Item Details

title

QUANTIFYING CHANGES IN THE CHLAMYDIA DEVELOPMENTAL CYCLE USING EXPANSION MICROSCOPY

author

Lee, Da Hee

abstract

Chlamydia trachomatis (Ct), an obligate intracellular bacterial pathogen, is known for its distinctive biphasic developmental cycle. The bacteria change their developmental form between two forms, named the elementary body (EB) and reticulate body (RB). The EB is non-metabolic and infectious, which enters into the host cell for initial infection as well as for multiple rounds of infection after the cell lysis. The RB is metabolically active and replicative for cell division during the developmental life cycle. Studying the developmental cycle is crucial in finding evidence to unravel the mechanism involved in the switching of genes. In this study, we sought to quantify the gene expression levels measured by the fluorescent intensity in the Ct developmental cycle using expansion microscopy (ExM). We hypothesized that the RB to EB conversion is concentration-dependent, where the gene expression levels increase or decrease in a gradient manner. To experimentally test this, developmentally specific promoters with fluorescent reporters were used to identify the two different body forms, the HctB enriched in EB and the Euo enriched in RB. Due to the minuscule nature of individual chlamydial bodies packed in inclusion, the ExM protocol with the optimized setup for the Chlamydia experiment physically increased the sample size which led to better resolution with the use of a conventional microscope alone. We observed that HctB levels increased as EB was differentiated from RB for another round of infection. The size of individual bodies measured shows that RB size was largely variable as well. In addition, penicillin and resveratrol were added tovalidate the effective use of expansion microscopy. We observed that the treatment of penicillin led to aberrant, large RBs which led to inhibiting the HctB expression as there were no detectable EBs at a later time point. On the other hand, the addition of resveratrol resulted in no significant decrease in both HctB and Euo at a later time point. Moreover, the RB sizes did not significantly change over two time points for all experiments tested. This suggests that the decrease in the RB size over time may not be the only signal leading to the differentiation of RB to EB. This study was able to quantify the gene expression levels of individual chlamydial bodies measured in fluorescent intensities using expansion microscopy and to validate its effective use using penicillin and resveratrol. The change in the gene expression levels at different time points provides evidence for the proposed concentration-dependent gene expression model in RB to EB conversion. Further experiments will lead to completing the pieces of puzzles and solving the unknown nature of the Ct developmental cycle. To conclude, expansion microscopy is a suitable and easy-to-follow protocol to investigate infection-related research like Chlamydia.

subject

Chlamydia trachomatis

Developmental Cycle

Expansion Microscopy

Gene Expression

Infectious Pathogen

Sexually Transmitted Disease

contributor

Brown-Harding, Heather (committee chair)

Pease, James (committee member)

Esstman, Sarah (committee member)

date

2022-09-17T08:35:50Z (accessioned)

2023-03-16T08:30:13Z (available)

2022 (issued)

degree

Biology (discipline)

embargo

2023-03-16 (terms)

identifier

http://hdl.handle.net/10339/101263 (uri)

language

en (iso)

publisher

Wake Forest University

type

Thesis