🦠 microbiology

Slow-growing human cell lines are a suitable alternative to rabbit Sf1Ep cells for in vitro cultivation of Treponema pallidum

This study establishes that slow-growing human epithelial cell lines, specifically CAL-39 and HepG2, serve as viable alternatives to rabbit Sf1Ep cells for the long-term in vitro cultivation of *Treponema pallidum*, enabling new insights into host-pathogen interactions and facilitating future research into syphilis pathogenesis.

Original authors: Capuccini, K., Govender, D., Goulding, D., Kyanya, C., Pasricha, S., Giacani, L., Thomson, N. R., Grillova, L.

Published 2026-02-20

📖 5 min read🧠 Deep dive

CC BY 4.0

Original authors: Capuccini, K., Govender, D., Goulding, D., Kyanya, C., Pasricha, S., Giacani, L., Thomson, N. R., Grillova, L.

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

The Great Syphilis Breakthrough: Finding a Human "Home" for a Stubborn Bacterium

Imagine trying to grow a very picky, slow-moving plant. For decades, scientists could only grow this plant in a specific type of rabbit pot. It worked, but it wasn't perfect. The plant was a bacterium called Treponema pallidum, the culprit behind syphilis. Because it only grew well in rabbit cells, studying how it actually interacts with humans was like trying to understand how a human feels by watching a hamster run on a wheel. It gave clues, but it wasn't the real thing.

This paper is about scientists finally finding the right human pots to grow this bacterium, opening a new door to understanding and curing syphilis.

Here is the story of how they did it, broken down simply:

1. The Problem: The "Rabbit Pot" Limitation

For a long time, the only way to grow T. pallidum in a lab was to use a specific rabbit skin cell line (called Sf1Ep).

The Analogy: Think of the rabbit cells as a very specific, high-end greenhouse. The bacteria loved it there. But because it was a rabbit greenhouse, scientists couldn't easily study how the bacteria would act inside a human body. They needed a human greenhouse.
The Challenge: The bacteria are incredibly slow growers (they take over 35 hours just to divide once) and they hate oxygen. Most human cells in a lab grow too fast and too aggressively, choking out the slow-moving bacteria before they can settle in.

2. The Search: Finding the Right "Human Neighbors"

The scientists decided to test six different types of human cells to see which ones could be good neighbors for the bacteria. They looked for human cells that were:

Slow growers: Like the bacteria, they needed to take their time.
Oxygen-tolerant: They needed to be comfortable in low-oxygen environments.
From relevant places: They picked cells from the vulva, liver, placenta, and kidney, because syphilis can infect all these areas.

3. The Discovery: Two New "Human Homes"

Out of the six human cell types they tested, two turned out to be perfect matches:

CAL-39: A cell line from the vulva.
HepG2: A cell line from the liver.

The Result: When the bacteria were put in these human cells, they didn't just survive; they thrived! They grew just as well as they did in the rabbit cells.

The Metaphor: Imagine the bacteria were like shy guests at a party. In the rabbit house, they were comfortable. In most human houses, they were too crowded and left early. But in the CAL-39 and HepG2 houses, the guests felt right at home, ate well, and stayed for the whole party.

4. The Secret Sauce: "Slow and Steady" Wins the Race

The paper found a fascinating pattern: The slower the human cell grew, the better the bacteria did.

The Analogy: Think of the human cells and the bacteria as roommates sharing a small apartment (the culture dish).
- If the human roommate is a "fast mover" (like the HEK-293 kidney cells), they run around, eat all the food, and change the atmosphere too quickly. The bacteria get stressed and die.
- If the human roommate is a "slow mover" (like CAL-39 and HepG2), they move calmly, don't hog the food, and keep the environment stable. This allows the slow-growing bacteria to survive and multiply.

5. The New Camera: Watching the Bacteria Dance

The most exciting part of this study wasn't just growing the bacteria; it was watching them for the first time using live cameras.

The Discovery: The scientists saw that the bacteria interact with human cells in two distinct ways:
1. The Crawler: The bacteria glide along the surface of the human cell, wiggling like a snake. This is how they move across tissues.
2. The Anchor: The bacteria stick to the cell with just one end (like a flagpole) and spin around, holding on tight.
Why it matters: They saw these behaviors in the successful human models (CAL-39 and HepG2) and the rabbit model. But in the failed human models, the bacteria eventually stopped moving and just floated away. This tells us that to study how syphilis invades the body, we need the models where the bacteria can actually "craw" and "anchor."

Why This Changes Everything

This paper is a game-changer for three main reasons:

Human Relevance: We can now study syphilis in human cells, not rabbit cells. This means the answers we find will actually apply to humans.
Vaccine Hope: To make a vaccine, we need to know exactly how the bacteria stick to and invade human cells. Now that we can watch them do this in a human dish, we can find the "locks" on the door to build a "key" (vaccine) to stop them.
Drug Testing: It's easier and cheaper to test new antibiotics in a human cell dish than in live rabbits.

In a nutshell: Scientists finally found the right human "neighborhoods" where the syphilis bacteria feel at home. By watching them live and move in these new homes, we are one giant step closer to understanding, treating, and eventually eradicating this ancient disease.

