Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains

Abstract

The bacterium that causes syphilis, Treponema pallidum subsp. pallidum, has now been cultured in vitro continuously for periods exceeding 3 years using a system consisting of coculture with Sf1Ep rabbit epithelial cells in TpCM-2 medium and a low-oxygen environment. In addition, long-term culture of several other syphilis isolates (SS14, Mexico A, UW231B, and UW249B) and the T. pallidum subsp. endemicum Bosnia A strain has been achieved. During in vitro passage, T. pallidum subsp. pallidum exhibited a typical bacterial growth curve with logarithmic and stationary phases. Sf1Ep cells are required for sustained growth and motility; however, high initial Sf1Ep cell numbers resulted in reduced multiplication and survival. Use of Eagle’s minimal essential medium as the basal medium was not effective in sustaining growth of T. pallidum subsp. pallidum beyond the first passage, whereas CMRL 1066 or M199 supported long-term culture, confirming that additional nutrients present in these more complex basal media are required for long-term culture. T. pallidum subsp. pallidum growth was dependent upon the presence of fetal bovine serum, with 20% (vol/vol) being the optimal concentration. Omission of reactive oxygen species scavengers dithiothreitol, d-mannitol, or l-histidine did not dramatically affect survival or growth. Additionally, T. pallidum subsp. pallidum can be successfully cultured in a Brewer jar instead of a specialized low-oxygen incubator. Phosphomycin or amphotericin B can be added to the medium to aid in the prevention of bacterial or fungal contamination, respectively. These results help define the parameters of the T. pallidum subsp. pallidum culture system that are required for sustained, long-term survival and multiplication. IMPORTANCE Syphilis is caused by the bacterium Treponema pallidum subsp. pallidum. Until recently, this pathogen could only be maintained through infection of rabbits or other animals, making study of this important human pathogen challenging and costly. T. pallidum subsp. pallidum has now been successfully cultured for over 3 years in a tissue culture system using a medium called TpCM-2. Here, we further define the growth requirements of this important human pathogen, promoting a better understanding of the biology of this fastidious organism.

Genetic engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete

“…Genetic manipulation strategies, however, particularly those that satisfy Koch's molecular postulates [31], have not been available for T. pallidum. The lack of genetic tools was not overly frustrating because, until 2018 [11][12][13], T. pallidum could not even be continually propagated in vitro. Understandably, this limitation hindered any attempt aimed at genetically engineering this 1 All peptides were recognized with high confidence, with a false discovery rate set at <0.01 2 Normalized score difference between the currently selected PSM and the highest-scoring PSM for this spectrum 3 Experimental mass (MH+), calculated mass of the peptide (Theo MH+), both in Da, and mass measurement error (ΔM) in parts per million (ppm).…”

“…Before the addition of the treponemal cells, Sf1Ep cells were incubated in a 5% CO 2 atmosphere in a HeraCell 150 incubator (Thermo Fisher). For transformation, treponemes were first sub-cultured into the wells of a 24-well plate as per protocol [11][12][13]. Briefly, the day before treponemal inoculation, a 24-well plate was seeded with 2x10 4 rabbit Sf1Ep cells/well in 2.5 ml of culture media.…”

“…A better understanding of T. pallidum biology and syphilis pathogenesis would help in devising more effective measures for disease control. Recently, Edmondson et al [11][12][13] described a method to continually propagate T. pallidum in vitro using a cell culture-based system previously pioneered by Fieldsteel et al [14]. This method represented a major advancement in the field, in that it provided investigators with an alternative to the propagation of treponemal strains in laboratory rabbits.…”

Comparison of transcriptional profiles of Treponema pallidum during experimental infection of rabbits and in vitro culture: Highly similar, yet different

“…The relative similarity in transcript levels in the two environments support previous work showing that T . pallidum metabolism and growth is similar during both experimental rabbit infection and the in vitro culture system [ 22 , 23 ]. However, the observed transcriptional differences may indicate important effects of these two environments on the organisms.…”

Selective whole genome amplification as a tool to enrich specimens with lowTreponema pallidumgenomic DNA copies for whole genome sequencing

“…In addition, studies have relied primarily on strains propagated in rabbits or DNA amplified directly from clinical specimens, because T. pallidum cannot be grown on routine laboratory media. However, advances have been made with in vitro tissue culture and the propagation of T. pallidum in rabbits from cryopreserved genital lesion specimens, which may make routine culture directly from clinical specimens a possibility in the near future (11)(12)(13).…”