Bacterial Toll/interleukin-1 Receptor Research Articles

Effects of TcpC’s NADase Activity on Inhibition of Innate Immune Signaling

Abstract The innate immune system is activated by pattern recognition receptors such as Toll-like receptors (TLRs), which differentiate between self and non-self by recognizing distinct molecular structures on pathogens. As this process threatens pathogen survival in the host, bacteria have developed mechanisms to combat this. Uropathogenic Escherichia coli (UPEC) express the Toll/Interleukin-1 Receptor (TIR) domain-containing protein TcpC, which inhibits the function of TLRs by binding to the signaling adapter protein myeloid differentiation factor 88 (MyD88). Bacterial TIR proteins, including TcpC, recently have been found to also exhibit NADase activity. We hypothesize that the ability of TcpC to block TLR signaling and its NADase function are interrelated and that the TcpC EA mutant, which has been shown to have diminished NADase activity will not bind efficiently to MyD88. We performed in vitro translation assays to first produce both WT and EA mutant versions of TcpC that contain both a streptavidin and histidine tag. Then we conducted pull-down experiments to determine if the TcpC EA mutant could successfully bind to streptavidin beads, as well as MyD88, potentially resulting in clustering with the TIR signaling protein and blocking the signaling pathway entirely. While we have successfully produced tagged WT and EA TcpC proteins and pulled down the proteins using streptavidin beads, we are working to optimize conditions to determine if the NADase-deficient TcpC EA mutant is able to bind MyD88, as has been shown previously for WT TcpC. The resulting information will give us a closer look into TcpC’s effect on virulence and the suppression of the innate immune response via TLR and MyD88 related signaling pathways. This research was partially funded by the USM LSAMP program, supported by NSF LSAMP Award #1619676, the Towson OSPR Seed Funding for Grant Seekers, and the Bridges to Doctorate Scholars Program, NIH-R25GM119970 04.

TcpC affects the grazing resistance of Escherichia coli to the social amoeba Dictyostelium discoideum

Abstract Uropathogenic Escherichia coli (UPEC) is among the most common causative agents of urinary tract infections (UTIs). UPECs survive in hosts using various mechanisms, including by secreting a protein known as TcpC, which is a Toll/interleukin-1 receptor (TIR) domain containing protein. In mammals, TIR domains are important components of Toll like receptor (TLR) signaling pathways that activate innate immune responses. TcpC interacts with components of TLR signaling, inhibiting activation of the immune system and preventing the detection and destruction of the bacteria in mammalian hosts. We hypothesize that TcpC also affects the survival of UPECs in model systems, including the social amoeba Dictyostelium discoideum and planaria flatworms. To test this hypothesis, we are using mutant strains of the UPEC CFTO73 that have altered expression of TcpC to analyze the effects of TcpC on bacterial growth and virulence in model organisms. Our initial tests with D. discoideum indicate that the presence of TcpC in UPEC strains may increase their resistance to grazing and phagocytosis by the social amoeba D. discoideum. If TcpC affects survival of UPECs in model systems such as D. discoideum, this may open new avenues for characterization of the molecular mechanisms used by bacterial TIR domains to evade innate immune cells. VA was supported by NSF LSAMP Award #1619676 and CP was supported by the NIH Bridges to Doctorate Scholars Program, NIH- R25 GM119970.

Results from a CURE course project reveal novel insights into the structure of bacterial Toll/interleukin-1 receptor (TIR) domain proteins

