In vitro culture method for treponema pallidum

Abstract

The strong host dependence of Treponema pallidum makes long-term in vitro culture difficult and greatly hinders efforts to elucidate infection mechanisms, to develop therapeutics, and to research vaccines. The purpose of the present invention is to provide a method for continuously and efficiently culturing Treponema pallidum in vitro. The present invention provides an in vitro culture method including a step for culturing Treponema pallidum in a medium containing an elastic fiber or a component thereof. This method makes it possible not only to solve the above-mentioned problem, but also to establish a platform technology for infection research and therapeutic development.

Description

In vitro culture method for Treponema pallidum. 【0001】 The present invention generally relates to the fields of microbiology and cell biology. More specifically, the present invention relates to a method for in vitro culture of Treponema pallidum, a culture composition containing Treponema pallidum, related kits, and a method for screening drugs against Treponema pallidum. 【0002】 Treponema pallidum, known as the causative agent of syphilis, is an extremely important pathogen in infectious disease research. Syphilis continues to spread worldwide, and the development of vaccines, treatments, and diagnostic technologies is urgently needed. There is no vaccine, and while penicillin is useful as a treatment, it kills intestinal bacteria, causing drug-induced enteritis, and also causes allergies in some patients. Diagnosis is based on a combination of multiple antibodies in the patient's blood rather than collecting the Treponema pallidum itself, which presents problems such as the possibility of not testing positive for up to a month after infection, false positives, and sometimes difficulty in interpretation. 【0003】 For the development of vaccines, diagnoses, and treatments, a highly versatile in vitro culture method for Treponema pallidum is desired, but such a method has not yet been established, and it is well known that in vitro culture is extremely difficult. In recent years, it has been reported that Treponema pallidum can be cultured in vitro using rabbit tissue under a special culture environment with low oxygen concentration (Patent Document 1, Non-Patent Document 1). However, unlike conventional cell culture methods, this method requires very specialized techniques, including the use of animal-derived tissue, and therefore has not yet been established as an efficient and stable in vitro culture method for Treponema pallidum. 【0004】 Furthermore, as mentioned above, conventional techniques use rabbit tissue, which differs from the natural growth conditions of Treponema pallidum in human tissue. Therefore, in the development of vaccines and diagnostic / treatment technologies, it is not possible to create products that reflect the characteristics of Treponema pallidum in human tissue, and the challenge of not being able to develop vaccines, diagnoses, and treatments that can be used in humans remains unresolved. In addition, the need to add expensive additives to the culture medium in order to establish a special culture environment is also a drawback, as it is expensive. 【0005】 U.S. Patent No. 11,130,937 【0006】Edmondson DG, Hu B, Norris SJ. Long-Term In Vitro Culture of the Syphilis Spirochete Treponema pallidum subsp. pallidum. mBio. 2018;9:e01153-18.Noda K, Dabovic B, Takagi K, Inoue T, Horiguchi M, Hirai M, Fujikawa Y, Akama TO, Kusumoto K, Zilberberg L, Sakai LY, Koli K, Naitoh M, von Melchner H, Suzuki S, Rifkin DB, Nakamura T. Latent TGF-beta binding protein 4 promotes elastic fiber assembly by interacting with fibulin-5. Proc Natl Acad Sci U S A. 2013;110:2852-7Wang C, Cheng Y, Liu B, Wang Y, Gong W, Qian Y, Guan Z, Lu H, Gu X, Shi M, Zhou P. Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay. Emerg Microbes Infect. 2018;7:83Wiegand SE, Strobel PL, Glassman LH. Electron microscopic anatomy of pathogenic Treponema pallidum. J Invest Dermatol. 1972;58:186-204Sell S, Salman J, Norris SJ. Reinfection of chancre-immune rabbits with Treponema pallidum. I. Light and immunofluorescence studies. Am J Pathol. 1985;118:248-55 【0007】 Currently, there is a need to promote the development of research and diagnostic technologies by realizing in vitro culture of Treponema pallidum and providing an efficient and stable culture method. Conventional techniques mainly require animal models, which differ from the natural culture environment in humans, and involve complicated procedures for the culture system. Therefore, the present invention aims to provide a new method for culturing Treponema pallidum. 【0008】 As a result of diligent research to solve the above problems, the present inventors have found that long-term culture of Treponema pallidum can be achieved by using a culture medium containing elastic fibers or components thereof, and / or by using feeder cells in the in vitro culture of Treponema pallidum, and have completed the present invention. 【0009】 The novel in vitro culture method for Treponema pallidum of the present invention is characterized by culturing Treponema pallidum in vitro under normal laboratory conditions and normal oxygen concentrations, without using special culture materials such as rabbits, but using, for example, commercially available human-derived cells. 【0010】In other words, the present invention is as follows: [1] A method for in vitro culture of Treponema pallidum, comprising the step of culturing Treponema pallidum in a culture medium containing elastic fibers or components thereof. [2] The in vitro culture method according to [1], wherein the elastic fibers or components thereof are derived from fibroblasts, dental pulp stem cells, or vascular smooth muscle cells. [3] The in vitro culture method according to [1] or [2], wherein the elastic fibers have an elongation rate of 50% or more relative to their initial length at maximum tension, and their length is restored to 110% or less of their initial length when the external force is removed. [4] The in vitro culture method according to any one of [1] to [3], wherein the components of the elastic fibers are selected from the group consisting of elastin, fibrillin, collagen, proteoglycan, hyaluronic acid, fibronectin, growth factors, cytokines, and microfibrils. [5] The in vitro culture method according to any one of [1] to [4], wherein the elastic fibers are derived from humans, rabbits, mice, rats, dogs, cats, or pigs. [6] An in vitro culture method for Treponema pallidum, comprising the step of adding Treponema pallidum to a culture system in which cells producing elastic fibers are seeded as feeder cells, wherein the cells are selected from the group consisting of fibroblasts, dental pulp stem cells, and smooth muscle cells. [7] The in vitro culture method according to [6], wherein the cells are derived from humans, rabbits, mice, rats, dogs, cats, or pigs. [8] The in vitro culture method according to [6] or [7], wherein the cells are selected from the group consisting of human fibroblasts, mouse fibroblasts, mouse fetal fibroblasts, human umbilical cord fibroblasts, human dental pulp cells, and human vascular smooth muscle cells. [9] The in vitro culture method according to any one of [6] to [8], further comprising the step of adding LTBP-4 protein and / or DANCE protein to the culture system.

[10] An in vitro culture method for Treponema pallidum, comprising the step of adding Treponema pallidum to a culture dish coated with elastic fibers or components thereof and culturing it.

[11] The in vitro culture method according to

[10] , wherein the components of the elastic fiber are selected from the group consisting of elastin, fibrillin, collagen, proteoglycan, hyaluronic acid, fibronectin, growth factors, cytokines, and microfibrils.

[12] The in vitro culture method according to

[10] or

[11] , wherein the elastic fiber is derived from humans, rabbits, mice, rats, dogs, cats, or pigs.

[13] A method for screening drugs against Treponema pallidum, characterized by using a culture system prepared using an in vitro culture method as defined in any of [1] to

[12] , comprising: (a) adding a candidate drug to the culture system; (b) measuring the viability, bacterial count, or bacterial concentration of Treponema pallidum; and (c) evaluating the effectiveness of the candidate drug based on the measurement results obtained in step (b).

[14] The screening method according to

[13] , wherein the candidate drug is selected from an antibacterial agent, an antiviral agent, a natural product, or a compound library.

