The bacteria Chlamydia trachomatis is responsible for one of the most common bacterial sexually transmitted infections (STIs) in the world - in 2020 alone there were 128.5 new infections worldwide. C. trachomatis can infect both men and women, and can be spread through vaginal, oral, and anal sex. While many cases of chlamydial infection are asymptomatic and easily treated with antibiotics (such as doxycycline, azithromycin, and levofloxacin), it can lead to serious problems if left untreated such as pelvic inflammatory disease, increased risk of HIV infection, and infertility.
C. trachomatis has a unique life cycle and mechanism of infection and replication: it alternates between two forms, the elementary body (EB) and reticulate body (RB). The EB is a metabolically inert form of C. trachomatis that is able to attach to and infect host cells, and when it attaches to receptors on the membrane of host cells it gets internalized into a membrane-bound vacuole called an inclusion. Since the inclusion is formed from host-cell membrane, it allows C. trachomatis to ‘hide’ from host recognition. Chlamydia actually got its name due to these inclusions - chlamydia comes from the Greek word “chlamys”, meaning “short cloak”, as scientists first observed these inclusions ‘cloaked’ around the nucleus of host cells!
Inside the inclusion, the EB is free to differentiate into the replicative body (RB) of C. trachomatis that can split into two equal daughter cells. It’s also been observed that C. trachomatis is able to re-route various host cell resources to its inclusion to aid in its replication and survival, almost ‘hacking’ its host cell. After reproducing, the RBs can re-differentiate back into the infectious EB form and are released, either by lysis of the host cell or exocytosis of the inclusion.
Figure 10 from Monahan et al: Life cycle of C. trachomatis. Essentially, dormant C. trachomatis EBs enter the host cell and ‘hide’ in an inclusion made of host-cell membrane. From there, they can ‘hack’ the system of the host cell to re-route resources to itself and replicate, before either destroying its host or ‘sneaking out’.
C. trachomatis created an ingenious system to hide from the host cell and create a safe space for itself to replicate, but this space is also very fragile. In order for the inclusion to stay intact, C. trachomatis needs to fortify it - it does so by taking advantage of the host-cell cytoskeleton, particularly actin. If the word ‘actin’ rings a bell about monomers and filaments, you’re on the right track! Actin is a highly conserved protein in many eukaryotic cells that exists in a monomeric (G-actin) form that can assemble into a filament (F-actin) form. In its filament form, actin plays a crucial role in the eukaryotic cytoskeleton.
Figure 2 from Dominguez and Homes: Structure of an actin filament (F-actin) where new monomers are added on the barbed end while monomers dissociate from the pointed end.
Actin is involved in many processes inside of eukaryotic cells, but it was shown in 2008 that C. trachomatis is able to recruit host cell actin, along with other cytoskeletal components such as intermediate filaments, to assemble around its inclusion in a protective barrier that provides structural stability.
Figure 1 from Scidmore: A membrane-bound chlamydial inclusion full of RBs and EBs, surrounded by a protective ‘cloak’ of actin and intermediate filaments.
But how does C. trachomatis actually recruit actin around itself? While it would make sense to think that C. trachomatis carries a protein or machinery to hijack actin, it turns out that C. trachomatis prefers to travel light; instead of carrying its own machinery, it hijacks a host cell protein. In the host eukaryotic cell, the only known motor proteins that interact with actin are called myosins. ‘Myosins’ are actually a huge superfamily of proteins that use ATP to “walk” along actin filaments, but we will focus on class I myosins which are involved in linking actin to membranes.
Graphic from Mechanobiology Institute: A class I myosin ‘head’ bound to actin (bottom grey filament) and ‘tail’ bound to a lipid membrane (top green membrane). The myosin walks towards the + end, or the ‘barbed’ end of the actin filament.
The researchers Cuervo et al. found in 2025 that C. trachomatis hijacks a class I myosin protein called Myosin 1C (MYO1C) to build the actin ‘cage’ around its inclusion. They built on the knowledge that C. trachomatis hijacks a different class of myosin protein (MYOII, class II) for other processes, such as ‘sneaking out’ of the host cell. Researchers also had previously identified that MYO1C is involved in stabilizing actin around the Golgi apparatus, so Cuervo et al. suspected that C. trachomatis might hijack it too.
