TYPE Review PUBLISHED 06 April 2023 DOI 10.3389/fimmu.2023.1126170 Syphilis vaccine: challenges, OPEN ACCESS controversies and opportunities EDITED BY Catherine Mary O’Connell, University of North Carolina at Chapel Hill, Carlos Ávila-Nieto 1,2*, Núria Pedreño-López 1, Oriol Mitjà 3,4,5, United States Bonaventura Clotet 1,4,5,6,7, Julià Blanco 1,5,6,7 REVIEWED BY Elizabeth De Gaspari, and Jorge Carrillo 1,6,7* Adolfo Lutz Institute, Brazil 1 IrsiCaixa AIDS Research Institute, Badalona, Spain, 2 Autonomous University of Barcelona, Cerdanyola Kelly Lynn Hawley, del Vallès, Catalonia, Spain, 3 Skin Neglected Tropical Diseases and Sexually Transmitted Infections University of Connecticut Health Center, Department, Germans Trias i Pujol Hospital, Badalona, Spain, 4 Fight Infections Foundation, Germans United States Trias i Pujol Hospital, Badalona, Catalonia, Spain, 5 Centre for Health and Social Care Research (CESS), *CORRESPONDENCE Faculty of Medicine, University of Vic – Central University of Catalonia (UVic – UCC), Vic, Jorge Carrillo Catalonia, Spain, 6 Germans Trias i Pujol Research Institute (IGTP), Badalona, Spain, 7 CIBERINFEC, jcarrillo@irsicaixa.es Instituto de Salut Carlos III (ISCIII), Madrid, Spain Carlos Ávila-Nieto cavila@irsicaixa.es SPECIALTY SECTION This article was submitted to Syphilis is a sexually or vertically (mother to fetus) transmitted disease caused by Vaccines and Molecular Therapeutics, the infection of Treponema pallidum subspecie pallidum (TPA). The incidence of a section of the journal Frontiers in Immunology syphilis has increased over the past years despite the fact that this bacterium is an obligate human pathogen, the infection route is well known, and the disease can RECEIVED 17 December 2022 ACCEPTED 27 March 2023 be successfully treated with penicillin. As complementary measures to preventive PUBLISHED 06 April 2023 campaigns and early treatment of infected individuals, development of a syphilis CITATION vaccine may be crucial for controlling disease spread and/or severity, particularly Ávila-Nieto C, Pedreño-López N, Mitjà O, in countries where the effectiveness of the aforementioned measures is limited. Clotet B, Blanco J and Carrillo J (2023) Syphilis vaccine: challenges, controversies In the last century, several vaccine prototypes have been tested in preclinical and opportunities. studies, mainly in rabbits. While none of them provided protection against Front. Immunol. 14:1126170. infection, some prototypes prevented bacteria from disseminating to distal doi: 10.3389/fimmu.2023.1126170 organs, attenuated lesion development, and accelerated their healing. In spite COPYRIGHT © 2023 Ávila-Nieto, Pedreño-López, Mitjà, of these promising results, there is still some controversy regarding the Clotet, Blanco and Carrillo. This is an open- identification of vaccine candidates and the characteristics of a syphilis- access article distributed under the terms of protective immune response. In this review, we describe what is known about the Creative Commons Attribution License (CC BY). The use, distribution or TPA immune response, and the main mechanisms used by this pathogen to reproduction in other forums is permitted, evade it. Moreover, we emphasize the importance of integrating this knowledge, provided the original author(s) and the in conjunction with the characterization of outer membrane proteins (OMPs), to copyright owner(s) are credited and that the original publication in this journal is expedite the development of a syphilis vaccine that can protect against cited, in accordance with accepted TPA infection. academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. KEYWORDS syphilis, treponema pallidum, vaccine, immune response, outer membrane proteins 1 Introduction Syphilis is a sexual transmitted infection caused by the spirochete Treponema pallidum subspecie pallidum (TPA). Although the origin of the syphilis epidemic is unclear, the first cases of the disease were reported in Europe in the late 15th century (1, 2). In 1905, Schaudinn and Hoffmann discovered the bacteria causing this venereal disease, and in Frontiers in Immunology 01 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 1943, the first cases of syphilis were successfully treated with identified to date, syphilis continues to be a significant global health penicillin (3). Over half a century later, penicillin remains one of problem. Therefore, the development of a syphilis vaccine is the most efficacious treatments (4). urgently needed to complement disease control and prevention Syphilis and the three nonvenereal treponematoses (yaws, bejel measures. In this review, we focus on: syphilis vaccine development, and pinta) were at first believed to be caused by the same agent, use of outer membrane proteins (OMPs) as putative immunogens, despite the fact that their clinical manifestations are different. Since anti-TPA immune responses, as well as TPA evasion mechanisms, pathogenic treponemes are morphologically and antigenically very to provide information about potential antigen selection in future similar (> 95% DNA homology), genomic sequencing was needed vaccine and immunization strategies. to identify distinct subspecies of Treponema pallidum that cause nonvenereal treponematoses: Treponema pallidum endemicum (causative agent of bejel), and Treponema pallidum pertenue (causative agent yaws). Pinta is caused by a different species of 2 Treponema pallidum subsp. spirochete bacteria, namely Treponema carateum (4, 5). All of them pallidum are obligate human pathogens characterized by its invasiveness and immune evasiveness (6–9). TPA is a flat-wave spirochete of 5-15 mm and 0.2 mm of A syphilis infection consists of three main stages (5). Primary diameter (20) that belongs to Spirochaetaceae family, particularly syphilis usually begins approximately 2-3 weeks after contact with Treponema genus. Treponemes are usually classified as Gram- the pathogen, and it is characterized by the presence of an ulcerated negative bacteria due to their double membrane structure (21). lesion, called chancre. Typically, this lesion appears on the genital However, the composition of their outer membrane (OM) is area, or other body parts related to sexual contact, usually noticeably different (22, 23). The TPA OM is characterized by the accompanied by regional lymphadenopathy (5, 10). In the paucity of surface-exposed proteins (24–26), the presence of absence of treatment, primary lesion resolves spontaneously in 3- phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, 6 weeks. Secondary syphilis develops as a result of bacteria and the lack of cardiolipins (23) and lipopolysaccharide (LPS) dissemination (10). Clinical manifestations include malaise, (27, 28). Interestingly, cardiolipins are present in the cytoplasmic headache, fever, diffuse lymphadenopathy, and maculopapular membrane (23), and represent the main lipid antigen targeted by rashes with either discrete or widespread body involvement (5, anti-TPA antibodies of infected individuals (23). However, other 10). Unless treated, secondary lesions can take up to several months constituents of the OM (e.g. glycolipids) do not present immune to resolve. The disease then enters in a latent stage without reactivity (23, 29). The lipid composition and poor protein content exhibiting clinical manifestations (5, 6, 10). It remains unclear of OM are key features of TPA, and may contribute to the poor how TPA establishes latency, and which tissues or organs act as immunogenicity of this pathogen (22–24, 26). reservoirs (6). A relapse of infection from these reservoirs can occur TPA is actively motile, although its motile system differs from in 25% of untreated patients within two years after secondary other flagellar bacteria (30). Axis filaments (known as endoflagella) syphilis resolution, and present new secondary-like clinical are found in the periplasmatic space and extend from cell poles manifestations (6, 10). After years or even decades, 15-40% of through the entire cell body length (31). These filaments comprise untreated and latently infected individuals will develop tertiary three core protein (FlaB1, FlaB2, and FlaB3) and an external syphilis. This stage involves serious cardiovascular, neurological, protein, which surround the filament core (FlaA) (28, 31). When bony and visceral affections that may eventually result on death of endoflagella rotate in one direction, the cell body moves in the infected individuals (5, 10). Remarkably, penicillin administration opposite one. This torsion results in a corkscrew-like motion that, prevents syphilis progression to its secondary and tertiary stages, together with the action of metalloprotease and adhesin proteins, and cures the infection. However, treated patients remain allows spirochetes to cross tissues and disseminate through the body susceptible to