[15] The screening method according to

[13] or

[14] , wherein in step (b), the viability, bacterial count, or bacterial concentration of Treponema pallidum is measured using RT-qPCR, ELISA, bacterial infectivity test, viral infectivity test, or cell proliferation assay.

[16] A kit for culturing Treponema pallidum in vitro, suitable for an in vitro culture method as defined in any of [1] to

[12] , comprising: (a) a culture device containing elastic fibers or components thereof, or cells that produce elastic fibers selected from the group consisting of fibroblasts, dental pulp stem cells, and smooth muscle cells; (b) a culture medium or buffer; and (c) instructions for use.

[17] The kit according to

[16] , further comprising (d) LTBP-4 protein and / or DANCE protein. A kit for screening drugs against Treponema pallidum, suitable for an in vitro culture method as defined in any of

[18] [1] to

[12] , comprising: (a) a reaction vessel containing elastic fibers or components thereof, or cells that produce elastic fibers selected from the group consisting of fibroblasts, dental pulp stem cells, and smooth muscle cells; (b) a culture medium or buffer; and (c) a reagent for measuring the viability, bacterial count, or bacterial concentration of Treponema pallidum.

[19] A method for determining whether Treponema pallidum is present in a biological sample obtained from a subject, comprising an in vitro culture method as defined in any of [1] to

