Date of Award

5-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Microbiology

Track

Molecular Sciences

Research Advisor

Thomas P. Hatch, Ph.D.

Committee

Robert J. Belland, Ph.D. James E. Bina, Ph.D. David Hasty, Ph.D. Mark A. Miller, Ph.D.

Abstract

Chlamydia spp. are prokaryotic obligate intracellular pathogens with a unique, biphasic developmental cycle in which an infectious, extracellular form termed the elementary body (EB) interconverts with a metabolically active intracellular reticulate body (RB) within host cells. Subsets of genes are differentially expressed during the developmental cycle, and these genes are believed to be responsible for the transitions between the EB and RB forms. The goal of these studies was to explore two potential mechanisms that may function in regulating developmental cycle stage‑specific gene expression in chlamydiae: a cascade of sigma factor expression and the binding of the early stage protein EUO to DNA.

The protein expression levels of each of the three sigma factors were monitored throughout the developmental cycle to determine if expression of the sigma factors is consistent with a role in directing temporal gene regulation via a cascade mechanism. Western blot and immunofluorescence assays revealed that the sigma 66, the major sigma factor, is present throughout the developmental cycle and, consequently, could direct temporal gene expression, but not via a simple cascade mechanism. The alternative sigma 28 protein was found to be highest during the late logarithmic phase, and it can be speculated that it plays a key role in the initial stages of RB‑to‑EB conversion through its regulation of the late DNA binding protein, histone HctB. Analysis of sigma 54 protein suggested it may exist in two forms: a cytoplasmic form, which most likely functions as a sigma factor during RB replication, and an outer membrane complex form, which is present in RBs and EBs and may have a structural function. Expression of the proposed functional sigma 54 protein throughout the logarithmic phase suggests that it plays no direct role in EB‑RB differentiation via a cascade mechanism.

EUO is a DNA-binding protein that is produced early in the developmental cycle but is also present during the logarithmic phase of growth. Because transcription studies have suggested that euo is the most actively expressed gene within the first few hours of infection, EUO is believed to be a potential transcriptional regulator. A ChIP‑to‑Chip protocol was performed with isolated genomic DNA and recombinant EUO to identify genes to which EUO may bind. DNA that co‑immunoprecipitated with the recombinant EUO was allowed to hybridize to a microarray that contained the intergenic regions and the open reading frames of the Chlamydia trachomatis serovar D genome. EUO binding was observed to be scattered throughout the genome, although with reproducible affinity for particular DNA fragments. However, direct evidence for regulation of key stage‑specific genes was not obtained by this study.

An immunoprecipitation-cloning protocol was developed to identify in vivo binding sites of EUO. Sixteen hour post-infection RBs were formaldehyde cross‑linked to preserve the physiological interactions between native EUO and DNA. EUO was immunoprecipitated from these cross‑linked reticulate bodies and the EUO‑bound DNA was cloned and sequenced. BLAST searches of these sequences identified thirty genes that may be in vivo targets of EUO, but the role of these genes in EB‑RB interconversion is unclear.

DOI

10.21007/etd.cghs.2011.0028