TNTs: The Chlamydia Transmission Route 66

By: Meredith Becher ‘23, Maddy Sewell ‘24, Hiba Malik ‘24

Background

Chlamydia trachomatis is a bacterial micro species that can cause several types of infections in humans, including sexually transmitted infections (STIs), eye infections, and respiratory infections. It is the most common bacterial STI in the world, with an estimated 127 million new cases each year. Chlamydia infections are typically spread through sexual contact with an infected partner, and can be transmitted through vaginal, anal, or oral sex. Symptoms of chlamydia may include pain during urination, discharge from the penis or vagina, and pain or bleeding during sex. However, many people with chlamydia do not experience any symptoms at all, which can lead to the infection going undetected and untreated. If left untreated, chlamydia infections can lead to serious health problems, including pelvic inflammatory disease (PID) in women, which can cause infertility and chronic pelvic pain. In men, chlamydia can cause epididymitis, an inflammation of the epididymis, which can lead to infertility. Chlamydia can also increase the risk of contracting HIV and can be transmitted from mother to child during childbirth, potentially causing pneumonia or conjunctivitis in the newborn.

Figure 1. Image of Chlamydia Trachomatis bacteria.

Chlamydia trachomatis is a small, gram-negative bacterium that has a unique anatomy that adapts for its intracellular lifestyle. This lifestyle allows it to evade the host immune system and establish a chronic infection in the host body. Chlamydia trachomatis’s microstructures include five major features: the outer membrane, the peptidoglycan layer, the elementary body, the reticulate body, and the inclusion body. The outer membrane is composed of lipopolysaccharides (LPS), which are thought to help the bacteria evade the host immune system. The outer membrane also contains several proteins that are important for bacterial attachment and invasion of host cells. Beneath the outer membrane, Chlamydia trachomatis has a thin layer of peptidoglycan. This layer provides some structural support to the bacterium. The elementary body (EB) is the infectious form of Chlamydia trachomatis. It is a small, spherical structure that is surrounded by a rigid outer membrane. The EB is able to survive outside of host cells and is resistant to environmental stressors such as heat, cold, and desiccation. The reticulate body is the replicative form of Chlamydia trachomatis. It is larger and more irregular in shape than the elementary body and is found within the cytoplasm of infected host cells. The RB is able to divide by binary fission, a form of asexual reproduction, eventually forming a large inclusion body that contains multiple bacteria. Lastly, the inclusion body is a large, distinct structure that is visible within the cytoplasm of infected host cells. It contains multiple reticulate bodies and is thought to be the site of bacterial replication.

Chlamydia trachomatis’ structure is an intracellular bacterium meaning that it cannot survive or replicate outside of a host cell. However, the bacterium is able to alternate between the infectious elementary body and the replicative reticulate body, allowing it to survive and replicate within the host cell. During the bacterium’s infectious cycle, chlamydia uses two distinct forms of the bacteria: the elementary body (EB) and the reticulate body (RB). The EB is the infectious form of the bacteria, which is able to enter host cells and establish an infection. Once inside the host cell, the EB differentiates into the RB, which is the replicative form of the bacteria. The RB divides by binary fission, which is where the bacteria are able to take up nutrients from the host cell and use them to synthesize new bacterial components (including DNA, RNA, and proteins), eventually forming a large inclusion body that contains multiple bacteria. After several days of replication, the RB differentiates back into the EB, which is then released from the host cell to infect new cells.

Figure 2. Image of the Chlamydia trachomatis life cycle.

Even though there has been a lot of information found on this infectious bacteria, there is still a vast amount of unknowns that researchers are trying to find to this day. The mechanism that allows EBs to enter the cells is still under study. For example, in order to enter the cell, EBs must interact with heparan sulfate-like glycosaminoflycans (GAGs). These are made of the disaccharide units Heparin and chondroitin sulfate. It has been found that heparin can block chlamydia from entering the cell, which leads researchers to wonder how chlamydia can still infect cells. From the information already known, and through experimental research, researchers have started speculating that TNTs (tunneling nanotubes) play a key role in Chlamydia trachomatis’s strong persistence and assist in the spread of this bacteria. In the particular research article based off this blogpost, the researchers were interested in knowing more about the different pathways in which this bacteria infects host cells, and what key components allow it to travel between different cells rapidly. The important findings from the experimental research found evidence that chlamydia cell to cell transmission was in fact mediated by the tunnleing nanotubes and that dyenin was a component that assists the bacteria in cell infection.