reinfection, because the previous one fails to (32, 33). produce a protective immune response (11–13). Until recently, TPA had not been grown in vitro (34), and According to WHO data, in 2016, 19.9 million people had bacteria propagation in rabbits was the only strategy to obtain syphilis, and there were 6.3 million of new cases per year (14). The sufficient live and infective organisms for experimental prevalence of congenital syphilis was 0.69% in 2016, with a rate of investigation (35). The lack of the tricarboxylic acid cycle and 473 cases per 100.000 live births (15). Syphilis is considered the microaerophilic requirements make this bacterium totally second cause of stillbirths after malaria (16). A high prevalence of dependent on host cells for the acquisition of purines, syphilis is observed in low-income countries, and it has been rising pyrimidines, and most amino acids (6, 8, 28). Therefore, TPA in high-middle income states during the last decade (5, 14, 17, 18). cannot survive outside the host, and loses its infectious capability Particularly, this increase has been observed in men who have sex within few hours (8, 36). with men (MSM) with multiple sexual partners, as well as sexual Besides humans, rabbits are one of the few mammalians that are networks of heterosexual individuals. In this setting, syphilis is also susceptible to TPA infection, and has become the reference animal associated with higher risk of HIV infection (5, 17–19). model to study syphilis immune protection. After infection, rabbits Importantly, although syphilis can be easily diagnosed, treated develop primary and secondary stage-like clinical signs, and a with an inexpensive antibiotic, and no animal reservoir has been humoral response similar to the one observed in humans (37–39). Frontiers in Immunology 02 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 Intradermal TPA inoculation induces dermal lesions that resemble TPA infection triggers a complex immune response that fails to human chancres, and bacteria can disseminate to distal organs, control the spread of the bacteria and the progression of the disease. mainly secondary lymphoid organs (i.e. spleen) (32, 40). However, In primary syphilis, polymorphonuclear leukocytes (PMNs) are the invasion of the central nervous system is not frequently observed first immune cells to infiltrate the infection site (59) (Figure 1A). and it depends on the utilized TPA strain (41). In addition to These cells may contribute to the initial control of the infection by rabbits, there are other species that are also susceptible to TPA (i.e. secreting anti-microbial peptides (60) and clearing spirochetes by non-human primates, hamsters, guinea pigs, and mice). While only phagocytosis (59, 61). However, the initial control of the infection is non-human primates and rabbits develop clinical signs similar to limited as bacteria dissemination occurs in virtually all cases. humans (40, 42), mice and other rodents may be useful to study Besides PMN, dendritic cells (DCs) (Figure 1A) can also bacteria dissemination (43). phagocyte whole or bacteria-derived fragments, thereby The TPA genome is a circular chromosome of approximately stimulating their maturation and antigen-presentation capacity. 1138 kilobase pairs that contains 1041 predicted ORFs (28). Its Thus, DCs increase the production of pro-inflammatory genome is small compared to other pathogenic bacteria (28). The cytokines, such as IL-12, IL-6 and IL-1B, and upregulate the genome sequence confirms that TPA cannot synthetize de novo expression of CD54, CD83, CD80, CD86, and HLA-DR (62, 63). enzyme cofactors, fatty acids, tricarboxylic pathway enzymes, or Mature DCs migrate to lymph nodes where they present nucleotides, even though it contains 57 ORFs encoding transport treponemal antigens to T cells, inducing antigen-specific T cells proteins and is able to use the glycolytic pathway (28). TPA lacks responses. These T cells can be detected in secondary lymphoid genes encoding superoxide dismutase, catalase, or peroxidase, organs of infected animals three days post-infection and which could explain its susceptibility to oxygen. Additionally, progressively accumulate at the infection site (64–66), coinciding several genes have also been identified that are involved in the with maximal TPA burden (67, 68). Rabbits infected with TPA synthesis of motile proteins and lipoproteins (4, 8, 28). Genome showed a rapid hyperplasia of T cell zones in secondary lymphoid comparison among pathogenic Treponema pallidum subspecies has organs (lymph node and spleen) (38, 66). Although Th1 CD4+ T shown that they are similar in size and structure, and differ less than cells dominate the T cell infiltrate (38, 61, 69)(Figure 1A), cytotoxic 0.2%-0.4% in their genome sequence (44, 45). T cells (CD8+) can also be found in primary lesions, where their role remains unclear. CD8+ T cell may be required for the elimination of TPA reservoir inside non-phagocytic cells in early syphilis lesions 3 Infection course and immune (69–71). In this sense, perforin and granzyme are detected in early response lesions, suggesting cytolytic activity (72). Furthermore, CD8+ T cells, as well as NK cells, could contribute to the secretion of Syphilis is generally transmitted through sexual contact or from interferon-g (IFN-g) (9, 73). Th1 cytokines can promote the mother to child. Spirochetes gain access to the host through migration and activation of macrophages (Figure 1A), whose epidermal micro-abrasions or directly penetrating mucosal number increase rapidly by day 10 after infection at the entry site membranes (46, 47). Once TPA has entered the body, it adheres (68). Activated macrophages phagocytose and destroy spirochetes to epithelial cells and extracellular matrix and locally multiplies with (64, 74) until they are completely cleared from the infection site. an estimate rate of once every 30-33 hours (48, 49). In vitro binding Interestingly, this process can be enhanced by TPA opsonizing studies have shown that laminin and fibronectin are among the antibodies and IFN-g, particularly through the Fc-Fcg receptor anchor molecules involved in these interactions (50–53). Since TPA interaction (73–77). However, other less efficient mechanisms, lacks cytotoxic toxins or other virulence factors (28), tissue such as non-opsonic phagocytosis and active direct invasion, may destruction and chancre lesions are probably caused by the also participate in TPA-macrophage interplay (78). In response to inflammatory response at the entry site (47). The initial immune TPA infection, macrophages polarize to M1 phenotype and secrete response clears bacteria locally, and the primary stage lesions proinflammatory cytokines (i.e. IL-1b, TNF-a, IL-12, and IL-15) resolve spontaneously within 3-6 weeks. Meanwhile spirochetes (77, 79), which can ultimately promote necrosis and ulcer spread throughout the body and the infection rapidly becomes formation typical of primary chancre lesions. systemic, triggering secondary syphilis. Afterward, spirochetes Regarding the humoral response, anti-TPA antibodies can be penetrate deeper tissues and induce the expression of detected as early as 6 days post-infection (80). However, the antibody inflammatory signals, promoting the migration of immune cells response kinetics vary based on the target protein and TPA strain. For to the infected tissues (47). In vitro studies have shown that TPA example, antibodies targeting Tpr Subfamily I and II rise between can induce the expression of the adhesion molecules ICAM-1, days 10 and 45 after infection (81). During primary infection, IgM VCAM-1 and E-selectin in endothelial cells (54–56). In addition, dominates the anti-TPA humoral response, while IgG levels gradually metalloprotease activity plays a major role in the penetration and increase (80, 82) (Figure 1A). Antibodies are not only involved in dissemination of TPA through extracellular matrix and intercellular opsonization processes, they can also block bacteria dissemination junctions (57, 58). In the final stage, infection and the associated and dermal lesions. Furthermore, the complement system is also immune response damage various organs (e.g. heart and brain), involved in limiting treponemal activity, specially working in resulting in debilitating health problems (47). conjunction with antibodies (83–85). TPA accumulates sialic acid Frontiers in Immunology 03 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 A B C FIGURE 1 Immune response to TPA infection. (A) During primary syphilis, a local lesion called chancre appear in the site of infection. After bacteria entry, the microorganisms proliferate and disseminate to distal organs. Bacteria are phagocyted by polymorphonuclear cells (PMN) and dendritic cells that migrate to draining lymph nodes to activate CD4+ Th cells. Th1 T cells migrate to the bacteria