1. Problem Statement

The Challenge: Treponema pallidum subsp. pallidum, the causative agent of syphilis, has historically been impossible to culture continuously in vitro. Research has relied on in vivo rabbit models, which are costly, ethically complex, and lack human-specific relevance.
Current Limitation: In 2018, a breakthrough allowed long-term co-culture of T. pallidum with rabbit skin epithelial cells (Sf1Ep). However, this system uses non-human cells, limiting the study of human-specific host-pathogen interactions, vaccine target discovery, and clinical relevance.
The Gap: Previous attempts to use human cell lines failed, largely because researchers selected fast-growing human lines (doubling time <20 hours). It was hypothesized that the rapid proliferation of these cells outcompetes the slow-growing bacterium (generation time >35 hours) for nutrients or causes rapid medium acidification, leading to bacterial death.

2. Methodology

The study employed a systematic approach to identify and validate human cell lines capable of supporting T. pallidum growth under microaerophilic conditions (1.5% O₂).

Cell Line Selection: Six human epithelial/epithelial-like cell lines were selected from the Cell Model Passports database, prioritizing:
- Slow growth rates: Doubling times >30 hours (mimicking Sf1Ep).
- Hypoxia tolerance: Ability to survive in low-oxygen environments.
- Tissue diversity: Vulva (CAL-39, SW954), Placenta (JAR, JEG-3), Liver (HepG2), and Kidney (HEK-293).
- Control: HEK-293 (fast-growing, <20h) was included as a negative comparator.
Cultivation Protocol:
- Cells were cultured in T. pallidum Cultivation Medium (TPCM-2) at 1.5% O₂.
- The SS14 strain of T. pallidum (engineered with GFP) was used.
- Experiments ran for up to 4 weeks with weekly passaging, and a separate 10-day longitudinal study monitored daily growth dynamics.
Quantification & Imaging:
- Growth Metrics: Total bacterial counts (attached to monolayer + floating in medium) and motility percentages were assessed via dark-field microscopy.
- Live-Cell Imaging: A specialized Leica THUNDER Imager with a gas-tight chamber (1.5% O₂, 34°C) was used to visualize real-time bacterial behavior.
- Electron Microscopy: Scanning (SEM) and Transmission (TEM) electron microscopy were used to characterize bacterial attachment structures.

3. Key Contributions

Establishment of Human Co-Culture Models: The paper successfully identifies two human cell lines, CAL-39 (vulvar squamous cell carcinoma) and HepG2 (hepatocellular carcinoma), that support sustained T. pallidum growth comparable to the gold-standard rabbit Sf1Ep cells.
Validation of the "Slow-Growth" Hypothesis: The study confirms that slow-growing human cell lines (doubling time >30h) are superior hosts for T. pallidum, likely due to reduced competition for nutrients and delayed medium acidification compared to fast-growing lines.
Discovery of Novel Interaction Behaviors: Using the first live-cell imaging platform for T. pallidum, the authors defined two distinct modes of host-cell interaction:
- Surface-associated crawling: Dynamic movement along the host membrane using an undulating waveform.
- Stable single-polar attachment: A stationary state where the bacterium anchors by one pole while the other remains free.
Differentiation of Permissive vs. Non-Permissive Environments: The study demonstrates that while T. pallidum can initially attach to various human cells, only permissive lines (CAL-39, HepG2, Sf1Ep) sustain these specific motility behaviors and bacterial replication over time.

4. Key Results

Growth Success: CAL-39 and HepG2 supported T. pallidum replication and long-term passage (up to 11 and 6 weeks, respectively, at the time of writing) with total bacterial yields statistically indistinguishable from Sf1Ep controls ( $p > 0.05$ ).
Failure of Other Lines:
- Fast-growing lines (HEK-293) and other slow-growing lines (JAR, JEG-3, SW954) failed to sustain growth; bacterial counts dropped below the threshold for passaging within 1–3 weeks.
- Non-supporting lines showed a higher proportion of non-attached (floating) bacteria and a loss of motility over time.
Motility Dynamics:
- In supporting lines (CAL-39, HepG2, Sf1Ep), motility remained high (~90%) throughout the 10-day observation period.
- In non-supporting lines, motility declined rapidly, and the "crawling" behavior disappeared, leaving only non-motile or single-polar attached cells.
Correlation with Host Division Time: A positive trend was observed where longer host cell division times correlated with higher T. pallidum yield (slope = 0.034 log₁₀ units/hour), though this did not reach strict statistical significance ( $p=0.147$ ) due to the limited sample size of cell lines.
Culture Duration: While Sf1Ep cultures remained stable beyond 10 days, CAL-39 and HepG2 cultures showed signs of acidification and bacterial decline after Day 9, suggesting a standard 7-day culture period is optimal for these human lines.

5. Significance

Clinical Relevance: Replacing rabbit cells with human cell lines (specifically vulvar and liver origins) provides a more physiologically relevant model for studying syphilis pathogenesis, as it reflects the actual human tissues the bacterium infects.
Mechanistic Insights: The ability to perform live imaging in human co-cultures allows for the first time the detailed observation of how T. pallidum invades and interacts with human cells, revealing specific motility mechanisms (crawling vs. attachment) previously unobserved.
Vaccine and Drug Development: These robust human models enable high-throughput screening for antibiotics and facilitate the identification of surface proteins essential for human cell attachment, accelerating vaccine target discovery.
Accessibility: By utilizing widely available human cell lines (CAL-39 and HepG2), this work lowers the barrier to entry for other laboratories, potentially democratizing T. pallidum research and moving the field away from reliance on rabbit models.