Chlamydia trachomatis: The Sneaky Takeover

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By: Tanya Karagiannis and Emma Li

Chlamydia trachomatis is a bacterium that causes millions of women each year to develop ectopic pregnancies and pelvic inflammatory disease (PID). In 2012, there were a reported 3,695.5 C. trachomatis infections in women between the ages of 20 and 24. However, this superbug can infect you without causing any visible symptoms! Symptoms that are connected to ectopic pregnancies and PID may possibly occur only several weeks post-infection. If left untreated, chlamydia (the sexually transmitted disease resulting from C. trachomatis infection) can be passed from a pregnant mother to her baby, causing conjunctivitis (an eye infection) and possibly pneumonia in the baby. In men, symptoms can include a burning sensation while urinating, pain and swelling in the testicles, and discharge from the penis. This sneaky pathogen is capable of surviving and propagating inside host cells to maintain an intracellular lifestyle.

Dynamin cleaves budding vesicles from the membrane of organelles.

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C. trachomatis first forms metabolically dormant elementary bodies (EBs) that can then differentiate into non-infectious reticulate bodies (RBs) enclosed in an inclusion (membrane-bound vacuole).  Finally RBs re-differentiate into EBs to be released from the host cell. EBs - due to their rigid outer membrane - are able to resist destruction by inhibiting endosome and lysosome fusion, and are able to bind to host cell surface proteins for infection. Contrary to conventional intuition, this microbe is actually infectious when it is in its EB form to infect host cells, and is metabolically active and replicating within the host cell in its RB form! We will take a further look into how dynamin, a GTPase responsible for the scission of budding vesicles from organelles, is necessary for homotypic fusion (fusion of multiple inclusions into one, large inclusion) of C. trachomatis inclusions and lipid acquisition for the pathogen’s development.

To determine host cells genes of importance in C. trachomatis infectivity, Gurumurthy et al. had to first identify target genes to generate a short hairpin RNA (shRNA) library. The shRNA library allowed for the researcher to “knock out” target gene function. A specific criterion of genes for this shRNA library included candidates that encode for proteins found on the surface of host cells, and molecules that were important in intracellular trafficking. After successful construction of the shRNA library, only some shRNAs were utilized to produce particles to be transduced into host HeLa cells (immortal - continuously dividing human cells). This allowed for successful knockdown (KD) - basically “knock out” - of targeted genes. These final constructs were then used for C. trachomatis infectivity screening.

KD Dyn-1 and KD Dyn-2 (black) reduce
C. trachomatis infectivity when compared
to control (grey).

Out of this screening, it was determined that KD dynamin 1 (Dyn-1) and KD dynamin 2 (Dyn-2) reduced the number of infectious C. trachomatis progeny. To further uncover the role of dynamin in the pathogen’s infectivity, an inhibitor of dynamin, dynasore, was used to inhibit the GTPase activity of both Dyn-1 and Dyn-2. The investigators found that treating HeLa cells with dynasore pre- and post- C. trachomatis infection reduced the pathogen’s infectivity. This was most effective when cells were treated with dynasore 1-2 hours pre-infection, thus determining that dynamin was important early in C. trachomatis infection. Then, to see if dynamin was important in host cell entry, cells with either dynasore treatment or dynamin-specific shRNAs were treated with the pathogen. A comparison between percentages of internalized EBs with total EBs were made. Compared to control cells, cells with dynamin inhibition did not show any difference in the quantity of C. trachomatis entry into the host cell. This concludes that while dynamin is necessary for C. trachomatis development, it is not vital for C. trachomatis internalization!

Dynasore investigating the role of dynamin in C. trachomatis infectivity! Source

Though dynamin inhibition caused lowered infectivity of C. trachomatis in host cells, the researchers saw that there was a higher percentage of smaller-sized C. trachomatis inclusions. Also, dynamin deficient cells exhibited smaller inclusion size than they normally would. It is a known property of individual inclusions to homotypically fuse early in C. trachomatis’s developmental cycle. With the evidence presented here, individual inclusions were assumed to not have been able to fuse to form a single, large inclusion within a cell. This was re-affirmed when the authors performed an experiment where they were able to compare and observe cells with and without dynamin inhibition through fluorescence microscopy. Cells that were not treated with dynasore possessed one, large C. trachomatis inclusion per cell. Cells that were treated with dynasore possessed multiple small, non-fused inclusions in each cell. To further evaluate this observation, transmission electron microscopy (TEM) was used to view infected cells that had been treated by dynasore 30 h post-infection. Dynamin-inhibited cells (again) possessed multiple, smaller inclusions. It can be concluded from these extensive experiments and results that functional dynamin is essential for homotypic fusion of multiple inclusions in each C. trachomatis-infected cell! Also, to obtain a glimpse into what part of the developmental cycle of C. trachomatis dynamin may effect, the authors found through TEM of the aforementioned experiment that cells treated with dynasore possessed more RBs and abnormal bodies, and less EBs. Thus, it can be determined that when re-differentiation between RBs and EBs occurs, functional dynamin seems to be necessary to facilitate this conversion.

Untreated cells possessed a single, homotypically fused C. trachomatis inclusion.

Cells treated with dynasore possessed multiple and small, non-fused inclusions per cell.

