Plasmid Regulation: The Superpower of Chlamydia trachomatis

By: Yacoba Zwennes

We have all most likely been told at one point by our doctor to be wary of chlamydia, a sexually transmitted disease caused by the bacterium Chlamydia trachomatis. When detected early, chlamydia can be treated fairly easily with antibiotics. Because of this, chlamydia may seem to be just as big of a deal as coming down with a cold to some. What many people do not know, however, is that some strains of C. trachomatis also cause trachoma, a disease that leads to blindness. Trachoma is classified as a Neglected Tropical Disease, and it has caused blindness in about 1.8 million people worldwide, making the disease the leading infectious cause of blindness in the world. Since trachoma – and chlamydia, for that matter – are very easily transmittable and can cause severe physiological complications I am sure we can all agree that the development of a vaccine to protect against these diseases is in order! Of course, to be able to develop one, we must first understand the characteristics of C. trachomatis and how it manages to have so much power in our bodies.

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C. trachomatis are Gram-negative bacteria. They always live in a host cell and may be pathogenic or live in symbiosis with the cell (Elwell et al. 2016). The human host is a highly hostile environment that is ready for defense with an arsenal of white blood cells that target foreign bodies and other mechanisms. This means that the bacteria must find ways to bypass the human host’s immune system in order to survive. The ability of the bacteria to complete their life cycle – which involves entering, moving around and exiting a host cell to infect other ones - is paramount to the success of the bacteria’s survival. The invade the host cell as small infectious cells known as elementary bodies. These bodies are metabolically inert (Yang et al. 2015), meaning that they cannot replicate or reproduce.They attach themselves to the host cell and eventually enter it. Upon entry, they are sequestered in a vacuole, called an inclusion. The inclusion gives the bacteria great protection because it quickly modifies to avoid fusing with the host cell’s lysozyme and thus potential degradation. In the inclusion, the elementary bodies differentiate into noninfectious reticulate bodies; they are much larger cells in comparison to the elementary bodies, and they are also metabolically active (they can replicate) (Yang et al. 2015). These again differentiate back into elementary bodies, after which the host cell lyses to release them to infect other cells. The entire life cycle can occur in 36 to 48 hours.

Computer-generated image of C. trachomatis inclusion. The red cells are reticulate bodies in the inclusion. Source.

Another way the bacteria are able to survive is by hijacking the host cell’s cytoskeleton. The elemental bodies secrete a translocated actin recruiting protein (TARP), which facilitates a signaling cascade to mobilize actin to the entry site. The actin polymerizes into filaments around the inclusion. Additionally, the inclusion is also moved along microtubules to the microtubule-organizing center so that it is able to position itself on the Golgi apparatus to obtain cholesterol. This helps for the inclusion to expand to accommodate the growth of the bacteria. Eventually, the inclusion (and the host cell) must lyse, or break down,so that the elementary bodies can infect other cells. This means that they must find their way out of the web of cytoskeletal filaments they have built around the inclusion. If they do not escape, their life cycle halts and the virulence of the infection is reduced! This is a great advantage since a less widespread infection is easier to treat and less likely to lead to worse complications. It also means that scientists could potentially work towards a vaccine that targets this part of the life cycle.

C. trachomatis life cycle. EB stands for elementary bodies, and RB stands for reticulate bodies. Source.

A study was conducted by Yang et al. which aimed to determine how the successful exit of C. trachomatis from host cells is genetically regulated (2015). Previous research had proven that the bacteria secrete a chlamydial protease activity factor (CPAF) to cleave vimentin (a protein in the cytoskeleton) which causes the relaxation of the membrane of the inclusion so that it can expand. However, there was a lack of previous research done to explain how the bacteria are able to destroy the web of actin around them in order to exit the cell. Thus, this study aimed to present the clever qualities of C. trachomatis are so clever at doing this.

The researchers first attempted to determine the role of the C. trachomatis plasmid – a circular DNA strand in bacteria that can replicate independently of its chromosome – in host to cell interactions. To do this, they tested for plaque-forming efficiency in C. trachomatis cells under various conditions (Yang et al. 2015).On an agar plate covered with an opaque field of bacteria, plaque is a clear region that indicates the inhibition of the bacterial cells by a virus or an antibiotic. Plasmids give bacteria resistance to antibiotics, and may also provide virulence. Testing for plaque-forming efficiency would tell whether the plasmids help C. trachomatis to grow intracellularly by making them resistant to certain conditions in the host cell, or by making them infectious in the cell. The scientists used L929 cells, a cell line from mice. They infected the L929 cells with either wild-type C. trachomatis cells (L2), plasmid-deficient cells, (L2R) or L2R cells that had been transformed into L2 cells with a shuttle vector known as pBRCT (L2RpBRCT). The L2R cells served as an extra control in testing for the role of the plasmid. A multiplicity of infection (MOI), which is the ratio of the infectious agent, was 0.0002.