proliferation foci and produce Th1 cytokines (IFN-g) for recruiting and activate macrophages. M1 macrophages phagocyte bacteria and produce TNF-a and IL-1b that contribute to chancre development and eventually necrosis. IgM antibodies and complement contribute to bacteria opsonization favoring their removal by macrophages. (B) During secondary syphilis, TPA expands from reservoirs with systematic affectation. A typical rash appears in most cases. Anti-TPA humoral responses are mainly IgGs and plasma cells are visible in the infiltrate of secondary cutaneous lesions. CD8+ T-cells are more represented than CD4 + T-cells in lesion infiltrates. (C) Tertiary syphilis affects deep organs like brain and heart. A Th1/Th2 switch progressively occur and the immune response is down regulated by the presence of regulatory T cells. Plasma cells are well represented. Gummas can be developed in different locations (i.e., liver, skin, brain). Gummas are a granuloma-like structure characterized by a necrotic hyaline nucleus surrounded by immune cells (i.e. plasma cells, macrophages and giant cells) and fibroblasts. on its surface, making it resistant to complement lysis by the histological appearance from primary syphilis. The histology of alternative pathway (86–89). Thus, classical complement activation secondary syphilis is variable but, in general, lymphocytes, pathway is key to bacterial clearance. This data suggest that the macrophages, and plasma cells are commonly present in immune response generated against TPA during primo-infection is a secondary lesions whereas polymorphonuclear and eosinophilic delayed type hypersensitivity (DTH) immune response, where infiltrates are found in a smaller proportion (99–101) (Figure 1B). sensitized T cells play a major role. In fact, it has been Furthermore, CD8+ T cells are highly represented (Figure 1B) (99). hypothesized that syphilis prognosis depends on the balance Contrary to primary syphilis, IgGs dominate the anti-TPA humoral between DTH and humoral responses. Thus, it has been proposed immune response during secondary syphilis (Figure 1B) (82, 102, that a strong DTH immune response may be required to clear the 103). Moreover, whereas IgG1 is the main IgG subclass in primary infection, whereas latency may result from intermediate DTH syphilis, both IgG1 and IgG3 subclass can be equally found in responses. Tertiary disease would be related to a weak DTH and a secondary syphilis (104). strong humoral response (68, 90). Despite this, antibodies may be Finally, tertiary syphilis is characterized by a multiorgan crucial for preventing infection (68, 91). Accordingly, rabbits affection and the development of gummas in different tissues receiving immune serum showed a delay in the appearance of (such as liver and skin). Gummas are granuloma-like structures lesions that were also less severe (92). Further, patients who have characterized by a necrotic nucleus surrounded by macrophages, been previously treated for syphilis remain susceptible to reinfection giant multinucleate cells, lymphocytes, and plasma cells (68) because they do not develop an effective anti-TPA humoral (Figure 1C). Neurosyphilis is characterized by an increase in response (68). the number of CD8+ T cells in blood (105). Moreover, a switch in Both cellular and humoral responses are maintained for months the production of Th1 to Th2 cytokines is observed during the after clearance of primary syphilis lesions (39, 93, 94), and their evolution of the disease. While Th1 cytokines are mainly detected magnitude correlate with the persistence of TPA as latent infection during primary syphilis, Th2 cytokines increase in late states of the (95–98). From this state, relapse of active infection can occur disease (106) (Figure 1C). This correlates with cell depletion in the leading to secondary and tertiary syphilis. Secondary syphilis diffuse cortex, and a follicular hyperplasia in lymph nodes with manifests as inflammatory cutaneous affection that differs in plasma cells accumulation within the interfollicular areas during Frontiers in Immunology 04 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 tertiary syphilis (68). These changes in the immune response during inability to genetically manipulate and cultivate TPA in vitro (until syphilis are closely related with disease progression. recently) (34), and the fragility of its OM, which generated a strong controversy in some studies. The intrinsic properties of the OM (i.e lack of LPS and low density of proteins) makes it can be easily 4 Immunization studies and vaccine damaged by common experimental manipulations (e.g. strategy centrifugation, resuspension, or using low concentration of non- ionic detergent) (22, 26). OM disruption leads to potential exposure Despite public health campaigns and the availability of of lipoproteins that are normally present in the periplasmic space efficacious treatment, syphilis prevalence has increased worldwide and cytoplasmic membrane, and could erroneously pinpoint in the last decade (17), suggesting the need for additional measures proteins as OMPs. Due to these technical limitations, the to control the transmission of the infection. The development of a identification of OMP was often based on prediction of their syphilis vaccine could be a valuable tool. However, after several sequence and structure or by comparison with functional decades of research, an effective vaccine for syphilis remains elusive. orthologs from other species (111). Thus, OMPs can be classified A variety of strategies have been tested, including inactivated in three main groups according to their putative functions: bacteria and subunit recombinant proteins, even though with limited success. Interestingly, Miller et al. demonstrated protection of rabbits against TPA infection following 60 5.1 Transport function immunizations with g-irradiated bacteria for 37 weeks (107). Although this experimental strategy is far from being applicable Since nutrient import and toxics efflux are crucial for bacteria to humans, it serves as a proof of concept that a syphilis vaccine is surveillance, several TPA OMPs have been involved in feasible. In addition, human challenge studies performed in the 50’s transport function. showed that latently infected patients were resistant to reinfection with a heterologous TPA strain (108). Accordingly, Marra and 5.1.1 Tpr protein family colleagues reported that previous syphilis infection may attenuate The Treponema pallidum repeat (Tpr) family of proteins the manifestation of subsequent TPA infection (109). Thus, those include 12 members that are divided into three subfamilies individuals that experimented three or more episodes of syphilis according to their amino acid composition: subfamily I (TprC, D, were more likely to develop latent early syphilis after subsequent F, I), subfamily II (TprE, G, J) and subfamily III (TprA, B, H, K, L). infections. These studies indicate that it will take a long time to Among them, subfamily I and TprK (subfamily III) are the most establish a protective immunity against TPA, further emphasizing widely studied. Regarding Tpr protein location, sequence analysis the low immunogenicity of this pathogen. predicted that TprB, TprC, TprD, TprE, TprG, TprH, TprI, TprJ, Until 2018, one of the main handicaps in the development of a TprK and TprL could be located in the OM (22). Supporting this syphilis vaccine was the inability to grow the bacteria in vitro (34). prediction, a putative cleavable signal peptide was identified in most This technical limitation shifted the focus of most studies towards of the Tprs superfamily members, including TprC, D, F, I, E, G, J, A, recombinant OMPs (110). Although none of these proteins B and TprK proteins (111, 115). provided complete protection against in vivo TPA challenge, The Tpr family is related to the major outer sheath protein promising results were observed; namely, the induction of a (MOSP) of Treponema denticola (115), a surface-exposed protein strong humoral response, less ulcerative lesions, faster recovery of with adhesive and porin function (116–118). Most Tprs include lesions, or inhibition of bacterial dissemination to distal organs. three main domains: 1) a N-terminal domain related to the N- These studies provided meaningful knowledge about OMPs, terminal domain of Treponema denticola MOSP, 2) a central region, immunization regimens, and potential vaccine targets. The and 3) a C-terminal domain related to the C-terminal domain of advancement of bioinformatic tools allowed the identification of Treponema dentiola MOSP, that is lacking in TprF and TprA additional putative OMPs, as well as the prediction of structure proteins. Recently, the presence of short molecular recognition models and B cell epitopes (111, 112). The employment of newly- features (MoRFs) was predicted in most Tprs. These motifs may