Abstract Toll-like receptors (TLRs) are critical for innate immune signaling, and various bacteria express Toll/interleukin-1 receptor (TIR) domains to evade this signaling. Bacterial TIRs exhibit NADase activity, although it is unclear what role NADase activity plays in evasion of innate responses. To better characterize the NADase activity of the TIR from Acenitobacter baumanii (AbTIR), we used site directed mutagenesis to identify amino acids predicted to be important for NADase activity. Studies were carried out in a Course-Based Undergraduate Research Experience (CURE) involving 16–20 undergraduate research students per year. Students used Chimera to analyze the AbTIR structure and hypothesize amino acids they predicted would play a role in enzymatic activity. They then performed site-directed mutagenesis to create plasmids for expression of mutant AbTIR. Mutant TIRs were expressed, purified and tested for NADase activity. So far, our mutagenesis results suggest a potential conformational change in the NAD binding site upon NAD binding. Identifying amino acids critical for enzymatic activity may inform drug development for diseases associated with dysregulated TIR activity. In addition, the application of the CURE class to carry out these studies has provided project-based research training experiences for approximately 80 undergraduate students, about half of whom are members of groups traditionally underrepresented in science. Several students, including some who had limited research experience before completing the course, are currently pursuing graduate studies and research-based careers, highlighting the value of CURE-based projects for facilitating the inclusion of students from diverse groups in research. TVS and TO were supported by the NIH Bridges to Baccalaureate Grant R25GM058264; CP was supported by the NIH Bridges to the Doctorate Grant R25GM119970, and the CURE course was supported by an HHMI Inclusive Excellence Grant

Cryo-EM structure of an active bacterial TIR–STING filament complex

Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes1–4. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide4–13, but the molecular basis of STING filament assembly and extension remains unknown. Here we use cryogenic electron microscopy to determine the structure of the active Toll/interleukin-1 receptor (TIR)–STING filament complex from a Sphingobacterium faecium cyclic-oligonucleotide-based antiphage signalling system (CBASS) defence operon. Bacterial TIR–STING filament formation is driven by STING interfaces that become exposed on high-affinity recognition of the cognate cyclic dinucleotide signal c-di-GMP. Repeating dimeric STING units stack laterally head-to-head through surface interfaces, which are also essential for human STING tetramer formation and downstream immune signalling in mammals5. The active bacterial TIR–STING structure reveals further cross-filament contacts that brace the assembly and coordinate packing of the associated TIR NADase effector domains at the base of the filament to drive NAD+ hydrolysis. STING interface and cross-filament contacts are essential for cell growth arrest in vivo and reveal a stepwise mechanism of activation whereby STING filament assembly is required for subsequent effector activation. Our results define the structural basis of STING filament formation in prokaryotic antiviral signalling.

NADase activity found in bacterial TIR proteins may aid in innate immune evasion

Abstract Pathogenic microbes have evolved various mechanisms to evade recognition by host immune responses, which can lead to the prevalence of disease and infection. Many strains of pathogenic bacteria contain genes encoding Toll/Interleukin-1 receptor (TIR) domain-containing proteins that function through the inhibition of TLR and myeloid differentiation factor 88 (MyD88) signaling. Previous research has also revealed that prokaryotic TIR domains may regulate bacterial and host metabolic pathways by acting as NAD+ glycohydrolases (NADases). We hypothesize that this NADase activity may contribute to the role of bacterial TIRs as virulence factors. In order to test this hypothesis, we observed the effects of TIR NADase activity on the binding interactions between bacterial TIRs and host MyD88, and on the ability of bacterial TIRs to block NF-kB signaling. For this, we have identified amino acids predicted to play a role in TIR NADase activity and have expressed and purified recombinant bacterial TIR proteins that have incorporated these mutations. We then tested the NADase activity of these mutants in comparison with that of wild-type TIR proteins and TIRs with a mutation known to inhibit NADase activity. We are now using TIRs with deficiencies in NADase activity in pull-down assays to test their binding to MyD88 and utilizing RT-PCR to analyze the effects of these mutant TIRs on innate cell activation. This information will allow us to gain a deeper understanding into the relationship between NADase activity in bacterial TIRs and their virulence activity and can potentially uncover new mechanisms for pathogenic evasion of host immune defenses. Supported by grant from NIH (R25 GM119970 04)

Products of gut microbial Toll/interleukin-1 receptor domain NADase activities in gnotobiotic mice and Bangladeshi children with malnutrition.

