The outer membrane protein Tp92 of Treponema pallidum induces human mononuclear cell death and IL‐8 secretion

Abstract Treponema pallidum is the pathogen that causes syphilis, a sexually transmitted disease; however, the pathogenic mechanism of this organism remains unclear. Tp92 is the only T. pallidum outer membrane protein that has structural features similar to the outer membrane proteins of other Gram‐negative bacteria, but the exact functions of this protein remain unknown. In the present study, we demonstrated that the recombinant Tp92 protein can induce human mononuclear cell death. Tp92 mediated the human monocytic cell line derived from an acute monicytic leukemia patient (THP‐1) cell death by recognizing CD14 and/or TLR2 on cell surfaces. After the stimulation of THP‐1 cells by the Tp92 protein, Tp92 may induce atypical pyroptosis of THP‐1 cells via the pro‐caspase‐1 pathway. Meanwhile, this protein caused the apoptosis of THP‐1 cells via the receptor‐interacting protein kinase 1/caspase‐8/aspase‐3 pathway. Tp92 reduced the number of monocytes among peripheral blood mononuclear cells. Interestingly, further research showed that Tp92 failed to increase the tumour necrosis factor‐α, interleukin (IL)‐1β, IL‐6, IL‐10, IL‐18 and monocyte chemotactic protein 1 (MCP)‐1 levels but slightly elevated the IL‐8 levels via the Nuclear Factor (NF)‐κB pathway in THP‐1 cells. The data suggest that Tp92 recognizes CD14 and TLR2, transfers the signal to a downstream pathway, and activates NF‐κB to mediate the production of IL‐8. This mechanism may help T. pallidum escape recognition and elimination by the host innate immune system.

the prescribed standards may suffer from chronic and persistent infections in the body. 3 Therefore, T. pallidum is likely to have some mechanisms that can affect the immune system, especially mechanisms for evading the innate immune response.
Appropriate killing of innate immune response cells that engulf pathogens would release the pathogens and expose them to the antibacterial machinery of the host; meanwhile, the infected innate immune response cells would be eliminated. 4 If these important innate immune response cells are eliminated in large quantities, the responsiveness of the host's innate immune response system to early infection will be greatly reduced. 5 Therefore, via this mechanisms, pathogens may induce the death of a large number of innate immune response cells, thereby evading elimination by the host's immune cells. The regulation of multiple celldeath-associated signalling pathways may be involved in pathogenic infection. For example, apoptosis, which depends on receptor-interacting protein kinase 1 (RIPK1)/caspase-8/caspase-3, and pyroptosis, which depends on caspase-1, are important cell-death-associated signalling pathways. 6,7 Some pathogenic Spirochaeta induce the death of innate immune response cells. For example, Borrelia burgdorferi, the pathogenic bacterium responsible for Lyme disease, induces the apoptosis of peripheral blood mononuclear cells (PBMCs). 8 The leptospiral lipoprotein LIC11207 increases the apoptotic rate of its host neutrophils. 9 However, to date, there has been no report on whether T. pallidum induces the apoptosis of innate immune response cells.
When Gram-negative bacteria invade hosts, bacterial antigens that are directly exposed to the external environment are the first to interact with the host's innate immune response system. These antigens, such as lipopolysaccharides (LPSs), outer membrane proteins and outer membrane lipoproteins, are instantly recognized by the innate immune response system, leading to a series of immunopathological effects and the activation of immune escape mechanisms. 10,11 T. pallidum lacks the key virulence factor LPS and other common virulence factors, such as exotoxin, that are secreted by other Gram-negative bacteria. 12 However, T. pallidum can still cause persistent infection and immune damage in patients who have not been treated at all or as prescribed. 3 It is believed that the outer membrane proteins and lipoproteins of T. pallidum play key roles.
There are seven variable regions in the open reading frame of the outer membrane protein TprK of T. pallidum, and the antigenic variability of this protein may be an immune escape mechanism of T. pallidum. 13 However, there have been no reports on whether other outer membrane proteins or lipoproteins of T. pallidum contribute similarly or otherwise to immune escape.
Tp92 is the only T. pallidum outer membrane protein that has structural features that are similar to those of the outer membrane proteins of other Gram-negative bacteria 14 ; however, its exact functions of this protein remain unclear. A study showed that the gene encoding the Tp92 protein may be associated with the pathogenesis of T. pallidum. 15 However, the pathogenic effect of Tp92 remains poorly understood. In a recent study, Jun et al demonstrated that Td92, a surface protein of the periodontal pathogen Treponema denticola and a homologue of the T. pallidum surface protein Tp92, activates caspase-4 and induces pyroptosis in primary cultured human gingival fibroblasts via cathepsin G activation. 16 In the present study, we investigated the potential pathogenic role of the outer membrane protein Tp92 by exploring the effect of Tp92 on the THP-1 innate immune response cells. The NF-κB inhibitor QNZ was purchased from MCE.

