TLRs constitute an essential family of pattern recognition molecules that, through direct recognition of conserved microbial components, initiate inflammatory responses following infection. In this role, TLR1 enables host responses to a variety of bacteria, including pathogenic species of mycobacteria. In this study, we report that I602S, a common single nucleotide polymorphism within TLR1, is associated with aberrant trafficking of the receptor to the cell surface and diminished responses of blood monocytes to bacterial agonists. When expressed in heterologous systems, the TLR1 602S variant, but not the TLR1 602I variant, exhibits the expected deficiencies in trafficking and responsiveness. Among white Europeans, the 602S allele represents the most common single nucleotide polymorphism affecting TLR function identified to date. Surprisingly, the 602S allele is associated with a decreased incidence of leprosy, suggesting that Mycobacterium leprae subverts the TLR system as a mechanism of immune evasion.

Experimental animals and transgenesAll fly lines were obtained from the Drosophila stock center except for the following. 981- Gal4 and SG18.1-Gal4 (Jhaveri et al., 2000) were kindly provided by V. Rodrigues. Or22a-Gal4, Or47a-Gal4 and Or47b-Gal4 (Vosshall et al., 2000) were gifts from L. Vosshall. MARCM (Lee and Luo, 1999) was carried out by heat-shocking thirdinstar larvae of the following genotypes: hs-FLP/+; dock Or47a-Gal4/dock (or +); FRT82 Gal80/FRT82 UAS-mCD8::GFP and hs-FLP/+; dock Or47b-Gal4/dock (or +); FRT82 Gal80/FRT82 UASmCD8::GFP at 37 o C for 40 minutes. Adult brains were dissected and processed as described below. GH146-Gal4 Clonal analysis ImmunohistochemistryAdult brains (from 1-to 2-day-old animals), pupal antennae and larval imaginal discs were dissected in phosphate buffered saline (PBS). Tissues were fixed in PLP (2% paraformaldehyde, 0.25% sodium periodate, 75 mM lysine-HCl and 37 mM sodium phosphate pH 7.4), washed with PBST (PBS with 0.5% Triton X-100) and subjected to antibody staining. nc82 mAb (1:20) (A. Hofbauer, PhD thesis, University of Wurzburg, 1991) was a gift from A. Hofbauer. Rabbit anti-Dock 1:500 (Clemens et al., 1996) was a gift from J. Dixon. Mouse 22C10 mAb (1:20) and rat anti-ELAV, 7E8A10 (1:20) were from Developmental Studies Hybridoma Bank. Rabbit anti-GFP polyclonal antibody (1:100) was from Clontech, and rat anti-CD8 α subunit mAb (1:100) was from Caltag. The secondary antibodies, FITC-conjugated goat anti-rabbit, Cy3-conjugated goat anti-mouse and FITC-conjugated goat anti-rat, were purchased from Jackson Laboratories and used at 1:200 dilutions. Cuticle preparationsAdult antennae from animals expressing Or-Gal4/UAS-lacZ nuclear were fixed in 25% gluteraldehyde for 1 hour, washed in PBST and stained in 0.2% X-Gal solution (Ashburner, 1989). The antennae were then cleared in Faure's mountant (34% v/v chloral hydrate, 13% glycerol, 20 mg/ml gum Arabic) and photographed with the SPOT-RT cooled CCD camera. Sensilla on the third antennal segment were counted by projecting images on a video monitor.

Innate recognition and signaling by Toll-like receptors (TLRs) is facilitated by functionally associated coreceptors, although the cooperativity mechanisms involved are poorly understood. As a model we investigated TLR2 interactions with the GD1a ganglioside binding subunit of type IIb Escherichia coli enterotoxin (LT-IIb-B 5 ). Both LT-IIb-B 5 and a GD1a binding-defective mutant (LT-IIb-B 5 (T13I)) could modestly bind to TLR2, but only the wild-type molecule displayed a dramatic increase in TLR2 binding activity in the presence of GD1a (although not in the presence of irrelevant gangliosides). Moreover, fluorescence resonance energy transfer experiments indicated that LT-IIb-B 5 induces lipid raft recruitment of TLR2 and TLR1 and their clustering with GD1a, in contrast to the GD1a binding-defective mutant, which moreover fails to activate TLR2 signaling. LT-IIb-B 5 -induced cell activation was critically dependent upon the Toll/IL-1 receptor domain-containing adaptor protein, which was induced to colocalize with TLR2 and GD1a, as shown by confocal imaging. Therefore, GD1a provides TLR2 coreceptor function by enabling the ligand to recruit, bind, and activate TLR2. These findings establish a model of TLR2 coreceptor function and, moreover, suggest novel mechanisms of adjuvanticity by non-toxic derivatives of type II enterotoxins dependent upon GD1a/TLR2 cooperative activity.Recent developments in the field of innate immunity support the concept that cellular activation by microbial molecules involves interactions with multiple cooperating host receptors within membrane lipid rafts, whereas single receptor-based interactions may often represent an oversimplified model (1-3). This concept is exemplified by the ability of patternrecognition receptors such as Toll-like receptor 4 (TLR4) 2 or TLR2 to functionally associate with accessory molecules or coreceptors for induction of intracellular signaling. TLR4 alone is not sufficient for inducing a vigorous innate response to lipopolysaccharide (LPS) and requires MD-2 and CD14, although additional components of the LPS recognition complex may play a role in modifying TLR4-mediated signaling (4, 5). TLR2 responds to microbial lipoproteins in association with TLR1 or TLR6 as signaling partners and with CD14 or CD36 as important coreceptors for robust activation of TLR2/1 or TLR2/6 complexes (1, 6). The formation of TLR complexes with coreceptors may serve to generate a combinatorial repertoire for discriminating among the abundant and diverse microbial molecules and thereby to appropriately tailor the host response. However, the mechanisms involved in TLR cooperativity with accessory receptors are currently poorly understood.We have recently shown that the B subunit of type IIb heatlabile enterotoxin of Escherichia coli (designated LT-IIb-B 5 ) activates TLR2 signaling, although the underlying mechanism was not addressed (7). Type IIb and related enterotoxins (types I and IIa) display an AB 5 oligomeric structure in which a toxic A subunit is noncovalently linked to a pe...