Methods and Materials
The authors employed a variety of methods to obtain the data necessary for analysis. The cells used in this study were human embryonic kiney (HEK) 293 cells (HEK293). These cells were also infected with Green Flourescent Protein (GFP)-expressing Chlamydia trachomatis LGV2, a strain of chlamydia. To control for external bacteria entry, the cells were kept in a medium that contained 5 ug/mL heparin. As mentioned previously, this prevents the bacteria from entering the cell from the outside.

The authors began their experiments through observing cells through live-cell imaging (LCI) but found that they were unable to see whether or not there was bacteria within the TNTs. To overcome this problem, the researchers switched to a higher resolution immunofluorescene microscopy. Once they did this, they were able to see bacteria throughout the cell and within the TNTs (Fig. 3). The researchers took data and photos at two time points: 12 hours post infection (hpi) and 48 hpi. Image types taken included phase contrast and fluorescent images. In the images, chlamydia fluoresces green due to the GFP and DNA fluoresces blue because the researchers stained the cells with DAPI.

To compare the presence of TNTs in chlamydia infected cells to those not infected, the researchers also had a control treatment of uninfected cells. TNT formation within these cells was also observed using the same higher resolution immunofluorescence microscopy. All treatments were also fixed using paraformaldehyde and measured for length and shape.

 

Figure 3. Shown above is Figure 2 from the research article displaying the presence of Chlamydia within the cytoplasm and interconnecting TNT’s of infected cells using immunoflueorescence and phase-contrast microscopy.

Figure Analysis

Figure 2, from the study (seen above), shows a working model of the flow cytometry results of the tunneling nanotube (TNT) formation between donor and acceptor cells. The figure illustrates how intracellular bacterial reticulate bodies (RBS) of Chlamydia trachomatis can pass through TNTs that are formed between infected and uninfected partner cells. The small image in the upper right hand corner shows interconnecting TNTS in the non infected control HEK293 cells. It can be seen that there is a drastic decrease in the number of green fluorescent cells in analyzed donor/acceptor pairs, alongside a corresponding increase in the number of double fluorescent cells. This suggests that the TNTs formed between the infected and uninfected cells allowing for the spread of Chlamydia trachomatis pathogen and its DNA to the uninfected cells.

Implications and Future Studies

The implications that can be deduced from this study are that nanotubes (TNTs) possibly play an important role when it comes to cell to cell communication and may facilitate the spread of certain pathogens, such as Chlamydia trachomatis, from one cell to another. The findings suggest that TNTs may be used to explain the observed rapid spread of chlamydia in multicellular tissues. Additionally, the study suggests that further research is necessary to determine the significance of this pathway in vivo and to explore whether it can be pharmacologically targeted. Future studies would help us find out more information by investigating whether or not chlamydia has an impact of TNT formation and function in infected malignant tissues, clarify the consequences of the cell death of chlamydia infected cells for the formation of TNTs and for the efficiency of chlamydial dissemination, and finally, to further study the direct effects of cell to cell transmission of chlamydia.


About the Authors:


Hiba Malik '24 is a junior at Mount Holyoke College that is currently pursuing a major in biological sciences and is on the pre-dental track. When she has some free time from school she enjoys to hangout with friends and go on road trips!


Maddy Sewell '24 is a junior at Mount Holyoke College that is currently pursuing a major in biological sciences and a minor in Psychology on the pre-medical track. She is excited to be working as an OR assistant this summer at her local hospital. When she has some free time from school she enjoys participating on the swim and dive team and hanging out with friends.


Meredith Becher '23 is a senior at Mount Holyoke college majoring in Biological Sciences. She is excited to start her new job at the UMASS Medical School this summer. With the free time she will have from no homework, she hopes to catch up on Netflix shows, read books, paint, and spend time with her friends and boyfriend.