[12] , or a kit as defined in

[16] or

[17] . 【0011】 According to the present invention, Treponema pallidum can be cultured in a normal laboratory using commercially available cells derived from humans or other organisms. Since the method of the present invention does not use rabbit-derived components, it is possible to observe the growth of Treponema pallidum under natural conditions that cause syphilis infecting humans. For this reason, the present invention is an extremely useful technology for elucidating the nature of Treponema pallidum, developing vaccines that do not yet exist, developing more effective diagnostic methods and drugs, and identifying infection routes in specific individuals. 【0012】 The drawings attached to this specification and identified by the following description constitute part of this specification and are included to further illustrate specific aspects of the invention. The invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein. 【0013】Figure 1 shows that in Example 1, normal human dermal fibroblasts (NHDFs) promoted the production of elastic fibers, according to previously reported information (Non-Patent Literature 2). Evidence of the promotion of elastic fiber production is the positive red color in immunostaining (middle panel) for elastin protein, a component of elastic fibers. When LTBP-4 was added to the DMEM / F12 medium recommended in the previously reported information, strong fibrous staining (arrows) was observed, clearly indicating an increase in the amount of elastic fiber produced. On the other hand, weak staining was also observed using other methods, indicating the production of elastic fibers. Scale bar: 50 μm. Figure 2 shows the results of protein analysis in Example 1, where normal human dermal fibroblasts (NHDFs) promoted the production of elastic fibers, according to previously reported information (Non-Patent Literature 2). Evidence of the promotion of elastic fiber production is the presence of elastin protein, a component of elastic fibers. Adding LTBP-4 to the previously recommended DMEM / F12 medium resulted in an increase in the amount of elastin protein. Figure 3 shows the relative amount of elastin protein, with the internal control protein (vinculin) as the denominator, based on the results from Figure 2. While adding LTBP-4 to the previously recommended DMEM / F12 medium resulted in an increase in the amount of elastin protein, the presence of elastin protein was also confirmed by other methods. Figure 4 shows the engraftment and growth of Treponema pallidum on cells in which the production of elastic fibers was shown to be promoted in Figures 1-3. Numerous rod-shaped Treponema pallidum are found. Warthin-Starly staining shows Treponema pallidum as positively brown. Scale bars: 200 μm (left and upper right figures), 50 μm (lower right figure). Figure 5 shows the results of immunohistochemical staining (higher specific than Warthin-Starly staining) of Treponema pallidum co-cultured with NHDF cultured in a medium supplemented with LTBP-4, similar to Figure 4 (top panel). Treponema pallidum is positive, appearing red. Scale bar: 50 μm. Figure 6 shows Treponema pallidum engrafting and growing on an elastin-coated petri dish, similar to the results shown in Figures 4 and 5. Scale bar: 20 μm. Figure 7 shows the growth curves of Treponema pallidum for both the results shown in Figures 4 and 5 and the results shown in Figure 6. In all cases, the horizontal axis represents days. The vertical axis shows the number of cells in the top panel, motility in the middle panel, and time required for subculturing in the bottom panel.Figure 8 shows the identification (arrow) of Treponema pallidum subsp. pallidum str. Nichols, complete sequence in the NIH's National Library of Medicine by PCR, using the same method as previously reported (Non-Patent Literature 3). Figure 9 shows the confirmation of Treponema pallidum by transmission electron microscopy in both the results shown in Figures 4 and 5, and Figure 6. Figure 10 shows the results of Treponema pallidum by transmission electron microscopy in both the results shown in Figures 4 and 5, and Figure 6, on shaved rabbit skin (left) and collected rabbit skin (right), similar to the results in Figure 5, for Treponema pallidum in rabbit infection experiments. Figure 11 is a summary table of the results in Figure 10. Figure 12 shows the immunohistochemical staining for Treponema pallidum (bottom left and bottom middle) and elastin (bottom right), as well as the corresponding hematoxylin-eosin (HE) staining (top) in rabbit skin obtained from the results in Figure 10. 【0014】 The following description may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments or specific examples. Unless otherwise specified, scientific and technical terms used herein should be interpreted as having meanings generally understood by those skilled in the art. The definitions of terms of particular importance in relation to the present invention are listed and explained in detail below as appropriate. 【0015】 Definition (1) Treponema pallidum "Treponema pallidum" (scientific name: Treponema pallidum subsp. pallidum) is a spiral-shaped, Gram-negative bacterium belonging to the Spirochetaceae family, and is known as a pathogen that causes syphilis in humans. This bacterium has an elongated spiral shape and characteristic motility, and is often observed under a dark-field microscope. It is very elongated, about 6-20 μm in length and about 0.1-0.2 μm in width, and its spiral shape contributes to its infectivity and motility. 【0016】Treponema pallidum, the bacterium that causes syphilis, is a strictly parasitic microorganism that uses humans as its sole natural host and is transmitted through direct contact between humans, especially sexual contact. It can also cause congenital syphilis through mother-to-child transmission. The infection mechanism of this bacterium is mainly through the entry of microscopic wounds in the skin and mucous membranes, and it spreads throughout the body. In the initial stage, it forms a hard ulcer (chancre) at the site of infection, and then spreads to organs and tissues throughout the body via the bloodstream. If left untreated, syphilis progresses through stages of early syphilis (primary syphilis), secondary syphilis, latent syphilis, and late syphilis, leading to a chronic and progressive disease affecting multiple organs. Therefore, early diagnosis of syphilis is essential. 【0017】 Physiologically, Treponema pallidum is an obligate anaerobic or microaerophilic bacterium that cannot grow under normal oxygen concentrations. Its slow replication rate and difficulty in vitro culture make it impossible to cultivate using conventional bacterial culture methods. Therefore, animal models such as rabbits are used for cultivation, and molecular biological techniques such as PCR are employed for diagnosis and research. Furthermore, Treponema pallidum differs from other Gram-negative bacteria in that, while it possesses an outer membrane, it lacks lipopolysaccharide (LPS). 【0018】 On the other hand, early diagnosis is considered particularly important for the detection of Treponema pallidum, and there is a need for a method to rapidly and accurately detect antibodies or antigens specific to Treponema pallidum. This includes rapid diagnostic kits using immunochromatography, or, as mentioned above, methods that directly detect the genes of the pathogen using PCR technology. To date, these technologies have enabled early detection of infection, contributing to faster treatment and prevention of infection spread. However, currently, due to the difficulty of in vitro culture of Treponema pallidum, general medical institutions diagnose the disease using combinations of multiple antibodies in the patient's serum, and diagnosis is difficult for up to one month after infection because antibodies do not become positive. 【0019】Furthermore, regarding the treatment of syphilis, since Treponema pallidum is highly susceptible to penicillin, antibiotic-based treatment is common. However, penicillin carries the risk of disrupting the gut microbiota and causing allergic reactions in some individuals. In recent years, research into new therapeutic drugs targeting other antibiotics and combination therapies has progressed to address the risk of developing penicillin-resistant Treponema pallidum, and related technological developments are also underway. For example, treatments using azithromycin and tetracycline are attracting attention, but since there is a possibility of drug-resistant strains emerging, the development of methods for screening more effective drugs is also needed. 【0020】 Recently, research has been conducted on vaccine development, but practical application of vaccines has not been achieved because Treponema genus bacteria possess sophisticated immune evasion mechanisms. The surface antigens of Treponema pallidum, which are prone to mutation, make it difficult to form immunological memory, which hinders vaccine development. Therefore, technical approaches aimed at stabilizing surface antigens or searching for novel antigens are needed. 【0021】 The present invention's method for culturing Treponema pallidum in vitro will aid in the development of novel antibiotics and vaccines against Treponema pallidum. 【0022】 (2) Elastic Fibers Elastic fibers are generally composed mainly of elastin and microfibrils (fine fibers), and are connective tissue components that provide elasticity and stretchability to tissues. Their structure consists of a microfibril layer surrounding the elastin inside, and the elastin forms a cross-linking structure with its hydrophobic amino acid residues, giving it high elasticity that allows it to return to its original shape even after repeated stretching. This double structure allows tissues to maintain their shape while being stretchable. The various components are intertwined in a complex manner, and they exhibit high functionality in tissues that stretch frequently, such as skin, blood vessels, lungs, and ligaments. 【0023】The modulus of elasticity of elastic fibers varies depending on the tissue and body part, but is generally in the range of 0.1 to 1.0 MPa. This indicates elasticity suitable for tissues that require high elasticity, such as skin, blood vessels, and lungs. This modulus of elasticity allows the fibers to withstand repeated stretching and contracting movements, supporting the maintenance of tissue shape and flexibility. Furthermore, within the body, the length of elastic fibers differs from tissue to tissue, generally ranging from a few micrometers to tens of micrometers. For example, elastic fibers in blood vessels need to expand and contract in response to blood pressure, so their length and arrangement are appropriately controlled. Similarly, the length and density of elastic fibers in the lungs and skin are adjusted according to the function of the tissue. 【0024】 On the other hand, elastic fibers can also be identified as connective tissue components that have the ability to stretch significantly beyond their original length and return to their original shape within a range that does not exceed the elastic limit. In one embodiment, it is preferable that the elastic fiber (or its components) has an elongation rate of 50% or more relative to its initial length at maximum tension. More preferably, the elongation rate is 50% to 300%, even more preferably 100% to 200%, and most preferably 150%. 【0025】 In another embodiment, it is preferable that the elastic fiber is restored to a length of 110% or less of its initial length after the external force is removed. More preferably, the restored length is 100% or more and 108% or less of the initial length, and even more preferably 101% or more and 105% or less. 【0026】The elastic fibers used in the in vitro culture method of Treponema pallidum of the present invention are not limited, but preferably have at least the above characteristics. Furthermore, the elastic fibers may be of biological origin, including cells, or synthesized in vitro, as long as they have the above characteristics. In the present invention, the method for obtaining elastic fibers from cells typically includes the following steps: preparing cells capable of secreting elastic fibers or their components (e.g., fibroblasts, smooth muscle cells, or genetically modified cells thereof) and maintaining the cells in a culture medium in an incubator under appropriate culture conditions; adding additives that promote the synthesis or secretion of elastin, a component of elastic fibers, or the formation of elastic fibers (ascorbic acid, copper ions, tropoelastin precursor, LTBP-4, and / or DANCE) to the culture medium; continuing to culture the cells; and isolating the elastic fibers produced (secreted) from the cells (and optionally from matrix components) by enzymatic treatment (e.g., collagenase treatment) or physical separation methods. 