The N‐terminal D1 domain of Treponema pallidum flagellin binding to TLR5 is required but not sufficient in activation of TLR5

Abstract Syphilis is a chronic bacterial infection caused by Treponema pallidum (T pallidum) and the pathogenesis that T pallidum infection induces immunopathological damages in skin and other tissues remains unclear. We have previously reported that recombinant flagellins of T pallidum can elicit IL‐6 and IL‐8 transcriptions via TLR5 pathway. To identify the domains which induced the pro‐inflammatory activity and the importance of the interactions between TLR5 and domains, homology‐based modelling and comparative structural analyses revealed that Tpflagellins can combine with TLR5 directly. Deletion mutations showed that the ND1 domain binding to TLR5 is required but not sufficient in TLR5 activation. Moreover, site‐directed mutagenesis analysis indicated that the arginine residue (Tpflagellins R89) of the ND1 domain and its adjacent residues (Tpflagellins L93 and E113) constitute a hot spot that elicits IL‐6, IL‐8 transcriptions and TLR5 activation, and affects the binding of Tpflagellins to TLR5. Taken together, these results give insight into the pathogenesis of T pallidum and may contribute to the future design of Tpflagellins‐based therapeutics and syphilis vaccine.


| INTRODUC TI ON
Treponema pallidum subsp pallidum (T pallidum) is the causative agent of syphilis, a multistage sexually transmitted disease with an estimated prevalence of 36 million individuals affected worldwide and 12 million new cases per year. 1 It is responsible for chronic infections in compromised individuals and disseminates throughout the entire body of patients through bloodstream, therefore leading to diverse clinical manifestations. 2 In spite of the great progress in the diagnosis of syphilis and long-term in vitro culture of the T pallidum, 3,4 few studies were performed on the pathogenesis of T pallidum. [5][6][7] Unlike other external flagellated bacteria, T pallidum, an obligate microaerophilic bacterial pathogen, 8 is motile via the endoflagella that undulating their bodies as travelling planar waves, thereby driving its movement to swim through viscous fluids, such as blood and interstitial fluid. 9,10 The flagellar filament of T pallidum is composed of three core protein (FlaB1, FlaB2 and FlaB3) and two outer layer proteins (FlaA1 and FlaA2). 11 The role of flagellar antigen-induced immune stimulation in the virulence of many pathogenic microorganisms, including Salmonella typhimurium and P aeruginosa, has been well established. [12][13][14] Flagellin, the major structural protein of bacterial flagella, can promote pathogens to adhere and invade into host cells as a virulence factor 15 and activate innate immunity through the TLR5 and NAIP5/NLRC4 pathway, [16][17][18][19] which will contribute to the immediate clearance of pathogens from the host. The flagellin molecule contains two to four domains, D0-D3. Of these, the D0 and D1 domains (N/C regions) are highly conserved between organisms, which are crucial for the immunostimulatory activity of flagellin. 17,20,21 However, the D2 or D3 domain (middle hypervariable region), which is essential for flagellin antigenicity 22 and could cause the undesirable toxicity of flagellin-based therapeutics, 23 has vast diversity in their sequence, size and composition in various bacterial strains and is even absent in some bacterial species, including T pallidum. Since flagellin was considered as an activator in shaping both the innate and adaptive arms of immunity in the first line of defence against flagellated pathogenic bacteria, it has been shown to enhance cross-protective response of related antigens against diverse infectious diseases as a vaccine carrier protein or a vaccine adjuvant, and some of the vaccines eventually made their way into human clinical trials. [24][25][26] More significantly, flagellin-activated TLR5 has also exhibited anti-tumour, anti-metastatic immunotherapy and radioprotective property, and has shown tremendous potential in resisting cancer cell growth and radiation-associated gastrointestinal tissue damage. 23,[27][28][29] TLR5, consisting of an extracellular domain with leucine-rich repeats (LRRs), a transmembrane domain, and an intracellular Toll/IL-1 receptor (TIR) domain, is a member of evolutionarily conserved type I transmembrane receptors located on the cell surface that provides a vital link between innate and adaptive immunity. 30 It has been reported that TLR5 could present on a variety of cells, including monocytes, macrophages, basophils, neutrophils, lymphocytes, NK cells, dendritic cells (DCs), endothelial cells and epithelial cells. [31][32][33] recognizes flagellin with the extracellular domain and recruits downstream adaptor molecules binding to the intracellular TIR domain, which will activate the MAPK and NF-κB signal pathway and further lead to the expression of a variety of gene, including pro-inflammatory cytokines, chemokines, nitric oxide (NO) and host-defence proteins. 34,35 Structural study has revealed that TLR5-flagellin interaction mediated through two distinct binding sites. The first facilitates formation of a 1:1 TLR5-flagellin complex, and the second guides the formation of a 2:2 complex necessary for signalling. 36 It has been identified that flagellins from Gram-negative Salmonella enterica subspecies enterica serovar Dublin, P aeruginosa and Gram-positive Bacillus subtilis are all binding to the TLR5 ectodomain with amino acid residues within the conserved D1 domain. [36][37][38] However, as a result of the variations in the sequences and domains of flagellins, flagellins from diverse bacterial species use the unequal TLR5-recognition mechanism. For instance, previous reports in FliC flagellin from Salmonella enterica serovar Enteritidis, S enterica serovar Typhimurium and Pseudomonas aeruginosa have shown that the hypervariable region is essential for the activation of TLR5. 39 In addition, some researchers found that the D1 domain of flagellins is essential for the activation of TLR5 in mammals. 38,40,41 However, a recent study revealed that the C-terminal D0 domain plays a crucial role in TLR5 activation. 42 Despite our knowledge of other flagellins and TLR5 binding interface based on the crystal structure, the contribution of each flagellin residue in flagellin-TLR5 binding and TLR5 activation has not been elucidated and therefore there is an urgent need for us to investigate the interactions between T pallidum flagellins (Tpflagellins) and TLR5.
We previously showed that Tpflagellins are capable of inducing IL-6 and IL-8 productions through TLR5-dependent MAPK and NF-κB signalling 43 and inhibiting the dissemination of T pallidum in rabbits. 26 The purpose of the present investigation was to further define the regions and the specific amino acid residues of T pallidum flagellins in receptor activation and pro-inflammatory activity, which will contribute to establishing the foundation work for the future design of Tpflagellins-based syphilis vaccine and therapeutics.

