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Factors and mechanisms contributing to the regulation of the developmental cycle in Chlamydia trachomatis
Baid, Srishti
Baid, Srishti
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Abstract
Chlamydia is an obligate intracellular bacterium causing one of the most commonly reported bacterial sexually transmitted infections in humans. It is asymptomatic in nature and exposure to infants can lead to neonatal blindness. Despite such serious consequences and high stakes, there are restricted treatment options and no available vaccines. Developing therapeutics has been challenging with chlamydia because it has been difficult to manipulate the organism and hence there is a paucity of available information about the basic biology of the organism. This is further complicated by 30% of the chlamydial genome being unannotated and defined as hypothetical protein coding genes.This dissertation attempts to bridge the gap of knowledge about chlamydial biology by using two different methods- 1) Through genetic studies by transposon mutagenesis and inter-species recombination in Chlamydia and 2) Through structure-function studies to characterize the role of 2 hypothetical proteins in Chlamydia trachomatis.The first aspect of the document emphasizes on how different kinds of unbiased genetic approaches can help us delve into the roles of gene products in chlamydial growth and infection capabilities. The genetic studies from transposon mutagenesis in Chlamydia trachomatis demonstrated the role of several chlamydial proteins in the developmental cycle of Chlamydia trachomatis by characterizing select mutants. Mutants obtained in this study can be individually characterized and insights to the regulation and role of different genes during the progression of the developmental cycle can be gathered. The second part of the genetic study concentrated on understanding the species-specific role of genes to understand the pathogenesis. In the context of this dissertation, once the chimeras were obtained around the region of differences between Chlamydia trachomatis and Chlamydia muridarum- called the Plasticity Zone (PZ), the growth phenotypes were assessed. These chimeras had the Chlamydia muridarum plasticity zone in a Chlamydia trachomatis background. Results support that the genes in the PZ region contribute to the growth and inclusion size expansion irrespective of the species but the genes that are directly contributing to these factors are part of the full PZ and the right partial PZ. The chimeras majorly had Chlamydia trachomatis-like growth and further validation of the genes crossed over in these recombinants is warranted.The second and major aspect of this dissertation focuses on understanding gene regulation and the role of hypothetical proteins in gene regulation during the developmental cycle of Chlamydia trachomatis. Here we attempted to characterize two hypothetical proteins- CT398 (CdsZ) and CT457 (YebC). These proteins had little sequence similarity with other bacteria but had no identified role in Chlamydia trachomatis. The protein structures of these proteins were crystalized, and the function was assessed using structural homology and in vivo over-expression of the protein approaches. Our data support that CdsZ over-expression had a profound impact on chlamydial RB to EB reorganization. RNA seq results showed down-regulation of a subset of late genes. Moreover, this regulation of conversion was mediated in a temporal fashion and most likely through the CdsZ interacting with RNA polymerase machinery during transcription. The data also suggests its regulatory role based on interaction with the Type III secretion system and its effect on pathogenesis. Further validation is needed to understand this mechanism. The last chapter focuses on the regulatory role of CT457 (YebC) during the developmental cycle. Although, we have transcriptional and translational data to show that it is constitutively present through the developmental cycle, over-expression had no effect on chlamydial growth or gene expression. Further optimization of assays is needed to decipher the exact role of CT457 given that it is a conserved protein and it could have the same role in different species of Chlamydia.
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Date
2023-05-31
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University of Kansas
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Keywords
Microbiology, chlamydia, developmental cycle, gene, regulation, transcription