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A TIR domain protein from E. faecalis attenuates MyD88-mediated signaling and NF-?B activation.

ABSTRACT: Toll-like receptor signaling, mediated by functional Toll/interleukin-1 receptor (TIR) domains, plays a critical role in activating the innate immune response responsible for controlling and clearing infection. Bacterial protein mimics of components of this signaling pathway have been identified and function through inhibition of interactions between Toll-like receptors (TLRs) and their adaptor proteins, mediated by TIR domains. A previously uncharacterized gene, which we have named tcpF (for TIR domain-containing protein in E. faecalis) was identified in the genome of Enterococcus faecalis V583, and predicted to encode a protein resembling mammalian and bacterial TIR proteins. We overexpressed and purified TcpF from E. coli and found that the recombinant protein could bind to phosphatidylinositol phosphates in vitro, suggesting a mechanism by which TcpF may be anchored to the plasma membrane in close proximity to TIR domains of TLRs and adaptor proteins. Purified TcpF was also found to interact specifically with the TIR adaptor protein MyD88, and this interaction was dependent on the BB loop domain in the Box 2 region of TcpF. Despite no evidence of TcpF being a secreted protein, recombinant TcpF was effectively able to enter RAW264.7 cells in vitro although the mechanism by which this occurs remains to be determined. Overexpression of TcpF in mammalian cells suppressed the NF-?B activation induced by bacterial lipoteichoic acid. A mutant lacking the tcpF gene was attenuated for survival in macrophages, with increased ability to activate NF-?B compared to the wild type strain. Complementation in trans restored growth, and inhibition of NF-?B, to that of wild type levels. No appreciable difference in bacterial persistence, dissemination or pathogenesis was observed between the wild type and mutant in a mouse peritonitis model however, which suggested either a subtle role for TcpF or functional overlap with other redundant factor(s) in this virulence model.

SUBMITTER: Zou J

PROVIDER: S-EPMC4219826 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A TIR domain protein from E. faecalis attenuates MyD88-mediated signaling and NF-κB activation.

Zou Jun J Baghdayan Arto S AS Payne Sarah J SJ Shankar Nathan N

PloS one 20141104 11

Toll-like receptor signaling, mediated by functional Toll/interleukin-1 receptor (TIR) domains, plays a critical role in activating the innate immune response responsible for controlling and clearing infection. Bacterial protein mimics of components of this signaling pathway have been identified and function through inhibition of interactions between Toll-like receptors (TLRs) and their adaptor proteins, mediated by TIR domains. A previously uncharacterized gene, which we have named tcpF (for TI ...[more]

PMID: 25369374

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Project description:BackgroundRed blood cells (RBCs) transfusion is related to perioperative neurocognitive disorders. The toxic effect of free heme has been identified in many pathologies. However, the underlying mechanisms of RBCs transfusion or free heme in cognitive impairment have not been clearly explored. Therefore, this research was conducted to determine the mechanism of free heme-induced neuroinflammation and cognitive impairment.MethodsRats were received intraperitoneal injection of hemin alone or combined with intracerebroventricular injection of Hemopexin (HPX), and MWM test was conducted to measure cognitive function. The amount of heme-HPX complexes was evaluated by flow cytometry for CD91 + cells. The microglial inflammatory response in rat brain was observed by immunofluorescence staining of Iba-1, and the inflammatory factors of TNF-α, IL-1β and IL-6 in rat brain and BV2 cells were detected by ELISA analysis. Furthermore, neuronal apoptosis in HT22 cells alone and in HT22 + BV2 coculture system was detected by flow cytometry and immunofluorescence staining. Finally, western blot was conducted to detect TLR4/MyD88/NF-κB proteins in rat brain and BV2 cells treated with hemin or combined with pathway inhibitors. Additionally, the M1 surface marker CD86 was observed in BV2 cells to further confirm neuroinflammation.ResultsIntraperitoneal injection of hemin induced cognitive impairment, increase of CD91 + cells, up-regulation of TNF-α and IL-1β, down-regulation of IL-6, activation of microglia, and activation of the TLR4/MyD88/NF-κB signaling pathway in rat brain. Significantly, intracerebroventricular injection of HPX reduced the above effects. Hemin induced boost of TNF-α, IL-1β and IL-6 in BV2 cells, as well as apoptosis in HT22 cells. Notably, when HT22 cells were cocultured with BV2 cells, apoptosis was significantly increased. Hemin also induced activation of the TLR4/MyD88/NF-κB signaling pathway and increased the M1 surface marker CD86 in BV2 cells, and inhibiting this pathway reduced the inflammatory responses.ConclusionsFree heme induces cognitive impairment, and the underlying mechanism may involve neuronal apoptosis and microglial inflammation via the TLR4/MyD88/NF-κB signaling pathway. HPX may have potential therapeutic effects. Video Abstract.

Dataset Information

A TIR domain protein from E. faecalis attenuates MyD88-mediated signaling and NF-?B activation.

Publications

A TIR domain protein from E. faecalis attenuates MyD88-mediated signaling and NF-κB activation.

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