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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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Characterization of intramacrophage survival of E. faecalis wild type (E99), TcpF mutant (SPB03) and complemented (SPB04) strains.Survival of E99, SPB03 and SPB04 based on colony-forming units per 105 RAW264.7 macrophages, infected at a MOI of 100, after 2, 24, 48, and 72 hours post infection. Experiments were performed in triplicate. Significant differences (*P<0.05) were found at 24, 48, and 72 hours between wild-type and mutant.
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pone-0112010-g007: Characterization of intramacrophage survival of E. faecalis wild type (E99), TcpF mutant (SPB03) and complemented (SPB04) strains.Survival of E99, SPB03 and SPB04 based on colony-forming units per 105 RAW264.7 macrophages, infected at a MOI of 100, after 2, 24, 48, and 72 hours post infection. Experiments were performed in triplicate. Significant differences (*P<0.05) were found at 24, 48, and 72 hours between wild-type and mutant.

Mentions: The role of TcpF in host persistence was investigated using an in vitro macrophage survival assay [26]. Bacterial survival rates were compared for the internalized wild type, TcpF mutant and complemented strains in macrophages infected at a multiplicity of infection of 100. At 2 hours postinfection, all the strains reached a density of about 104 CFU per 105 macrophages. The number of viable bacteria for the wild-type and complemented strains is significantly higher than that of the TcpF mutant strain at 24, 48 and 72 hours postinfection (Figure 7). An approximately one log difference in bacterial numbers was observed between the wild-type and mutant at 72 hours postinfection.


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)

Characterization of intramacrophage survival of E. faecalis wild type (E99), TcpF mutant (SPB03) and complemented (SPB04) strains.Survival of E99, SPB03 and SPB04 based on colony-forming units per 105 RAW264.7 macrophages, infected at a MOI of 100, after 2, 24, 48, and 72 hours post infection. Experiments were performed in triplicate. Significant differences (*P<0.05) were found at 24, 48, and 72 hours between wild-type and mutant.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112010-g007: Characterization of intramacrophage survival of E. faecalis wild type (E99), TcpF mutant (SPB03) and complemented (SPB04) strains.Survival of E99, SPB03 and SPB04 based on colony-forming units per 105 RAW264.7 macrophages, infected at a MOI of 100, after 2, 24, 48, and 72 hours post infection. Experiments were performed in triplicate. Significant differences (*P<0.05) were found at 24, 48, and 72 hours between wild-type and mutant.
Mentions: The role of TcpF in host persistence was investigated using an in vitro macrophage survival assay [26]. Bacterial survival rates were compared for the internalized wild type, TcpF mutant and complemented strains in macrophages infected at a multiplicity of infection of 100. At 2 hours postinfection, all the strains reached a density of about 104 CFU per 105 macrophages. The number of viable bacteria for the wild-type and complemented strains is significantly higher than that of the TcpF mutant strain at 24, 48 and 72 hours postinfection (Figure 7). An approximately one log difference in bacterial numbers was observed between the wild-type and mutant at 72 hours postinfection.

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