| Cell cultures
The human monocytic leukaemia cell line, THP-1 (American Type Culture Collection) was cultured in complete medium RPMI 1640 (Gibco, NY, USA), containing 10% foetal bovine serum (Gibico) supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C in 5% CO 2 . For experimental purposes, cells were resuspended in serum-free medium and then seeded at 1 × 10 6 cells/well in 6-well tissue culture plates and then cultivated overnight.

| Construction of wild-type and mutant flagellin plasmid DNAs
The construction of a bacterial expression plasmid pET28a-FlaB containing T pallidum flagellins-coding sequences has been described previously. 44 These plasmids were used as the template for subsequent mutagenesis. Deletion mutant plasmids were generated by one-step PCR and cloned into the expression vector pET28a via restriction sites to give expression of N-terminal His-tag proteins.
The chimeric NC without the hypervariable region was constructed using overlap extension PCR. 45 Point mutations in the flagellin gene were created by circular PCR with DpnI digestion to eliminate background wild-type plasmid. All primers used are given in Tables S1 and S2. All mutants were confirmed by DNA sequence analysis.

| qRT-PCR
Total RNA was extracted from human THP-1 cells using TRIzol Reagent conditions were as follows: one cycle of initial denaturation (5 minutes at 95°C) and 40 amplification cycles (10 seconds at 94°C, 20 seconds at 58°C and 10 seconds at 72°C). Each RNA sample was performed in triplicate. The results normalized by using the reference gene GAPDH.
Normalized relative quantities were calculated using the comparative threshold cycle (2 -△△Ct ) method.