| Recombinant protein expression and purification
After removing the signal peptide (1-18aa), the Tp92 fragment was expressed in Escherichia coli and purified with Ni-NTA columns (Darmstadt, Germany) as described previously. 17

| Hoechst33342 staining
THP-1 cells (5 × 10 4 /well) were plated in 24-well plates and stimulated with different concentrations of Tp92 for 12 hours. Then, the cells were harvested and washed with PBS twice before being resuspended in 100 μL of PBS. Hoechst33342 dye (5 g/mL; Thermo Fisher Scientific) was added before incubation at 37°C for 15 minutes. After washing with PBS twice, the cells were resuspended in PBS and plated in fresh 24-well plates for observation of blue fluorescence under a fluorescence microscope (magnification, 400×; Nikon).

| Cell counting kit-8 assay
Total cell death rate was examined using cell counting kit-8 (CCK- 8) assay. The samples were processed according to the protocol of the CCK-8 assay kit (Beyotime, Shanghai, China) before determination of absorbance on a reader. After subtracting the absorbance of the blank control, the absorbance values of the samples were used to calculate the total cell death rate.

| Lactate dehydrogenase release assay
The samples were processed according to the protocol for the lactate dehydrogenase (LDH) release assay kit (Beyotime) before determination of the absorbance on a reader. After subtracting the absorbance of the blank control, the absorbance values of the samples were used to calculate the LDH release rate.

| Western blotting
Cells were centrifuged and collected before lysis with radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific).
The supernatant was used to determine protein concentration by the BCA protein concentration determination kit (Thermo Fisher Scientific). Then, loading buffer was added to the samples before boiling at 100°C for 5 minutes. Then, 10% or 12% sodium dodecyl LUO ET AL.

| Treponema pallidum propagation
The propagation of T. pallidum was performed in accordance with the method described by Lukehart and Marra. 19 Subsequently, T. pallidum was extracted from testes under the appropriate conditions.

| Statistical analysis
The results were analysed using SPSS 18.0 statistical software (IBM, Armonk, NY, USA). All data were expressed as means ± SDs. Student's t test was used for comparisons between two groups. A nonparametric statistical test was used for comparisons across three conditions. Differences with P-values less than 0.05 were considered statistically significant. All tests were performed in triplicate.

THP-1 cell death
To test whether Tp92 induces THP-1 cell death, the cells were subjected to AO, EB or Hoechst33342 staining. The results of AO/EB staining showed that cell membrane damage was dependent on the concentration and duration of Tp92 treatment, and treatment with a concentration of 10 μg/mL ( Figure 1A) or for a duration of 48 hours ( Figure 1B) resulted in the maximum number of damaged cells. Upon increasing the Tp92 concentration, the nucleus also appeared to be broken and slightly shrunken ( Figure 1C). In addition, the total cell death rate was also dependent on the concentration and duration of Tp92 treatment, and a concentration of 10 μg/mL ( Figure 1D) or a duration of 48 hours ( Figure 1E) led to the highest total death rate of THP-1 cells. In contrast, the total cell death rate in the live and dead Tp groups, inactivated Tp92 (heat inactivation) group and Tp0663 group did not increase. These results suggested that stimulation by the Tp92 protein induces THP-1 cell death.

| The Tp92 protein may induce atypical pyroptosis of THP-1 cells via the pro-caspase-1 pathway
To examine whether Tp92 induces pyroptosis, caspase-1 enzyme activity was tested. The data showed that different concentrations or durations of Tp92 treatment failed to alter the caspase-1 enzyme activity (no significant differences were observed); however, the caspase-1 enzyme activity in the LPS+Nig group was significantly higher than that in the control group (P < 0.05) (Figure 2A and B). Activated caspase-1 can be released from cells through the secretory pathway.
Western blotting showed that the caspase-1 protein level in the supernatant in the Tp92 group was not different from that in the control group, but the levels in the LPS and LPS+Nig groups were higher than the level in the control group. However, the pro-caspase-1 protein levels in the Tp92, LPS and LPS+Nig groups were not significantly different from the level in the control group ( Figure 2C). Notably, GSDMD P30 levels in the Tp92, LPS and LPS+Nig groups were higher than the level in the control group, suggesting that GSDMD was cleaved in the three groups ( Figure 2D). The ELISA showed that different concentrations or durations of Tp92 treatment did not alter the concentration of IL-1β or IL-18 (no significant differences were observed); however, the IL-1β and IL-18 levels in the LPS+Nig group were significantly higher than those in the control group (P < 0.05) ( Figure 2E-G). Furthermore, the cells were treated with the caspase-1specific inhibitor VX-765 or pan-caspase inhibitor Z-VAD-FMK before examining LDH release, total death rate and caspase-1 activity. After treatment with Tp92 for 4 or 12 hours, the LDH release rates in the Tp92+VX-765 and Tp92+Z-VAD-FMK groups were significantly lower than the rate in the Tp92 group (P < 0.05) ( Figure 2H). In addition, the total cell death rates in the Tp92+VX-765 and Tp92+Z-VAD-FMK groups were significantly lower than the rate in the Tp92 group (P < 0.05) ( Figure 2I). Similarly, caspase-1 activity in the Tp92+VX-765 and Tp92+Z-VAD-FMK groups was significantly lower than that in the Tp92 group (P < 0.05) ( Figure 2J). These results indicated that the Tp92 protein may induces atypical pyroptosis of THP-1 cells via the pro-caspase-1 pathway.