【0027】LTBP-4 (Latent Transforming Growth Factor Beta Binding Protein 4) is a protein involved in the regulation of TGF-β, particularly in the production of the extracellular matrix (ECM), including fibroblasts. LTBP-4 is a member of a family that plays a role in regulating TGF-β activity. TGF-β is an important cytokine that regulates cell proliferation, differentiation, and ECM formation. It is secreted in an inactive (latent) form and is retained in the extracellular matrix by binding to LTBP-4. This binding is necessary to maintain a stable state until TGF-β is activated as needed. LTBP-4 contributes to elastin formation and fiber formation, and plays a role in stabilizing the structure of elastic fibers. By assisting elastin synthesis, it contributes to maintaining the elasticity of the skin, lungs, and blood vessel walls. According to the present invention, in the culture of Treponema pallidum, the addition of LTBP-4 to the culture system significantly enhances the production of elastic fibers containing elastin protein by fibroblasts (see Figure 3). The amount of elastic fibers produced is increased by at least 1.1 times, preferably 1.2 times, more preferably 1.3 times, even more preferably 1.4 times, and still more preferably 1.5 times compared to cultures without the addition of LTBP-4. In this specification, the above term "enhancement" may be used synonymously with "promotion." 【0028】 DANCE, also known as Del1 (Developmental endothelial locus-1), is a protein and a type of extracellular matrix protein. DANCE indirectly supports the production of the extracellular matrix (ECM) by fibroblasts in particular and regulates cell adhesion and migration by acting as a component of the ECM. DANCE is known to interact with integrins, which are cell adhesion molecules, and to influence the migration, proliferation, and differentiation of fibroblasts and other cell types. 【0029】On the other hand, the components of the elastic fibers used in the in vitro culture method of Treponema pallidum of the present invention may include, but are not limited to, elastin, fibrillin, collagen, proteoglycan, hyaluronic acid, fibronectin, growth factors, cytokines, and microfibrils selected from among these. 【0030】 The term "elastin," as described above, is a protein that constitutes the core of elastic fibers and contains a large amount of hydrophobic amino acids (glycine, alanine, proline, etc.). Elastin is extremely flexible and, by forming crosslinks between molecules, it exhibits elasticity that allows it to return to its original shape even after repeated stretching and contraction. Once synthesized, elastin molecules are difficult to decompose and maintain a very stable structure. 【0031】 The term "microfibril" refers to fine fibers present in the outer layer of elastin, providing the overall structure and stability of the fiber. The main component of microfibrils is a glycoprotein called fibrillin, which functions as a scaffold during elastin formation. The arrangement of fibrillin in a network allows elastin to be deposited in an orderly manner, ensuring the high elasticity of the elastic fibers. 【0032】 (3) Feeder Cells "Feeder cells" refer to auxiliary cells that provide direct nutrient supply and support functions in bacterial culture outside of a living organism. In particular, when culturing bacteria that are highly host-dependent or bacteria that are difficult to grow in standard artificial media, using feeder cells can indirectly create the environment necessary for the growth of bacteria. Generally, mammalian cells such as fibroblasts are often used as examples of feeder cells. In this invention, "feeder cells" can be used synonymously with "feeder layer". 【0033】When using feeder cells in bacterial culture, they are first seeded in a culture dish and allowed to grow until they reach a certain confluence. Then, bacteria are added for co-culture, allowing the feeder cells to create a suitable environment for bacterial growth. Feeder cells can also have their proliferation stopped by radiation or chemical treatment, thus providing a sustained support function without inhibiting bacterial growth. 【0034】 The culture medium used for co-culture is not limited, but it should contain components suitable for both feeder cells and bacteria, and in some cases, components to promote bacterial growth may be added. Furthermore, incorporating metabolites from feeder cells may facilitate bacterial growth. The culture environment for feeder cells can also be appropriately adjusted to suit the bacteria, including oxygen and carbon dioxide concentrations. 【0035】 Description of Exemplary Embodiments The following describes exemplary embodiments of the present invention. Research on Treponema pallidum subsp. pallidum, the bacterium that causes syphilis, has been hampered despite more than a century of effort because it is extremely difficult to continuously and stably culture Treponema pallidum in vitro. 【0036】 In one embodiment, this disclosure addresses the above-mentioned problems by providing an in vitro culture method that involves incorporating elastic fibers or components thereof into a culture system of Treponema pallidum, or by adding Treponema pallidum to a culture system in which cells producing elastic fibers are seeded as feeder cells and then culturing the cells. Furthermore, by using a culture system utilizing the above-mentioned in vitro culture method, drugs (candidate drugs) against Treponema pallidum can be easily and efficiently identified and screened. In addition, a kit that enables the in vitro culture method of the present invention to be easily implemented at various research institutions and hospitals can also be provided. 【0037】 (1) In vitro culture method The present invention provides an in vitro culture method for Treponema pallidum, which includes elastic fibers or components thereof in the culture system, or uses cells that produce elastic fibers as feeder cells. 【0038】 Method for culturing Treponema pallidum in a medium containing elastic fibers or their components According to the present invention, there is provided a method for in vitro culturing of Treponema pallidum, which includes a step of culturing Treponema pallidum in a medium containing elastic fibers or their components. The "elastic fibers" can be those described in the above-mentioned definition. In addition, as is well known to those skilled in the art, the components of elastic fibers include elastin, fibrillin, proteoglycan, glycosaminoglycan, and collagen, etc. In addition to those derived from living organisms or cells, polymers and functional substances synthesized in vitro may also be included. 【0039】 The content of elastic fibers or their components (hereinafter sometimes simply referred to as "elastic fibers") in the medium can be appropriately adjusted. For example, as the final concentration in the medium, it is preferably 0.1 to 500 μg / ml. According to the present invention, depending on the initial concentration or state (such as growth ability) of the cultured Treponema pallidum, for example, it can also be used at concentrations divided into low concentration (0.1 to 10 μg / ml), medium concentration (10 to 50 μg / ml), and high concentration (50 to 500 μg / ml). 【0040】 The elastic fibers can be derived from living organisms, cells, etc. as described above. For example, if they are cell-derived, they may be derived from fibroblasts, dental pulp stem cells, or vascular smooth muscle cells. 【0041】 As used herein, the term "fibroblast" refers to the cells inherent in the tissue that constitutes connective tissue. More specifically, fibroblasts do not have particularly prominent functions in normal tissues, but when damage occurs, they migrate to the damaged area and secrete elastin, collagen, hyaluronic acid, etc., thereby starting the production of extracellular matrix (ECM) and enhancing the accumulation of ECM. In addition, it plays an important role in the wound healing process, such as inducing wound contraction. 【0042】As used herein, "dental pulp stem cells (DPSCs)" refer to pluripotent stem cells present in dental pulp tissue, which play a particularly important role in the regeneration of tooth tissue, such as dentin and periodontal ligament. These cells originate from the neural crest and possess mesenkim-like properties, giving them the ability to differentiate into diverse cell lineages, including bone, cartilage, fat, and nerve cells. Dental pulp stem cells also possess the ability to synthesize and secrete elastic fibers, which is thought to contribute to tissue regeneration. Dental pulp stem cells can be relatively easily collected from extracted teeth such as deciduous teeth, permanent teeth, and wisdom teeth, and their high proliferative and differentiation capabilities have attracted attention as a useful cell source in regenerative medicine and tissue engineering. 【0043】 As used herein, “vascular smooth muscle cells (VSMCs)” refer to cells that are normally located in the media of the vascular wall, but may also migrate to the intima, and that play a role in regulating blood flow and blood pressure through vasoconstriction and relaxation. These cells are primarily responsible for vasoconstriction, but under certain conditions they have the ability to synthesize and secrete extracellular matrix (ECM) components, particularly elastic fibers or their components. Elastic fibers are important components that maintain the flexibility and elasticity of blood vessels and contribute to their durability and resilience. By producing elastic fibers, vascular smooth muscle cells are involved in stabilizing the vascular structure and maintaining its function. 【0044】 In this invention, fibroblasts isolated from living organisms using well-known methods, or commercially available fibroblasts, can be used. The animal species from which the fibroblasts are derived are not limited, but include humans, rabbits, mice, rats, dogs, cats, and pigs, and fibroblasts derived from these animal species may also be used. 【0045】(1-2) Method for culturing Treponema pallidum in a culture system seeded with cells that produce elastic fibers as feeder cells The present invention provides a method for in vitro culture of Treponema pallidum, comprising the step of adding Treponema pallidum to a culture system seeded with cells that produce elastic fibers as feeder cells and culturing the cells, wherein the cells are selected from the group consisting of fibroblasts, dental pulp stem cells, and smooth muscle cells. "Cells that produce elastic fibers" refers to cells that have the function of synthesizing, secreting, and maturing elastic fibers, which are one of the components of the extracellular matrix (ECM). Details of the term "elastic fiber" are as described above. The "cells that produce elastic fibers" that can be used in the in vitro culture method of Treponema pallidum of the present invention are not limited as long as they have the above characteristics, but typical examples include fibroblasts, dental pulp stem cells, or vascular smooth muscle cells. These cells are as described above. 【0046】 When culturing Treponema pallidum using "cells that produce elastic fibers" as a feeder layer (feeder cells), the following culture conditions can typically be used as a guideline: (a) Number of cells seeded: The cell density in the culture of seeded elastic fiber-producing cells should be 1 × 10⁻¹⁶ so that a uniform layer is formed on the culture surface of the culture dish by the cells, and the Treponema pallidum can make sufficient contact and depend on it. ４ ~5 x 10 ４ cells / cm ２ It is preferable that the cells are seeded in this manner. This provides a stable feeder cell layer that promotes interaction with Treponema pallidum. 【0047】 (b) Culture days After seeding cells that produce elastic fibers as feeder cells, culture for 1 to 2 days until confluent (occupying approximately 80-90% of the culture dish surface). Depending on the amount of elastic fiber produced, the culture period may be extended for another 1 to 3 weeks. After replacing with an antibiotic-free medium, seed Treponema pallidum. It is preferable to add Treponema pallidum when the cell density and viability are stable so that the cells do not overgrow. In this case, the concentration of Treponema pallidum added is typically 10 ４ ~10 ６The concentration is cells / ml. Generally, culturing Treponema pallidum for 3 to 7 days allows feeder cells to produce sufficient elastic fibers while maintaining a state in which the Treponema pallidum can be supplied with the necessary components. 【0048】 (c) Passaging of feeder cells is desirable to be performed before the cells age, depending on their proliferation rate. Since the objective is to produce elastic fibers, it is preferable to passage the feeder cells while confirming the production of elastic fibers. For example, cell passaging can be performed while confirming the production of elastic fibers, i.e., the maintenance of the function of cells that produce elastic fibers, using the amount of elastin detected by the use of anti-elastin antibodies as an indicator. At the same time, the amount of Treponema pallidum added to the culture system can be qualitatively measured by PCR, and it is possible to maintain a culture system containing Treponema pallidum for a long period of time while detecting mutations and disruption in the genes of Treponema pallidum. 