developed approaches (e.g. genetic engineering), and refined interact with specific proteins and undergo disorder-to-order bioinformatic tools enable to delve further into OMPs knowledge, transition upon binding (111). Sequence analysis has shown that benefiting future vaccine studies (113, 114). the N- and C-terminal domains of the Tpr proteins from subfamily I and II are relatively conserved, whereas central domains are variable in length and sequence (6). Terminal domains present 5 The outer membrane proteins amphipathic regions and flank hydrophilic regions of the central domains. According to Hawley et al., the N- and C-terminal One of the major features that differentiate TPA from other domains from all Tprs adopt a b-stranded structure, while the bacteria is the paucity of surface-exposed OMPs (24–26). It has central regions may be mostly a-helices (111). been calculated that the density of OMP in TPA is approximately Structural analysis of the subfamily I has shown that these 100-fold less than that in E. coli (22). The identification and proteins form a b-barrel trimeric channel. However, unlike classical characterization of TPA OMPs has been challenging due to the porins, which the entire polypeptide forms a b-barrel, Tpr Frontiers in Immunology 05 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 subfamily I possesses bipartite topology. The C-terminal domain controversies on the role of anti-TprK antibodies, and TprK forms a b-barrel structure and is surface-exposed, while the N- protective efficacy as immunogen and its location. Centurion-Lara terminal and central domains anchor the b-barrel into the and colleagues described that TprK is located on the OM, and peptidoglycan sacculus of the periplasmatic region (119, 120). TprK-specific antibodies had opsonization capacity (115). Accordingly, while the expression of TprC/D in E. coli is surface- Interestingly, rabbits immunized with this protein did not develop exposed, TprF lacks the C-terminal domain, and therefore, it is ulcerative lesions. These lesions healed faster than those observed in entirely periplasmatic (22). The immunodetection of Tpr proteins unvaccinated control animals and contained fewer spirochetes, by electron microscopy using purified rabbit anti-Tpr I when analyzed by darkfield microscopy (115). Additionally, immunoglobulins, confirmed the presence of Tpr proteins in OM Morgan et al. identified the N-terminal region of TprK (37-273 and periplasmic spaces, which could be due to the bipartite amino acids) and, to a lesser extent, the C-terminal portion (349- structure previously described or because the anti-Tpr I antibody 478 amino acids), as the parts of the protein that induced the recognized proteins with distinct location (121). More recently, previously-described protective immune response (134). Hawley et al., confirmed that the bipartite membrane topology Conversely, Hazlett and colleagues described that TprK was identified in subfamily I is common to all Tprs with three full located in the periplasmic space, anti-TprK antibodies lacked domains (111). Based on sequence and structure analysis, it has opsonization activity, and immunization with recombinant TprK been shown that the Tpr family may be involved in the import of did not induce a protective immune response nor TprK sequence small soluble molecules. In this sense, the integration of some Tpr variation (135). Further investigation would be needed to clarified proteins in liposomes increased their permeability (119, 120). these discrepancies. Therefore, changes in protein expression and/or variation of their Regarding the immunity elicited against other Tprs members, channel-forming b-barrel region could be used to adapt TPA anti-TprI antibodies were detected in 98% of syphilis-affected nutritional requirements to the environment. Accordingly, the individuals. These antibodies mainly targeted the conserved N- expression of Tpr proteins can vary among strains, and in the terminal region. The lack of reactivity was associated with early same strain overtime (122), which may explain why the anti-Tpr infection (121). Remarkably, experiments performed in rabbits humoral response differ among TPA isolates (81). This observation showed that TprI immunization did not protect rabbits from suggests the existence of mechanisms that can regulate the infection (136). However, cutaneous lesions did not ulcerate and expression of these genes. Thus, a hypervariable homopolymeric healed faster than those generated in unvaccinated animals. This guanosine (poly-G) tract and a cAMP receptor protein (CRP) protein elicited strong humoral and cellular responses mainly binding motif have been identified upstream of the transcription targeting the conserved N-terminal region of the protein during start site of the Tpr subfamily II and III (123–125). In fact, Tp0262 TPA infection (81, 136). Not surprisingly, immunization with the (a TPA CRP homologue) can bind to these promoters and regulate conserved N-terminal region of TrpF, which is also common to all the surface expression of these proteins during infection (123). As a subfamily I members, did not protect rabbits from TPA infection result, the specificity of the humoral response elicited against Tpr after challenge. However, TprF vaccination attenuated lesion proteins differ among TPA isolates (81). development, prevented ulceration, and reduced bacterial burden Of the Tpr superfamily, TprK is one of the best characterized in skin lesions (136). In regards to TprC and TprD, epitope proteins. It has been previously showed that recombinant TprK is a mapping has shown that the humoral response detected in rabbit monomeric porin by negative-staining electron microscopy (126). sera is directed against exposed loops (112). Moreover, the N- and Anti-TprK antibodies can be detected early during infection (81). C- terminal regions contain the most reactive epitopes. Surprisingly, However, TprK exhibits sequence variation that contributes to Anand et al. showed that sera from infected individuals do not immune escape. Thus, several alleles have been identified among recognize TprC, while TPA-infected rabbits develop antibodies with TPA strains (127, 128). Moreover, this protein shows seven variable opsonization activity (119). One explanation could be the low d o m a i n s w h o s e s e q u e n c e c h a n g e s by n o n - r e c i p r o c a l expression of TprC and the different magnitude of the humoral recombination, mainly with regions downstream of the TprD response found among various syphilis strains and subspecies. gene (127, 128), a mechanism that is enhanced under immune pressure (129, 130). The existence of this mechanism could explain why TprK variability was higher in TPA isolates from patients 5.1.2 Tp0515 affected with secondary syphilis than those with primary syphilis Tp0515 is a structural ortholog of LptD protein, which is part of (128). However, TprK genetic variation can also occur in the a multiprotein complex involved in LPS trafficking to the OM in absence of immune pressure (131). Interestingly, it has been Gram-negative bacteria (22). Thus, since LptD is embedded in the reported that humoral response in vivo targets variable regions, OM, Tp0515 is inferred to be an OMP. However, sequence analysis while cellular response recognizes conserved TprK epitopes (132, of TPA genome failed to identify LPS biosynthetic pathway (28). 133). Moreover, animal challenges using TPA with TprK Notably, orthologous proteins for several components of the LPS homologous to the one used in immunization regimen show transport pathway have been found in TPA (111), suggesting that better protection than those animals challenged with a strain even without LPS, these proteins could be involved in translocation expressing heterologous TprK (132). Despite that, there are of other cellular products to the OM, as glycolipids (22). Tp0515 Frontiers in Immunology 06 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 structural modelling found that this protein is a 26-stranded b- uptake of long-chain fatty acids, such as the FadL (Long-chain barrel embedded in OM with exposed extracellular loops (111). fatty acid transport protein). TPA is unable to synthesize long-chain Moreover, Hawley et al. identified B cells epitopes inside these fatty acids (28). However, since TPA OM is more permeable to fatty extracellular loops using bioinformatic analysis (111). However, the acids than other Gram-negative bacteria (142), membrane diffusion immunogenicity and capacity of this protein to induce protective may be one of the mechanisms that this microorganism uses to immune response still need to be empirically demonstrated. uptake these molecules. Nevertheless, five FadL orthologs (Tp0548, Tp0856, Tp0858, Tp0859, and Tp0865) have been identified in TPA, suggesting that passive diffusion of fatty acids