| 3D structure prediction
The 3D models of the Tpflagellins were predicted by Phyre 2. 46 The structure comparisons of various flagellins were performed by using UCSF Chimera 1.12 program. 47

| Statistical analysis
Results are expressed as means ± SD of three experiments. GraphPad Prism 6.0 (GraphPad Software, Inc, La Jolla, CA) was used for statistics. Student's t test was used for statistical comparison of the data. Differences were considered statistically significant when the P value was less than .05. Tpflagellins were structurally similar to its orthologs (Stflagellin, Paflagellin, Ssflagellin, Sdflagellin and Bsflagellin) in that they contained three α-helices bundle structure ( Figure 1B,C). [36][37][38][48][49][50] The

| Modelling of Tpflagellins structures and structural comparison of various flagellins
Tpflagellin structures were the same as the majority of flagellins except for Sdflagellin, which contained two helices in the C-terminal of the D1 domain (CD1 domain) ( Figure 1C,D); however, the CD1 domain in the BcFlg-hTLR5 complex structure was also discontinued to two α-helices. 51 This comparative analysis of various flagellin structures suggested that the CD1 domain was structurally diverse. To further ensure that the Tpflagellins were able to combine with TLR5, the structural analysis of a complex formation between TpFlaB1 and human TLR5 (hTLR5) was performed in silico ( Figure 1E,F). It was consistent with previous reports that TpFlaB1 formed a 1:1 complex with TLR5 through three α-helices (αND1a, αND1b and αCD1) in D1 domain.

| Pro-inflammatory mediators induced by the deletion mutant flagellins
To begin the molecular analysis of mutant Tpflagellins, we first made

| Mutant flagellins containing only ND1 domains bind to human TLR5
The previously reported crystal structure of zebrafish TLR5 in complex with the Salmonella flagellin FliC visualized the D1 domain is a conserved TLR5 binding hot spot. 36 To confirm that the Tpflagellinsmediated TLR5 activation occurs through a direct interaction between flagellin and TLR5, and to examine whether Tpflagellins induce TLR5 activation through the common binding site with other species of flagellins, we used co-immunoprecipitation to detect the combination between mutant flagellins and human TLR5. As shown in Figure 4A  Taken together, these results indicated that the regions in flagellin required for pro-inflammatory activity were also required for its functional activation of the TLR5 receptor.

| Identification of residues in the Nterminal D1 domain of Tpflagellins that affect inflammation response
Considering the fact that the D1 domain in N-terminal was important for the pro-inflammatory activity of Tpflagellins, we next sought to make clear the contribution of individual amino acids in inducing pro-inflammatory response. Previous studies have suggested that the TLR5 LRR9 loop residues generate a cavity that accommodates the residues R89, L93 and E113 of flagellins through conserved key interactions. 36,38 The comparative structural analyses revealed that Tpflagellins possessed a common conserved molecular pattern for the TLR5 interaction with Bsflagellin ( Figure 1C). Thus, we generated three different site-specific mutations, single, double or triple (corresponding primers were listed in Table S2) in ND1 domain whereby arginine (Arg89) was changed to alanine (R89A), leucine (Leu93) was changed to alanine (L93A), and glutamic acid (Glu113) was changed to alanine (E113A) (Figure 6 and Figure S2B-D). In addition, the phylogenetic tree displayed that the Tpflagellins shared a close genetic relationship with Bsflagellin ( Figure S2A) and the three residues were highly conserved in TLR5-activated flagellins, but not in TLR5 non-activated flagellins ( Figure 7A). All site-directed mutant flagellins were prepared in recombinant form ( Figure S2E)