【0049】 In certain embodiments, the culture may be subcultured or replenished with culture medium at intervals of approximately 2 to 14 days, such as every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, subculturing consists of treating the culture with trypsin and adding fresh culture medium. In further embodiments, such subculturing can be repeated to allow Treponema pallidum to survive for at least about one month. The survival period of Treponema pallidum in culture is at least one month, three months, six months, one year, or longer. 【0050】 (d) Other culture conditions: For feeder cell culture, 10% fetal bovine serum (FBS) is added to a standard synthetic medium (e.g., DMEM, RPMI, etc.), but antibiotics must be removed before seeding of Treponema pallidum, and the medium and its components must be adjusted to ensure that both Treponema pallidum and feeder cells can survive. Culture temperature and CO2 ２ Concentration: Same as normal cell culture, 37°C, 5% CO2 ２ The environment is suitable, and the co-cultured Treponema pallidum can be controlled by different temperatures and CO2 levels.２ When concentration is required, the culture conditions may be adjusted at the stage when the feeder layer is stable. Radiation treatment and mitomycin treatment: In order to prevent the feeder cells from over-proliferating, a treatment to stop proliferation (for example, radiation irradiation, mitomycin C treatment, etc.) may be performed. As a result, the feeder layer can be stabilized and an environment more suitable for the culture of Treponema pallidum can be provided. 【0051】 Note that the above conditions are typical, and those skilled in the art can select more appropriate culture conditions while making choices according to the differences of various culture facilities and culture apparatuses for the culture conditions suitable for co-culture. 【0052】 In an alternative embodiment related to the above co-culture, Treponema pallidum can be present in the co-culture in a ratio in the range of about 1:1 to 10,000:1 with respect to the cells that produce elastic fibers. In certain examples, Treponema pallidum can be present in the co-culture in a ratio between about 1:1 and 1,000:1; between 1:1 and 100:1; or between 10:1 and 100:1 with respect to the cells that produce elastic fibers. In another example, the co-culture is an adherent cell culture system and a culture dish (for example, wells, plates, etc.) is used. Thus, it is preferred that the co-culture is not a suspension culture. 【0053】 Standard media that can be used for co-culture include, but are not limited to, typically DMEM / F12; 10% FBS; 2 mM L-glutamine; 100 unit / ml penicillin; 100 μg / ml streptomycin. In the initial stage (for example, two weeks), fibroblasts (for example, 1 - 10 × 10 ４ cells / ml) can be cultured in this standard medium. Subsequently, an appropriate reagent such as LTBP-4 (for example, LTBP-4 at a final concentration of 30 nM) may be added to the culture system. Subsequently, when further culturing Treponema pallidum in the above culture system, after appropriate washing, fresh medium from which penicillin and streptomycin have been removed from the standard medium can be used. 【0054】(1-3) Method for culturing Treponema pallidum on a culture dish coated with elastic fibers or its components The present invention provides a method for in vitro culture of Treponema pallidum, comprising the step of adding Treponema pallidum to a culture dish (Petri dish, well, dish, etc.) coated with elastic fibers or its components and culturing it. Details of the elastic fibers and its components can be defined as described above. Furthermore, coating the culture dish with elastic fibers (or its components) can be done based on the method used by those skilled in the art when performing cell culture. In the method for in vitro culture of Treponema pallidum of the present invention, commercially available culture dishes coated with elastic fibers (or its components) may be used. Examples of such commercially available coated culture dishes include, but are not limited to, elastin-coated Petri dishes (Extracellular Matrix Research Institute Co., Ltd. (Tsu City, Mie Prefecture, Japan), catalog number: SE-60). 【0055】 The concentration of Treponema pallidum added to a culture dish coated with elastic fibers (or its components) and the culture conditions (including the subculturing method) may be the same as those used when using a culture system in which cells producing the aforementioned elastic fibers are seeded as feeder cells. 【0056】 (2) Detection and viability confirmation of Treponema pallidum The in vitro culture method of the present invention can be used to culture not only pathogens related to syphilis but also Treponema species related to a wide range of pathological conditions. Examples of Treponema species, described in relation to specific diseases, include syphilis (Treponema pallidum subsp. pallidum), frambezia (T. pallidum subsp. pertenue), begel (T. pallidum subsp. endemicum), and pinta (T. pallidum subsp. carateum). By using the in vitro culture method of the present invention, at least 60% (e.g., 70%, 80%, 90%, 95%, or 100%) of these Treponema species are viable. In another embodiment, at least 60% (e.g., 70%, 80%, 90%, 95%, or 100%) of the Treponema species exhibit motility and / or infectivity. 【0057】Methods well known to those skilled in the art can be used to measure the abundance of Treponema pallidum in culture. Examples of measurement methods, though not limited to them, include qPCR (quantitative PCR), RT-qPCR, fluorescence microscopy, flow cytometry, live imaging using fluorescence labeling, ELISA (enzyme immunoassay), and ATP measurement. In qPCR, primers targeting DNA sequences specific to Treponema pallidum (e.g., tpp47 gene, polA gene, etc.) can be used. The use of qPCR allows for the continuous and highly sensitive quantification of Treponema pallidum. RT-qPCR is a method for quantifying specific mRNA of Treponema pallidum (e.g., genes related to metabolism) using qPCR. The results obtained by this method indicate the amount of viable Treponema pallidum, making it easy to understand the dynamics based on the RNA state. That is, it is characterized by the ability to evaluate active bacteria independently of the amount of remaining DNA. In "fluorescence microscopy," immunofluorescence staining using antibodies specific to Treponema pallidum, or fluorescent dyes with affinity for Treponema pallidum (e.g., DAPI, SYBR Green), are used. By using a fluorescence microscope, Treponema pallidum can be directly observed under a microscope and their number can be counted. In "flow cytometry," Treponema pallidum can be measured in cell suspension using fluorescently labeled antibodies or DNA stains. "Live imaging using fluorescent labeling" is based on adding a fluorescent probe that binds to the membrane or DNA of Treponema pallidum and observing it with a live imaging device. This method allows for real-time observation of living Treponema pallidum. "ELISA (enzyme immunoassay)" is a method that measures the amount of antigen or metabolite (e.g., outer membrane protein) derived from Treponema pallidum using specific antibodies. "ATP measurement method" is a method that measures ATP derived from living Treponema pallidum based on the luciferase reaction. This allows us to indirectly obtain the survival rate of Treponema pallidum, the bacterium that causes syphilis. 【0058】(3) Infectious Treponema pallidum The infectivity of cultured Treponema pallidum can be measured using an animal model. Rabbits are preferred as such an animal model. A typical method involves intradermally injecting serial dilutions of a sample (culture, bacterial suspension) containing Treponema pallidum into the backs of shaved rabbits. Dilution is performed using conventional synthetic media (e.g., DMEM, RPMI, etc.) or TpCM-2, a medium described in a previously published document (Non-Patent Literature 1), and each dilution is inoculated into overlapping sites on each rabbit. The inoculation sites are then shaved, and the development of erythema and induration is observed daily for a predetermined period. Needle aspirates from representative lesions (two per administration) are observed under a dark-field microscope to check for the presence of motile Treponema pallidum indicating active Treponema infection. During the experiment, rabbits are fed antibiotic-free diet and water, kept at 16-18°C, and shaved daily. The median infectious dose (ID) is used. ５０ This can be determined by the Reed and Munch method (Reed and Munch, 1938). 【0059】 (4) Screening Method The present invention provides a method for screening drugs against Treponema pallidum, characterized by using a culture system used in a method for culturing Treponema pallidum in vitro as disclosed herein. The screening method of the present invention typically includes the following steps: (a) adding a candidate drug to the culture system; (b) measuring the viability, bacterial count, or bacterial concentration of Treponema pallidum; and (c) evaluating the effectiveness of the candidate drug based on the measurement results obtained in step (b). 【0060】 Candidate drugs in step (a) above may include antibacterial agents, antiviral agents, natural products, or compound libraries. In step (b) above, the viability, bacterial count, or bacterial concentration of Treponema pallidum can be measured using RT-qPCR, ELISA, bacterial infectivity tests, viral infectivity tests, or cell proliferation assays. These measurement methods may be well known to those skilled in the art. 【0061】(5) Kits The present invention provides a kit suitable for a method of culturing Treponema pallidum in vitro as disclosed herein. The kit of the present invention typically includes: (a) elastic fibers or components thereof, or cells that produce elastic fibers selected from the group consisting of fibroblasts, dental pulp stem cells, and smooth muscle cells; (b) culture medium or buffer; and (c) instructions for use. 【0062】 Furthermore, the above kit may also contain (d) LTBP-4 protein and / or DANCE protein. 【0063】 In another embodiment, the present invention provides a kit for screening drugs against Treponema pallidum, suitable for the in vitro culture method of the present invention. The kit of the present invention typically comprises: (a) a reaction vessel containing elastic fibers or components thereof, or cells that produce elastic fibers selected from the group consisting of fibroblasts, dental pulp stem cells, and smooth muscle cells; (b) a culture medium or buffer; and (c) a reagent for measuring the viability, bacterial count, or bacterial concentration of Treponema pallidum, and may further comprise (d) Treponema pallidum. 【0064】 (6) Method for determining the presence of Treponema pallidum in a biological sample The present invention provides a method for determining whether Treponema pallidum is present in a biological sample obtained from a subject (for example, blood, serum, transudate, exudate, mucus, tissue, cerebrospinal fluid, nucleic acid, etc.). The method is characterized by using an in vitro culture method or kit described herein. 【0065】 The following non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of the representative embodiments intended herein. These examples are intended to be merely a subset of all possible contexts in which the components of the present invention can be combined. Accordingly, these examples should not be construed as limiting any of the embodiments described herein, including those relating to the types and quantities of components of the present invention, and / or their methods and uses. 【0066】The commercially available reagents used in the following examples were used according to their respective protocols unless otherwise specified. 【0067】 [Example 1] Human fibroblasts were cultured in a dish to promote the production of elastic fibers, and then Treponema pallidum was inoculated onto them and allowed to grow. Treponema pallidum was then subcultured onto fibroblasts in which the production of elastic fibers had been promoted using the same procedure. 【0068】 (1) Production of elastic fibers by human fibroblasts on a culture dish. Normal human dermal fibroblast (NHDF) juvenile foreskin cell line (catalog number: c-12300) was purchased from Promocell (Heidelberg, Germany). The cells were cultured in DMEM / F12 (1:1) medium (Invitrogen, California, USA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine (Invitrogen, California, USA), and 100 units / ml penicillin / 100 μg / ml streptomycin (Invitrogen), or in D12046 medium / fibroblast growth medium kit (500 ml) (Takara Bio Inc., product code: C23110). The cells were cultured without changing the medium in 5% CO2. ２ The cells were maintained at 37°C in a humidified incubator. After two weeks, laten TGF-beta protein 4 (LTBP-4) (R&D systems, Minnesota, USA) was added at a final concentration of 30 nM, and the cells were cultured for a further 4 days. Before inoculation with Treponema pallidum, the culture medium was replaced with an antibiotic-free medium. 