is not the only 5.1.3 Tp0126 and other OmpW/AlkL orthologs mechanism (111). Structural modelling of these orthologs predicted Four proteins that are orthologs of OmpW (Tp0126 and that all TPA FadL-like proteins form a 14-stranded b-barrel with N- Tp0733) and OprG (Tp0479 and Tp0698) have been identified in terminal in the barrel lumen (111). Moreover, all five proteins TPA (111). Both E. coli OmpW and Pseudomonas aeruginosa OprG contain one or more predicted B cell epitopes (111). Interestingly, proteins are members of the OmpW/AlkL family of proteins, which Delgado et al. recently described the presence of IgG antibodies and have been suggested to be involved in bacterial adaption to IgG + B cells in TPA-infected rabbits that recognized the environment stress and nutrient acquisition (137, 138). OmpW extracellular loops 2 and 4 from Tp0856 and Tp0858 (143), and OprG have an eight-stranded b-barrel architecture and form a indicating that these proteins could be useful in vaccine design. peculiar hydrophobic channel, which allows the transport of hydrophobic molecules directly to the membrane, bypassing the hydrophilic periplasmatic space (111, 139). Thus, Tp0126 could be 5.1.5 TolC-like proteins involved in fatty acid transport (unable to be synthesized by TPA TolC is an OM protein from E. coli, which is part of an efflux alone) (140). Interestingly, this protein is highly conserved among pump complex involved in the removal of toxic substances (144). To TPA strains. Its transcription is regulated by the presence of date, four TolC orthologs (Tp0966, Tp0967, Tp0968, and Tp0969) guanidine homopolymers of various length located upstream of have been described in TPA (145). Structural modelling of these its promoter, which is consistent with phase variation (141). proteins identified that they have a TolC-like topology based on four Remarkably, Tp0126 may show low immunogenicity. Anti- b-strands with two large extracellular loops and six a-helices (111). Tp0126 antibody levels in serum samples from patients with Furthermore, Hawley et al. predicted that all four TolC-like TPA latent syphilis were lower than those targeting Tp0574, a highly proteins, particularly Tp0969, enclose surface-exposed B cells epitopes expressed and immunogenic TPA protein (141). In addition, anti- (111). Thus, these predicted OMPs could be targeted by the immune Tp0126 antibodies were detected in Tp0126-immunized rabbits system. However, further in vivo analysis of their immunogenicity is only after the third boost, highlighting the potentially low required to determine their potential in vaccine development. immunogenicity of this protein (141). Interestingly, these antibodies targeted Tp0126-external loops and were able of opsonize TPA. However, immunization with Tp0126 showed 5.1.6 Treponema rare outer membrane proteins poor protective capacity, if any, in rabbits. In fact, treponemes (TROMPs) isolates from lesions of immunized and challenged rabbits showed TROMP family includes three proteins: TROMP-1 (31 kDa), lower transcription levels of Tp0126, which was associated with a TROMP-2 (28 kDa) and TROMP-3 (65 kDa) (146). TROMP-1 longer polyG region (≥ 9 G’s), when compared to treponemes (also called TroA or Tp0163) was firstly described as a surface- obtained from the challenge inoculum (141). These results suggest exposed protein with porin-like properties (147). However, it was that the expression of Tp0126 may be reduced during infection, later identified to be a periplasmatic metalloprotein anchored to the which could explain the absence of protection observed in cytoplasmatic membrane (148–150). TROMP-2 (also called immunization and challenge experiments. Tp0663) was localized on the OM when it was expressed as Like Tp0126, other members of the TPA OmpW/AlkL ortholog recombinant protein in E. coli. However, the isolation of group (Tp0733, Tp0479, and Tp0698) (111) are predicted to form a TROMP-2 from E. coli OM showed low porin insertional events, membrane-channel and being involved in transport of small molecules. casting doubts on its porin function (29). Interestingly, both Hawley et al. predicted multiple surface-exposed B cell epitopes in their TROMP-1 and TROMP-2 were targeted by antibodies present in extracellular loops, although the density of these epitopes differs from sera from infected rabbits and humans (146). In fact, TROMP-2 one to another protein (111). Future experimental studies with these could be a potential candidate for serodiagnosis of all syphilis stages OMPs are envisaged to confirm their in silico predictions, and to (151). TPA challenge experiments performed in Tp0663- inform about potential immunogenicity and capacity of inducing immunized rabbits showed partial protection, with high titers of protective immune responses. anti-Tp0663 antibodies. The authors observed attenuated lesions with an increase cellular infiltration. Importantly, no bacteria dissemination to distal organs was detected in immunized 5.1.4 FadL-like proteins animals (152). Of note, TROMP-3 is expressed at a lower Due to the low OM permeability of Gram-negative bacteria, concentration than the other two members of this family, and its these bacteria have an OM protein machinery involved in the function and structure remain unknown (29). Frontiers in Immunology 07 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 5.2 Adhesion function on the OM (155). This result was in line with the fact that TDE2318, a fibronectin-binding protein with high homology to Tp0155, was found Adhesion to cells and extracellular matrix is crucial to the in the surface of T. denticola according to immunofluorescence establishment of infection and the dissemination of TPA. microscopy data (156). However, immunofluorescence studies using agarose microencapsulated treponemes showed that Tp0155 is not 5.2.1 Tp0136 exposed on the OM of TPA (157). According to this observation, Tp0136 has been identified as a fibronectin-binding TPA OMP Tp0155-immunized rabbits, which elicited high antigen-specific that binds more efficiently to cellular than plasma fibronectin (50, antibody titers, were not protected against an intradermal challenge 51). This selective binding involves different domains of this with live treponemes (157). Thus, further investigation will be needed to protein. Thus, the conserved N-terminal region is mainly determine whether Tp0155 would be a bona fide OMP or not. responsible for binding to plasma fibronectin. Residues in the C- Remarkably, studies performed with human sera showed that terminal end, and also in the central portion of the protein, approximately 70% of syphilis patients did not show antibodies participate in binding to the cellular form of fibronectin (51). against Tp0155, indicating that the expression of this protein or its Interestingly, Tp0136 shows sequence variability among TPA immunogenicity may be low in natural infection (157, 158). strains, and its transcription is regulated during infection (50, 51). The interaction of Tp0136 with fibronectin expressed on cell surface 5.2.3 Tp0435 can promote bacteria attachment to tissues, facilitating body Tp0435 is a highly immunogenic 14 kDa lipoprotein with dissemination and favoring the colonization of distal endothelial adhesin function, also known as Tp17. To identify the location of tissues, central nervous system, or placenta (153). Tp0435 in TPA, Chan and colleagues performed a study that In addition to its adhesion function, Tp0136 may also play an analyzed the Tp0435 surface expression in TPA and Tp0435- important role in chancre healing by promoting fibroblast and transformed B. burgdorferi cells (159). The authors concluded microvascular endothelial cell migration, and the activation and that Tp0435 may be post-translationally modified, generating aggregation of platelets (154). several variants that can be differentially located between the Immunization and challenge experiments performed in rabbits surface and the periplasmic space. Contrarily, Cox et al., did not using recombinant Tp0136 produced in E. coli showed a delay in find Tp0435 surface-exposure evidences (160). Structurally, Tp0435 lesion ulceration but not in their development, indicating no encompasses eight-stranded anti-parallel b-barrel with a basin-like protection against infection (50). Remarkably, sera obtained from domain located at one end (161). According to Chan et al., Tp0435 immunized animals reduced the attachment of the bacteria to favors the attachment of the spirochetes to mammalian cell lines fibronectin, although at lower levels than sera obtained from (159). Interestingly, human sera from infected patients are reactive unvaccinated and infected rabbits (50). Interestingly, Xu et al. against Tp0435 (158), and immunization of rabbits with a Tp0435- showed that rabbits immunized with Tp0136 elicited high levels of expressing B. burgdorferi