Team Members

Abstract

The silent epidemic caused by chlamydia can be resolved by producing efficient ways to diagnose patients with the disease; however, small discoveries must be made first. This research project used the fluorescent label, C5-BODIPY-ceramide, to distinguish between chlamydia-infected HeLa cells and uninfected HeLa cells. By using this label, we sorted and analyzed infected and uninfected cells, and also maintained their viability. The viability of these host cells is extremely important in future research because it allows researchers to examine chlamydia in an environment that more closely resembles their natural environment. Additionally, researchers can compare protein expression between infected and uninfected cells, thereby allowing us to measure the impact of chlamydia on their host cells. Therefore, researchers will be able to precisely examine the interactions between chlamydia and its host cell, which has the potential to further improve chlamydia testing and diagnostics. In this experiment, we used Chlamydia psittaci and worked on optimizing the variables, label concentration, and incubation duration, to ensure the effectiveness of labeling and viability of cells. We hypothesized that by working with these variables, we would be able to determine an optimized labeling process that would ensure proper cell sorting and analysis in the future. The results of this research indicated the potential fluorescent labeling has on the accurate diagnosis of Chlamydia infected cells. However, due to BODIPY’s accessibility constraints, it is beneficial to explore other labeling options. As a result, the potential to improve chlamydia testing, diagnosis, and analysis of its interactions with host cells is high. Ultimately, this research should help future researchers discover more efficient ways to diagnose chlamydia.

Introduction

Chlamydia is the most prevalent sexually transmitted bacterial infection globally (dos Santos et al., 2024). Trachoma, the leading cause of preventable blindness, is a condition that can result in infants born to mothers with chlamydia (Rockey & Matsumoto, 2014). Due to the high prevalence rates of chlamydia and the potential serious consequences those with the disease may have, billions of dollars are spent towards studying chlamydia and attempting to develop innovations for prevention and treatment of the disease (Rockey & Matsumoto, 2014). There are many strains of chlamydia, with this experiment focusing on Chlamydia psittaci, but all causative agents for diseases resulting from chlamydia exhibit a nearly identical developmental cycle (Rockey & Matsumoto, 2014).

Chlamydia is an obligate intracellular bacterium that uses the host cell's nutrients for growth and survival (Brunham & Rey-Ladino, 2005). Although the exact pathogenesis of chlamydia is not entirely understood, the developmental cycle of chlamydia has undergone much research, and its nature has been determined (Brunham & Rey-Ladino, 2005; Rockey & Matsumoto, 2014). The entire development of chlamydia occurs in an inclusion, which is a membrane-bound vesicle that has the ability to link with the exocytic branch of the host cell’s vesicular trafficking pathway (Rockey & Matsumoto, 2014). The infectious form of chlamydia is called elementary bodies (EB), and in this stage, the bacterium is able to survive for a few hours outside of the host cell (Becker, 1996). The early chlamydia inclusion can be engulfed by a host cell, where it migrates near the Golgi apparatus of the target cell and then differentiates into a reticulate body (RB) (Rockey & Matsumoto, 2014). During this time, the chlamydia inclusions will grow in size and quantity until the transition point when the RBs condense back into EBs (Rockey & Matsumoto, 2014). Once the inclusion expands to fill the entire cytoplasm, the host cell often undergoes lysis, and consequently, EBs will be released to infect other cells (Rockey & Matsumoto, 2014).

One problem in chlamydia research is the inability to label chlamydia and still keep the infected cells alive. Current research has suggested that C5-BODIPY-ceramide, a Golgi-specific label, is able to effectively label the inclusions in infected cells while also keeping them alive (Alzhanov, 2007). Therefore, by using this fluorescent label, infected cells will appear green when viewed with a UV microscope, which will provide a distinction between infected and uninfected cells. In fact, infected cells that are highly concentrated with chlamydia will glow red due to the concentration of this label. Being able to distinguish between infected and uninfected cells is important because it allows for continued research into the molecular processes of chlamydia. In this experiment, we will be manipulating two variables, concentration of C5-BODIPY-ceramide and duration of incubation, to see what is most effective in maintaining viability, while also precisely labeling Chlamydia psittaci. After testing these variables, flow cytometry can be used to sort the cells into categories based on whether or not they have been infected.

