Bacterial TIR containing proteins and host innate immune system evasion

Rohini R. Rana1+, Minghao Zhang1, Abigail M. Spear2, Helen S. Atkins2, Bernadette

Byrne1*

Division of Molecular Biosciences, Imperial College London, London SW7 2AZ,

UK1 and Department of Biomedical Sciences, Defence Science and Technology

Laboratory, Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK2.

*Corresponding

Dr Bernadette Byrne,

Division of Molecular Biosciences

Imperial College London

South Kensington

London

SW7 2AZ.

Fax: +44 20 7594 3022

E-mail: b.byrne@imperial.ac.uk

+ Now based at MRC National Institute for Medical Research, The Ridgeway, Mill

Hill, London NW7 1AA, UK.

@ Crown Copyright 2011. Published with the permission of the Defence Science and

Technology Laboratory on behalf of the Controller of HMSO.

1

Abstract

The innate immune system provides the first line of host defence against

invading pathogens. Key to upregulation of the innate immune response are Toll-like

receptors (TLRs) which recognise pathogen-associated molecular patterns (PAMPs)

and trigger a signaling pathway culminating in the production of inflammatory

mediators. Central to this TLR signaling pathway are heterotypic protein-protein

interactions mediated through Toll/Interleukin-1 receptor (TIR) domains found in

both the cytoplasmic regions of TLRs and adaptor proteins. Pathogenic bacteria have

developed a range of ingenuous strategies to evade the host immune mechanisms.

Recent work has identified a potentially novel evasion mechanism involving bacterial

TIR domain proteins. Such domains have been identified in a wide range of

pathogenic bacteria and there is evidence to suggest that they interfere directly with

the TLR signaling pathway and thus inhibit the activation of NFB. The individual

TIR domains from the pathogenic bacteria Salmonella enterica serovar Enteritidis,

Brucella sp, uropathogenic E. coli and Yersinia pestis have been analysed in detail.

The individual bacterial TIR domains from these pathogenic bacteria seem to differ in

their modes of action and their roles in virulence. Here we review the current state of

knowledge on the possible roles and mechanisms of action of the bacterial TIR

domains.

Keywords: TLR signaling pathway, TIR domain, innate immunity, pathogenic

bacteria, bacterial TIR domain, virulence

2

The innate immune system and TLRs

Upregulation of the innate immune system provides the first line of host

defence against invading pathogens. Key components of the innate immune system

are pattern recognition receptors (PRRs) which recognise pathogen-associated

molecular patterns (PAMPs) and initiate an intracellular signaling cascade

culminating in a host immune response [1]. The best-studied PRRs are the Toll-like

receptors (TLRs), type I transmembrane sensory receptors that recognise a range of

PAMPs from pathogenic bacteria, fungi, viruses and parasitic-protozoa [1]. The

principal role of the TLRs is in regulation of the innate immune system however there

is evidence to show that they also play important functions in shaping adaptive

immune responses, particularly in combination with other PRRs [2].

TLRs are expressed on immune cells such as phagocytes, B cells and certain T

cells, and on non-immune cells including fibroblasts and epithelial cells. They can be

located on the cell surface (TLR1-2, TLR4-6 and TLR10) or in intracellular

compartments such as endosomes (TLR3 and TLR7-9) [1,3].

Ten TLRs are encoded in the human genome, each of which has evolved to

recognise certain PAMPs from a broad range of microbes. For instance, TLRs signal

the presence of triacyl lipopeptides (Pam3CSK4; TLR1/2), peptidoglycan and zymosan

(TLR2), double-stranded ribonucleic acid (ds RNA; TLR3), lipopolysaccharides

(LPS) of Gram-negative bacteria (TLR4), flagellin (TLR5), single-stranded RNA (ss

RNA; TLR7 and TLR8) and unmethylated CpG DNA (TLR9) [3,4]. The ligand for

TLR10 is currently unknown. The expression of the different TLRs is subject to

change and is regulated by rapid cellular responses to pathogens, various cytokines

and environmental stresses [1].