strain induced a strong immune response antigen-specific antibodies, attenuated lesion development with against Tp0435. However, this immune response failed to protect increased cellular infiltration, and limited treponemal dissemination from infection or lesion development after experimental challenge to distant organs (152). Although there are discrepancies between (162). Thus, further studies may be needed to determine the both studies that could be explained by differences in adjuvant, location of Tp0435 and its potential as vaccine candidate. immunization schedule, and the amount of live inoculated bacteria, these results suggest that antibodies developed against Tp0136 might 5.2.4 Tp0483 be protective. However, the fact that antibodies elicited during Tp0483 can bind both extracellular matrix and soluble experimental infection blocked more efficiently TPA binding to fibronectins (155). The C-terminal portion of Tp0483 (179-374 fibronectin than vaccine-induced antibodies elicited against amino acids) also showed reactivity against laminin (163). Little is Tp0136, suggest that, during vaccine design, other specificities known about the structure of Tp0483. However, a peptide should be also targeted to more efficiently block bacteria from encompassing 316-333 amino acids inhibited binding of Tp0483 binding to the extracellular matrix (51). to fibronectin, suggesting that this peptide, or a near region of Tp0483, should be responsible for binding to adhesive glycoprotein 5.2.2 Tp0155 (164). Similar to Tp0155, Cameron et al. indirectly suggested that It has been described that Tp0155 preferably binds to Tp0483 was an OMP (155). Contrarily, Tomson and colleagues did fibronectin matrix and, therefore, it might be involved in the not find evidences of the Tp0483 OM location. Even though rabbits dissemination of TPA (155). Tp0155 shows two lysin motifs immunized with this protein elicited high antibody titers, the (LysM) domains in its N-terminal portion that can play a major authors did not detect protection after challenge (157). role in binding to fibronectin and peptidoglycans, and a M23 peptidase domain in the C-terminal region, which might show 5.2.5 Tp0750 peptidoglycan lytic activities (156). Tp0750 binds to fibrinogen and the fibrinolytic receptor complex There is some controversy regarding Tp0155 surface location. protein Annexin A2. Tp0750 shows serine metalloprotease activity, Inhibition assays showed that recombinant Tp0155 reduced live TPA and it is able to degrade fibrinogen and fibronectin, inhibiting the binding to fibronectin-coated slides, suggesting that Tp0155 is expressed coagulation cascade, but cannot degrade fibrin clots (57). Structurally, Frontiers in Immunology 08 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 Tp0750 shows metal ion-dependent adhesion site (MIDAS)- that Tp0954-transformed B. burgdorferi B314 bacteria, which is containing von Willebrand Factor type A domains (region V29- known to be a poorly adherent strain, gained binding to T147). The MIDAS domains bind to primary calcium. This protein, mammalian epithelial cell lines, including a human placental cell as well as Tp0751 (see section 5.2.6), are co-transcribed and might line [i.e. BeWo (CCL-98)] (169). Unlike other TPA adhesins, coexist as heterodimeric complex. However, unlike Tp0751, Tp0750 Tp0954 can mediate adhesion and bacteria dissemination through shows a limited laminin binding capacity (57). Sequence analyses of binding to glycosaminoglycans present in human placenta, such as both proteins have shown that Tp0750 is highly conserved among dermatan sulfate, heparin, and heparan sulfate, which are pathogenic treponemes species, whereas Tp0751 is less conserved, components of the extracellular matrix (169). Dermatan sulfate is except for the most invasive treponemes species. Interestingly, Tp0750 associated with fetal blood vessels and syncytial surface, whereas and Tp0751 orthologs from the less invasive treponemes species do heparan sulfate is located in trophoblast layers (170–173). Thus, it is not bind or degrade host proteins, which strengthen the idea that both possible that Tp0954 might facilitate congenital infection through adhesion function as well as degradative capabilities are important to binding to placental glycosaminoglycans. syphilis progression (165). 5.2.6 Tp0751 5.3 Other functions While there is a degree of agreement in regards to the structure of Tp0751, there are discrepancies regarding its function, location, A number of proteins in the OM of TPA are not related with and the relevance of the immune response elicited against this neither transport or adhesion function. protein. From the structural point of view, Tp0751 presents a bipartite topology with a disorganized N-terminal region that is 5.3.1 Tp0326 joined through an a-helix domain to a C-terminal portion arranged Tp0326 (or Tp92) shows homology with BamA orthologs from in eight anti-parallel b-sheets (52, 166). other Gram-negative bacteria. BamA-like proteins are characterized It has been previously described that Tp0751 binds to different by a bipartite structure with a b-barrel domain located in the C- forms of laminin in a dose-dependent manner, and can work as terminal portion, and at least one polypeptide-transport-associated vascular adhesin, promoting bacterial dissemination (53, 167). (POTRA) domain in the N-terminal end. Particularly, Tp0326 was Additionally, Tp0751 showed metalloprotease activity, and can predicted to contain five N-terminal POTRA and one C-terminal b- degrade fibrinogen and laminin, promoting clot dissolution, and barrel domains (174, 175). According to Desrosiers et al., the N- favoring bacterial dissemination (58). However, Luthra and terminal POTRA domains are periplasmic, whereas the C-terminal b- colleagues showed that Tp0751 is located in the periplasmic space barrel is embedded in the OM forming a pore (175).The N-terminal and its main role is binding to small molecules along the barrel rim, end can bind to multiple periplasmatic proteins through POTRA such as hemes (166). domains, establishing a protein complex that is crucial for membrane While Cameron and colleagues described that Tp0751 is synthesis. As other BamA-like proteins, Tp0326 is suggested to be part recognized by antibodies in patient and infected rabbit sera (163), of a protein machinery involved in the translocation and insertion of and these antibodies can opsonize the bacterium, Luthra et al. proteins from the periplasmatic space to the TPA OM (175). observed opposite results. In their study, Tp0751 induced a weak Interestingly, human antibodies are mainly directed against the antibody response in infected human and challenged rabbits, and Tp0326 periplasmic segment, while rabbit antibodies target both N- anti-Tp0751 antibodies lack opsonization capacity, probably due to terminal and C-terminal regions (175). Indeed, Luthra et al. found that the low levels of Tp0751 expression on the OM (166). POTRA domains are immunodominant over the b-barrel domains in Immunization and challenge experiment in rabbits also provided both rabbits and humans (174). In addition, these authors described contradictory results. Lithgow and colleagues showed that Tp0751- that the Tp0326 central pore exposes eight loops to the extracellular immunized animals showed attenuated lesions with low bacteria space. Among them, loop 4 is specially targeted by antibodies with burden. Although immunization did not prevent infection, it opsonizing capabilities from infected rabbits and humans with successfully inhibited bacteria dissemination (168). Conversely, secondary syphilis (174). By contrast, Tomson and colleges failed to Luthra et al. showed no protection against local or disseminated detect the expression of Tp0326 on the OM of treponemes infection following intradermal TPA challenge in Tp0751- encapsulated within agarose gel microdroplets using indirect immunized animals (166). Therefore, further research will be immunofluorescence (157). In addition, they did not observe needed to clarify these discrepancies. protection in Tp0326-immunized animals after TPA challenge. Conversely, Cameron et al. showed that, although all Tp0326- 5.2.7 Tp0954 immunized rabbits were infected after challenge, there was some Tp0954 has been recently described as a surface lipoprotein, and degree of protection in the vaccinated animals that had high antigen- may have a major role in congenital syphilis (169). According to its specific antibody titers (176). structure, this protein is predicted to have a tetratricopeptide repeat (TPR) structural motif with tandem a-helices. Moreover, Tp0954 5.3.2 Tp0257 sequence is conserved among several TPA strains (e.g. Nichols, Tp0257 (or GpD) was identified using a treponema genetic Chicago, Mexico A and Amoy) (169). Primus et al. also described expression library and opsonized