【0069】(2) Confirmation of elastic fibers by immunohistochemistry Cells were cultured on culture slides (BD Falcon Japan, Osaka, Japan) in the same manner as above, fixed on ice with 100% MeOH for 10 minutes, and washed twice with PBS. The slides were incubated with anti-elastin polyclonal antibody (1:200, rabbit, bs-1756r, Bioss, Massachusetts, USA) at room temperature (RT) for 1 hour, followed by incubation with Alexa Fluor 555-conjugated donkey anti-rabbit IgG antibody (1:200, A31572, Invitrogen, Massachusetts, USA) at RT for 30 minutes. Cell nuclei of NHDF were counterstained with DAPI. Primary antibody was omitted in the negative control experiment. The results are shown in Figure 1. 【0070】 The top panel of Figure 1 shows the results of Elastica van Gieson staining, a dye staining method for elastic fibers. Elastic fibers show a purplish-black positive image in Elastica van Gieson staining. In this experiment, no clear positive image was detected, making it difficult to identify elastic fibers using this staining method. The middle panel shows the results of fluorescent antibody staining using an antibody against elastin; elastin was stained red in a fibrous pattern as a positive image (see DMEM / F12 + LTBP-4). The bottom panel shows an image where only a secondary antibody was reacted, without the elastin antibody, as a negative control. The arrows in the figure indicate particularly strong expression of elastin protein. 【0071】(3) Confirmation of elastic fiber formation by protein analysis Cells cultured as described in (1) above were suspended together with the extracellular matrix in RIPA buffer containing protease inhibitors (Complete Mini and phosSTOP, Roche, Mannheim, Germany) and homogenized with VP-5s (TAITEC, Saitama, Japan). After homogenization, the samples were left to stand on ice for 30 minutes. The supernatant was centrifuged at 15,000 rpm at 4°C for 30 minutes and collected. Protein concentration was measured by ultraviolet absorption at 280 nm. The samples were subjected to electrophoresis on a 10% SDS-polyacrylamide gel (TGX Gel #450-1036, Bio Rad, California, USA). After electrophoresis, the samples were transferred to a PVDF membrane (#1704156, Bio Rad) using TransBlot Turbo (Bio Rad, California, USA). The transferred membrane was reacted with an anti-elastin polyclonal antibody (1:500, rabbit, bs-1756r, Bioss, Massachusetts, USA). An anti-vinculin polyclonal antibody (1:4,000, rabbit, E1E9V, Cell Signaling Technology, Massachusetts, USA) was used as an internal control. The reaction products were reacted with an HRP-labeled anti-rabbit IgG antibody (1:5000, NA934v, GE Cytiva, Tokyo, Japan) and identified using Chemi Doc (Bio Rad). The obtained signals were measured and quantified using Image Lab (Biorad). The results are shown in Figures 2 and 3. "TPC-1" in the figures is a papillary thyroid cancer cell line, and was included as a preliminary experiment to compare elastin expression. 【0072】 Figure 2 shows the protein analysis results obtained by promoting elastic fiber production with NHDF. The amount of elastin protein was enhanced by adding LTBP-4 to DMEM / F12 medium. Figure 3 shows the signal intensity of the elastin band obtained in Figure 2 and the signal intensity of the vinculin band used as an endogenous control, measured using Image Lab. The elastin / vinculin ratio was calculated and its value is shown. 【0073】(4) Inoculation and Passaging of Treponema pallidum Nichols strain. The Treponema pallidum Nichols strain was provided courtesy of the National Institute of Infectious Diseases. It was thawed and inoculated into culture dishes with NHDF adsorbed to start culturing. After 3 to 4 days, the culture dishes were treated with trypsin to recover the Treponema pallidum and NHDF. Centrifuge was performed at 1,000 rpm at room temperature for 5 minutes, and the precipitate was recovered. This precipitate was suspended in 500 μl of PBS. 10 μl of this suspension was further suspended in 40 ml of PBS. 10 μl of this suspension was inoculated into culture dishes with NHDF adsorbed in DMEM / F12 medium supplemented with LTBP-4, and the Treponema pallidum was passed through and engrafted. The results are shown in Figure 4. Furthermore, as shown in Figures 1 and 3, engraftment of Treponema pallidum was also confirmed in "other methods" where a lower amount of elastin protein was observed (specifically, NHDF cultured in DMEM / F12 medium but without LTBP-4 supplementation). Treponema pallidum cultured in DMEM / F12 medium with LTBP-4 supplementation on NHDF could be cultured for more than 117 days by subculturing 32 times. 【0074】 (5) Identification of Treponema pallidum by nuclear staining and immunohistochemistry As described in (4) above, Treponema pallidum and NHDF were cultured on culture slides (BD Falcon Japan, Osaka, Japan), fixed on ice with 100% MeOH for 10 minutes, and washed twice with PBS. Warthin-Stalin staining, which is used in standard pathological diagnostic procedures, was performed on these slides. In addition, anti-treponema pallidum polyclonal antibody (1:200, rabbit, ab20923, Abcam) was incubated at room temperature (RT) for 1 hour, followed by incubation with Alexa Fluor 555-conjugated donkey anti-rabbit IgG antibody (1:200, A31572, Invitrogen, Massachusetts, USA) at RT for 30 minutes. Cell nuclei were counterstained with DAPI. Primary antibody was omitted in the negative control experiment. The results are shown in Figures 4 and 5. 【0075】Figure 4 shows the engraftment and growth of Treponema pallidum on cells that showed enhanced elastic fiber production in Figures 1-3. Numerous rod-shaped Treponema pallidum are found. After inoculation, Treponema pallidum proliferated and formed colonies (upper left), but after subculturing, they proliferated in a dispersed manner (upper right). Furthermore, in Warthin-Starly staining performed after subculturing, Treponema pallidum was detected as a positive brown color (lower right). In addition, in Figure 5, immunohistochemical staining detected the Treponema pallidum observed in Figure 4 as a positive red color (upper middle). DAPI staining mainly detected the cell nuclei of NHDF (upper left, lower left). The positive appearance of Treponema pallidum did not necessarily coincide with the location of the cell nuclei of NHDF (upper right). 【0076】 [Example 2] Treponema pallidum was inoculated onto elastin-coated petri dishes and grown, then subcultured. Elastin-coated petri dishes (catalog number: SE-60) were purchased from Cellular Matrix Research Institute Co., Ltd. (Tsu City, Mie Prefecture, Japan). DMEM / F12 (1:1) medium (Invitrogen) supplemented with 10% fetal bovine serum and 2 mM L-glutamine (Invitrogen, California, USA) was used. Treponema pallidum, which had been co-cultured with NHDF, was inoculated into this medium at a 1,000,000-fold dilution. A micrograph taken on the third day after inoculation is shown in Figure 6. On the elastin-coated petri dishes, Treponema pallidum could be cultured for at least 175 days after 50 subcultures. 【0077】 Figure 6 (right) shows the engraftment and growth of Treponema pallidum on an elastin-coated petri dish. Similar to when cultured on cells that showed enhanced elastic fiber production (Figure 6 (left)), Treponema pallidum also adhered and grew on the elastin-coated petri dish. The middle panel shows medium magnification under a phase-contrast microscope, and the bottom panel shows high magnification under a dark-field microscope. The bar in both images is 20 μm. 【0078】 (6) Growth curves of Treponema pallidum. Treponema pallidum was continuously subculturised every 3 or 4 days on NHDF producing elastic fibers and on elastin-coated petri dishes. The growth curves are shown in Figure 7. In both cases, the horizontal axis represents the number of days. The upper panel shows the number of cells counted using a hemocytometer. 3 mm３ If confluence occurs in the culture medium, 10 ７ It is weak, and is passed down at a rate of about 1 / 20th of the original rate. ５ The activity level remains high. The middle section shows two microscope images taken one second apart in the same field of view to measure cell movement. Approximately 97.5-99% of the cells showed movement, indicating that the ability to move is maintained even after subculturing. The bottom section shows the time required for subculturing. Subculturing was continued alternately every three days and every four days, resulting in approximately 70-75 hours (3 days) and 90-100 hours (4 days). Similar findings were obtained to those reported previously (Non-Patent Literature 1). 【0079】 (7) Confirmation of Treponema pallidum subsp. pallidum str. by PCR: Following the manufacturer's manual, DNA extraction was performed by detaching NHDF sheets that had been inoculated with Treponema pallidum and passed through 30 times, or full sheets without Treponema pallidum, using TaKaRa Ex Taq (TaKaRa Bio Inc.) on NHDF that produces elastic fibers. Similarly, DNA extraction was performed by detaching tissue from elastin-coated petri dishes after 30 passages. To amplify the PolA region gene, known to be specific to Treponema pallidum, the National Library of Medicine of the NIH was referenced for Treponma pallidum subsp. pallidum str. PCR amplification was performed using 5'-TGGCGCGTGTGCGAATGGTG-3' (sense) (SEQ ID NO: 1) and 5'-ACAGTGCTCAAAAACGCCT-3' (antisense) (SEQ ID NO: 2) as primers, referring to Nichols, complete sequence. The PCR reaction was performed in a total volume of 25 μl with 1 μl of DNA (100 ng / μl), 12.5 μl of 2× PCR master mix (Roche), and 0.6 μM primers. The PCR reaction was performed on a TP 800 thermocycler Cycler Dice (Takara, Shiga Prefecture, Japan) under the following cycle conditions: 5 minutes at 94°C, 10 seconds at 94°C, 30 seconds at 61°C, and 20 seconds at 72°C for 40 cycles. The PCR products were examined by electrophoresis on a 2.5% Nusive 3:1 agarose gel. The results are shown in Figure 8. As indicated by the arrows in lanes 3 and 4 of Figure 8, a band specific to Treponema pallidum at 376 bp was detected as a PCR product. 【0080】 (8) Confirmation of Treponema pallidum by transmission electron microscopy Following the manual of the Electron Microscopy Section, Kawagoe Pathology Laboratory, PCL Japan Co., Ltd., tissue samples were removed from elastin-coated petri dishes after 30 passages following inoculation with Treponema pallidum on NHDF, which produces elastic fibers, and also after 30 passages. After solidification with agarose gel, secondary fixation, dehydration, and substitution were performed using phosphate buffer, 2% osmium tetroxide aqueous solution, ethanol, QY-1 (n-butylglycidyl ether), and epoxy resin. The resin-polymerized samples were then ultrathin sectioned, electron-stained, and observed under an electron microscope. The results are shown in Figure 9. Structures similar to those described in previous reports (Non-Patent Literature 4), such as granular surfaced bodies, large dense objects attached to the outer envelope, nose pieces, large balloon-shaped bodies, concentric dense bodies, and “Intestine-like” structures, were observed. 【0081】(9) Rabbit infection experiment: Tissue samples were removed from elastin-coated petri dishes after 30 passages following inoculation with Treponema pallidum on NHDF producing elastic fibers, and also after 30 passages. The undiluted solution, a 10-fold dilution, and a 100-fold dilution were each inoculated at two locations on the shaved back skin of rabbits (strain kbl: NZW, 9 weeks old at arrival, 1 week later). Subsequently, the same areas were shaved and observed every week. After 1-2 weeks, typical redness, characteristic of syphilis infection symptoms, was observed at many of the inoculation sites for the undiluted solution and the 10-fold dilution (Figure 10 left). After tissue collection six weeks later, the tissues were fixed in formalin, embedded in paraffin, and incubated with anti-Treponema pallidum polyclonal antibody (1:200, rabbit, ab20923, Abcam) at room temperature for 1 hour, followed by incubation with Alexa Fluor 555-conjugated donkey anti-rabbit IgG antibody (1:200, A31572, Invitrogen, Massachusetts, USA) at room temperature for 30 minutes. Dark-field microscopy images revealed numerous cells thought to be Treponema pallidum, described as spiral-shaped bacilli ranging from 6 to 20 μm (Figure 10, right). Figure 11 shows the results table of the rabbit infection experiment. Cells thought to be Treponema pallidum were observed at many inoculation sites for the undiluted solution, 10-fold dilution, and 100-fold dilution. 【0082】To determine which tissues were infected by the bacteria, presumably Treponema pallidum, found in rabbit infection experiments, the secondary antibody used in the immunostaining described in