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The TLRs contain an extracellular leucine-rich repeat (LRR) domain that

recognises and binds to specific microbial ligands [5]. Linked to the LRR domain by a

single transmembrane domain [6] is a cytoplasmically located Toll/Interleukin-1

receptor (TIR) domain that mediates downstream signaling [7]. TLRs are thought to

exist as dimers, which can be heterotypic, homotypic or both depending of the

receptor [8,9]. Upon interaction with a PAMP the TLR dimer undergoes a molecular

rearrangement of the intracellular TIR domains to form an active interaction domain

allowing recruitment of adaptor molecules [4,8,9]. TLR signalling pathways converge

to activate transcription factors that regulate the induction of proinflammatory

cytokines and co-stimulatory molecules [5]. This in turn elicits innate immune

responses against the invading pathogens.

One of the key features of TLR signaling is that the signal is both initiated and

propagated by heterotypic TIR-TIR interactions (Fig 1). In addition to the TIR

domains of the TLR receptors there are five TIR containing adaptor proteins [10];

myeloid differentiation factor 88 (MyD88), TIR domain containing adaptor protein

including interferon- (TRIF), MyD88-adaptor like (MAL, also called TIRAP),

TRIF-related adaptor molecule (TRAM) and sterile - and armadillo-motif-

containing protein (SARM). The MyD88-mediated pathway leads to the activation of

NFκB and MAP kinase pathways inducing the production and release of

proinflammatory cytokines [5]. The TRIF-mediated pathway leads to activation of

interferon response factors (IRFs) inducing the production of type I interferon [11].

All TLRs and IL-1Rs, except TLR3, signal through the MyD88-dependent pathway

while TLR3 and TLR4 function through the MyD88-independent pathway (TRIF;

[12]). TLR1, TLR2, TLR4 and TLR6 additionally recruit the TIR domain bridging

adaptor MAL, which mediates the interactions between the TIR domains of TLRs and

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MyD88 [13]. Similarly, TLR4 recruits the alternative TIR domain bridging adaptor,

TRAM, in addition to TRIF to link between the TIR domain of TLR4 and TRIF [14]

(Fig 1). The final TIR containing adaptor, SARM, acts as a negative regulator of

TRIF signaling [15].

TIR domains

The cytoplasmic TIR domains of TLRs typically contain 150-200 amino acid

residues [16]. As mentioned the principal role of TIR domains is the mediation of

heterotypic protein-protein interactions between receptor and adaptor molecules in the

signal transduction process. TIR domains share common sequence motifs, called box

1 (F/Y)-DAFISY), box 2 (GYKLC-RD-PG) and box 3 (a conserved W surrounded by

basic residues), of which boxes 1 and 2 are vital for mediating signalling [12,17] (Fig

2). One of the most functionally important regions of the TIR domains is the BB loop

(Fig 2 and Fig 3), part of the Box 2 region. This motif is comprised of the sequence

RDxPG (where x is any residue and is a hydrophobic residue). Site-directed

mutagenesis studies have revealed that several residues within this loop are important

for TLR signal transduction. This is most notable in the case of the Pro residue.

Mutation of this residue, Pro712, to His in TLR4-TIR results in a loss of TLR

signalling in response to lipopolysaccharide [18].

A summary of all the current solved structures of TIR domains is given in

Table 1. The structure of the human TIR domain of TLR1 reveals a central five-

stranded parallel β-sheet (βA - βE) surrounded by five helices (A - E) and the

connecting loops (Fig 2 and Fig 3) [19]. The functionally relevant BB loop (Fig 2 and

Fig 3) connects strand βB and helix B, which protrudes out from the main core of

the protein in most structures of TIR domains [16,19-22]. Whilst the core structure is

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maintained in most TIR domains, some conformational differences are observed even

in the case of the TIR domains of TLR1 and TLR2 which share 50% sequence

identity [19]. The regions of the proteins that exhibit the largest variation include the

helices B and D and the loops BB, CD and DD. Such structural diversity is

suggested to be crucial for the specificity of signal transduction from different

receptors [19,23]. The recent structure of MAL lacks the helix connecting the B and

C strands. This region of the protein is present as a long flexible loop which links the

A helix and the B strand [22]. It is not clear precisely why there is less order in this

region of MAL compared to the other TIR domains however it seems likely that this

is important for function.