Methods

Each trial of the experiment took place over 2 days. The first day began by culturing HeLa cells, a well-studied human cervical epithelial cell, on coverslips in 24-well plates, in a mixture of MEM (minimal essential media), 10% fetal bovine serum, and antibiotics. The purpose of these antibiotics was to prevent the HeLa cells from being infected with other forms of bacteria. The cells were then washed with 5 mL of phosphate-buffered saline (PBS), which was aspirated and followed by 1 mL of trypsin for the 100 mm HeLa cell culture monolayer. The cells were incubated for another 10 minutes to allow time for trypsin, a digestive enzyme, to detach the HeLa cells from the plate surface. The plate was resuspended in 9 mL of MEM for a total of 10 mL of suspension. The cell suspension and pellet were collected by spinning at 500xg for 10 minutes at 4°C on the tabletop centrifuge. The supernatant was aspirated, and the cell pellet was then resuspended in 10 mL of MEM. This new suspension was transferred to a 50 mL centrifugation tube. A hemocytometer was used to count the cells to determine the number of cells per mL in the tube (n). From here, the density was adjusted to 105 cells per milliliter, and 1 mL of the suspension was added to each well. The plates were centrifuged for 5 minutes at 300xg to promote cell adhesion. Afterward, the 24-well plates were transferred to a 5% CO2 incubator and incubated for 24 hours at 37°C to ensure HeLa cell adhesion. 

After the 24-hour incubation, the cells were inoculated with one ID50 of Chlamydia psittaci using dilutions. Compared to Chlamydia trachomatis, Chlamydia psittaci is much easier to work with due to the fact that it has fewer nutrient requirements, can grow more efficiently, and requires less preparation during experimentation. The ID50 is the quantity of chlamydia that is sufficient to infect 50% of the HeLa cells. There was a minimum of four different groups for each trial of the experiment. Some experimental trials had more than 4 groups depending on the variables being tested. 

• The negative control wells were not infected with chlamydia but were abeled with C5-BODIPY-ceramide.

• Another set of negative control wells was not infected with chlamydia but were stained with Giemsa stain. 

• The infection control groups had HeLa cells infected with chlamydia, not labeled, but stained with the Giemsa stain. The purpose of this group being stained was to provide a baseline measure of infection to compare the detection of infection levels by fluorescence. 

• The experimental group contained HeLa cells infected with Chlamydia psittaci and labeled with C5-BODIPY-ceramide.

The experimental wells had the suspension aspirated and replaced with a medium containing the calculated, appropriate quantity of chlamydia and HBSS. The plates were centrifuged at 22°C, room temperature, for 10 minutes at 1000 g. At this point, a 24-hour timer was started, which was used to indicate how long the plates needed to incubate. After centrifuging for 10 minutes at room temperature, the plates were transferred to the 5% CO2 incubator, where they were incubated at 37°C for 2 hours. After incubation, the inoculum from each well was aspirated and replaced with 1 mL of MEM media. Referring to the time remaining on the 24-hour timer started after centrifugation, the plates were incubated for the remaining designated time. 

On the third day, after the plates had incubated for 24 hours, the media was aspirated. The infection control groups were stained with Giemsa. Their medium was aspirated, and then the cells were rinsed with PBS. The PBS was aspirated, and enough methanol was added to cover the bottom of the plates. This sat for 10 minutes, during which a 1/20 Giemsa stain/water solution was prepared. The methanol was aspirated, and 1 mL of the Giemsa stain solution was added to each well. After 20-30 minutes, the stain was aspirated and the plates were washed twice with high-quality water. The coverslips in these plates were then extracted using forceps and mounted onto microscope slides using 15 microliters of Permount while still wet. Then the cells were observed under the UV light microscope and assessed along with the negative control and experimental groups. 

