMDMs stimulated with Shigella iLPS or aLPS. Cytokine release in BMDMs stimulated with Shigella iLPS, aLPS, and E. coli LPS: TNF-α, IL-1β, IL-6, KC, CXCL-10, CCL-5, and IL-18 released by BMDMs after stimulation with 1 and 10 ng/mL of LPS derived from intracellular shigellae (iLPS) and shigellae grown in TSB medium (aLPS) and E. coli LPS, measured by ELISA at 6 h (A) and 18 h (B). For TNF-α, IL-6, KC, CXCL-10, and CCL-5, mean values (±SEM) of three representative experiments are shown; for IL-1β, data shown are mean values (±SEM) of six representative experiments. Significant difference between iLPS-generated values and the corresponding aLPS values are indicated (iLPS vs. aLPS; *P < 0.05, **P < 0.01, and ***P < 0.001).
Fig. 4.
Fig. 4.
MyD88- and TRIF-mediated signaling is involved in cytokine production upon stimulation with Shigella iLPS and aLPS. WT, myd88−/−, or trif−/− BMDMs stimulated with Shigella iLPS, aLPS, and E. coli LPS at 6 h (A) and 18 h (B). (A and B) TNF-α, IL-1β, IL-6, KC, CXCL-10, and CCL-5 released by BMDMs after stimulation with 1 and 10 ng/mL of LPS purified by intracellular shigellae (iLPS), shigellae grown in TSB (aLPS), and E. coli LPS measured through ELISA at 6 h and 18 h, respectively. Mean values (±SEM) of three representative experiments are shown. Significant difference between values WT cells and the corresponding values in defective cells are indicated (WT vs. defective; *P < 0.05, **P < 0.01, and ***P < 0.001).
Fig. 5.
Fig. 5.
Influence of the iLPS and aLPS on the Shigella-mediated inflammasome in BMDMs. (A) (Left) Representative cytofluorimetric output of PI analysis of unstimulated BMDMs or BMDMs pretreated with 10 ng/mL of iLPS or aLPS or E. coli LPS (4 h) and infected with M90T at an MOI of 10 (3 h). (Right) LDH results of BMDMs stimulated and infected as described earlier. (B) (Upper) Representative cytofluorimetric output of caspase-1 activation through the FLICA approach under conditions as described earlier (caspase-1–negative BMDMs in R1 quadrant; caspase-1–positive BMDMs in the R2 quadrant). (Lower) Immunoblot analysis of caspase-1 maturation in cell lysates and supernatants of M90T-infected BMDMs treated as described earlier. The blot is probed with the protein Hsp70 as a loading control. (C) (Upper) IL-1β (Left) and IL-18 (Right) release of Shigella-infected BMDMs measured through ELISA under conditions as described before. Mean values (±SEM) of three representative experiments are shown. Significant differences between iLPS-generated values and the corresponding aLPS values are indicated (iLPS vs. aLPS; **P < 0.01). (Lower Left) qPCR of il-1β mRNA following stimulation in BMDMs with 10 ng/mL LPS at 3 h as described earlier and in BMDMs unstimulated or pretreated with LPS (4 h) as described earlier and infected with M90T at an MOI of 10 (3 h). Results are normalized to the internal gapdh gene control and are presented as the ratio of gene expression between stimulated and unstimulated BMDMs. (Lower Right) Western immunoblot analysis of IL-1β maturation in cell lysates and supernatant of M90T-infected BMDMs treated as described earlier. The blot is probed with the protein Hsp70 as a loading control.
Fig. 6.
Fig. 6.
iLPS fails to effectively prime human neutrophils. Human neutrophils were isolated and primed with Shigella aLPS, iLPS, or S. typhimurium S-form TLR-grade LPS at 100, 33, 11, 4, or 1 ng/mL. Neutrophils were then stimulated with 100 nM fMLF, and ROS levels were measured continuously for 15 min by chemiluminescence by using HPR and luminol. Peak ROS production occurred at 1 min 30 seconds after fMLF addition, and these data are the average of triplicate wells from this time point. Data are displayed as relative light units (RLU). The experiment was performed three times. Means and SDs from a representative experiment are shown. Significant differences between iLPS generated values and the corresponding aLPS values are indicated (*P < 0.05, **P < 0.01, and ***P < 0.001).
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