Shigella flexneri type III secreted effector OspF reveals new crosstalks of proinflammatory signaling pathways during bacterial infection
Introduction
During bacterial infection, the ability of a host organism to mount an innate immune response is critical to limit bacterial colonization. Such a response is initiated by the recognition of bacterial components including lipopolysaccharide (LPS), peptidoglycan and nucleic acids by the Toll-like receptors (TLRs) and Nod-like receptors, which in turn activate downstream proinflammatory cascades that orchestrate innate immunity [1], [2], [3]. Despite a large spectrum of innate defense mechanisms, many pathogens can effectively colonize their host. Successful infection often results from evolutionary selected strategies that circumvent or manipulate the host response to infection [4]. A widespread stratagem developed by pathogenic bacteria consists of injecting effector proteins into the cytoplasm or the nucleus of a target cell to inhibit or usurp specific cellular processes or signaling pathways [5]. This strategy is successfully exploited by the bacterium Shigella flexneri to invade and colonize the intestinal epithelium of humans, causing an acute mucosal inflammation called shigellosis or bacillary dysentery [6]. Once in contact with the basolateral surface of epithelial cells, these bacteria inject, by means of a type III secretion (T3S) apparatus, a set of effector proteins that promote their internalization. In addition, S. flexneri interferes with multiple host signaling pathways to dampen the inflammatory response of infected epithelial cells [7]. This response is initiated by initial sensing of bacterial invasion via a pool of membrane-localized Nod1 proteins, recruited at the site of bacterial entry and remaining on cell membrane fragments after rupture of the internalization vacuole [8], [9]. This early recognition is potentiated by Nod1-mediated sensing of peptidoglycan-derived peptides released by bacteria multiplying in the cytoplasm of infected epithelial cells [10]. Peptidoglycan recognition is generally followed by the homo-dimerization of Nod1, the recruitment and polyubiquitination of the kinase RICK/RIPK2 and the sequential activation of TGF-β-activated kinase-1 (TAK1), a member of the mitogen-activated protein (MAP) kinases kinase kinase (MAP3K) family [11]. TAK1 forms with the proteins TAK1-binding protein 1 (TAB1), TAB2 and/or TAB3 a complex that controls the activation of downstream key signaling pathways that lead to activation of the transcription factors nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) and induce proinflammatory gene expression [12], [13]. Once activated, TAK1 phosphorylates inhibitor of NF-κB (IκB) kinase-β (IKKβ) at key serine residues in the activation loop, resulting in its activation and leading to the phosphorylation and degradation of IκB and the nuclear translocation of NF-κB [14] . It has been recently reported that both IKKα and IKKγ/NEMO also contribute to NF-κB activation during S. flexneri infection [15]. In addition, TAK1 phosphorylates members of the MAP kinase kinase (MAP2K) family, which in turn phosphorylate and activate the MAP kinases Jun N-terminal kinase (JNK) and p38 [12].
Interestingly, S. flexneri has developed a set of T3S effectors that interfere with inflammation signaling and downregulate the expression of the inflammatory chemokine interleukin-8 (IL-8) IL-8, which recruits polymorphonuclear cells to the site of infection [7]. Among these effectors, OspF harbors a phosphothreonine lyase activity, which leads to the irreversible dephosphorylation of p38 and ERK in the nucleus of infected cells [16], [17]. The elimination of a phosphate by OspF suppresses MAPK activity by changing the conformation of the activation loop. It has been reported that a defect in MAPK activation is associated with inhibition of histone H3 phosphorylation and gene-specific repression of a subset of NF-κB regulated genes, including IL-8 [16]. A recent study from our laboratory, monitoring IL-8 expression at the single-cell level, confirms the role of OspF on IL-8 expression in infected cells and shows that, uninfected bystander epithelial cells constitute the main source of IL-8 secretion during S. flexneri infection [18].
Whereas the phosphothreonine lyase activity of OspF on MAPKs has been well characterized [16], [17], the impact of OspF on other host signaling pathways remains unknown. Here we report that, in addition to the dephosphorylation of p38 and ERK, OspF potentiates the activation of the JNK and NF-κB pathways triggered by S. flexneri infection. This unexpected effect of OspF was dependent on p38 and resulted from the disruption of a negative feedback loop regulation between p38 and TAK1 mediated via the phosphorylation of TAB1. Interestingly, potentiated JNK activation was not associated with enhanced c-Jun signaling as OspF also inhibits c-Jun expression at the transcriptional level. Altogether, our data reveal the broad impact of OspF on the activation of NF-κB, JNK and c-Jun and the crosstalks that connect these signaling pathways during bacterial infection.
Section snippets
Cell lines and reagents
Mouse embryonic fibroblasts (MEFs) from wild-type and p38α knockout mice were kindly provided by Prof. A.R. Nebreda (CNIO, Madrid, Spain). MEFs and HeLa Kyoto cells [19] were cultured in DMEM, supplemented with 10% FCS, antibiotics and L-glutamine. Antibodies against TAK1, MAPK p38, the phosphorylated form of MAPK p38, JNK, TAK1 and c-Jun were obtained from Cell Signaling Technology. Total c-Jun antibody was obtained from BD Transduction Laboratories. Antibodies against IκBα, NF-κB p65 and
OspF dephosphorylates p38 and potentiates the activation of JNK
The activation of the JNK signaling pathway is critical to mount an inflammation response against S. flexneri infection [22]. Therefore, in order to characterize the impact of OspF on inflammation signaling, we examined whether OspF affected the activation of JNK during infection. The effect of OspF on the activation of p38 was analyzed in parallel for comparison. To avoid secondary invasion resulting from intercellular bacterial motility that would complicate the analysis of time course
Discussion
Immune responses to bacterial infection are controlled by the complex molecular interactions that occur between the host signaling network and various bacterial products present at the surface of bacteria or released during infection. Structural components including LPS, peptidoglycan and lipoproteins as well as numerous bacterial proteins interact with specific receptors or signaling pathways and trigger or shape specific facets of host defense. In the case of S. flexneri infection, multiple
Conclusion
In conclusion, our data show that OspF has a complex impact on host signaling during infection. We found that, whereas OspF dephosphorylates p38, this effector potentiates the activation of JNK and NF-κB. This unexpected effect results from the disruption of a negative feedback loop between p38 and TAK1, which is mediated via the phosphorylation of TAB1 during infection. In addition, OspF inhibits c-Jun expression at the transcriptional level. Taken together, our data reveal new insights into
Acknowledgments
We thank Prof. A.R. Nebreda (CNIO, Madrid, Spain) for the generous gift of wt and p38−/− MEFs. We thank Dr. Hesso Farhan for comments on the manuscript. This work was funded by the Swiss National Science Foundation (grants 3100A0-113561 and 310030_1273361 to C.A.). C. Kasper was supported by the Werner-Siemens Foundation.
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