| D ISCUSS I ON
We recently reported that purified T pallidum flagellins played an essential role in eliciting pro-inflammatory cytokines in vitro. 43 In the current study, we demonstrated that the ND1 domain of Tpflagellins binding to TLR5 receptor was required but not sufficient for TLR5 activation and IL-6 and IL-8 transcriptions. This finding gives insight into the pathogenesis of T pallidum and contributes to designing effective therapeutics and vaccines in the future.
Several studies have indicated that the release of inflammatory mediators caused by flagellins occurred through the interaction between flagellin and TLR5. [52][53][54] However, as one of the smallest flagellin consisting only of the D0 and D1 domains, it was necessary to understand the TLR5 Tpflagellin interaction in more detail since the conflicts dated from different researchers in which some claimed that a central hypervariable domain was essential for activation of pro-inflammatory gene expressions, 39 others revealed that the conserved N-terminal domain mediated the pro-inflammatory response, 38,40 still other researcher indicated that the D0 domain of flagellin was required to produce a maximum TLR5-mediated response. 42 However, our data showed that the conserved D1 domain was responsible for inducing pro-inflammatory response. This was consistent with the results of previous researchers. 21,40,55 Moreover, F I G U R E 4 Mutant flagellins containing only ND1 domains bind to human TLR5. (A-C) Co-immunoprecipitation was performed to assess the binding of the mutated proteins to human TLR5. 30 μg of each protein was added to lysed THP-1 cells (or lysis), then incubated with anti-hTLR5 antibody at 4°C for 16 h, half of the lysates were incubated with 40 μL of beads for 1 h at room temperature and washed 5 times with wash buffer, and then, they were eluted and boiled for Western blotting analysis; the other half of the lysates were boiled and separated by SDS-PAGE for Western blotting analysis. TLR5 polyclonal antibody was used for the detection of TLR5. Anti-His antibodies were used for the detection of wild-type flagellin and mutants in the experiment of mutant flagellins binding to TLR5, we found that although mutant flagellins △C and N lack CD1, which was shown to combine directly with TLR5 in a previous reported SdFlg-zfTLR5 structure 36 and our TpFlaB1-hTLR5 complex ( Figure 1E,F This further indicated that although ND1 domain was involved in the primary binding, the △C and N may lack residues to guide receptor dimerization for TLR5 activation. Thus, the ND1 domain of F I G U R E 5 Identification of the D1 domain of Tpflagellins that affect TLR5 signalling. THP-1 cells were transfected with psiRNA-hTLR5 (psiRNA-LucGL3 was used as a control) for 28 h, then stimulated with 1 μg/mL FlaB1 mutant flagellins (A), 10 μg/mL FlaB2 mutant flagellins (B), or 5 μg/mL FlaB3 mutant flagellins (C) for 1 h to analyse the expressions of nonphosphorylated and phosphorylated forms of ERK1/2 (p42-44MAPK), p38MAPK, and IκBα, stimulated with 1 μg/mL FlaB1 mutant flagellins (D), 10 μg/mL FlaB2 mutant flagellins (E), or 5 μg/ mL FlaB3 mutant flagellins (F) for 24 h to measure the transcription levels of IL-6 and IL-8 mRNA in the cells by using qRT-PCR. Data are presented as mean ± SD of three independent experiments. *P < .05, **P < .01 versus corresponding control The gene transcription levels of IL-6 and IL-8 were analysed by qRT-PCR. Data are presented as mean ± SD of three independent experiments. *P < .05, **P < .01 as compared with wild-type flagellins. E, Co-immunoprecipitation was performed to assess the binding of the mutated proteins to human TLR5. 30 μg of each protein was added to lysed THP-1 cells (top) or control lysis (bottom) and then incubated with anti-hTLR5 antibody at 4°C for 16 h, and the complexes were then incubated with 40 μL of beads for 1 h at room temperature and washed 5 times with wash buffer and eluted for Western blotting analysis. Anti-His antibodies were used for the detection of wild-type flagellin and mutants Tpflagellins binding to TLR5 receptor was required but not sufficient for TLR5 activation.
Tpflagellins subdomain deletions revealed that the D1 domain played a key role in TLR5 activation, while the D0 domain has a minimal effect on TLR5 activation. This was the same as Stflagellin, 40 however, different from Sdflagellin. 36 It may be related to the differences among species in the D0 domain. In addition, despite the phylogenetic tree ( Figure S2A) revealed that Tpflagellins shared a close genetic relationship with Bacillus subtilis flagellin (Bsflagellin) and alignment through ClustalW showed that significant sequence homology existed in the conserved N-and C-terminal between Tpflagellins and Bsflagellin ( Figure 1A) and Bsflagellin cent -rTLR5 N14 complex structures was derived from zebrafish and displayed only the N-terminal region of zfTLR5, and the D0 domain was not located in the complex structure. Furthermore, a recent study indicated that flagellin was recognized by TLR5 through a more extensive interaction surface than crustal structure shown. 56 Therefore, the key domains to guide receptor dimerization for TLR5 activation needed to be identified in the future.
Our mutational analyses of Tpflagellins-mediated TLR5 activation displayed that R89 is especially critical for TLR5 signalling and its adjacent residues L93 and E113 seemed to play assistant roles for the interactions. Sequence analyses indicated that the three residues were highly conserved in TLR5-activated flagellins, but not in TLR5 non-activated flagellins. This was consistent with previous structural model of Sdflagellin D1-D2 -rTLR5 N14 and Bsflagellin cent -rTLR5 N14 complexes, in which TLR5 formed a cavity to interact with R89, L93, and E113. 38 These site-directed mutations not only had varying degrees of impact on TLR5 activation, but also affected the binding efficiency of those flagellin molecules to TLR5. This demonstrated that the three sites are essential for the combination of Tpflagellins and TLR5 in the primary binding site. Moreover, predicted 3D structures of Tpflagellins were similar to Bsflagellin in TLR5-binding area ( Figure 6). Taken together, we concluded that Tpflagellins R89 and its adjacent residues played a critical role for TLR5 activation in a conserved mechanism.
As a vaccine adjuvant, the potent antigenicity of flagellin might affect its potency and induce side effects, thus efforts should be made to reduce potential adverse effects induced by flagellin while maintaining its adjuvanticity. Many studies have proved that the middle hypervariable region of flagellin was replaced with the antigen, which could reduce the side effects. However, the fusion process may introduce inter-molecular steric clashes or unexpected modifications, such as proteolysis or destabilization, which could reduce the efficacy of the flagellin-antigen fusion vaccine. Moreover, the D2 and D3 domains could induce an unwanted toxic immune response.
Therefore, it would be useful to identify the smallest naturally occurring flagellin containing only the D0 and D1 domains.
T pallidum flagellins contain only D0 and D1 domains and are one of the shortest flagellins. Although T pallidum flagellins lack the hypervariable domains, it exhibits similar TLR5-binding affinity and TLR5 signalling activity to other flagellins. Flagellin, as the only TLR5-specific agonist, has been developed as a vaccine adjuvant and the adjuvant activity of flagellin is mainly mediated by TLR5activating capacity. 57 The induction of pro-inflammatory cytokines and chemokines in a number of innate and non-immune cell 33,[58][59][60] mediated by MyD88-dependent NF-κB signalling is critical for the activation and development of antigen-specific adaptive immune responses, which could promote cell-mediated immunity via stimulation of Th1 cells and a marked recruitment in T and B lymphocytes to draining lymph nodes, and thus maximizes the chances of antigen-specific lymphocytes encountering their cognate antigens. 61 In addition, flagellin, a vaccine adjuvant, has the ability to induce macrophages to produce a variety of cytokines, which were central to the role of macrophages that mediate the transition from innate to adaptive immunity and enhance the microbicidal ability. 62 For instance, IL-6 secreted by macrophages could promote differenti-

