Name: Video: In Vitro Cultivation of Treponema pallidum in Co-Culture with Sf1Ep Cells
Uploaded: 2025-01-24T12:20:00
Duration: 5 min 21 s
Description: 103 Views. In Vitro Cultivation of Treponema pallidum in Co-Culture with Sf1Ep Cells

in vitro cultivation of treponema pallidum in co-culture with sf1ep cells

Scalable In Vitro Cultivation of T. pallidum Using Mammalian Cell Co-Culture

Treponema pallidum is a species of spiral-shaped bacteria (called spirochetes) that causes syphilis and related infections in humans and other primates. Syphilis is a serious disease with long-term effects on infected individuals, and it is estimated that over 8 million new cases of syphilis occur worldwide each year1. T. pallidum has been subdivided into three subspecies based on the diseases they cause in humans as well as minor genetic differences: subspecies pallidum (which causes the sexually transmitted disease syphilis), subspecies pertenue (yaws), and subspecies endemicum (causing bejel or endemic syphilis)2,3. T. pallidum subsp. pertenue also causes infections in baboons, chimpanzees, and other primates. A closely related organism called Treponema paraluiscuniculi (also called Treponema paraluisleporidarum) causes an infection in rabbits and hares4,5. All of these bacteria are very closely related, with greater than 98% DNA sequence identity at the genome level6,7,8. They each have a single, small circular chromosome about 1.14 million base pairs (Mb) in size. The members of this T. pallidum group are found only in association with their mammalian hosts; as such, they are obligate pathogens that are dependent on their host species for survival and growth9,10.
Attempts to culture T. pallidum in vitro began shortly after its identification by Schaudinn and Hoffman in 190511,12. However, these efforts failed to lead to consistent, reproducible growth of the organism. As a result, T. pallidum research studies required propagation of the organism through the experimental infection of laboratory animals, most commonly the rabbit13,14. In 1981, Fieldsteel et al.15 introduced a tissue culture system that promoted the multiplication of T. pallidum strains for a period of up to 2 weeks. This system involved co-culture of T. pallidum with Sf1Ep cottontail rabbit epithelial cells in a modified tissue culture medium (T. pallidum Culture Medium 1, TpCM-1) based on Eagle&#39;s Minimum Essential Medium (MEM) and 20% fetal bovine serum (FBS). Other culture conditions required were incubation at 34 &#176;C in an atmosphere containing 1.5% O2 and 5% CO29,16. In this system, T. pallidum attaches to the Sf1Ep cells and multiplies when in close association with the mammalian cell surface. Despite many subculture attempts and other modifications, the Fieldsteel et al. system failed to promote continuous in vitro growth.
In 2018, our laboratory reported that the use of a modified medium called TpCM-2 (in which Eagle&#39;s MEM was replaced with a more complex tissue culture medium, CMRL 1066) provided T. pallidum the required nutrients to permit consistent long-term culture17. To date, this modification has led to a consistent, continuous culture of at least 5 strains of T. pallidum subsp. pallidum (Nichols, SS14, Mexico A, UW231B, and UW249B) and one strain of T. pallidum subsp. endemicum (Bosnia A)18,19. As an example, the Nichols strain has now been cultured continuously in vitro for over 6 years. Thus far, attempts to culture yaws isolates (T. pallidum subsp. pertenue) or T. paraluiscuniculiin vitro have been unsuccessful18. The TpCM-2 system still requires the presence of Sf1Ep cells, low oxygen concentrations, and incubation at 34 &#176;C, making the system more complex than most bacterial culture techniques. However, this modified T. pallidum culture system has been useful for further defining the bacterium&#39;s growth requirements18, determining minimal inhibitory concentrations (MICs) of antimicrobial compounds and peptides20,21,22,23,24,25, propagating new strains from patient tissue aspirates26, isolating clonal populations of the organism27, characterizing the tprK antigenic variation system27,28, examining gene expression29, and performing mutational analysis30,31,32.
Here, we describe the current methods for cultivating T. pallidum in vitro. We hope this information will help facilitate the more widespread application of this in vitro culture technique to the improved diagnosis, treatment, and prevention of syphilis and related treponemal infections.

Author Spotlight: Advancing Syphilis Research &#8212; Innovations in Treponema pallidum Cultivation and Genetic Engineering

Procedures for In Vitro Cultivation of Treponema pallidum, the Syphilis Spirochete

In Vitro Cultivation of Treponema pallidum in Co-Culture with Sf1Ep Cells

in-vitro-cultivation-treponema-pallidum-co-culture-with-sf1ep

Research

JoVE Journal

Immunology and Infection

103 Views. In Vitro Cultivation of Treponema pallidum in Co-Culture with Sf1Ep Cells

Video: In Vitro Cultivation of Treponema pallidum in Co-Culture with Sf1Ep Cells

For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and harvest of the organisms from rabbit testes. In 2018, we described a method to continuously cultivate T. pallidumin vitro. This system utilizes co-culture with rabbit epithelial cells (Sf1Ep cells) in a serum-containing tissue culture medium called TpCM-2. The T. pallidum doubling time in culture is similar to that estimated to occur during natural infection (about 33-45 h). The organism can be cultured continuously with a standard passage time of 1 week in a low oxygen (1.5%) environment at 34 &#176;C. This article contains the protocols for culturing T. pallidum, methods for growing and maintaining the required tissue culture cells, and the technique for generating isogenic strains by limiting dilution. The ability to grow T. pallidum in vitro provides new experimental avenues to study and understand this enigmatic organism.

This protocol describes in vitro cultivation of the syphilis pathogen Treponema pallidum subsp. pallidum in co-culture with mammalian cells. The method is scalable; it can be used to produce large quantities of T. pallidum and to generate clonal cultures.

For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and ...