First, the researchers used fluorescence microscopy to look at HeLa cells infected with C. trachomatis bacteria to observe the localization of endogenous MYO1C, the class I myosin of interest, and MYOII, the class II myosin that was previously shown to interact with C. trachomatis. They used a blue stain called DAPI that binds to DNA to stain the cells’ nuclei blue. The pink color is due to an engineered version of C. trachomatis that expresses a fluorescent molecule called mCherry, so the pink indicates a chlamydial inclusion. The HeLa cells had been genetically modified so that their endogenous MYO1C and MYOII had a green fluorescent protein (GFP) attached, so these myosins appear green:
Figure 1A: HeLa cells infected with C. trachomatis where nuclei are stained in blue, C. trachomatis inclusions are pink, and endogenous MYO1C (top row) or MYOII (bottom row) are green. The inset panel on the far right shows the localization of these endogenous myosins around the chlamydial inclusion.
The furthest right panels zoom in on an area around a chlamydial inclusion and only show the myosins in green. You can see for both MYO1C (top) and MYOII (bottom) that their concentration is greater near the inclusion and that there is a little ‘ring’ around the inclusion, indicating that both of these myosin proteins localize to C. trachomatis inclusions.
To further investigate the role of MYO1C in inclusion formation, Cuervo et al. inhibited host cell expression of MYO1C using multiple different methods. They used small interfering RNA (siRNA) to silence the gene before it was translated, and they also used a drug called PCIP to inhibit the ATPase and motor activity of the MYO1C protein. They performed what they call an Inclusion Forming Units (IFU) assay: essentially, C. trachomatis was introduced to either control HeLa cells or HeLa cells that underwent MYO1C inhibition treatment, then the number of chlamydial inclusions that formed was calculated:
Figures 2E and 2F: Results from IFU assays where either MYO1C RNA was silenced (left) or the ATPase and motor activity of MYO1C protein was inhibited (right). Both methods of inhibiting MYO1C showed a decrease in the number of chlamydial inclusions formed compared to control treatments.
What they observed was a decrease in the number of C. trachomatis inclusions that formed when the myo1C gene was knocked down compared to the knock down of an unrelated gene (Luc). They also saw a decrease in inclusions formed when ATPase and motor activity of MYO1C protein was inhibited. Together, this suggests that MYO1C and its activity as a class II myosin is very important for C. trachomatis’ ability to form inclusions.
To confirm that the specific interaction of MYO1C with actin is what allows the C. trachomatis inclusion to surround itself in actin, they mutated the region of MYO1C that interacts with actin (MYO1C ΔABL) and imaged the protein along with actin around a chlamydial inclusion:
Adapted from figures 3E and 3G: HeLa cells with wild type MYO1C (left) or mutated MYO1C that’s lost its ability to interact with actin (right), infected with C. trachomatis inclusions. MYO1C is green, the chlamydial inclusion is red, and actin is grey. The inset panels show the localization of MYO1C around the inclusion (top) and the organization of actin around the inclusion (bottom). The mutated MYO1C shows a decrease in MYO1C recruitment and actin organization around the inclusion.
They observed that the mutated version of MYO1C did not localize around the chlamydial inclusion as much as the wild type did, and that there was a loss of actin organization around the inclusion in cells with the mutant MYO1C. In fact, there doesn’t appear to be a visible actin ‘cage’ around the inclusion at all with the mutant MYO1C, suggesting that the interaction of MYO1C with actin is essential for the formation of the actin cage around C. trachomatis inclusions!
So it was clear that MYO1C is very important for the formation of the actin cage, but the researchers wondered whether MYO1C alone was enough to form that cage. In an in vitro experiment, Cuervo et al. simulated an inclusion with an artificial vesicle, then added varying concentrations of MYO1C and ATP, as well as the inhibitor of MYO1C activity (PCIP):
Figure 4C: The top row shows the vesicle in red, which is present in all samples. The bottom rows show different views of the actin cage (green) around the vesicle. When MYO1C and ATP are present, an actin cage can form around the vesicle (middle columns), but when MYO1C is inhibited by PCIP, no actin cage forms (right column).
They observed that adding MYO1C and ATP was enough to induce actin cage formation around the vesicle, that adding more MYO1C and ATP allowed a more robust actin cage to form, and that adding a MYO1C inhibitor prevented actin cage formation entirely. This demonstrates in vitro that MYO1C alone is sufficient to form an actin cage, and gives support to the proposal that MYO1C plays a vital role in C. trachomatis inclusion formation.
In short, the researchers Cuervo et al. showed that C. trachomatis hijacks a class I myosin protein called MYO1C and uses its interaction with actin filaments to recruit a protective ‘cage’ of actin around its inclusion inside of host cells. Actin cage formation is an absolutely vital part of the chlamydial lifecycle as without proper inclusion stability, C. trachomatis would be discovered by the host cell and destroyed before it got a chance to replicate. However, rather than carrying around the machinery to build its protective cage, C. trachomatis instead hijacks machinery already in its host cell, a true embodiment of the saying “work smarter, not harder”.
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