| The Tp92 protein causes apoptosis of THP-1 cells via the RIPK1/caspase-8/caspase-3 pathway
To study how Tp92 affects the apoptosis of THP-1 cells, flow cytometry was performed. The data showed that the Tp92 protein induced the apoptosis of THP-1 cells in a concentration-and time-  Figure 3A and B). In addition, the activities of caspase-3 and caspase-8 in the presence of high concentrations of the Tp92 protein were significantly higher than those in the control (P < 0.05) ( Figure 3C and E), and treatment with Tp92 (5.0 μg/mL) for 12 hours resulted in peaks corresponding to caspase-3 and caspase-8 activities (P < 0.05) ( Figure 3D and F). Similarly, caspase-9 activity was also increased upon treatment with the Tp92 protein ( Figure 3G and H). Furthermore, pretreatment with the caspase-3 inhibitor Z-DEVD-FMK or caspase-8 inhibitor Z-IETD-FMK reduced the apoptotic rates of the THP-1 cells (P < 0.05) ( Figure 3I). However, the cell death rates in the other groups treated with neutralizing antibodies were not significantly different from the rate in the Tp92 group ( Figure 7A). Pretreatment with the TLR2 dominant negative plasmid or TLR4 interference plasmid showed that the cell death rate in the Tp92+TLR2 siRNA group was significantly lower than that in the Tp92 group (P < 0.01); however, the cell death rate in the Tp92+TLR4 siRNA group was not significantly different from that in the Tp92 group (P > 0.05) ( Figure 7B). Western blotting showed that the siRNA reduced the expression of TLR2 and TLR4 ( Figure 7C).

GSDMD
These results indicate that Tp92 mediates THP-1 cell death by recognizing CD14 and/or TLR2 on the cell surface.

| Tp92 reduces the number of monocytes and induces the secretion of IL-8 from PBMCs
To study whether the Tp92 protein affects PBMCs in the body,  with TLR2. [24][25][26][27][28] In addition, CD14 is a common receptor of TLR7 and TLR9. 29 When the LPS concentration is low, CD14 helps TLR4   During pathogenic infection, the main mechanisms of host cell death are pyroptosis and apoptosis. 6,7 Pyroptosis is a type of inflammation-related cell death that includes typical pyroptosis and atypical pyroptosis. In typical pyroptosis, activated NLRP3 binds with ASC to accumulate pro-caspase-1, which is then cleaved into active caspase-1. Activated caspase-1 cleaves the precursors of IL-1β and IL-18 to facilitate the maturation and secretion of these proteins. On the other hand, activated caspase-1 cleaves GSDMD, and the resultant GSDMD-N is then transferred to the cell membrane, where nonselective pores are formed and the cell membrane is damaged, finally leading to cell death. 34,35 In atypical pyroptosis, the inflammasome NLRC4 directly activates pro-caspase-1 without cleaving the precursors of IL-1β and IL-18. Then, GSDMD is directly cleaved, and GSDMD-N is transferred to the cell membrane, leading cell membrane damage and cell death. 36,37 In the present study, Tp92 treatment failed to elevate the caspase-1 levels and enzyme activity.
Moreover, the secretion of IL-1β and IL-18 was not elevated. However, GSDMD was cleaved. After pretreatment with the caspase-1 inhibitor VX-765 and caspase inhibitor Z-VAD-FMK, the cell death rate was reduced, suggesting that the caspase-1 inhibitor inhibits cell death. Meanwhile, the THP-1 membrane was disrupted and LDH release was elevated when Tp92 induced THP-1 cell death, suggesting that Tp92 possibly causes atypical pyroptosis via pro-caspase-1.
However, it remains unclear whether Tp92 induces atypical pyroptosis by activating NLRC4.
Apoptosis is a type of cell death that is not related with inflammation but is dependent on caspase. It is believed that caspase-1, -4,