For the infected and uninfected experimental groups of BODIPY incubated-cells, the plates were rinsed with a wash of Hank’s balanced salt solution (HBSS) buffered HEPES to maintain cell vitality and neutral pH. The remainder of the experiment utilized a fluorescent probe and therefore had to take place in a dark room in order to prevent photobleaching. The cells were incubated in 1.0 mL of HBSS/HEPES for 30 minutes at 4℃. This allowed the cells to become loaded with the fluorescent label. The cells were removed from the incubator and washed multiple times with a cold solution of HBSS/HEPES. They were returned to the 5% CO2 incubator for 150 minutes in a fresh MEM solution. The purpose of this incubation was to allow for back exchange, reduce non-specific labeling, and allow for the probe to be transported to the appropriate compartments. In this way, the chlamydia inclusions were identified more easily because there was no outside, excess staining. The cells were again rinsed with HBSS/HEPES. The coverslips containing the cells were removed using forceps and mounted onto microscope slides using 15 microliters of HardSet VectaShield. The cells were assessed under a UV light microscope to observe for green and red fluorescence of the inclusion. This allowed assessment of the quality of the labeling, and the results were used to determine the optimal C5-BODIPY-ceramide concentration for observation of chlamydia inclusions in HeLa cells. 

This project studied the effects of different variables. The first two experimental trials focused on perfecting the experimental methodology so that experimentation of variables in future trials could be performed and analyzed as accurately as possible. This involved some corrections to the methodology, including recalculating the titer of chlamydia and the ID50, adjusting the cell monoculture density, adjusting when the cells were mounted, and the amount of mounting medium used. During the third experimental trial, the C5-BODIPY-ceramide concentration was optimized. This included analysis under three different concentrations: 1.58 μM, 5 μM, and 0.5 μM. Additionally, one well was double-stained in an attempt to see if the cells could be viewed through Giemsa staining and BODIPY stain. 

Results

After the first trial, which was attempted in the fall of 2024, the Chlamydia psittaci infectivity rate was too high. Based on the resulting cells, the titer, which is from the stock chlamydia vials, and ID50 were recalculated. The second trial, which was also completed in the fall of 2024, had similar problems with an infectivity rate that was too high. However, this was because the HeLa cell density was too low, not because the ID50 was incorrect. After ensuring proper HeLa cell density, 10^5 monoculture layer, another trial was performed in the spring of 2025. This trial included some variable changes, including two different BODIPY concentrations and a test of whether the Chlamydia could be stained with both BODIPY and Giemsa.  After this successful third trial, the calculations were shown to be accurate and set as the standard for future trials. This trial resulted in the visibility and analysis of uninfected HeLa cells stained with Giemsa through a compound microscope. Figures 1 and 2 show the view through the compound microscope of both uninfected and infected Giemsa-stained HeLa cells. The HeLa cells infected with Chlamydia psittaci have obvious inclusions, indicating a successful infection. Figures 3 and 4 demonstrate the process of using the UV-light microscope to visualize the HeLa cells, labeled with C5-BODIPY-ceramide, in the dark room and adjust the light intensity and exposure in order to have the clearest observation of the cells for effective analysis. This microscope and the visualization conditions were important to ensure that the C5-BODIPY-ceramide was not photobleached. BODIPY fluorophores have lower photobleaching rates than some other fluorophores, like fluorescein, which is why this material was favored for the project. However, there is still a risk of photobleaching of C5-BODIPY-ceramide through light exposure, so all analysis of the labeled HeLa cells had to be completed in a dark room. Figures 5, 6, and 7 show the view through the UV-light microscope of C5-BODIPY-ceramide-labeled HeLa cells infected with Chlamydia psittaci under either green or red fluorescent light. These slides show that C5-BODIPY-ceramide was successful at labeling chlamydia inclusions and allowing for differentiation between the inclusions and the Golgi Apparatus, which also fluoresced. This allowed for effective detection and comparison between uninfected and infected cells. This successful trial was completed again, but with a few modifications to optimize the concentration of C5-BODIPY-ceramide. Both uninfected and infected HeLa cells were labeled with a normal concentration of C5-BODIPY-ceramide and a half concentration of C5-BODIPY-ceramide. Under the normal concentration of C5-BODIPY-ceramide, distinction between uninfected HeLa cells and cells infected with Chlamydia psittaci is possible through both green and red fluorescence using the UV-light microscope. A brighter fluorescing cell indicating an infected one, which was about half of the total HeLa cells. Under the half concentration of C5-BODIPY-ceramide, distinction between uninfected HeLa cells and cells infected with Chlamydia psittaci is possible only through green fluorescence using the UV-light microscope. The red fluorescence is too dark, even at the highest light intensity and exposure settings, to visualize any inclusions in the HeLa cells. 

