Infection by Toxoplasma gondii Induces Amoeboid-Like Migration of Dendritic Cells in a Three-Dimensional Collagen Matrix

Toxoplasma gondii, an obligate intracellular parasite of humans and other warm-blooded vertebrates, invades a variety of cell types in the organism, including immune cells. Notably, dendritic cells (DCs) infected by T. gondii acquire a hypermigratory phenotype that potentiates parasite dissemination by a ‘Trojan horse’ type of mechanism in mice. Previous studies have demonstrated that, shortly after parasite invasion, infected DCs exhibit hypermotility in 2-dimensional confinements in vitro and enhanced transmigration in transwell systems. However, interstitial migration in vivo involves interactions with the extracellular matrix in a 3-dimensional (3D) space. We have developed a collagen matrix-based assay in a 96-well plate format that allows quantitative locomotion analyses of infected DCs in a 3D confinement over time. We report that active invasion of DCs by T. gondii tachyzoites induces enhanced migration of infected DCs in the collagen matrix. Parasites of genotype II induced superior DC migratory distances than type I parasites. Moreover, Toxoplasma-induced hypermigration of DCs was further potentiated in the presence of the CCR7 chemotactic cue CCL19. Blocking antibodies to integrins (CD11a, CD11b, CD18, CD29, CD49b) insignificantly affected migration of infected DCs in the 3D matrix, contrasting with their inhibitory effects on adhesion in 2D assays. Morphological analyses of infected DCs in the matrix were consistent with the acquisition of an amoeboid-like migratory phenotype. Altogether, the present data show that the Toxoplasma-induced hypermigratory phenotype in a 3D matrix is consistent with integrin-independent amoeboid DC migration with maintained responsiveness to chemotactic and chemokinetic cues. The data support the hypothesis that induction of amoeboid hypermigration and chemotaxis/chemokinesis in infected DCs potentiates the dissemination of T. gondii.     

Lipopolysaccharide induces cell volume increase and migration of dendritic cells

Migration of dendritic cells (DCs) plays an important role in T‐cell‐mediated adaptive immune responses. Lipopolysaccharide (LPS) sensed by Toll‐like receptor 4 (TLR4) serves as a signal for DC migration. We analyzed LPS‐induced DC volume changes preceding the directed movement towards chemoattractants. Treatment with LPS resulted in rapid, prolonged cell swelling in wild‐type (WT), but not in TLR4^?/? bone marrow‐derived (BM) DCs indicating that TLR4 signaling is essential for LPS‐induced swelling. As a consequence, LPS‐treatment enhanced the migratory activity along a chemokine (CCL21)‐gradient in WT, but not in TLR4‐deficient BMDCs suggesting that the LPS/TLR4‐induced swelling response facilitates DC migration. Moreover, the role of calcium‐activated potassium channels (K_Ca3.1) as putative regulators of immune cell volume regulation and migration was analyzed in LPS‐challenged BMDCs. We found that the LPS‐induced swelling of K_Ca3.1‐deficient DCs was impaired when compared to WT DCs. Accordingly, the LPS‐induced increase in [Ca²⁺]_i detected in WT DCs was reduced in K_Ca3.1‐deficient DCs. Finally, directed migration of LPS‐challenged K_Ca3.1‐deficient DCs was low compared to WT DCs indicating that activation of K_Ca3.1 is involved in LPS‐induced DC migration. These findings suggest that both TLR4 and K_Ca3.1 contribute to the migration of LPS‐activated DCs as an important feature of the adaptive immune response.