Microbial targeting of the TLR signaling pathway

Research over the last decade or so has highlighted that the TLR signaling pathway is

a key target of pathogen mechanisms of host immune system evasion. One of the

strategies for successful host evasion involves manipulation of the TLR signaling

pathway via modification of microbial PAMPs so that they are less effective at

stimulating the TLRs. An example of this can be seen with the modification in Lipid

A content of Yersinia pestis with changing growth temperature [24]. Alternatively, by

co-stimulating host carbohydrate binding receptors as well as TLRs it has been shown

that Mycobacterium tuberculosis [25] is able to initiate anti-inflammatory

mechanisms counteracting the effects of TLR signaling. Viruses have been shown to

target various levels of the TLR signaling pathway. The A46R and A52R proteins

from vaccinia virus reduce TLR signaling by forming interactions with a range of host

TIR containing proteins including MyD88, MAL, TRIF and TRAM [26] as well as

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TLR4 [27]. Based on sequence homology to the Box2 region of TIR domains it was

thought that A46R and A52R function as negative regulators of the heterotypic TIR-

TIR interactions involved in upregulating the TLR signaling pathway through

competitive binding with host TIR domain proteins. However A52R has also been

shown to form direct association with Interleukin-1 receptor-associated kinase-like 2

(IRAK2) [28], a component of the TLR signaling pathway which does not contain a

TIR domain, so it may be that the role of this protein is more general. The suggestion

that the viral proteins are not TIR containing proteins, is supported by analysis using

bioinformatics tools which were unable to find known TIR domains using A46 as a

search protein or find TIR domains using A46 as a search protein [29]. There are

proteins in other viruses such as Hepatitis B that also interact with components of the

TLR signaling pathways probably in a similar way to the vaccinia proteins [30]. In

contrast, the Karposi’s sarcoma virus replication and transcription activator (RTA)

causes degradation of TRIF [31].

Bacterial TIRs and their prevalence throughout bacterial species

A new potential host evasion mechanism involving TLRs came to light with the

identification of bacterial TIR homologues. The first report described the

identification >200 TIR homologues in a wide range of bacterial species including

Staphylococcus aureus, Brucella melitensis and Salmonella enterica serovar

Enteriditis [32]. Sequence alignment of the so-called TIR-like protein A (TlpA) from

S. enterica serovar Enteriditis with the TIR domains of human TLR6, TLR4 and

MyD88, revealed high sequence homology in the box 1 region [32]. Subsequent work

identified Paracoccus denitrificans TIR-like protein (PdTLP) as a homologue of S.

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enterica serovar Enteriditis tlpA, sharing 39% sequence similarity over 255 residues

[33]. Cirl et al. [34] identified bacterial TIR domains in the CFT073 strain of

uropathogenic Escherichia coli and in Brucella species, which were termed TIR

domain containing proteins C (TcpC) and B (TcpB, also known as Btp1). A more

comprehensive bioinformatics analysis by Spear et al. [29] identified 922 bacterial

TIR domains in a wide range of both pathogenic and non-pathogenic bacterial species

including the plague-causing bacterium, Yersinia pestis. More recently a study

identified a substantially lower number of 483 bacterial TIR domains proteins [35]. It

is possible that this is due to subtle differences in the search methodologies used to

find the proteins. An intriguing finding was revealed by recent research by Zhang et al

[35] demonstrating that animal SARM is more closely related to the bacterial TIR

domain proteins than it is to the other animal TIR adaptors suggesting a common

origin for these proteins.

The bacterial TIR proteins are approximately 230 to 310 amino acids long

with the conserved TIR domain comprised of 150-200 amino acids. The TIR domain

can be located in either the C-terminal or the N-terminal region and the remaining

region of the protein can be highly variable. In the TlpA and PdTLP proteins the TIR

domain is located within the C-terminal region with an N-terminal region postulated

to comprise a highly -helical coiled coil domain. The presence of a coiled coil

domain was supported by circular dichroism analysis on the N-terminal region of

PdTLP indicating a high -helical content [33]. In contrast in the case of TcpC the N-

terminal region has been annotated as a putative transmembrane segment while the N-

terminal domain of TcpB has been identified as a lipid binding domain with

specificity for phosphoinositide phosphates. The recent bioinformatic analysis

highlights the co-occurrence of the TIR domains with a range of different domains

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[29]. Examples include the presence of an N-terminal TIR domain followed by C-

terminal tetratricopeptide repeats (TPR), common protein-protein interaction

domains, in Frankia sp. [29]. Bacterial TIR containing proteins in Beggiatos sp.

contain an N-terminal Mettallophos domain involved in phosphorylation, while in

Clostridium thermocellum it contains C-terminal trypsin, a serine-protease domain,

and in Mariprofundus ferroxydans it contains a C-terminal Mrr-cat, a type IV

restriction endonuclease domain [29].