SummaryPerturbed gut microbiome development has been linked to childhood malnutrition. Here, we characterize bacterial Toll/interleukin-1 receptor (TIR) protein domains that metabolize nicotinamide adenine dinucleotide (NAD), a co-enzyme with far-reaching effects on human physiology. A consortium of 26 human gut bacterial strains, representing the diversity of TIRs observed in the microbiome and the NAD hydrolase (NADase) activities of a subset of 152 bacterial TIRs assayed in vitro, was introduced into germ-free mice. Integrating mass spectrometry and microbial RNA sequencing (RNA-seq) with consortium membership manipulation disclosed that a variant of cyclic-ADPR (v-cADPR-x) is a specific product of TIR NADase activity and a prominent, colonization-discriminatory, taxon-specific metabolite. Guided by bioinformatic analyses of biochemically validated TIRs, we find that acute malnutrition is associated with decreased fecal levels of genes encoding TIRs known or predicted to generate v-cADPR-x, as well as decreased levels of the metabolite itself. These results underscore the need to consider microbiome TIR NADases when evaluating NAD metabolism in the human holobiont.

Antiviral activity of bacterial TIR domains via immune signalling molecules.

The Toll/interleukin-1 receptor (TIR) domain is a canonical component of animal and plant immune systems1,2. In plants, intracellular pathogen sensing by immune receptors triggers their TIR domains to generate a molecule that is a variant of cyclic ADP-ribose3,4. This molecule is hypothesized to mediate plant cell death through a pathway that has yet to be resolved5. TIR domains have also been shown to be involved in a bacterial anti-phage defence system called Thoeris6, but the mechanism of Thoeris defence remained unknown. Here we show that phage infection triggers Thoeris TIR-domain proteins to produce an isomer of cyclic ADP-ribose. This molecular signal activates a second protein, ThsA, which then depletes the cell of the essential molecule nicotinamide adenine dinucleotide (NAD) and leads to abortive infection and cell death. We also show that, similar to eukaryotic innate immune systems, bacterial TIR-domain proteins determine the immunological specificity to the invading pathogen. Our results describe an antiviral signalling pathway in bacteria, and suggest that the generation of intracellular signalling molecules is an ancient immunological function of TIR domains that is conserved in both plant and bacterial immunity.

Contribution of the NADase function of bacterial TIR domain proteins to the inhibition of innate immune responses

Abstract Pathogenic microbes have evolved various mechanisms to evade recognition by host immune responses, which can lead to the prevalence of disease and infection. Many strains of pathogenic bacteria contain genes encoding Toll/Interleukin-1 receptor (TIR) domain-containing proteins that function through the inhibition of TLR and myeloid differentiation factor 88 (MyD88) signaling. Previous research has also revealed that prokaryotic TIR domains may regulate bacterial and host metabolic pathways by acting as NAD+ glycohydrolases (NADases). We hypothesize that this NADase activity may contribute to the role of bacterial TIRs as virulence factors. In order to test this hypothesis, we observed the effects of TIR NADase activity on the binding interactions between bacterial TIRs and host MyD88, and on the ability of bacterial TIRs to block NF-kB signaling. For this, we have identified amino acids predicted to play a role in TIR NADase activity and have expressed and purified recombinant bacterial TIR proteins that have incorporated these mutations. We then tested the NADase activity of these mutants in comparison with that of wild-type TIR proteins and TIRs with mutations already known to inhibit NADase activity. We are now using mutant TIRs with deficiencies in NADase activity in pull-down assays to test their binding to MyD88 and utilizing reporter assays to analyze the effects of these mutant TIRs on NF-kB signaling. This information will allow us to gain a deeper understanding into the relationship between NADase activity in bacterial TIRs and their virulence activity, and can potentially uncover new mechanisms for pathogenic evasion of host immune defenses.

In silico structural homology modeling and characterization of multiple N-terminal domains of selected bacterial Tcps.