     

Induction of IL-12p40 and type 1 immunity by Toxoplasma gondii in the absence of the TLR-MyD88 signaling cascade

Toxoplasma gondii is an orally acquired pathogen that induces strong IFN-γ based immunity conferring protection but that can also be the cause of immunopathology. The response in mice is driven in part by well-characterized MyD88-dependent signaling pathways. Here we focus on induction of less well understood immune responses that do not involve this Toll-like receptor (TLR)/IL-1 family receptor adaptor molecule, in particular as they occur in the intestinal mucosa. Using eYFP-IL-12p40 reporter mice on an MyD88^-/- background, we identified dendritic cells, macrophages, and neutrophils as cellular sources of MyD88-independent IL-12 after peroral T. gondii infection. Infection-induced IL-12 was lower in the absence of MyD88, but was still clearly above noninfected levels. Overall, this carried through to the IFN-γ response, which while generally decreased was still remarkably robust in the absence of MyD88. In the latter mice, IL-12 was strictly required to induce type I immunity. Type 1 and type 3 innate lymphoid cells (ILC), CD4⁺ T cells, and CD8⁺ T cells each contributed to the IFN-γ pool. We report that ILC3 were expanded in infected MyD88^-/- mice relative to their MyD88^+/+ counterparts, suggesting a compensatory response triggered by loss of MyD88. Furthermore, bacterial flagellin and Toxoplasma specific CD4⁺ T cell populations in the lamina propria expanded in response to infection in both WT and KO mice. Finally, we show that My88-independent IL-12 and T cell mediated IFN-γ production require the presence of the intestinal microbiota. Our results identify MyD88-independent intestinal immune pathways induced by T. gondii including myeloid cell derived IL-12 production, downstream type I immunity and IFN-γ production by ILC1, ILC3, and T lymphocytes. Collectively, our data reveal an underlying network of immune responses that do not involve signaling through MyD88.     

The unicellular eukaryotic parasite Toxoplasma gondii hijacks the migration machinery of mononuclear phagocytes to promote its dissemination

Toxoplasma gondii is an obligate intracellular protozoan with the ability to infect virtually any type of nucleated cell in warm-blooded vertebrates including humans. Toxoplasma gondii invades immune cells, which the parasite employs as shuttles for dissemination by a Trojan horse mechanism. Recent findings are starting to unveil how this parasite orchestrates the subversion of the migratory functions of parasitised mononuclear phagocytes, especially dendritic cells (DCs) and monocytes. Here, we focus on how T. gondii impacts host cell signalling that regulates leukocyte motility and systemic migration in tissues. Shortly after active parasite invasion, DCs undergo mesenchymal-to-amoeboid transition and adopt a high-speed amoeboid mode of motility. To trigger migratory activation – termed hypermigratory phenotype – T. gondii induces GABAergic signalling, which results in calcium fluxes mediated by voltage-gated calcium channels in parasitised DCs and brain microglia. Additionally, a TIMP-1-CD63-ITGB1-FAK signalling axis and signalling via the receptor tyrosine kinase MET promotes sustained hypermigration of parasitised DCs. Recent reports show that the activated signalling pathways converge on the small GTPase Ras to activate the MAPK Erk signalling cascade, a central regulator of cell motility. To date, three T. gondii-derived putative effector molecules have been linked to hypermigration: Tg14-3-3, TgWIP and ROP17. Here, we discuss their impact on the hypermigratory phenotype of phagocytes. Altogether, the emerging concept suggests that T. gondii induces metastasis-like migratory properties in parasitised mononuclear phagocytes to promote infection-related dissemination.     

Natural killer cell intrinsic toll-like receptor MyD88 signaling contributes to IL-12-dependent IFN-γ production by mice during infection with Toxoplasma gondii

Myeloid differentiation factor 88 (MyD88)-dependent IL-12 secretion by dendritic cells is critical for natural killer cell-mediated IFN-γ production and innate resistance to Toxoplasma gondii. Although MyD88^−/− mice challenged with T. gondii have defective IL-12 responses and succumb to infection, administration of IL-12 to MyD88^−/− mice fails to prevent acute mortality, suggesting that MyD88 may mediate signals within natural killer cells important for IL-12-dependent IFN-γ production and innate resistance to this parasite. In this study, we found that T. gondii antigens and IL-12 could synergistically trigger IFN-γ secretion by natural killer cells, which was dependent on toll-like receptor-MyD88 signaling. Further analysis showed that p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, c-Jun N-terminal kinase and NF-κB multiple pathways downstream of MyD88 contributed to IFN-γ production by natural killer cells. Moreover, the well-established toll-like receptor agonists, T. gondii profilin (Tgprofilin) and T. gondii heat shock protein 70 (TgHSP70) could evoke a similar IFN-γ secretory response in natural killer cells to that evoked by T. gondii antigens. In vivo adoptive transfer experiments showed that, upon challenge with T. gondii, NOD/SCID-β2 microglobulin null (NOD/SCID-β2m^−/−) mice injected i.v. with MyD88^−/− natural killer cells had reduced serum IFN-γ levels and increased splenic tachyzoite burdens compared with those injected i.v. with wild-type natural killer cells. Taken together, these findings demonstrate a critical role for natural killer cell intrinsic toll-like receptor-MyD88 signaling in IL-12-dependent early IFN-γ production and innate resistance to T. gondii.     