rabbit immune sera. This protein Frontiers in Immunology 09 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 presents homology with H. influenzae GplQ protein, a and angiogenesis characterize the lesions of this stage (189). Thus, glycerolphosphodiester phosphodiesterase protein, which is Tp1038 is an interesting target for vaccine development since it is partially expressed on the surface (177). Thus, it is possible that engaged in all syphilis stages. Tp0257 may be an OM protein. Accordingly, Cameron et al. reported indirect evidences of its surface location by immunoblot analysis of TPA lysates. The authors observed positive staining in 6 Immune evasion strategies the preparation that included the OM, but not in the OM-removed TPA lysate fraction (178). Moreover, GpD was isolated from OM While an immune response is triggered against TPA, it does not preparations (179). Interestingly, rabbits immunized with protect against bacterial dissemination and disease progression to recombinant Tp0257 exhibited a reduction in lesions the second or third stage. In addition, reinfections are quite development and bacterial proliferation after TPA challenge common, with an incidence around 5-22% (190–192), indicating (178). Contrarily, Shevchenko et al., showed that Tp0257 could be that TPA developed some mechanisms to evade the immune a periplasmic protein, since they did not identify surface-exposed system (Figure 2). evidences by multiple assays, and Tp0257-immunization failed to protect rabbits from TPA challenge (180). More recently, a DNA vaccine encoding a Tp0257-IL-2 fusion protein showed a decrease 6.1 Phase variation or switch on/off in ulcerative lesions, as well as, in the number of lesions containing TPA (181). The inclusion of IL-2 in the vaccine enhanced anti-GpD TPA can modulate the expression of several OMPs during its humoral responses and the levels of IFN-g. Remarkably, rabbits life cycle (122). However, the mechanisms controlling the immunized with a Tp0257-IL2 DNA prime and intranasal boost expression of those proteins are not well characterized. In the with recombinant protein adjuvanted with CpG-ODN induced case of subfamilies I and II of Tpr proteins, guanosine mucosal and systemic immunity, and showed a faster lesion homopolymer (polyG) repeats have been identified upstream of recover after TPA inoculation (182). the transcriptional start site. The length of the polyG sequences Interestingly, Tp0257 is conserved among TPA strains, as well differ among treponemal isolates and modulate the expression of as in Treponema pallidum endemicum and Treponema pallidum Tpr proteins at the transcriptional level (124). PolyG signals have pertenue (183). These results make this protein an attractive also been found in other OMPs, such as the Tp0126 protein (140). immunogen candidate for vaccine design. Phase variation based on hypervariable homopolymeric repeats have been previously reported in several other bacteria such as 5.3.3 Tp1038 Neisseria meningitidis, Helicobacter pylori, or Haemophilus Tp1038, also known as TpF1 or antigen 4D, is a bacterioferritin influenzae, and it is crucial for the survival of pathogens facing with ferroxidase activity playing a major role in iron uptake (184). host immunity (193–195). Moreover, other transcriptional Radolf et al. showed that Tp1038 is surface-exposed since sera from regulatory mechanisms have been identified, such as the G4FS Tp1038-immunized rabbits was reactive to immobilized TPA in the cis-acting DNA elements, which form guanine-quadruplexes and presence of complement (185). induce recombination and gene conversion of Tp0136 gene (196). Tp1038 is able to induce diverse immune responses. For Using these mechanisms, TPA can modify its OMP repertoire to example, this protein might be involved in the proinflammatory adapt to the environment conditions and evade the immune response during primary syphilis, since it can activate the response. Consequently, the specificity of the humoral response inflammasome complex in monocytes, and thereby, induces the against Tpr proteins may vary between animals or humans that are release of IL-1b, IL-6, and TNF-a (186, 187), which could result in infected with the same or different TPA strain, thereby supporting tissue damage associated with this stage. However, Babolin et al. the existence of a variable repertoire of OMPs (81). described that Tp1038 may also drive a T regulatory response (186). CD4+ CD25+ Foxp3+ T cells from patients with secondary syphilis produce TGF-b under Tp1038 stimulation, and Tp1038-stimulated 6.2 Functional redundancy monocytes produce IL-10 and TGF-b, two cytokines linked to Treg cell differentiation (186). Therefore, TpF1 might be a virulence Several OMPs present similar functions in transport or cell factor involves in the persistence of TPA infection through the adhesion. Functional redundancy may be advantageous since downregulation of the immune response (186). Regarding the the replacement of one protein by a functional homologous can humoral response, anti-Tp1038 antibodies are detectable in all guarantee that phase variation and post-translational modifications syphilis stages (186, 188). Finally, Pozzobon and colleagues will not adversely affect cell homeostasis (22). As an example, identified that patients with tertiary syphilis have Tp1038-specific Salmonella has been reported to have two distinct flagellin genes, T cells, which stimulate monocyte and human umbilical vein fliC and fliB, with the same function but different structure. By endothelial cells to produce tissue factor, IL-8, and CCL-20 (189). switching their expression from one flagellin protein to the other, These cytokines are involved in angiogenesis, thus Tp1038 may also the immune system becomes ineffective against assembled be implicated in tertiary syphilis, in which vascular inflammation flagella (197). Frontiers in Immunology 10 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 A B G F C E D FIGURE 2 Immune evasions mechanisms use by TPA. (A) During its adaptation to environment changes, including the immune system pressure, TPA modulates the expression of certain genes by a switch on/off mechanism. (B) Linked with the prior mechanism; function redundancy ensure that bacterial homeostasis is not compromised by changing the expression of proteins with vital functions. (C) OM of TPA is characterized by a paucity of protein compare with the inner membrane (IM), the lack of LPS and the presence of anti-inflammatory phospholipids (i.e., phosphatidylserine and phosphatidylcholine). (D) Some proteins experience post-translational modifications that result in variants with different location preference. (E) Proteins can accumulate sequence variations by point mutation, gene conversion or recombination. (F) TPA can persist in immune privileged organs, such as central nervous system or nerve fibers, and prolong their survival by remaining hidden to the immune system. (G) TPA may be coated by host serum proteins, reducing its immunogenicity. 6.3 Outer membrane composition phospholipids (e.g. phosphatidylserine and phosphatidylcholine), these characteristics may also hamper the development of an TPA is characterized by a low number of OMPs, which may efficient immune response (200, 201). encompass an advantage by limiting the number of exposed antigens to the immune system. Moreover, the spacing distribution of these proteins may promote that antibodies bind to them just with one arm, 6.4 Post-translational modifications reducing the avidity of the interaction. This mechanism has been previously described in Human Immunodeficiency Virus (HIV) Post-translational protein modifications play a crucial role in infection (198), which also presents low density of Envelope different processes of prokaryote cells, including persistence and glycoprotein on the virion surface. In HIV, polyreactivity of virulence (202). These modifications have been found in the TPA neutralizing antibodies may increase binding avidity by proteome. For example, there are several variants of Tp0435 that heteroligation with membrane components (198). However, the may coexist and were generated by palmitoylation (159). development of these antibodies could be limited by tolerance Palmitoylation consists in the addition of lipid chains to terminal mechanisms (199). Whether this phenomenon is also related to cysteine residues. This process has been previously reported in TPA immunity requires further investigation, but it could partially other bacteria and contributes to their infectivity and recognition of explain the low antibody efficacy at controlling infection. Since the the immune system (203). Tp0435 variants may be located on the OM lacks LPS (27, 28), and it is enriched in anti-inflammatory surface or into periplasmatic spaces, being the latter one the most Frontiers in Immunology 11 