Several bacterial pathogens produce Toll/interleukin-1 receptor (TIR) domain-containing protein homologs that are important for subverting the Toll-like receptor (TLR) signaling cascades in hosts. Consequently, promoting the persistence and survival of the bacterial pathogens. However, the exact molecular mechanisms elucidating the functional characteristics of these bacterial proteins are not clear. Physicochemical and homology modeling characterization studies have been conducted to predict the conditions suitable for the stability and purification of these proteins and to predict their structural properties. The outcomes of these studies have provided important preliminary data for the drug discovery pipeline projects. Here, using in silico physicochemical and homology modeling tools, we have reported the primary, secondary and tertiary structural characteristics of multiple N-terminal domains of selected bacterial TIR domain-containing proteins (Tcps). The results show variations between the primary amino acid sequences, secondary structural components and three-dimensional models of the proteins, suggesting the role of different molecular mechanisms in the functioning of these proteins in subverting the host immune system. This study could form the basis of future experimental studies advancing our understanding of the molecular basis of the inhibition of the host immune response by the bacterial Tcps.

Development of a CURE course aimed at characterization of bacterial Toll/interleukin 1 receptor (TIR) domain-containing proteins

Abstract Course-based undergraduate research experiences (CUREs) can engage a large, diverse group of students in authentic research, promote student research skills, student content knowledge, scientific literacy, critical thinking and analysis skills, support student self-efficacy, and at the same time provide a means to advance faculty research productivity. These varied goals of a CURE course, at times, can prove to be competing. For example, courses aimed at providing advanced levels of student input in design of research questions, hypotheses and protocols can veer from faculty research goals. In an effort to balance these competing objectives, we designed a CURE course engaging students at Towson University with research ongoing at the University of Maryland, School of Medicine aimed at characterizing the function of bacterial Toll/interleukin 1 receptor (TIR) domain containing proteins. Bacterial TIR proteins were originally characterized as virulence factors that impede mammalian innate immune signaling, but recently also have been shown to exhibit enzymatic NADase activity. Students in the CURE course used bioinformatics analysis of modeled structures of bacterial TIRs to predict sites for NADase function and hypothesized the effects of mutations of these sites on TIR functions. Students then designed and created recombinantly-expressed proteins mutated at their predicted sites and tested their mutant proteins for both NADase function and potential inhibition of mammalian innate immune signaling. This course outline thus allowed student engagement in project design and hypothesis formation, while also maintaining projects aimed at advancing a specific faculty research program.

TIR domains of plant immune receptors are NAD+-cleaving enzymes that promote cell death.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors activate cell death and confer disease resistance by unknown mechanisms. We demonstrate that plant Toll/interleukin-1 receptor (TIR) domains of NLRs are enzymes capable of degrading nicotinamide adenine dinucleotide in its oxidized form (NAD+). Both cell death induction and NAD+ cleavage activity of plant TIR domains require known self-association interfaces and a putative catalytic glutamic acid that is conserved in both bacterial TIR NAD+-cleaving enzymes (NADases) and the mammalian SARM1 (sterile alpha and TIR motif containing 1) NADase. We identify a variant of cyclic adenosine diphosphate ribose as a biomarker of TIR enzymatic activity. TIR enzymatic activity is induced by pathogen recognition and functions upstream of the genes enhanced disease susceptibility 1 (EDS1) and N requirement gene 1 (NRG1), which encode regulators required for TIR immune function. Thus, plant TIR-NLR receptors require NADase function to transduce recognition of pathogens into a cell death response.