Involvement of the MyD88‐independent pathway in controlling the intracellular fate of Burkholderia pseudomallei infection in the mouse macrophage cell line RAW 264.7

Burkholderia pseudomallei is a facultative intracellular Gram‐negative bacterium which is capable of surviving and multiplying inside macrophages. B. pseudomallei strain SRM117, a LPS mutant which lacks the O‐antigenic polysaccharide moiety, is more susceptible to macrophage killing during the early phase of infection than is its parental wild type strain (1026b). In this study, it was shown that the wild type is able to induce expression of genes downstream of the MyD88‐dependent (iκbζ, il‐6 and tnf‐α), but not of the MyD88‐independent (inos, ifn‐β and irg‐1), pathways in the mouse macrophage cell line RAW 264.7. In contrast, LPS mutant‐infected macrophages were able to express genes downstream of both pathways. To elucidate the significance of activation of the MyD88‐independent pathway in B. pseudomallei‐infected macrophages, the expression of TBK1, an essential protein in the MyD88‐independent pathway, was silenced prior to the infection. The results showed that silencing the tbk1 expression interferes with the gene expression profile in LPS mutant‐infected macrophages and allows the bacteria to replicate intracellularly, thus suggesting that the MyD88‐independent pathway plays an essential role in controlling intracellular survival of the LPS mutant. Moreover, exogenous IFN‐γ upregulated gene expression downstream of the MyD88‐independent pathway, and interfered with intracellular survival in both wild type and tbk1‐knockdown macrophages infected with either the wild type or the LPS mutant. These results suggest that gene expression downstream of the MyD88‐independent pathway is essential in regulating the intracellular fate of B. pseudomallei, and that IFN‐γ regulates gene expression through the TBK1‐independent pathway.     

MyD88 and IFN-alphabeta differentially control maturation of bystander but not Salmonella-associated dendritic cells or CD11cintCD11b+ cells during infection

The interface between dendritic cells (DCs) and T cells is critical to elicit effective immunity against pathogens. The maturation state of DCs determines the quality of the interaction and governs the type of response. DCs can be matured directly through activating Toll-like receptors (TLRs) or indirectly by cytokines. We explore the role of the TLR adaptor MyD88 on DC maturation during Salmonella infection. Using Salmonella expressing GFP, we also examine the phenotype and function of bacteria-associated DCs matured in the absence of bacteria-mediated TLR signalling. MyD88 was required for upregulation of CD80 on DCs during infection, whereas CD86 and CD40 were upregulated independently of MyD88, although requiring a higher bacterial burden in the MLN. MyD88-independent upregulation was mediated by IFN-αβ produced during infection. In infected MyD88^−/−IFN-αβR^−/− mice, which lack most bacteria-driven TLR signalling, indirect DC maturation was abolished. In contrast, DCs containing Salmonella upregulated co-stimulatory molecules independently of MyD88 and IFN-αβ, revealing a pathway of phenotypic maturation active in infected DCs. However, despite high co-stimulatory molecule expression, Salmonella -containing DCs from MyD88^−/− or MyD88^−/−IFN-αβR^−/− mice had a compromised capacity to activate T cells. Thus, bacterial stimulation of TLRs influences DC function at multiple levels that modulates their capacity to direct antibacterial immunity.     