FIG 1 Chlamydial plaque-forming efficiency is plasmid and pgp4 dependent. (A) PFU on L929 cell monolayers infected with a multiplicity of infection (MOI) of 0.0002 of wild-type (L2), plasmidless (L2R), or L2RpBRCT cells. (B) One-step growth curves were prepared for L929 cells infected with L2, L2R, and L2RpBRCT cells at an MOI of 5. Recoverable IFU were determined at various times post-infection. Each time point represents the mean rIFU from triplicate cultures.

There was a significant reduction in the number of plaques formed in L2R cells, as seen in Figure 1A. The L2R cells that were transformed into L2 cells with pBRCT also yielded a higher number of plaques, similar to that of the wild-type cells. It was important to look at the growth rate of all three C. trachomatis cell types because it could play a factor in their ability to form plaques. Figure 1B shows that all three strains grew at an identical rate (rIFU indicates recoverable inclusion forming unit). This means that the plasmid did not play a role in the cells’ ability to grow in the host cell. Thus, the scientists concluded that the varied plaque-forming efficiencies seen in L2, L2R and transformed L2R cells were more likely due to the fact that the plasmid may have a part to play in the lysis of infected cells. This lysis causes the release of infectious elementary bodies so they begin a new infection cycle; this is what would result in the formation of plaques. Further experimentation also determined that the plasmid gene that controls cell lysis is pgp4.

Following this portion of the experiment, the scientists aimed to determine exactly how plasmids mediate the exit of C. trachomatis elementary bodies from the inclusion and the host cell. They had previously determined that cells that were plasmid-deficient – that is L2R cells and L2RpΔpgp4 cells (cells without the plasmid gene that controls cell lysis) – were not able to exit their host cells. Thus, they hypothesized that if these cells could not escape, they should be able to ‘rescue’ them by treating them with actin and vimentin inhibitors. First, they tested their hypothesis on actin polymerization around the inclusions. They injected human cervical epithelial cells (HeLa 229) with the various C. trachomatis cell types and incubated them for 48 hours. They replaced the medium around the cells with a medium containing latrunculin B, an actin inhibitor. Some cells were also treated with dimethyl sulfoxide (DMSO) as a form of control. They incubated the cells for another 8 hours and performed an assay on the culture medium to test for inclusion-forming units they could recover (rIFU).

FIG 3 L2R and L2RpΔpgp4 strains can exit cells when treated with latrunculin B. latrunculin B. A indicates actin, I, the inclusion, and N, the nucleus. , (A) Note the assembly of actin at the inclusion membrane. (B) The number of actin-positive inclusions were quantified at various times postinfection. (C) The number of inclusion-forming units (rIFUs) recovered are shown here. Adapted from original paper.

In the Figure 3A above, actin polymerization around the inclusions is observed. In 3C, it is evident that more inclusions from plasmid-deficient C. trachomatis cells treated with Lantruculin B were recovered. However, fewer inclusions were recovered from cells with plasmids (L2 and L2RpBRCT) that were treated with latrunculin B than from those treated with DMSO, indicating that they were probably able to rescue themselves and lyse before the aid of the actin inhibitor.

This same experiment was repeated with Withaferin A, a vimentin inhibitor. The actin polymers remained intact while vimentin polymerization was inhibited.

FIG 4 Vimentin is not essential for chlamydial exit from host cells. V indicates vimentin, I, the inclusion, and N, the nucleus. (B) Assembly of vimentin filaments increased with time. (C) The number of inclusion forming units (rIFUs) are shown here. Adapted from paper.

In comparing this experiment to that of the former, there was a relatively low rIFU in cells treated with Withaferin A. This means that even though vimentin polymerization was inhibited, the inclusions still could not be rescued as actin had still polymerized around them. Thus, the scientists concluded that plasmids indeed regulate the exit of elementary bodies from the host cell, and they do this by altering facilitating actin disposition from inclusions.

In conclusion, a C. trachomatis infection in a human cell cannot spread to other cells if the inclusion cannot lyse. Since this experiment showed that plasmids and the pgp4 gene regulate the lysis of the inclusion in the bacteria, future experiments could target these factors in order to avoid the spread of the infection. Perhaps, they could determine the protein that results from the gene and develop a drug to alter it; this way, lysis can never occur, and the bacteria eventually die. I wondered why the scientists did not conduct the first experiment to examine host and cell interactions in Hela 229 cells too; I thought that would have given a more accurate representation of how plasmids play a role in human cells. Nevertheless, I’m excited to see how their findings will be beneficial in the ongoing research to develop a vaccine against C. trachomatis!

References

Elwell, C., Mirrashidi, K., & Engel, J. (2016). Chlamydia cell biology and pathogenesis. Nature Reviews Microbiology,14(6), 385-400. doi:10.1038/nrmicro.2016.30

Yang, C., Starr, T., Song, L., Carlson, J. H., Sturdevant, G. L., Beare, P. A., Caldwell, H. D. (2015). Chlamydial Lytic Exit from Host Cells is Plasmid Regulated. MBio,6(6), 1-9. doi:https://doi.org/10.1128/mBio.01648-15