TIR Domain Proteins Are an Ancient Family of NAD+-Consuming Enzymes

The Toll/interleukin-1 receptor (TIR) domain is the signature signaling domain of Toll-like receptors (TLRs) and their adaptors, serving as a scaffold for the assembly of protein complexes for innate immune signaling [1, 2]. TIR domain proteins are also expressed in plants, where they mediate disease resistance [3, 4], and in bacteria, where they have been associated with virulence [5-9]. In pursuing our work on axon degeneration [10], we made the surprising discovery that the TIR domain of SARM1 (sterile alpha and TIR motif containing 1), a TLR adaptor protein, has enzymatic activity [11]. Upon axon injury, the SARM1 TIR domain cleaves nicotinamide adenine dinucleotide (NAD+), destroying this essential metabolic co-factor to trigger axon destruction [11, 12]. Whereas current studies of TIR domains focus on their scaffolding function, our findings with SARM1 inspired us to ask whether this enzymatic activity is the primordial function of the TIR domain. Here we show that ancestral prokaryotic TIR domains constitute a new family of NADase enzymes. Using purified proteins from a cell-free translation system, we find that TIR domain proteins from both bacteria and archaea cleave NAD+ into nicotinamide and ADP-ribose (ADPR), with catalytic cleavage executed by a conserved glutamic acid. A subset of bacterial and archaeal TIR domains generates a non-canonical variant cyclic ADPR (cADPR) molecule, and the full-length TIR domain protein from pathogenicStaphylococcus aureus induces NAD+ loss in mammalian cells. These findings suggest that the primordial function of the TIR domain is the enzymatic cleavage of NAD+ and establish TIR domain proteins as a new class of metabolic regulatory enzymes.

Inhibition of TLR4 signaling by Brucella TIR-containing protein TcpB-derived decoy peptides

Brucella spp. avoid host immune recognition and thus, weaken the immune response to infection. The Toll/interleukin-1 receptor (TIR) domain-containing protein (TcpB/Btp1) of Brucella spp. is thought to be involved in blocking host innate immune responses by binding to adaptors downstream of Toll-like receptors. In this study, based on the observation that TcpB binds to the host target proteins, MAL, through the TIR domain, we examined decoy peptides from TcpB TIR domains and found that TB-8 and TB-9 substantially inhibit lipopolysaccharide (LPS)-induced signaling in vitro and in vivo. Both these peptides share a common loop, the DD loop, indicating a novel structural region mediating TIR interactions. The inhibition of LPS signaling by TB-8 and TB-9 shows no preference to MyD88-dependent cytokines, such as TNF-α and IL-1β or TRIF-dependent cytokines including IFN-β and IL-6. Furthermore, these two peptides rescue the virulence of Brucella ΔtcpB mutants at the cellular level, indicating key roles of the DD loop in Brucella pathogenesis. In conclusion, identification of inhibitors from the bacterial TIR domains is helpful not only for illustrating interacting mechanisms between TIR domains and bacterial pathogenesis, but also for developing novel signaling inhibitors and therapeutics for human inflammatory diseases.

Innate immune signaling inhibition via disruption of adapter interactions.

Abstract Toll-like receptors (TLR) form homo- or heterodimers in response to pathogen-associated molecular patterns. The TLR homo- or heterodimers establish an intracellular signaling platform that recruits the signaling adapter protein Toll-Interleukin 1 Receptor (TIR) domain containing adaptor protein (TIRAP). This receptor-mediated platform initiates a signaling cascade which leads to NF-kB activation, cytokine production, and inflammation. Inflammation leads to pathogen clearance; however, excessive stimulation of the innate immune pathway can lead to injury and death. We are investigating mechanisms to disrupt TIRAP-mediated TLR signaling via two mechanisms: the bacterial TIR domain protein, TcpB from Brucella sp., and the decoy peptide, 2R9. We have previously determined the structures of TIRAP and TcpB. Presumably via molecular interactions with TIRAP, TcpB inhibited NF-kB activation. We now report surface plasma resonance binding of TIRAP with TcpB. The decoy peptide, 2R9, was derived from the TLR2 TIR domain D-helix. We have previously described interactions between 2R9 and TIRAP via surface plasma resonance. Based on the ability of 2R9 peptide to protect against lethality in response to acute influenza infection, 2R9 peptide could highlight a novel drug target to prevent injury and death caused by innate immune over activation. Using recombinant TIRAP with TcpB and 2R9 peptide, we are performing structural studies to characterize molecular interactions to identify putative sites for therapeutic intervention.