[0081] above was not Alexa Fluor 555-conjugated donkey anti-rabbit IgG antibody. Instead, an anti-rabbit immunoglobulin-conjugated peroxidase-labeled polymer (Envision K4003, DAKO, Glostrup, Denmark) was used as the secondary antibody and incubated at room temperature for 1 hour. After washing the slides, a color reaction was performed using liquid 3,3'-diaminobenzidine for 5 minutes. The results, compared with the HE-stained images, are shown in Figure 12. Infection with Treponema pallidum was conspicuously observed around hair follicles and peripheral nerves, consistent with previously reported findings (Non-Patent Literature 5). In elastin immunostaining of rabbits, the anti-elastin polyclonal antibody used in Figure 1 was not used because it is a rabbit antibody. Instead, an anti-elastin monoclonal antibody (1:100, mouse, SAB4200718, Sigma-Aldrich, St. Louis, Missouri, USA) was incubated for 1 hour, and then an anti-mouse immunoglobulin-conjugated peroxidase-labeled polymer (Envision K4001, DAKO) was used as a secondary antibody and incubated at room temperature for 1 hour. After washing the slides, a color reaction was performed using liquid 3,3'-diaminobenzidine for 5 minutes. As a result, a positive image corresponding to the hair follicles was obtained. This result suggests that Treponema pallidum infects elastin not only in humans but also in rabbits (Figure 12 right). 【0083】The in vitro culture technology for Treponema pallidum according to the present invention has broad applications in the fields of medicine, pharmacy, and microbiology. Conventionally, Treponema pallidum is highly host-dependent and difficult to culture long-term in vitro, posing many challenges in the study of infection mechanisms, the development of therapeutic drugs, and vaccine design. In the present invention, by using a culture medium containing elastic fibers or components thereof, sustained in vitro culture of Treponema pallidum becomes possible, and the present invention can contribute to pathogen research, the development of novel diagnostic technologies, the research and development of therapeutic drugs and vaccines, and the improvement of public health. In particular, from this embodiment, as a development of novel diagnostic technologies, for example, if a needle is inserted into a skin lesion of a patient suspected of syphilis infection and fluid is aspirated, and as described in this application, the fluid can be placed on an elastin-coated dish, and Treponema pallidum itself can be confirmed after three days. 【0084】 All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for any purpose. However, no reference to any references, articles, publications, patents, patent publications, or patent applications cited herein constitutes, and should not be construed as, an endorsement or any suggestion that they constitute valid prior art or form part of common technical knowledge in any country of the world.