ation of B cells into plasma cells, activate cytotoxic T cells and lead
to the recruitment of monocytes to the inflammation site 63 ; IL-8 is a potent chemoattractant for neutrophils, in which it also induces degranulation and morphological changes. 64 Both of them could help to eliminate pathogens. CBLB502, as an anti-radiation therapeutic, has been used to protect against gastrointestinal and haematopoietic acute radiation syndromes by removing the D2 and D3 domains from sdflagellin. 23 However, given that CBLB502 protein seems to be unstable during purification and storage, and the D2 and D3 domains are not necessary for TLR5-mediated immune response and could induce an unexpected adaptive immunity and cellular toxicity in the host; thus, the naturally Tpflagellins containing only the D0 and D1 domains are useful for the development of flagellin-based anti-pathogenic vaccines and anti-radiation therapeutics.
All in all, our findings demonstrated that the ND1 domain of Tpflagellins binding to TLR5 is required but not sufficient for TLR5 activation and IL-6 and IL-8 transcriptions, which are consistent with the TLR5-Tpflagellin complex structure analysis. Site-directed mutational analyses revealed that at least three distinct sites (R89, L93 and E113) on flagellin were required in Tpflagellins-mediated TLR5 activation. All of these will contribute to establishing the foundation work for the future design of Tpflagellins-based syphilis vaccine.