Using functional genomics to discovery Chlamydia virulence factors
Genome-wide discovery and characterization of virulence determinants for Chlamydia remains a major unaddressed research area. Through recent landmark advances in genetic manipulation of Chlamydia, and a rich collaboration with Scott Hefty and Dan Rockey that exploits new methodologies for genetic engineering in Chlamydia, we use forward genetic approaches to discover the Chlamydia genes and host protein targets that play critical roles in Chlamydia infection and pathogenesis.
Genetic tool development
In order to fully dissect the mechanisms used by Chlamydia to infect and cause disease in hosts, new tools for genetic manipulation of Chlamydia need to be developed and/or adapted, and optimized. We currently leverage two major strategies:
We actively use the transposon mutagenesis system to generate isogenic, single gene disruption mutants in C. trachomatis and C. muridarum. Over 250 mutant clones have been produced and genotyped using an expanding array of selectable markers and insertion sequences. Mutant strains are available for the Chlamydia research community upon request.
Interspecies chimeric Chlamydia strains
Leveraging the fact that Chlamydiae are naturally competent and capable of DNA exchange by homologous recombination, we have produced the largest library of interspecies chimeric Chlamydia strains in existence. This rich biological resource (2000+ strains) allows deep evaluation of the multifactorial contribution of Chlamydia genome-encoded factors to infection and host tropism.
Broadly, we are interested in elucidating the cell and molecular biology of Chlamydia-host interactions, specifically as it relates to strategies used by Chlamydia to control host signaling pathways and cell function. This work often encompasses target-based experimentation as well as large scale proteomic strategies.
We have looked at Chlamydia entry and exit mechanisms into and out of host cells. Our detailed investigations of chlamydial exit mechanisms helped to establish a new paradigm for our understanding of chlamydial cell to cell spread within a host.
Human chlamydial infections
We are increasingly interested in connecting our molecular and genetic findings (performed in model systems) to the context of human infections. We are currently exploring this research direction on three fronts. First, we use whole genome sequencing and molecular genetics in an effort to identify the key Chlamydia genetic factors responsible for governing pathogen–host tropism, at distinct mucosal sites. Second, we are working to determine the human immunological correlates of protection and clearance of Chlamydia infections. Finally, we have begun to use a peptide-based assay to perform molecular serology on patient sera.
Structure-function analysis of Chlamydia proteins
Through ongoing collaborations with Scott Hefty, Scott Lovell, and the Seattle Structural Genomics Center for Infectious Disease (SSGCID), we are working to discover the structures and functions of the sizable pool of uncharacterized, hypothetical proteins encoded by C. trachomatis. This work incorporates X-ray crystallography, structure modeling, molecular genetics, biochemical, and cell biological approaches.
ChlamBase: a curated model organism database for the Chlamydia research community
Together with the Su Lab, experts in aggregating community acquired research knowledge into open source databases, we developed and launched ChlamBase.org. ChlamBase is a fork of WikiGenomes.org, a Wikidata-backed database of over 100 NCBI microbial reference genomes. The goal of ChlamBase is to provide a streamlined, researcher-curated database of genetic and proteomic knowledge for the Chlamydia research community. This database uniquely offers information not available on other services, such as:
- Current gene annotations
- Orthologous gene comparisons and alignments
- Annotations of Chlamydia mutants
- Developmental expression timing
- RB/EB specific protein expression
- Host and bacterial protein interactions