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

Zou J, Baghdayan AS, Payne SJ, Shankar N - PLoS ONE (2014)

Bottom Line: 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.A mutant lacking the tcpF gene was attenuated for survival in macrophages, with increased ability to activate NF-κB compared to the wild type strain.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.

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-κB activation induced by bacterial lipoteichoic acid. A mutant lacking the tcpF gene was attenuated for survival in macrophages, with increased ability to activate NF-κB compared to the wild type strain. Complementation in trans restored growth, and inhibition of NF-κB, 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.

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Related in: MedlinePlus

Expression, purification and characterization of MBP-TcpF.(A) Cell lysates from uninduced and induced cultures of C41 (DE3) E. coli harboring plasmid pOU1811 were separated on a 10% SDS-PAGE gel and stained with Coomassie blue R-250. (B) Purified MBP-TcpF (lane 1), MBP-TcpF digested with enterokinase (lane 2) and purified TcpF (lane 3) were run on a 10% SDS-PAGE gel and stained with Coomassie blue R-250. (C) RAW264.7 cells incubated with increasing concentrations of MBP-TcpF or MBP alone for 5 h. After washing and treatment with trypsin, the lysate was subjected to Western blot and probed with antibodies to TcpF.
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pone-0112010-g002: Expression, purification and characterization of MBP-TcpF.(A) Cell lysates from uninduced and induced cultures of C41 (DE3) E. coli harboring plasmid pOU1811 were separated on a 10% SDS-PAGE gel and stained with Coomassie blue R-250. (B) Purified MBP-TcpF (lane 1), MBP-TcpF digested with enterokinase (lane 2) and purified TcpF (lane 3) were run on a 10% SDS-PAGE gel and stained with Coomassie blue R-250. (C) RAW264.7 cells incubated with increasing concentrations of MBP-TcpF or MBP alone for 5 h. After washing and treatment with trypsin, the lysate was subjected to Western blot and probed with antibodies to TcpF.

Mentions: Employing the pMAL-c5E vector, we were successful in purifying MBP-TcpF fusion protein that was expressed in the cytoplasm (Figure 2A). Efficient cleavage of the MBP tag was achieved by controlled digestion with recombinant enterokinase, and pure TcpF obtained by employing ion-exchange chromatography was checked on an SDS-PAGE gel following Coomassie Blue staining (Figure 2B). Identity was confirmed by limited N-terminal sequencing.


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

Zou J, Baghdayan AS, Payne SJ, Shankar N - PLoS ONE (2014)

Expression, purification and characterization of MBP-TcpF.(A) Cell lysates from uninduced and induced cultures of C41 (DE3) E. coli harboring plasmid pOU1811 were separated on a 10% SDS-PAGE gel and stained with Coomassie blue R-250. (B) Purified MBP-TcpF (lane 1), MBP-TcpF digested with enterokinase (lane 2) and purified TcpF (lane 3) were run on a 10% SDS-PAGE gel and stained with Coomassie blue R-250. (C) RAW264.7 cells incubated with increasing concentrations of MBP-TcpF or MBP alone for 5 h. After washing and treatment with trypsin, the lysate was subjected to Western blot and probed with antibodies to TcpF.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219826&req=5

pone-0112010-g002: Expression, purification and characterization of MBP-TcpF.(A) Cell lysates from uninduced and induced cultures of C41 (DE3) E. coli harboring plasmid pOU1811 were separated on a 10% SDS-PAGE gel and stained with Coomassie blue R-250. (B) Purified MBP-TcpF (lane 1), MBP-TcpF digested with enterokinase (lane 2) and purified TcpF (lane 3) were run on a 10% SDS-PAGE gel and stained with Coomassie blue R-250. (C) RAW264.7 cells incubated with increasing concentrations of MBP-TcpF or MBP alone for 5 h. After washing and treatment with trypsin, the lysate was subjected to Western blot and probed with antibodies to TcpF.
Mentions: Employing the pMAL-c5E vector, we were successful in purifying MBP-TcpF fusion protein that was expressed in the cytoplasm (Figure 2A). Efficient cleavage of the MBP tag was achieved by controlled digestion with recombinant enterokinase, and pure TcpF obtained by employing ion-exchange chromatography was checked on an SDS-PAGE gel following Coomassie Blue staining (Figure 2B). Identity was confirmed by limited N-terminal sequencing.

Bottom Line: 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.A mutant lacking the tcpF gene was attenuated for survival in macrophages, with increased ability to activate NF-κB compared to the wild type strain.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.

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-κB activation induced by bacterial lipoteichoic acid. A mutant lacking the tcpF gene was attenuated for survival in macrophages, with increased ability to activate NF-κB compared to the wild type strain. Complementation in trans restored growth, and inhibition of NF-κB, 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.

Show MeSH
Related in: MedlinePlus