After this discovery, the researchers were curious as to how dynamin plays a role in homotypic fusion. As mentioned before, dynamin is a GTPase that releases vesicles from different organelles such as the Golgi apparatus. Vesicles that bud off of the Golgi complex contain sphingomyelin, a type of lipid that is essential in the fusion of multiple inclusions. Since inhibiting dynamin would keep vesicles from budding from the Golgi apparatus, Gurumurthy et al. then realized that the inability of C. trachomatis inclusions to homotypically fuse could possibly be due to the lack of lipid availability to facilitate fusion.

In order to confirm this idea, Gurumurthy et al. infected HeLa cells with C. trachomatis and treated the samples with and without dynasore for 30 hours. They tagged the lipids to show their presence and location within the cells. In the untreated cells, the inclusions absorbed lipids overtime. Interestingly, the cells treated with dynasore were shown to have lipids concentrated in one area outside of the inclusions! This demonstrates that inhibiting dynamin prevents vesicles from breaking off of the Golgi and prevents lipids from being transported and taken up by inclusions.

Untreated cells possessed lipids within inclusion. Dynasore-treated

cells possessed lipids outside inclusions.

During infection, C. trachomatis is believed to fragment the Golgi complex to promote development. Gurumurthy et al. wondered whether the retention of the Golgi’s structure is also involved in reducing infectivity and lipid transport into inclusions. Similarly, HeLa cells were infected with C. trachomatis for 30 minutes with and without dynasore. This time, the Golgi apparatus and C. trachomatis inclusions were detected in the cells using antibodies. In dynasore-treated cells, there was no fragmentation of the Golgi. In contrast, untreated cells had fragmentation of the Golgi appartus occurring in only a few cells. Intriguingly, not all untreated cells with single inclusions had a fragmented Golgi apparatus. This suggested that lipid uptake by C. trachomatis inclusions does not depend on Golgi fragmentation.

Untreated cells did not possess fragmented Golgi. Cells treated with dynasore did.

Since fragmentation does not seem to play a role in the uptake of lipids by the inclusion, could the fragmentation they were seeing be from cells undergoing mitosis? In untreated cells infected with C. trachomatis, the number of Golgi elements within the cells and the number of mitotic cells were significantly higher than in dynasore-treated cells. Therefore, the fragmentation of the Golgi apparatus was in connection to mitotic cells, re-affirming that lipid uptake does not depend on the Golgi apparatus.

Do I get fragmented or not?! Source

Now that the researchers have uncovered the mechanisms by which C. trachomatis acquires lipids, they wanted to determine whether Golgi apparatus fragmentation is important for C. trachomatis growth.  They used the agents, aphidocolin and nocodazole, to inhibit replication and arrest mitotic stages respectively in order to show that the morphology of the inclusions of C. trachomatis were similar whether the Golgi complex was intact or fragmented. In addition, nocodazole-treated cells had significantly more Golgi fragmentation than aphidicolin treated cells, showing that the increased Golgi fragmentation occurs due to the increase in mitotically-dividing cells. Hence, Golgi fragmentation occurs later during C. trachomatis infection.  However, aphidicolin and nocodazole did not reduce regular infectivity. Thus, Golgi apparatus fragmentation is not important for C. trachomatis development!

In order to support their findings, Gurumurthy et al. looked whether the cell cycle and Golgi apparatus status could lead to reduced lipid transportation due to dynamin inhibition. They looked at whether treating the cells with aphidicolin or nocodazole would prevent dynasore from lowering lipid intake of inclusions and infectivity. Interestingly, they found in dynamin-inhibited cells that nocodazole and aphidicolin did not affect the reduced lipid uptake into inclusions and did not change the loss of infectivity. The status of the Golgi structure and the cell cycle phase did not affect lipid uptake into inclusions and C. trachomatis development. This confirmed that dynamin is essential for lipid uptake and development.

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The authors concluded that dynamin is responsible for homotypic fusion of C. trachomatis inclusions and lipid uptake. This is necessary to form a single inclusion per cell and for the development of C. trachomatis. These processes are integral for the microbe to infect the host cells and replicate. Contrary to prior assumptions, Golgi fragmentation and cell cycle do not affect lipid uptake and development. With further studies in C. trachomatis lipid acquisition, it may be possible to synthesize drugs targeting various steps in this process. The experiments performed through Gurumurthy et al.’s research are important in understanding this pathogen’s infectivity. The microbe is able to invade and propagate itself incognito, within the host cell, and has the ability to hijack host cell machinery as well. Due to these properties, this pathogen remains “silent” during initial infection and is able to spread until symptoms arise a few weeks later (with severe infections). Even though there are many treatments against chlamydia, it is still easily spread through sexual contact. The best way to protect oneself from getting an infection from this sly microbe is to use protection during sexual interactions, limit the number of sexual partners, and regularly get screened. Your body will thank you!

Tanya and Emma are Biological Sciences majors at Mount Holyoke College. Outside of studying microbiology, they share common hobbies including drinking bubble tea and trying diverse foods.

References

Chlamydia - CDC Fact Sheet. (2014, January 7). Retrieved December 6, 2014, from http://www.cdc.gov/std/chlamydia/stdfact-chlamydia-detailed.htm.

Gurumurthy RK, Chumduri C, Karlas A, Kimmig S, Gonzalez E, Machuy N, Rudel T, and Meyer TF. (2014). Dynamin-mediated lipid acquisition is essential for Chlamydia trachomatis development. Molecular Microbiology 94: 186-201.