Rapid cytoskeleton remodelling in dendritic cells following invasion by Toxoplasma gondii coincides with the onset of a hypermigratory phenotype

Host cell manipulation is an important feature of the obligate intracellular parasite Toxoplasma gondii. Recent reports have shown that the tachyzoite stages subvert dendritic cells (DC) as a conduit for dissemination (Trojan horse) during acute infection. To examine the cellular basis of these processes, we performed a detailed analysis of the early events following tachyzoite invasion of human monocyte‐derived DC. We demonstrate that within minutes after tachyzoite penetration, profound morphological changes take place in DC that coincide with a migratory activation. Active parasite invasion of DC led to cytoskeletal actin redistribution with loss of adhesive podosome structures and redistribution of integrins (CD18 and CD11c), that concurred with the onset of DC hypermotility in vitro. Inhibition of parasite rhoptry secretion and invasion, but not inhibition of parasite or host cell protein synthesis, abrogated the onset of morphological changes and hypermotility in DC dose‐dependently. Also, infected DC, but not by‐stander DC, exhibited upregulation of C‐C chemokine receptor 7 (CCR7). Yet, the onset of parasite‐induced DC hypermotility preceded chemotactic migratory responsesin vitro. Collectively, present data reveal that invasion of DC by T. gondii initiates a series of regulated events, including rapid cytoskeleton rearrangements, hypermotility and chemotaxis, that promote the migratory activation of DC.     

Characterization of secreted recombinant <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> surface antigen 2 (SAG2) heterologously expressed by the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The surface antigen 2 (SAG2) gene of the protozoan parasite, Toxoplasma gondii, was cloned and extracellularly expressed in the yeast Pichia pastoris. The effectiveness of the secreted recombinant SAG2 (rSAG2-S) as a serodiagnosis reagent was assessed by western blots and ELISA. In the western blot assay, rSAG2-S reacted with all Toxoplasma-antibody positive human serum samples but not with Toxoplasma-negative samples. In the ELISA, rSAG2-S yielded sensitivity rates ranging from 80% (IgG negative, IgM positive) to 100% (IgG positive, IgM negative). In vivo experiments showed that serum from mice immunized with rSAG2-S reacted specifically with the native SAG2 of T. gondii. These mice were protected when challenged with live cells of T. gondii.     

Direct evidence of Toxoplasma-induced changes in serum testosterone in mice

Latent toxoplasmosis is known to influence the morphology of infected persons and also increases the probability of the birth of male offspring in both humans and mice. All these traits can be related to the observed differences in the concentration of testosterone between Toxoplasma-infected and Toxoplasma-free subjects. However, it is not possible to decide, using the Toxoplasma-human model, whether toxoplasmosis influences the level of testosterone in the infected host or whether individuals with different levels of testosterone vary in the probability of toxoplasma infection. Here we studied changes in the testosterone levels in the latent phase of toxoplasmosis in laboratory mice artificially infected with cystogenic but relatively virulent strain T38 of T. gondii. We observed decreased testosterone levels in both female and male mice with latent toxoplasmosis in comparison to uninfected controls (P = 0.001). The present results indicate that Toxoplasma infection changes the concentration of serum testosterone in mice and human rather than changed concentration of testosterone influences the probability of the Toxoplasma infection. It is possible that the decrease of testosterone is an adaptive mechanism of infected mice aimed to compensate toxoplasmosis-induced immunosuppression observed during latent Toxoplasma infection.     

Toxoplasma gondii Is Dependent on Glutamine and Alters Migratory Profile of Infected Host Bone Marrow Derived Immune Cells through SNAT2 and CXCR4 Pathways