Bacterial TIR proteins and the TLR signaling pathway

The majority of studies on bacterial TIR proteins have focused on their

potential role as virulence factors that directly subvert host TLR signaling. Newman

et al. [32] in the first studies on a bacterial TIR showed that S. enterica serovar

Enteritidis TlpA suppresses the induction of NFB activation by the mammalian TIR-

containing proteins TLR4, IL-1R and MyD88 but does not suppress NFB activation

induced by the cytokine TNF in cultured mammalian cells. Most notably, mice

infected with genetic knockouts of S. enterica serovar Enteritidis lacking the TlpA

encoding gene showed increased time to death compared to mice infected with the

wild-type strain. The TlpA knockout strain also demonstrated a reduced ability to

survive in cultured mammalian cells [32]. These data strongly suggested that the TlpA

was important for virulence of S. enterica serovar Enteritidis via a mechanism of

inhibition of the TLR signaling pathway responsible for activation of NFB.

Support for both a role in virulence and the general mechanism of action of

bacterial TIR proteins came from further studies on homologues from Brucella

melitensis (TcpB [also known as Btp1]; [34,36]) and the uropathogenic E. coli strain

CFT073 (TcpC; [34]). One study revealed that both TcpB and TcpC suppress TLR2-

9

and TLR4-mediated activation of NFB but do not suppress the activation of NFB

induced by the TIR-independent cytokine TNF and that a TcpC knockout strain

showed a reduced ability to survive in mouse RAW264.7 macrophage cells compared

to wild-type CFT073 [34]. It was possible to achieve wild-type accumulation rates in

RAW264.7 cells when the TcpC knockout was complemented with a plasmid borne

copy of TcpC. In addition, a comparison using a mouse model of urinary tract

infections revealed that the TcpC knockout strain had reduced virulence compared to

wild-type [34]. A further in-depth analysis of the precise sites of activity of the

bacterial TIR proteins revealed that TcpB and the purified TIR domain of TcpC

(TcpC-TIR) interacted with endogenous MyD88 in mammalian cell lines [34]. Pull-

down assays confirmed the interaction of TcpC-TIR directly with MyD88 but

revealed a lack of interaction with two other TIR containing mammalian proteins

TLR2-TIR and TRIF, or two non-TIR domain containing components of the TLR

signaling pathway, IRAK1 and IRAK4 [34]. More recently there is some evidence to

suggest that TcpC also interacts with TLR4 [37]. Other TIR containing proteins

involved in TLR signaling, for example MAL and TRAM, have not been tested so far

for interaction with TcpC.

Based on sequence similarity, plasma membrane localization profile and the

ability to inhibit MAL-induced NFkB activation it has been suggested that TcpB acts

as a MAL mimic and competes with MAL for binding of MyD88 [36]. However more

recent research suggests that TcpB only interacts with MAL and not MyD88 and does

not block the interaction between these two proteins [38]. Instead, data indicate that

TcpB interaction results in a reduction in the cellular levels of MAL in mammalian

cells expressing TcpB [38]. It has been suggested that interaction with TcpB results in

ubiquitination and targeting of MAL for degradation. Since TcpB is unlikely to act

10

directly as a ubiquitin ligase it is possible that TcpB binding causes degradation by

indirect means [38]. Studies on TcpB from Brucella abortus have identified a role in

inhibition of maturation of infected dendritic cells, suggested to act as key cellular

environments for pathogen proliferation [39].

Most recently research has focused on the TIR protein from the plague causing

bacterium, Yersinia pestis (YpTdp). In an in vitro reporter assay YpTdp inhibits

activation of NFB, although the TIR domain only (YpTIR) has no effect [40].

