Recombination, resistance, and drug discovery in Chlamydia Public Deposited


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  • The intracellular life cycle of Chlamydia has been difficult to study due to the inability to genetically manipulate the bacteria. As a result, indirect methods have been employed to study the pathogen and the aspects that contribute to its unique intracellular life cycle. A wide variety of laboratory adapted strains, animal strains, and clinical strains have been used to model different interactions with the host cell and to highlight, in the face of extreme genetic conservation, the molecular subtleties that distinguish each of these strains from one another. The use of sequencing, traditional biochemical analyses of proteins, and expression of chlamydial proteins in heterologous systems have been the stand by approaches for studying the biology in this system. Here we have explored utilities, both inherent to the chlamydial system, and with the aid of high through put technologies, to develop genetic tools in a system without genetics. Presented here is the early work that lead to the development of a recombination system and establishment of a recombinant strain library that allows for large scale genotype-phenotype analyses in Chlamydia. We have characterized the recombination events between two interspecies crosses with different antibiotic resistant markers and pioneered the use of next gen sequencing to do full genome characterization to identify regions targeted by recombination. Through these studies, we show that large regions of the chlamydial genome recombine at regions specific to the antibiotic selection markers, tetracycline, rifampin, and ofloxacin. These studies also show that large fragments of DNA both insert into recipient genomes, resulting in duplication events, or recombine at homologous regions, resulting in functional replacement of the genetic sequence. In partnership with SIGA technologies, we conducted a high throughput screen against Chlamydia caviae GPIC to try to identify compounds that inhibited chlamydial growth, blocked host processes important in chlamydial development, and to develop new research tools that can be used in intracellular pathogen research. Five compounds were identified that blocked chlamydial growth and two of these compounds also inhibited the growth of Coxiella or Staphylococcus aureus. To understand the resistance determinants associated with the inhibitory properties of these compounds, a chemical genetics approach was utilized in the chlamydial system to select for resistance and genome sequence to identify the resistance-associated mutations. Chlamydia develop resistance to certain antibiotics through the accumulation of mutations when exposed to increasing concentration of drugs in vitro. In these studies, we show that they also mutate in response to stress induced by unknown chemical inhibitors and that the mutations associated with the resistance phenotype suggests potential resistance determinants of the uncharacterized inhibitors. We have also shown the utility of the chlamydial system as a model for characterizing host cell processes important to intracellular pathogen replication and used this system to aid in the development of a host-specific, broad-spectrum anti-infective compound. The data from these studies also suggests that lipid droplets are widely exploited by both viral and bacterial organisms to support their intracellular replication. Collectively, this work highlights the genetic tools that are available to study a system without traditional genetic techniques and the use of the chlamydial intracellular life cycle to dissect host processes universally important to intracellular pathogen development.
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