A TIR domain protein from E. faecalis attenuates MyD88-mediated signaling and NF-κB activation.

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.

Molecular analysis of a novel Toll/interleukin-1 receptor (TIR)-domain containing virulence protein of Y. pseudotuberculosis among Far East scarlet-like fever serotype I strains

Pathogenicity of Yersinia pseudotuberculosis is determined by an arsenal of virulence factors. Particularly, the Yersinia outer proteins (Yops) and the Type III secretion system (T3SS) encoded on the pYV virulence plasmid are required for Yersinia pathogenicity. A specific group of Y. pseudotuberculosis, responsible for the clinical syndrome described as Far East scarlet-like fever (FESLF), is known to have an altered virulence gene cluster. Far East strains cause unique clinical symptoms for which the pYV virulence plasmid plays apparently a rather secondary role. Here, we characterize a previously unknown protein of Y. pseudotuberculosis serotype I strains (TcpYI) which can be found particularly among the FESLF strain group. The TcpYI protein shares considerable sequence homology to members of the Toll/IL-1 receptor family. Bacterial TIR domain containing proteins (Tcps) interact with the innate immune system by TIR-TIR interactions and subvert host defenses via individual, multifaceted mechanisms. In terms of virulence, it appears that the TcpYI protein of Y. pseudotuberculosis displays its own virulence phenotype compared to the previously characterized bacterial Tcps. Our results clearly demonstrate that TcpYI increases the intracellular survival of the respective strains in vitro. Furthermore, we show here that the intracellular survival benefit of the wild-type strain correlates with an increase in tcpYI gene expression inside murine macrophages. In support of this, we found that TcpYI enhances the survival inside the spleens of mice in a mouse model of peritonitis. Our results may point toward involvement of the TcpYI protein in inhibition of phagocytosis, particularly in distinct Y. pseudotuberculosis strains of the FESLF strain group where the pYV virulence plasmid is absent.

Adaptors in Toll-Like Receptor Signaling and their Potential as Therapeutic Targets

To initiate the innate immune response, Toll-like receptors (TLRs) associate with cytoplasmic adaptor proteins through TIR (Toll/interleukin-1 receptor) domain interactions. The four principal signaling adaptor proteins include MyD88, MAL, TRIF and TRAM, and the fifth protein SARM, involved in negative regulation of TLR pathways, is usually considered a part of the TIR domain-containing adaptor protein group. Other TIR domain-containing proteins have also been shown to regulate these signaling pathways, including ST2 and SIGIRR, as well as several bacterial and viral TIR domain-containing proteins that modulate these pathways as virulence factors. TLR pathways and the adaptor proteins are associated with a number of diseases, including infection, sepsis, inflammatory, allergic and autoimmune diseases and cancer. We review our current understanding of the structure and function of adaptor proteins and their regulatory proteins, their association with disease and their potential as therapeutic targets in human disease.

Subversion of Innate Immune Signaling Through Molecular Mimicry

Innate immune signaling is mediated by a number of membrane-anchored or cytosolic receptor or sensor molecules. Several receptor families utilize conserved signaling domains such as the Toll/interleukin-1 receptor (TIR) domain and Pyrin domain (PYD) to link microbe recognition to induction of proinflammatory cytokines and interferons. Recent studies have identified a number of bacterial and viral TIR domains and PYD domains that directly target the signaling function of their host homologues. Emerging biochemical and structural studies of these microbial TIR and PYD domains suggest that they are mimics of their host counterparts at the sequence and structure levels. Unraveling the mechanisms of such molecular mimicry is crucial to our understanding and clinical intervention of infectious diseases and inflammatory disorders.

