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Abstract
Abstract
Members of the Chlamydiae family are bacterial pathogens that can cause sexually transmitted infection, respiratory infection, and ocular infection. While symptomatic infection is widespread in the U.S. and around the world, many more people have asymptomatic chronic infections. Chronic infection in women can cause tubal factor infertility and pelvic inflammatory disease, and can transmit to a child during vaginal birth. Chlamydia sequesters immune signaling molecules called ceramides, which may modulate an immunologically protective mechanism that breaks down tryptophan. We modified existing colorimetric essays to measure the breakdown of tryptophan in this process. Using this assay, we attempted to establish a dose-response curve that measured the amount of tryptophan breakdown in response to IFN-𝛾 stimulation. Our goal is to eventually quantify the activity of the enzyme responsible for catalyzing the breakdown of tryptophan and demonstrate that chlamydia directly interferes with it.
Introduction
Introduction
Chlamydia is an obligate intracellular bacterial pathogen responsible for respiratory, ocular, and sexually transmitted infections around the world1. Chlamydia is known to be capable of causing persistent asymptomatic infections, possibly because it has the ability to evade innate immune defenses2. One of the pathways that is activated in response to chlamydial infection is the tryptophan depletion pathway. Activated by the immune signaling molecule interferon gamma (IFN-𝛾), this pathway leads to the expression of an intracellular enzyme indoleamine 2, 3-dioxygenase 1 (IDO1). IDO1 breaks down intracellular supplies of tryptophan to prevent pathogens from using it to grow and reproduce within tissues3,4. Chlamydia is known to sequester ceramide, another immune signaling molecule that may have a role in regulating this pathway.
We propose that ceramide has an excitatory effect on tryptophan depletion as an explanation for its uptake by chlamydia. and designed an assay that was capable of measuring relative levels of the breakdown product of tryptophan, kynurenine. We aim to measure the concentration of kynurenine, the product of IDO1-mediated tryptophan breakdown, in cells treated with IFN-𝛾. After measuring the tryptophan breakdown response to IFN-𝛾, we will measure the effect of ceramide exposure on this process. If ceramide is capable of increasing IDO1-mediated tryptophan breakdown, it would be logical to conclude that Chlamydia is uptaking ceramide to protect from tryptophan depletion.
Methods
Methods
HeLa cells were used because they are cervical epithelial cells of the same type usually infected with Chlamydia. Cells were cultured in minimal essential media (MEM) completed with antibiotics and fetal bovine serum. Cells were plated in a 24-well plate and allowed to incubate for 24 hours to reach a concentration of 4x105 cells/mL. More MEM with concentrations of interferon gamma ranging from 0ng/mL to 20ng/mL were added, creating wells with concentrations of interferon at 0, .1, .316, 1, 3.16, and 10 ng/mL. Cells were allowed to incubate for 48 hours with the interferon-containing media. After 48 hours, the media was removed and replaced with .2mL of tryptophan in Hank’s Balanced Salt Solution and allowed to incubate for six hours.
The resulting tryptophan-containing media was placed in replicates in a 96-well plate, with 150𝜇L/well. 25 𝜇L of trichloroacetic acid in Hanks (in a 2:3 dilution) was also added to each well. The plate was incubated at 50 C for 30 minutes and then centrifuged for ten minutes at 3500 rpm. The resulting supernatant was transferred to a new 96-well plate with 100𝜇L/well. Then, 100𝜇L of p-DMAB in acetic acid (2% weight/volume) was added to each well. The resulting solutions were analyzed at 480 nm in a spectrometer alongside standard concentrations of kynurenine.
Results
Results
Figure 1. Absorbance of Tryptophan Samples
Figure 1. Absorbance of Tryptophan Samples
Figure 2. Absorbance of Samples with Kynurenine and Tryptophan
Figure 2. Absorbance of Samples with Kynurenine and Tryptophan
Figure 3. Absorbance of Samples by IFN Treatment Concentration
Figure 3. Absorbance of Samples by IFN Treatment Concentration
Discussion
Discussion
Figures 1 and 2 demonstrate the direct relationship between the amount of kynurenine present in a sample and the absorbance of that sample after treatment. They also demonstrate that the presence of unreacted tryptophan does not change the relationship between absorbance and kynurenine concentration. This is demonstrated, specifically, by the unchanged absorbance of the tryptophan only solutions. Figure 2 clearly shows that the relationship between absorbance and kynurenine concentration is both positive and linear. Not only that, the r2 value of the graph indicates that this assay can produce results with high precision.
Figure three shows a functionally nonexistent correlation between the level of IFN treatment and the absorbance of the solution. This indicates that changing the concentration of interferon treatment doesn’t increase the rate of tryptophan breakdown. In fact, the graphs are similar to controls produced with no kynurenine.
Conclusion
Conclusion
The assay used in these trials shows the ability to measure the variable under study without interference from any confounding factors. Unfortunately, we did not see a tryptophan conversion as a result of interferon stimulation. The existing literature on this topic suggests that epithelial cells upregulate IDO1 activity when stimulated with interferon gamma. This suggests that there is some aspect of experimental design that is flawed. It is likely that either the cells are being prevented from activating IDO1, or the production of kynurenine from tryptophan is not being detected.
Multiple strategies are undergoing testing to identify the issue(s). First, the tryptophan concentration in Hank’s Balanced Salt Solution is being increased from 50 micromolar to 200 micromolar to ensure that the IDO1 enzyme has sufficient substrate. Because the absorbance values are similar to samples without any kynurenine, the interferon concentration is being increased up to 100 fold. To ensure that the assay itself is not the issue, samples are going to be verified using HPLC-MS measurement. This will provide a measurement of absolute tryptophan concentration.
Works Cited
Works Cited
“Detailed STD Facts - Chlamydia.” CDC, The Center for Disease Control and Prevention, 31 December 2021, https://www.cdc.gov/std/chlamydia/stdfact-chlamydia-detailed.htm. Accessed 10 February 2022.
AbdelRahman, Yasser M., and Robert J. Belland. “The chlamydial developmental cycle.” FEMS Microbiology Reviews, 2005.
Byrne, Gerald I., et al. “Induction of Tryptophan Catabolism Is the Mechanism for Gamma-Interferon-Mediated Inhibition of Intracellular Chlamydia psittaci Replication in T24 Cells.” Infection and Immunity, vol. 53, no. 2, 1986, pp. 347-351. American Society for Microbiology, https://journals.asm.org/doi/epdf/10.1128/iai.53.2.347-351.1986. Accessed 7 March 2022.
Moffett, John R., and MA Aryan Namboodiri. “Tryptophan and the Immune Response.” Journal of the Australian and New Zealand Society for Immunology, vol. 81, no. 4, 2003, pp. 247-265. Wiley Online Library, https://onlinelibrary.wiley.com/doi/10.1046/j.1440-1711.2003.t01-1-01177.x. Accessed February 2022.
*Images Courtesy of Emily Davis and Avery B., the Carlin Lab
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