Claims

1. A method for in vitro culture of Treponema pallidum, comprising the step of culturing Treponema pallidum in a culture medium containing elastic fibers or components thereof.

2. The in vitro culture method according to claim 1, wherein the elastic fiber or its components are derived from fibroblasts, dental pulp stem cells, or vascular smooth muscle cells.

3. The in vitro culture method according to claim 1 or 2, wherein the elastic fiber has an elongation rate of 50% or more of its initial length at maximum tension, and its length is restored to 110% or less of its initial length when the external force is removed.

4. An in vitro culture method according to any one of claims 1 to 3, wherein the components of the elastic fiber are selected from the group consisting of elastin, fibrillin, collagen, proteoglycan, hyaluronic acid, fibronectin, growth factors, cytokines, and microfibrils.

5. The in vitro culture method according to any one of claims 1 to 4, wherein the elastic fiber is derived from humans, rabbits, mice, rats, dogs, cats, or pigs.

6. An in vitro culture method for Treponema pallidum, comprising the step of adding Treponema pallidum to a culture system in which cells producing elastic fibers are seeded as feeder cells, wherein the cells are selected from the group consisting of fibroblasts, dental pulp stem cells, and smooth muscle cells.

7. The in vitro culture method according to claim 6, wherein the cells are derived from humans, rabbits, mice, rats, dogs, cats, or pigs.

8. The in vitro culture method according to claim 6 or 7, wherein the cells are selected from the group consisting of human fibroblasts, mouse fibroblasts, mouse fetal fibroblasts, human umbilical cord fibroblasts, human dental pulp cells, and human vascular smooth muscle cells.

9. The in vitro culture method according to any one of claims 6 to 8, further comprising the step of adding LTBP-4 protein and / or DANCE protein to the culture system.

10. A method for in vitro culture of Treponema pallidum, comprising the step of adding Treponema pallidum to a culture dish coated with elastic fibers or components thereof and culturing it.

11. The in vitro culture method according to claim 10, wherein the components of the elastic fiber are selected from the group consisting of elastin, fibrillin, collagen, proteoglycan, hyaluronic acid, fibronectin, growth factors, cytokines, and microfibrils.

12. The in vitro culture method according to claim 10 or 11, wherein the elastic fiber is derived from humans, rabbits, mice, rats, dogs, cats, or pigs.

13. A method for screening drugs against Treponema pallidum, characterized by using a culture system prepared using an in vitro culture method defined in any one of claims 1 to 12, comprising: (a) adding a candidate drug to the culture system; (b) measuring the viability, bacterial count, or bacterial concentration of Treponema pallidum; and (c) evaluating the effectiveness of the candidate drug based on the measurement results obtained in step (b).

14. The screening method according to claim 13, wherein the candidate drug is selected from an antibacterial agent, an antiviral agent, a natural product, or a compound library.

15. The screening method according to claim 13 or 14, wherein in step (b), the viability, bacterial count, or bacterial concentration of Treponema pallidum is measured using RT-qPCR, ELISA, bacterial infectivity test, viral infectivity test, or cell proliferation assay.

16. A kit for culturing Treponema pallidum in vitro, suitable for an in vitro culture method as defined in any one of claims 1 to 12, comprising: (a) a culture device containing elastic fibers or components thereof, or cells that produce elastic fibers selected from the group consisting of fibroblasts, dental pulp stem cells, and smooth muscle cells; (b) a culture medium or buffer; and (c) instructions for use.

17. The kit according to claim 16, further comprising (d) LTBP-4 protein and / or DANCE protein.

18. A kit for screening drugs against Treponema pallidum, suitable for an in vitro culture method as defined in any one of claims 1 to 12, comprising: (a) a reaction vessel containing elastic fibers or components thereof, or cells that produce elastic fibers selected from the group consisting of fibroblasts, dental pulp stem cells, and smooth muscle cells; (b) a culture medium or buffer; and (c) a reagent for measuring the viability, bacterial count, or bacterial concentration of Treponema pallidum.

19. A method for determining whether Treponema pallidum is present in a biological sample obtained from a subject, comprising using an in vitro culture method as defined in any one of claims 1 to 12, or a kit as defined in claim 16 or 17.

Images