Molecular Mimicry in Innate Immunity: CRYSTAL STRUCTURE OF A BACTERIAL TIR DOMAIN

Macrophages detect pathogen infection via the activation of their plasma membrane-bound Toll-like receptor proteins (TLRs). The heterotypic interaction between the Toll/interleukin-1 receptor (TIR) domains of TLRs and adaptor proteins, like Myeloid differentiation primary response gene 88 (MyD88), is the first intracellular step in the signaling pathway of the mammalian innate immune response. The hetero-oligomerization of the TIRs of the receptor and adaptor brings about the activation of the transcription factor NF-kappaB, which regulates the synthesis of pro-inflammatory cytokines. Here, we report the first crystal structure of a bacterial TIR domain solved at 2.5 A resolution. The three-dimensional fold of Paracoccus denitrificans TIR is identical to that observed for the TIR of human TLRs and MyD88 proteins. The structure shows a unique dimerization interface involving the DD-loop and EE-loop residues, whereas leaving the BB-loop highly exposed. Peptide amide hydrogen-deuterium exchange mass spectrometry also reveals that the same region is used for dimerization in solution and in the context of the full-length protein. These results, together with a functional interaction between P. denitrificans TIR and MyD88 visualized in a co-immunoprecipitation assay, further substantiate the model that bacterial TIR proteins adopt structural mimicry of the host active receptor TIR domains to interfere with the signaling of TLRs and their adaptors to decrease the inflammatory response.

Bacterial TIR Domains

Bacteria need to evade the first line of immune defense, the innate immune response, to survive in their hosts. Signaling in response to foreign molecules by Toll-like receptors (TLRs) is an important part of the innate immune response, so pathogens have evolved mechanisms to disrupt this pathway (see O’Neill). Cirl et al . identified proteins containing the Toll/interleukin-1 receptor (TIR) domains in several human bacterial pathogens, including Escherichia coli strain CFT073, which causes urinary tract infections, and Brucella melitensis , which causes brucellosis, a disease with symptoms similar to those associated with flu. The authors named these proteins Tcps, for TIR domain-containing proteins; the E. coli protein was named TcpC, and the B. melitensis protein was named TcpB. Cells infected with a TcpC-deficient mutant of CFT073 showed greater production of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) and increased bacterial load compared to cells infected with wild-type CFT073. Cells transfected with plasmids encoding TLR signaling components and the TcpC or TcpB proteins, along with reporter plasmids, exhibited decreased signaling through TLRs that are coupled to the adaptor MyD88--TLR4 and TLR2--but not those coupled to other adaptors. CFT073 infection of MyD88-deficient cells did not show a decrease in TNF secretion. Thus, TcpC and TcpB appear to selectively interfere with MyD88-dependent TLR pathways. The TIR domain of TcpC or full-length TcpB bound to MyD88 in pull-down assays. TcpC was secreted from the bacteria and was detectable inside bone marrow-derived macrophages even when the macrophages were separated from the bacteria in a transwell system. Uptake of a purified peptide of the TIR domain of TcpC was inhibited if cholesterol was depleted from the membranes of the host cells, which suggests that lipid rafts may be important for uptake. Finally, the importance of TcpC in disease was determined by infecting mice with the wild-type CFT073 or the TcpC-deficient strain, and those with the wild-type strain had more aggressive disease. In human urinary tract isolates, more severe disease (acute pyelonephritis) was associated with a higher frequency of the presence of the TcpC-positive strains, whereas TcpC-positive strains were less frequently detected in samples from patients with less serious (cystitis) or asymptomatic infection. Thus, as with some viral pathogens, bacteria also appear to target TLR signaling to enhance virulence. C. Cirl, A. Wieser, M. Yadav, S. Duerr, S. Schubert, H. Fischer, D. Stappert, N. Wantia, N. Rodriguez, H. Wagner, C. Svanborg, T. Miethke, Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins. Nat. Med. 14 , 399-406 (2008). [PubMed] L. A. J. O’Neill, Bacteria fight back against Toll-like receptors. Nat. Med. 14 , 370-372 (2008). [PubMed]