Table 1.
Enterococcus faecalis strains and plasmids used in this work.
Table 2.
Oligonucleotide primers used in this work.
Figure 1.
Comparison of TcpF to other known microbial TIR domain proteins and mammalian adaptor molecules.
The top panel (A) shows alignment and domain organization of the predicted TIR domain of E. faecalis TcpF with representative TIR domains of bacteria (TlpA, TcpB, TcpC), human TLRs (TLR2, TLR4, TLR6) and adaptor molecules (MyD88, TIRAP, TRIF). Significant sequence similarity is observed in the Box 1 region with more sequence divergence evident in the Box 2 region, both of which are important signatures of all TIR domain proteins. The alignment was constructed with T-Coffee:: advanced server from EMBnet (http://www.ch.embnet.org/). The color scheme represented in the figure is indicative of the reliability of the alignment, with red corresponding to highest probability of correct alignment. Highly conserved identical residues including the glycine in the Box 2 region are denoted by an asterisk (*). (B) Superposition of the predicted TcpF-TIR (green) and E. coli TcpC-TIR (pink) domains (left) and TcpF-TIR (green) and human TLR2-TIR (pink) domains (right). The divergent BB-loop that defines specificity of TIR domain interactions is highlighted. Comparative structure modeling was done using 3D-JIGSAW for PDB generation and UCSF Chimera for visualization.
Figure 2.
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.
Figure 3.
Analysis of the binding of TcpF to phospholipids.
Western blot analysis of the interaction between various phosphorylated derivatives of phosphoinositol: (LPA, lysophosphatidic acid; LPC, lysophosphocholine; PtdIns, phosphoinositide phosphates; PE, phosphatidylethanolamine; PC, phosphatidylcholine, S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidyl serine) and MBP (A) or MBP-TcpF fusion protein (B) using PIP strip binding assay. (C) The binding of Ptdlns(3,4,5)p and Ptdlns(3)p to TcpF protein was confirmed by Poly PIPosome pull-down assay using anti-MBP-TcpF rabbit serum.
Figure 4.
(A) Interaction between TcpF and MyD88. The immunoprecipitates obtained from incubation of MBP, MBP-TcpF or MBP-TcpFm (BB-loop mutant form) with lysates of RAW264.7 cells were analyzed by Western blot. (B & C) MEF cells were transiently transfected with NF-κB-luciferase and a plasmid expressing TcpF, and with pTK-Rel as an internal control. After 24 hours, transfected cells were stimulated with LTA (B) or TNF-α (C). Luciferase activity was measured and normalized to the activity of internal control, and fold activations relative to untreated samples were determined. Shown are averages and standard deviations of 3 independent experiments performed in triplicate. Significance * P<0.05.
Figure 5.
Characterization of TcpF wild type, deficient and complemented strains.
(A) E99 was cultured in THB medium overnight and TcpF was detected in cell lysate by Western blot. The mutant and complemented strains were used as control. (B) Phagocytosis of E. faecalis by RAW264.7 cells. E. faecalis E99, SPB03 and SPB04 transformed with plasmid pMV158GFP were incubated with RAW264.7 cells for 45 minutes. Free bacteria were removed by washing and cells were analyzed by flow cytometry (BD Accuri, Ann Arbor, MI). These data are from a representative experiment that was repeated with similar results.
Figure 6.
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.
Figure 7.
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.