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 abundant (159), which could explain why there is no consensus 7 Discussion about the Tp0435 location. In fact, this protein has been the first description of post-translationally modified protein variants in Syphilis is a multistage disease caused by TPA, for which an TPA. However, other OMPs could be also susceptible to post- effective vaccine remains elusive. Despite several decades of research, translational modifications. the impossibility to genetically manipulate and grow TPA in vitro, the paucity of OMPs, the OM fragility, and the lack of an appropriate animal model that recapitulates all stages of human syphilis have 6.5 Sequence variation hampered the field progress. In addition, many controversies remain unresolved with respect to OMPs identification, location, function, the Genetic variation of several OMPs has been described in TPA, potential of these proteins to induce protective immune responses, or including laboratory-adapted or primary isolated strains (127, 129, the characteristics of these responses. Researchers have hypothesized 204). Some of these variations have been reported in the Tpr family, that a DTH immune response is essential to control and clear TPA including TprC, TprD, TprG and TprK proteins (127, 204, 205). infection, but it is also responsible for the development of tissue Interestingly, TprK accumulates sequence variation in variable damage, including an ulcerative lesion that forms chancres. As DTH is regions (particularly in V6) by non-reciprocal recombination with primarily mediated by Th1 CD4+ T cells, the presentation of antigens silent cassettes, which is enhanced under immune pressure (129, by DCs, macrophages, or other professional antigen-presenting cells is 130). Additionally, several tprk alleles have been identified in crucial. Of note, this process is independent of antigen location, and different TPA strains (127), which contributes to increase the both intracellular and surface-exposed antigens can be indistinctly variability of this protein. Besides Tpr proteins, others OMPs, processed and presented to T cells. Therefore, both antigens might such as Tp0326 (BamA), also show genetic variants (204, 205). generate protective Th1 CD4+ T cell responses. Thus, rabbits This protein has accumulated mutations on the extracellular immunized with intracellular endoflagellar antigens that have been hydrophilic loops predicted to contain B cell epitopes (204). directly isolated from TPA showed faster development of lesions Sequence variation was also confirmed in FadL orthologs proteins (215). However, bacteria were not detected in these lesions, which may ranging from fully conserved Tp0856 to deeply variable Tp0548, as be explained by an accelerated memory DTH response. In contrast, well as in Tp0136, Tp0868, Tp0966 and Tp0967 (206–208). rabbits immunized with a plasmid coding for FlaB3 showed attenuated lesion development that was associated with an enhanced cellular infiltration, and an inhibition of bacteria dissemination to 6.6 Colonization of immune privileged distal organs (216). In line with this observation, rabbits immunized organs with TprF, a periplasmic protein, displayed attenuated skin lesions with reduced bacteria burden (136). The reasons behind these TPA can colonize distal immune privileged organs, such as the different outcomes are not completely understood, but differences in central nervous system, placenta, or eyes. In these tissues, the action vaccine formulation and delivery routes, the magnitude of vaccine- of the immune system may be limited, contributing to the induced T cell responses, and the relative representation of antigens persistence of this pathogen (209–211). In fact, studies performed (flagellin and TprF) within the whole TPA proteome could be using the rabbit model showed that TPA can be detected in nerves involved. Similar results to TprF immunization were also observed early after infection (68). when OMPs were used as antigens. Therefore, DTH could be a protective immune response that modifies lesion development and clears bacteria from infection sites. However, none of TPA proteins 6.7 Host proteins coat tested as immunogens to date protected against infection, indicating that additional immune responses are likely to be required. Similarly to other bacteria; for example, some streptococcal Despite some controversy (68), strong evidence supports a protective species (212), TPA immune evasion may be associated with host role of antibodies, facilitating bacteria opsonization and killing by innate proteins forming a surface coat, also known as antigenic disguise. immune cells, as well as blocking their interaction with components of Alderete and Baseman described that both rabbit and human the extracellular matrix, such as laminin and fibronectin. These functions albumins, as well as other host proteins, could be adsorbed on the strictly depend on the exposure of antigens on the OM and, in some surface of TPA (213). Egesten et al. showed that Group G cases (e.g. bacteria phagocytosis), the interaction with Fcg receptors streptococci express protein G on their surface, and it is able to expressed on the surface of innate immune cells, such as macrophages. bind human albumin, and therefore, inhibit anti-microbial action of IgG-FcgR interaction may also improve antigen presentation by antigen- CXCL9 (214). Accordingly, it has been proposed that the capacity of presenting cells and the generation of Th1 cellular responses (217). Tp0483 to bind soluble fibronectin could be related to the evasion of Therefore, to develop a protective TPA vaccine, it is crucial to define the humoral response (164). However, Cox and colleagues did not which types of antigens can induce synergic anti-TPA immune detect serum proteins on the surface of TPA by immunoelectron responses. It is also essential to considerer immune evasion strategies microscopy (26), stating that the low density of OMP might be the developed by TPA, such as functional redundancy and phase variation. main immune evasion mechanism of TPA. In this sense, targeting several proteins with two or more functions, such Frontiers in Immunology 12 frontiersin.org Ávila-Nieto et al. 10.3389/fimmu.2023.1126170 as transport and adhesion, could increase vaccine efficacy, as it was Innovació en Salut (Generalitat de Catalunya) (projects SLD0015 previously observed (218, 219). and SLD0016), the Carlos III Health Institute (PI17/01518 and The ideal syphilis vaccine should provide protection against TPA PI18/01332). JB is supported by the Health Department of the infection. However, even if the vaccine is unable to completely Catalan Government (Generalitat de Catalunya). CÁ -N was prevent infection, it could still worth considering as a means of supported by a predoctoral grant from Generalitat de Catalunya limiting bacteria dissemination and tissue invasiveness, blocking the and Fons Social Europeu (2020 FI_B_0742). This study was also establishment of latency, or preventing the disease from progressing supported by CIBER - Consorcio Centro de Investigació n to secondary and tertiary stages (113). Additionally, a vaccine that Biomé d ica en Red (CB 2021), Carlos III Health Institute, reduces bacterial persistence or prevents TPA transplacental invasion Ministerio de Ciencia e Innovació n and Unió n Europea – might also reduce the number of congenital syphilis in endemic areas, NextGenerationEU.Funders had no role in study design, data where the number of non-diagnosed infected individuals might be analysis, decision to publish, or manuscript preparation. high, and the access to health care may be limited. Moreover, a syphilis vaccine has to be effective against possible reinfections with similar or different TPA strains or isolates, indicating the importance Acknowledgments of using conserved antigens. Recently, there has been evidence that TPA can be cultured in vitro and that genetic manipulation of this Figure 1 and Figure 2 were created with BioRender.com. pathogen can be accomplished (114, 220, 221). These scientific advances are likely to open the gateway to new research lines that can shed light on the current controversies. Conflict of interest The authors declare that the research was conducted in the Author contributions absence of any commercial or financial relationships that could be construed as a potential conflict of interest. CÁ -N, OM, BC, JB and JC designed the scientific content. CÁ - N, NP-L and JC drafted the manuscript and figures. OM, BC and JB edited the manuscript and participate in content discussion. All Publisher’s note authors have read and approved the content of the manuscript. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated Funding organizations, or those of the publisher, the editors and the reviewers. 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