However, pull-down assays show that YpTIR does form interactions with MyD88-

TIR [40] suggesting that in this case the N-terminal, non-TIR, domain of the YpTdp

also plays a role in inhibition of the TLR signaling pathway. Both inhibition of NFκB

by YpTdp and interaction of YpTIR with MyD88-TIR are dependent upon the proline

residue located on the BB loop [40]. This indicates that YpTdp mediates the

downregulation of the innate immune system through competitive interaction with

MyD88 as described for TcpC [34].

Studies with a genetic deletion mutant of Y. pestis indicate that YpTdp does

not have a role in virulence of the organism. It is possible that any effects on virulence

in this case are minor. Y. pestis employs a very sophisticated system of infection and

evasion mechanisms. For example YopJ, a protease delivered into host cells via the

type III section system (TTSS) is known to block NFB activation by TLR receptors

[41]. Since it appears that in Y. pestis there is some redundancy in the mechanisms

employed to inhibit NFB activation it is possible that the YopJ is masking the effects

of YpTdp on the virulence of Y. pestis.

The data reported so far on bacterial TIR proteins strongly indicates that there

is no one clear mechanism by which they act to subvert the host immune system.

11

TcpC and TlpA show direct interactions with components of the TLR signaling

pathway and are likely to directly block the pathway whereas TcpB seems to trigger

the degradation of one key component and thus reduce signaling. While it seems clear

that in some cases the bacterial TIR domains play a role in virulence in the case of

YpTdp, the protein lacks any clear relevance for virulence despite inhibiting NFB

activation. It seems likely that the TIR proteins from these pathogenic bacteria have

different roles which means that only by studying a range of these can we really

understand the full breadth of functions.

TIR domains in non-pathogenic bacteria

As mentioned above TIR domains have been identified in a range of both pathogenic

and non-pathogenic bacterial species [29]. To account for this it has been suggested

that the TIR domain is a generic protein-protein interaction domain which has

subsequently been adapted to function as a mechanism of host immune system

evasion [29]. However, whilst the roles of the TIR domains in the non-pathogenic

bacteria remain uncharacterized there is evidence to show that these can be used to

understand the mechanism of action of the TIR domains from pathogenic bacteria.

This is illustrated by research carried out on the TIR domain protein from Paracoccus

denitrificans (PdTdp). Intriguingly this protein interacts with both MyD88 and TLR4

[33] and is the only bacterial TIR protein for which there is a high resolution structure

([42]; see below for a more detailed discussion). This has allowed the first insights

into the domains involved in bacterial TIR domain interaction with MyD88.

Anecdotal reports and our own experiences indicate that bacterial TIR domain

proteins are challenging to express and purify. However the data from the P.

denitrificans studies indicates that PdTdp is a suitable homologue for studying some

aspects of the function of TIR domains in pathogenic bacteria.

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How do the bacterial TIR proteins localise to the host cell cytoplasm

Sequence analysis of the regions around tlpA revealed a phage origin for tlpA

as well as links with pilin-related domains [32]. Since pilins are extracellular proteins

playing roles in cell adhesion, it was suggested that proteins encoded within this

genomic region including TlpA are secreted [32]. However further links with the type

III or type IV secretion systems could not be made on the basis of gene location due

to discontinuous genome sequence of S. enterica in this region [32]. To date, no more

direct evidence of secretion of TlpA has been reported.

TcpC has been reported to be secreted into the media of cultured bacterial

cells and taken up into host macrophages in a cholesterol-dependent manner to

interfere with TLR-mediated TNF induction [34]. The secretion mechanism has yet

to be identified for TcpC as the protein does not contain a recognizable signal

sequence. As mentioned previously, TcpB has been shown to contain an N-terminal

lipid binding domain which is suggested to important for cell permeability. Indeed

purified MBP-TcpB internalizes into mouse macrophages in a dose dependent manner

[43]. The YpTdp does not contain any known signal sequences and no mechanism of

secretion has yet been identified [40].

As yet there is no clear consensus on the mechanism by which TIR domains

localize to the host cytosol and the data so far indicates that there may be different

mechanisms depending on the pathogen. Further studies are needed to clarify this

issue perhaps by performing real-time imaging and co-localisation studies.