Control
Rosup Tp92 Fas-associated death domain and induces apoptosis by activating caspase-3, -6 and -7 via the activation of caspase-8. 43,44 In the present study, ROS levels were seen to be elevated during the induction of THP-1 apoptosis by Tp92. It has been reported that RIPK1

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induces the production of ROS, 45 and ROS are associated with apoptotic signal transduction. 46 Our data showed that the RIPK1 inhibitor necrostatin-1 blocks the apoptosis that is induced by Tp92 and inhibits the activities of caspase-3 and caspase-8. These results suggested that Tp92 induces THP-1 cell apoptosis by activating the RIPK1/caspase-8/caspase-3 pathway. The results of the present study also show that caspase-9 is activated and the mitochondrial membrane potential is decreased, suggesting that the mitochondrial apoptosis pathway may also be involved.
RIPK1 is a key molecule in programmed cell death. 47   (2 μg/mL) or TLR4 (2 μg/mL) interference plasmids for 24 hours. The cells were treated with Tp92 (5 μg/mL) for 12 hours, and the CCK-8 assay was carried out to measure the total cell death rate. Data are expressed as the means ± standard deviations (n = 3). *P < 0.05 and **P < 0.01. C, Expression of TLR2 and TLR4 proteins in THP-1 cells pretreated with TLR2 and TLR4 interference plasmids for 24 hours. Western blotting was used to determine protein expression and TLR4, and activates the downstream MyD88 pathway or the TRIF pathway. 59 Yang et al discovered that stimulation with LPS enhances the TRIF, RIPK1, TRAF3, NF-κB, IRF7, TNF-α, IL-1β and IL-6 levels in pulmonary alveolar macrophages after CD14 binds with LPS. 60 The mechanism by which Tp92 induces THP-1 apoptosis may be that Tp92 recognizes CD14 and CD14 transfers the signal to TLR2, which then activates the RIPK1/caspase-8/caspase-3 pathway. It is also possible that CD14 directly activates the TRIF pathway after recognizing Tp92, finally leading to apoptosis. For example, the YopJ protein of Yersinia pestis inhibits the NF-κB and MAPK inflammatory pathways to reduce the production of inflammatory factors. 62 The T. pallidum antigen TpF1 promotes the secretion of the anti-inflammatory factor IL-10 to resist elimination by inflammation. 63 Interestingly, further research showed that Tp92  64 On the other hand, IL-8 promotes the release of elastase by neutrophils, damages endothelial cells, and facilitates the transport of T. pallidum across the endothelial barrier and into the blood. 65 The TpF1 protein induces the secretion of IL-8 by hosts, promotes skin and mucosal vascular proliferation in patients with syphilis, increases vascular permeability, and helps the spread of T. pallidum throughout the body via the blood vessels. Simultaneously, vascular proliferation leads to the accumulation of nutrients and energy, providing material support for the survival of T. pallidum. 66 Further study is required to determine whether IL-8 secretion induced by Tp92 has a similar effect.
Our data regarding the effect of Tp92 on PBMCs showed that Tp92 induces the death of monocytes among PBMCs. However, the effects of this protein on T cells or dendritic cells are small, probably because monocytes, but not T cells or dendritic cells, exhibit abundant CD14 and TLR2 expression on their surfaces. 67,68 In conclusion, the present study demonstrates that the Tp92 protein induces the death of human mononuclear cells and the secretion of IL-8 at the early stage of T. pallidum infection (Figure 12). This mechanism may help T. pallidum escape recognition and elimination by the innate immune response system of the host. (10 μg/mL) or TLR4 (10 μg/mL) for 1 hour before treatment with Tp92 (5 μg/mL), PGN (10 μg/mL), or LPS (1 μg/mL) for 12 hours. *P < 005. B, The cells were pretreated with the siRNA of TLR2 (2 μg/ mL) and TLR4 (2 μg/mL) for 24 hours before treatment with Tp92 (5 μg/mL) for 12 hours. *P < 005 F I G U R E 1 2 The outer membrane protein Tp92 of Treponema pallidum induces human mononuclear cell death and IL-8 secretion

CONF LICT OF I NTEREST
All authors declare no financial competing interests. All authors declare no nonfinancial competing interests.

ETHICAL APPROVAL
All procedures performed in this study involving human participants were approved by the Human Ethics Committee of University of South China and were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.