Discussion

Overall, the experiment was successful in fluorescently labeling Chlamydia psittaci-infected cells with BODIPY. Although a few adjustments were made to the procedure throughout the process, the final procedure resulted in successful results with accurate cell infectivity rates for both staining procedures. Once the procedure was finalized, a new concentration of BODIPY was tested. Under the green light, this new concentration did not show significantly different results. Adjusting the exposure of the photos assisted with the ease of the visualization of the results, just like with the normal concentration. However, there was a big difference under the red light. Under the red light, the BODIPY staining was extremely light; therefore, almost impossible to visualize regardless of the various ways of adjusting the photos. As a result, it is evident that using a lower concentration of BODIPY could be beneficial since it still allows for visualization under the green light, but the lower concentration would not be visible under the red light. 

In order to test whether both BODIPY and Giemsa stains could be used at the same time on Chlamydia-infected cells, we had to adjust the initial procedure. We started with the Giemsa stain, but removed the cell fixation step with methanol. Then, we performed the BODIPY staining procedure. This test was unsuccessful as the cells could not be seen under white light, green light, or red light. This test likely failed due to the removal of the methanol step, which did not allow the cells to adhere to the slides. In future experiments testing double staining, more research would need to be done in order to determine the best way to label with both fluorescent labels and labels that could be seen under normal lighting conditions. 

Although our results for the BODIPY staining were successful, there are still a few areas of improvement necessary in order to make a quick and accessible way to diagnose Chlamydia. To start, BODIPY is a very expensive labeling agent that requires a lot of consideration when using in order to prevent photobleaching. In order to work with BODIPY, there can be no white light, but only red light or no light at all. This is an issue because this requires a lot of practice and skill. When the cells are finally labeled, the BODIPY cannot be observed under normal lighting, but rather can only be observed in the dark using a UV-light microscope under green or red light, depending on the concentration. A UV-light microscope is not an accessible tool; therefore, this type of testing could only be done at a hospital lab or where the correct equipment is available. As a result, this would not be accessible to the general public for rapid testing of Chlamydia. To add, the cells have to be observed at most a few hours to a day after being tested in order to get the best results. This is common for many other at-home rapid tests, such as pregnancy tests and COVID-19 tests, but again, BODIPY would require a UV light to visualize. Ultimately, while we were able to successfully label viable Chlamydia using BODIPY, it does not provide the most accessible and ease of use procedure. Future research would need to be done in order to find a staining solution that is not only capable of maintaining HeLa cell viability, but also can be visualized under white light so it can be viewed easily. In addition, the labeling agent would need to be less expensive in order to increase accessibility to the general public. 

References

Alzhanov, D. T., Suchland, R. J., Bakke, A. C., Stamm, W. E., & Rockey, D. D. (2007).Clonal isolation of chlamydia-infected cells using flow cytometry. Journal of Microbiological Methods,68(1), 201-208. doi:10.1016/j.mimet.2006.07.012.   

Becker, Y. (1996). Chapter 39. In Medical Microbiology. 4th edition. essay, University of Texas Medical Branch at Galveston. 

Brunham, R. C., & Rey-Ladino, J. (2005a). Immunology of chlamydia infection: Implications for a chlamydia trachomatis vaccine. Nature Reviews Immunology, 5(2), 149–161. https://doi.org/10.1038/nri1551  

dos Santos, L. M., Vieira, M. R., Vieira, R. C., Silva, L. B., de Macêdo, G. M., Miranda, A. E., Brasiliense, D. M., e Guimarães, R. J., Sousa, E. C., Ferrari, S. F., Pinheiro, H. H., Ishikawa, E. A., & de Sousa, M. S. (2024). Prevalence and circulant genotypes of chlamydia trachomatis in university women from cities in the Brazilian Amazon. PLOS ONE, 19(1). https://doi.org/10.1371/journal.pone.0287119  

Rockey, D. D., & Matsumoto, A. (2014). The chlamydial developmental cycle. Prokaryotic Development, 403–425. https://doi.org/10.1128/9781555818166.ch20