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Biophysical characterization of bacterial TIR proteins

Preliminary biophysical characterization of bacterial TIR proteins by

molecular modelling indicated that they share considerable structural homology with

the TIR domain of TLR1 [34]. This was confirmed by the first crystal structure of a

bacterial TIR protein, PdTIR (Table 1), which revealed an overall fold identical to the

known structures of the human TIR domains [42]. The asymmetric unit of the PdTIR

crystals contains four molecules that form two equivalent dimers [42]. Unlike the

homodimer interface observed in the structure of the human TLR10-TIR [20]

involving mainly the BB loops (Figs 2 and 3; Table 1), the dimer interface in PdTIR

involves the D-helix, the DD- and EE-loops leaving the BB-loops largely exposed

on the surface of the molecules [42]. A large network of hydrogen bonds mediates

dimerisation of the PdTIR protomers in the crystal structure.

The crystal structures of a number of TIR domains [16,19,20,22,42] indicates

the formation of dimers in the crystal lattice. In contrast, the purified proteins are all

present as monomers in solution although there is some evidence to suggest that TIR

domains function as weakly associated dimers [44].

In contrast to all the other biochemically characterized TIR domains YpTIR

forms dimers in solution which are likely to be disulphide bond dependent [45].

Mutation of one of the two Cys residues to Ser present in YpTIR retains the dimeric

form of the protein whereas mutation of both results in highly unstable protein not

suitable for analysis. Interestingly this study showed that mutation of Cys132 to Ser

abolished interaction with MyD88 suggesting that the disulphide bonds may be key in

maintaining an active confirmation of YpTIR [45].

14

The structure of PdTIR indicated that the highly exposed BB loop is the region

of PdTIR which interacts with human TIR proteins of the TLR signaling pathway

[42]. The importance of the BB loop region in interactions between bacterial TIR

domains and human adaptor proteins has been demonstrated by mutagenesis studies.

Pull-down studies on a Pro173His mutant of YpTIR shows no detectable interaction

with GST-MyD88-TIR in contrast to the wild-type protein [40]. In addition, mutation

of another highly conserved residue of the BB loop Gly158 to an Ala resulted in a

TcpB with reduced activity in a NFkB reporter assay [36]. However it should be noted

that there is variability in this region of the bacterial TIR domains with TcpB and

YpTdp lacking the conserved Pro and Gly residues respectively. While these studies

strongly suggest that the BB loop is essential for bacterial TIR activity only a

structure of a bacterial TIR domain in complex with a human adaptor protein will

provide definitive characterization of the interaction domain.

Other roles of bacterial TIR domains

While the majority of studies have focused on the role of bacterial TIR

domains in direct inhibition of the TLR signaling pathway through competitive

binding of human adaptor proteins recent research has highlighted another possible

function. Radhakrishnan and colleagues [46] have shown that exposure of

microtubules to TcpB expression results in improved nucleation and growth of

microtubules in vitro. Preliminary results show that this activity is mediated by the

BB loop. The significance of this effect of TcpB in terms of immune system evasion

remains to be clarified although the authors of the study speculate that this may be a

15

further mechanism by which the upregulation of the NFkB signaling pathway is

inhibited.

The first study on bacterial TIR proteins indicated that expression of TlpA

from S. enterica serovar Enteritidis resulted in activation of caspase-1, a host protease

[32]. Activation of this protease results in cleavage, activation and secretion of the

proinflammatory cytokine IL-1. An increase in IL-1 secretion was observed in

cultured cells expressing TlpA and in macrophages infected with wild-type S. enterica

serovar Enteritidis compared to the tlpA deletion strain. This rather surprising finding

suggests that TlpA is capable of both simulating and inhibiting proinflammatory

cytokine production. Recent research has indicated that YopJ from Y. pestis also has

such a dual role [47]. It has been suggested that the upregulation of caspase-1 may

represent a mechanism of host defence against pathogens able to inhibit the NKB

pathway. It will be interesting to see if bacterial TIR domain proteins in addition to

TlpA are able to stimulate this host cell response.

The YpTdp deletion mutant shows some interesting phenotypes including

increased auto-aggregation and reduced ability to survive in high salt conditions when

compared to wild-type Y. pestis [40]. These features may contribute to the ability of

the Y. pestis to survive in the host and thus increase pathogenicity. They also suggest

functions of the YpTdp in addition to inhibition of TLR signaling.

