A Mal functional variant is associated with protection against invasive pneumococcal disease, bacteremia, malaria and tuberculosis

Abstract

Toll-like receptors (TLRs) and members of their signaling pathway are important in the initiation of the innate immune response to a wide variety of pathogens. The adaptor protein Mal (also known as TIRAP), encoded by TIRAP (MIM 606252), mediates downstream signaling of TLR2 and TLR4 (refs. 4-6). We report a case-control study of 6,106 individuals from the UK, Vietnam and several African countries with invasive pneumococcal disease, bacteremia, malaria and tuberculosis. We genotyped 33 SNPs, including rs8177374, which encodes a leucine substitution at Ser180 of Mal. We found that heterozygous carriage of this variant associated independently with all four infectious diseases in the different study populations. Combining the study groups, we found substantial support for a protective effect of S180L heterozygosity against these infectious diseases (N = 6,106; overall P = 9.6 x 10(-8)). We found that the Mal S180L variant attenuated TLR2 signal transduction.

Figure 1

Macrophage-activating lipopeptide 2 (Malp2) induced, TLR2 signalling assay as measured by degradation of IκB-α at various time points. First panel: Malp2 (5nM) -stimulated wild-type murine embryonic fibroblast (MEF) cells. Second panel: Malp2-stimulated Tirap knockout cells. Third panel: Malp2-stimulated Tirap knockout cells transfected with Tirap 180-Ser. Fourth panel: Malp2-stimulated Tirap knockout cells transfected with Tirap 180-Leu. Samples were immunoblotted with an anti-I IκB-α antibody. Protein loading was controlled for using the β-actin housekeeping gene. Results shown are representative of 3 separate experiments.

Figure 2

Wild-type MEFs or Tirap knockout MEFs transfected with empty vector or plasmids encoding Tirap 180-Ser or Tirap 180-Leu as indicated, were treated with LPS (1μg/ml) or Malp2 (5nM). After 24h, supernatants were removed and assayed for IL-6 by ELISA. Expression levels of Tirap S180 and Tirap L180 in the cells analysed (untreated or treated with LPS or Malp2 for 24h) are shown in the lower panel. Results shown are mean ± s.e.m. of triplicate determinations, similar results being obtained in 2 further experiments.

Figure 3

Tirap knockout MEFs were transfected with 100ng empty vector, or plasmids encoding Tirap 180-Ser or Tirap 180-Leu (50ng of each, supplemented to 100ng with 50ng of empty vector), or a combination of plasmids encoding Tirap 180-Ser and Tirap 180-Leu (50ng of each) as indicated, each in combination with luciferase reporter gene (80 ng per well 5 x NF-κB-luciferase) with Renilla-luciferase reporter gene (40 ng) as an internal control. Cell lysates were prepared as previously described. Results shown are mean + S.D. from triplicate determinations.

Figure 4

Molecular models of wild-type and mutant TIRAP. The computational utility Molecular Operating Environment (MOE2006.02) (www.chemcomp.com) was used to generate the models. Electrostatic surface potentials are highlighted in blue (positive) and red (negative) (upper panel). Structural features are highlighted in the lower panel. Box 1, the TIRAP BB loop and the position of Serine 180 are all indicated.

Figure 5

HEK-293 cells (1×10⁶) were transfected with 3 μg of Flag-tagged TLR2, HA-Tirap or AU1-tagged MyD88. Lysates were incubated with purified glutathione-coupled GST, GST-Tirap and GST-Tirap S180L for 2 hrs. After washing, the complex was analysed by SDS-PAGE and western blotting. Results shown are representative of 3 separate experiments.