The obligate intracellular parasite, Toxoplasma gondii, disseminates through its host inside infected immune cells. We hypothesize that parasite nutrient requirements lead to manipulation of migratory properties of the immune cell. We demonstrate that 1) T. gondii relies on glutamine for optimal infection, replication and viability, and 2) T. gondii-infected bone marrow-derived dendritic cells (DCs) display both “hypermotility” and “enhanced migration” to an elevated glutamine gradient in vitro. We show that glutamine uptake by the sodium-dependent neutral amino acid transporter 2 (SNAT2) is required for this enhanced migration. SNAT2 transport of glutamine is also a significant factor in the induction of migration by the small cytokine stromal cell-derived factor-1 (SDF-1) in uninfected DCs. Blocking both SNAT2 and C-X-C chemokine receptor 4 (CXCR4; the unique receptor for SDF-1) blocks hypermotility and the enhanced migration in T. gondii-infected DCs. Changes in host cell protein expression following T. gondii infection may explain the altered migratory phenotype; we observed an increase of CD80 and unchanged protein level of CXCR4 in both T. gondii-infected and lipopolysaccharide (LPS)-stimulated DCs. However, unlike activated DCs, SNAT2 expression in the cytosol of infected cells was also unchanged. Thus, our results suggest an important role of glutamine transport via SNAT2 in immune cell migration and a possible interaction between SNAT2 and CXCR4, by which T. gondii manipulates host cell motility.     

Effects of myeloid differentiation primary response gene 88 (MyD88) activation on Helicobacter infection in vivo and induction of a Th17 response

Background:  Helicobacter pylori is a spiral‐shaped Gram‐negative microaerophilic bacterium associated with a number of gastrointestinal disorders, including gastritis, peptic ulcers, and gastric cancer. Several studies have implicated a Th17 response as a key to protective immunity against Helicobacter. Materials and Methods:  Wild type (WT) and MyD88‐deficient (MyD88^?/?) mice in the C57BL/6 background were infected with H. felis for 6 and 25 weeks and colonization density and host response evaluated. Real‐time PCR was used to determine the expression of cytokines and antimicrobial peptides in the gastric tissue of mice. Results:  mRNA expression levels of the Th17 cytokines interleukin‐17A (IL‐17A) and IL‐22 were markedly up‐regulated in WT compared with MyD88^?/? mice both at 6 and at 25 weeks in response to infection with H. felis, indicating that induction of Th17 responses depends on MyD88 signaling. Furthermore, reduction in the expression of Th17‐dependent intestinal antimicrobial peptide lipocalin‐2 was linked with increased bacterial burden in the absence of MyD88 signaling. Conclusion:  We provide evidence showing that MyD88‐dependent signaling is required for the host to induce a Th17 response for the control of Helicobacter infection.     

Sodium fluoride induces podosome formation in endothelial cells

Background information. Fluoride is a well‐known G‐protein activator. Exposure of cultured cells to its derivatives results in actin cytoskeleton remodelling. Podosomes are actin‐based structures endowed with adhesion and matrix‐degradation functions. This study investigates actin cytoskeleton reorganization induced by fluoride in endothelial cells. Results. Treatment of cultured endothelial cells with sodium fluoride (NaF) results in a rapid and potent stimulation of podosome formation. Furthermore, we show that Cdc42 (cell‐division cycle 42), Rac1 and RhoA activities are stimulated in NaF‐treated cells. However, podosome assembly is dependent on Cdc42 and Rac1, but not RhoA. Although the sole activation of Cdc42 is sufficient to induce individual podosomes, a balance between RhoGTPase activities regulates podosome formation in response to NaF, which in this case are often found in groups or rosettes. As in other models, podosome formation in endothelial cells exposed to NaF also involves Src. Finally, we demonstrate that NaF‐induced podosomes are fully competent for matrix protein degradation. Conclusions. Taken together, our findings establish NaF as a novel inducer of podosomes in endothelial cells in vitro.     

Intracellular parasitism with Toxoplasma gondii stimulates mammalian‐target‐of‐rapamycin‐dependent host cell growth despite impaired signalling to S6K1 and 4E‐BP1

