| 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.LPS is a glycolipid located in the outer membrane of Gram-negative bacteria. It is composed of three covalently linked domains: lipid A, which is embedded in the outer membrane; the oligosaccharide core; and the O-polysaccharide or O-antigen, which cover the bacterial surface. During infections sustained by Gram-negative bacteria, detection of LPS initiates an acute inflammatory response as LPS, mainly by the lipid A, which is the real pathogen-associated molecular pattern (PAMP), is sensed by the innate immune system, through the binding to the pattern recognition receptor (PRR) complex of myeloid differentiation protein 2 (MD-2) and Toll-like receptor (TLR) 4 (TLR4) (1–3). The downstream effects of LPS recognition elicit effector mechanisms aimed at pathogen eradication. However, LPS can also elicit an host reaction because it is a major mediator of pathologic processes (4). The strength of the innate immune response to LPS can be modulated by its chemical structure; specifically, a fine tuning of the lipid A structure can significantly affect the immunostimulatory properties of the whole LPS molecule (5, 6). There is a strong correlation between the number of acyl chains of lipid A and the immunological response via the TLR4 pathway. In general, hexaacylated lipid A species are agonists, whereas tetraacylated species are antagonists with a weak inflammatory potential (7). Gram-negative bacteria can synthesize a range of differentially acylated LPSs as a result of the LPS biosynthesis. Changes in lipid A acylation underlie the adaptation of pathogens to different hosts, such as Yersinia pestis (8), or to different phases of pathogenesis such as Salmonella typhimurium (9) or in the establishment of chronic infection such as Pseudomonas aeruginosa (10, 11).Shigella flexneri is a Gram-negative pathogen that infects humans. The ingestion of as few as 100 bacteria is sufficient to cause bacillary dysentery, a severe rectocolitis caused by the dramatic inflammatory reaction induced by Shigella invasion on the colonic and rectal mucosa (12). Shigella enters epithelial cells by injecting effectors via a type III secretion system (T3SS) (13), escapes from the phagocytic vacuole, and actively proliferates within the cytosol of infected cells (14, 15). Bacterial proliferation is a potent signal to initiate inflammation because intracellular shigellae activate NF-κB following recognition of peptidoglycan (PGN) by the PRR Nod1, leading to IL-8 production (16, 17). IL-8 attracts neutrophils that are required for the clearance of shigellae, but also participates in epithelial barrier destruction (18). In macrophages, Shigella is able to trigger the assembly of the inflammasome, an important defense mechanism that is part of the innate immune system (19). The inflammasome is a multiprotein complex that mediates activation of caspase-1, which promotes the secretion of the proinflammatory cytokines IL-1β and IL-18 as well as a cell death process called pyroptosis (20, 21). Different PRRs, i.e., TLRs and nucleotide-binding oligomerization domain-like receptors (NLRs) contribute to the inflammasome assembly (22). In Shigella-infected macrophages, the activation of the NLRC4-mediated inflammasome triggers cell death and release of IL-1β and IL-18 (19, 23). Indeed, production of IL-1β is a paradigm of shigellosis: the chief role of this cytokine has been highlighted in vivo in several studies (24–26).In tissues of animals and in ex vivo human samples infected with Shigella (27), a huge amount of LPS is usually observed, reflecting the presence of living bacteria and/or of processed molecules. However, whether, how, and at to what extent this mass of LPS present in Shigella-infected tissues could play a role in the inflammation remains largely unknown.In 2002, D’Hauteville et al. reported that, in S. flexneri, the lack of msbB genes, msbB1 and msbB2, both encoding the enzyme myristoyl transferase, reduces lipid A acylation degree along with TNF-α production and epithelial lining inflammatory destruction in a rabbit model of Shigella infection (28, 29). This study suggests that LPS composition can greatly influence the degree of inflammation induced by Shigella.In line with these issues, here we intend to contribute to the understanding of the role played by LPS in Shigella pathogenesis. Hence, we addressed the question of whether Shigella could adapt the LPS structure to the host thereby exploiting the mechanism of LPS modification to hijack the innate immune response. With this aim, we extracted, purified, and analyzed the LPS of shigellae resident in epithelial cells. We detailed the immunopotential of this structure and compared it to that of conventionally grown bacteria. Together our results point to a key role for LPS during the Shigella invasive process.We report 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 conventionally grown (aLPS) differ in the number of acyl chains in lipid A, with iLPS being hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of aLPS; (iv) both LPS forms signal mainly through the TLR4/MyD88 pathway; (v) iLPS influences the inflammasome-mediated production of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime PMNs for an oxidative burst. |