Overall, the widespread distribution of TIR domains in non-pathogenic

bacterial species as well as the diversity of the associated protein domains [29] is

strongly indicative that these TIR containing proteins have roles other than in immune

system evasion. However it remains to be seen what these other possible roles are.

16

Microbial TIR domains as therapeutic agents

Upregulation of the innate immune response is highly advantageous to host

survival although anomalous upregulation has been implicated in a number of

autoimmune and inflammatory diseases including multiple sclerosis and rheumatoid

arthritis [48]. In addition, research has also highlighted the role of NFB in cancer.

Overactivation of NFB as the result of mutagenesis of proteins in the TLR signaling

pathway may contribute to cell proliferation [49]. Recently, highly potent TLR

signaling inhibitors have been generated based on the A46R protein from vaccinia

virus [50]. Previous studies have shown it is possible to make cell-penetrating

peptides based on the BB-loops of the TIR domains [51] of both adaptor proteins and

TLRs which inhibit TLR signaling pathways. The wide variety of ways in which

bacterial TIR domains modulate the TLR signaling pathway means that it may be

possible to design peptides which target different components of the pathway and thus

have subtly different effects on the innate immune system. These represent a means of

tuning the innate immune response to possibly alleviate some of the symptoms of

debilitating auto-immune diseases and potentially treat some cancers.

Conclusion

The current evidence strongly suggests that bacterial TIR domains represent a novel

mechanism by which pathogens can subvert the host immune system. However

questions remain regarding how the bacterial TIR domains are taken up into host cells

and at what points in the TLR signaling pathway they can form negatively regulating

interactions. Some of the studies seem to indicate that while the bacterial TIR

domains share some common features there is a certain amount of individuality in

17

their interaction profiles and precise functions. Further research is required in order to

elucidate the precise mechanism of action of these interesting molecules.

Acknowledgements

This work was funded by the UK Ministry of Defence.

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Figure Legends

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Figure 1 Overview of TLR/ IL-1R signalling through TIR domain containing

adaptors The receptors IL-1R, TLR1, TLR2, TLR4, TLR5 and TLR6 are expressed

on the cell surface while receptors TLR3, TLR7, TLR8 and TLR9 are expressed on

endosomal membranes. The cytoplasmic TIR domain containing adaptors MyD88,

Mal, TRIF and TRAM are differentially recruited by TLR/ IL-1R complexes to

positively regulate the activation of transcription factors NF-κB and IRFs while

SARM negatively regulates TRIF. The evidence so far indicates that bacterial TIR

proteins can also act as negative regulators through interaction with MyD88, MAL

and the TIR domain of TLR4 (indicated by the white asterisks; see main text for more

details). However the interaction profile differs for individual bacterial TIR domains.

Figure 2 Primary sequence alignment of a range of bacterial TIR domains with some

mammalian TIR domains of known structure. The regions corresponding to the TIR

motifs, Box1, 2 and 3, are indicated by the solid rectangles above the alignment. The

regions of the sequence of TLR1-TIR corresponding to secondary structure elements

as determined by X-ray crystallographic analysis (Pdb accession code: 1FYV) are

shown below the alignment. Some of the residues involved in dimer formation are

shown in bold for both the TIR domain of TLR10 and PdTIR. The alignment was

generated using ClustalW.

Figure 3 Structure of a) the TIR domain of TLR1 (Pdb accession code, 1FYV) and b)

PdTIR (Pdb accession code, 3H16). The positions of the BB (red), DD (green) and EE

(cyan) loops with key roles in dimer formation are indicated. c) Superposition of the

TIR domain of TLR1 (dark blue) and PdTIR (magenta). Space-filling model of the

structure of the dimer of d) TIR domain of TLR10 (Pdb accession code, 2J67) and e)

PdTIR. In each case monomer 1 is shown in dark blue with the region forming the

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interaction in red while monomer 2 is shown in cyan with the regions forming the

interaction in gold. Some of the key residues involved in dimer formation are

individually labeled. In d) the residues indicated all form part of the BB loop while in

e) the residues are part of the DD and EE loops as indicated in a) and b). See also

Figure 2 for more details.

Figure 1

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Figure 2

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Figure 3

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