The Ser/Thr kinase mammalian‐target‐of‐rapamycin (mTOR) is a central regulator of anabolism, growth and proliferation. We investigated the effects of Toxoplasma gondii on host mTOR signalling. Toxoplasma invasion of multiple cell types rapidly induced sustained mTOR activation that was restricted to infected cells, as determined by rapamycin‐sensitive phosphorylation of ribosomal protein S6; however, phosphorylation of the growth‐associated mTOR substrates 4E‐BP1 and S6K1 was not detected. Infected cells still phosphorylated S6K1 and 4E‐BP1 in response to insulin, although the S6K1 response was blunted. Parasite‐induced S6 phosphorylation was independent of S6K1 and did not require activation of canonical mTOR‐inducing pathways mediated by phosphatidylinositol 3‐kinase–Akt and ERK. Host mTOR was localized in a vesicular pattern surrounding the parasitophorous vacuole, suggesting potential activation by phosphatidic acid in the vacuolar membrane. In spite of a failure to phosphorylate 4E‐BP1 and S6K1, intracellular T. gondii triggered host cell cycle progression in an mTOR‐dependent manner and progression of infected cells displayed increased sensitivity to rapamycin. Moreover, normal cell growth was maintained during parasite‐induced cell cycle progression, as indicated by total cellular S6 levels. The Toxoplasma‐infected cell provides a unique example of non‐canonical mTOR activation supporting growth that is independent of signalling through either S6K1 or 4E‐BP1.     

Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis

Invasive tumor dissemination in vitro and in vivo involves the proteolytic degradation of ECM barriers. This process, however, is only incompletely attenuated by protease inhibitor-based treatment, suggesting the existence of migratory compensation strategies. In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of beta 1 integrins and MT1-matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks. Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates. Sustained protease-independent migration resulted from a flexible amoeba-like shape change, i.e., propulsive squeezing through preexisting matrix gaps and formation of constriction rings in the absence of matrix degradation, concomitant loss of clustered beta 1 integrins and MT1-MMP from fiber binding sites, and a diffuse cortical distribution of the actin cytoskeleton. Acquisition of protease-independent amoeboid dissemination was confirmed for HT-1080 cells injected into the mouse dermis monitored by intravital multiphoton microscopy. In conclusion, the transition from proteolytic mesenchymal toward nonproteolytic amoeboid movement highlights a supramolecular plasticity mechanism in cell migration and further represents a putative escape mechanism in tumor cell dissemination after abrogation of pericellular proteolysis.     

BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming

The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4⁺ T cells at that site. We found EpCAM^low CD11b^high migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4⁺ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4⁺ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration.     

R753Q Polymorphism Inhibits Toll-like Receptor (TLR) 2 Tyrosine Phosphorylation,Dimerization with TLR6, and Recruitment of Myeloid Differentiation Primary Response Protein 88

The R753Q polymorphism in the Toll-IL-1 receptor domain of Toll-like receptor 2 (TLR2) has been linked to increased incidence of tuberculosis and other infectious diseases, but the mechanisms by which it affects TLR2 functions are unclear. Here, we studied the impact of the R753Q polymorphism on TLR2 expression, hetero-dimerization with TLR6, tyrosine phosphorylation, and recruitment of myeloid differentiation primary response protein (MyD) 88 and MyD88 adapter-like (Mal). Complementation of HEK293 cells with transfected WT or R753Q TLR2 revealed their comparable total levels and only minimal changes in cell surface expression of the mutant species. Notably, even a 100-fold increase in amounts of transfected R753Q TLR2 versus WT variant did not overcome the compromised ability of the mutant TLR2 to activate nuclear factor κB (NF-κB), indicating that a minimal decrease in cell surface levels of the R753Q TLR2 cannot account for the signaling deficiency. Molecular modeling studies suggested that the R753Q mutation changes the electrostatic potential of the DD loop and results in a discrete movement of the residues critical for protein-protein interactions. Confirming these predictions, biochemical assays demonstrated that R753Q TLR2 exhibits deficient agonist-induced tyrosine phosphorylation, hetero-dimerization with TLR6, and recruitment of Mal and MyD88. These proximal signaling deficiencies correlated with impaired capacities of the R753Q TLR2 to mediate p38 phosphorylation, NF-κB activation, and induction of IL-8 mRNA in transfected HEK293 cells challenged with inactivated Mycobacterium tuberculosis or mycobacterial components. Thus, the R753Q polymorphism renders TLR2 signaling-incompetent by impairing its tyrosine phosphorylation, dimerization with TLR6, and recruitment of Mal and MyD88.