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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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Role of TcpF in NF-κB activation.(A) The translocation of the p65 subunit of NF-κB to the nucleus of RAW264.7 macrophages infected (MOI = 10) with wild type, TcpF mutant (SPB03) and complemented strain (SPB04) for 1 hour or without infection (Control), was analyzed by subjecting the nuclear fraction to Western blot with p65 antibody. (B) The NF-κB transcriptional activity was assessed during infection of MEF cells by wild type, TcpF mutant and complemented strains. MEF cells were transfected with NF-κB-luciferase and Renilla-luciferase reporter constructs. After 24 hours, the medium was changed and cells were challenged with enterococci at a MOI of 10 or with LPS (0.5 µg/ml) as positive control. The data represent the mean values of three independent experiments and error bars indicate the standard deviations. Significance * P<0.05.
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pone-0112010-g006: Role of TcpF in NF-κB activation.(A) The translocation of the p65 subunit of NF-κB to the nucleus of RAW264.7 macrophages infected (MOI = 10) with wild type, TcpF mutant (SPB03) and complemented strain (SPB04) for 1 hour or without infection (Control), was analyzed by subjecting the nuclear fraction to Western blot with p65 antibody. (B) The NF-κB transcriptional activity was assessed during infection of MEF cells by wild type, TcpF mutant and complemented strains. MEF cells were transfected with NF-κB-luciferase and Renilla-luciferase reporter constructs. After 24 hours, the medium was changed and cells were challenged with enterococci at a MOI of 10 or with LPS (0.5 µg/ml) as positive control. The data represent the mean values of three independent experiments and error bars indicate the standard deviations. Significance * P<0.05.

Mentions: NF-κB is a transcription factor which is activated by Toll-like receptor signaling through TIR domain-containing adaptor molecules. To test the role of TcpF in NF-κB activation during enterococcal infection, we first compared the nuclear translocation of NF-κB-p65 (a subunit of the NF-κB transcription complex) during infection of mouse RAW264.7 macrophages with strains E99, SPB03 or SPB04, by Western blot. The results revealed that deletion of TcpF increases the abundance of p65 in the host nucleus during enterococcal infection compared with wild type and complemented strains (Figure 6A). To further confirm the role of TcpF in interfering with NF-κB activation, NF-κB reporter plasmid was used to transfect MEF cells to monitor the NF-κB transcriptional activation during enterococcal infection. The results from these experiments showed that the TcpF mutant SPB03 stimulated a much higher level of NF-κB activation compared to wild type and complemented strains (Figure 6B). Although the expression of TcpF did not totally abolish NF-κB activation, the diminished NF-κB activation during enterococcal infection may serve as one way to evade the host immune response.


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)

Role of TcpF in NF-κB activation.(A) The translocation of the p65 subunit of NF-κB to the nucleus of RAW264.7 macrophages infected (MOI = 10) with wild type, TcpF mutant (SPB03) and complemented strain (SPB04) for 1 hour or without infection (Control), was analyzed by subjecting the nuclear fraction to Western blot with p65 antibody. (B) The NF-κB transcriptional activity was assessed during infection of MEF cells by wild type, TcpF mutant and complemented strains. MEF cells were transfected with NF-κB-luciferase and Renilla-luciferase reporter constructs. After 24 hours, the medium was changed and cells were challenged with enterococci at a MOI of 10 or with LPS (0.5 µg/ml) as positive control. The data represent the mean values of three independent experiments and error bars indicate the standard deviations. Significance * P<0.05.
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Related In: Results  -  Collection

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

pone-0112010-g006: Role of TcpF in NF-κB activation.(A) The translocation of the p65 subunit of NF-κB to the nucleus of RAW264.7 macrophages infected (MOI = 10) with wild type, TcpF mutant (SPB03) and complemented strain (SPB04) for 1 hour or without infection (Control), was analyzed by subjecting the nuclear fraction to Western blot with p65 antibody. (B) The NF-κB transcriptional activity was assessed during infection of MEF cells by wild type, TcpF mutant and complemented strains. MEF cells were transfected with NF-κB-luciferase and Renilla-luciferase reporter constructs. After 24 hours, the medium was changed and cells were challenged with enterococci at a MOI of 10 or with LPS (0.5 µg/ml) as positive control. The data represent the mean values of three independent experiments and error bars indicate the standard deviations. Significance * P<0.05.
Mentions: NF-κB is a transcription factor which is activated by Toll-like receptor signaling through TIR domain-containing adaptor molecules. To test the role of TcpF in NF-κB activation during enterococcal infection, we first compared the nuclear translocation of NF-κB-p65 (a subunit of the NF-κB transcription complex) during infection of mouse RAW264.7 macrophages with strains E99, SPB03 or SPB04, by Western blot. The results revealed that deletion of TcpF increases the abundance of p65 in the host nucleus during enterococcal infection compared with wild type and complemented strains (Figure 6A). To further confirm the role of TcpF in interfering with NF-κB activation, NF-κB reporter plasmid was used to transfect MEF cells to monitor the NF-κB transcriptional activation during enterococcal infection. The results from these experiments showed that the TcpF mutant SPB03 stimulated a much higher level of NF-κB activation compared to wild type and complemented strains (Figure 6B). Although the expression of TcpF did not totally abolish NF-κB activation, the diminished NF-κB activation during enterococcal infection may serve as one way to evade the host immune response.

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