Multifunctional analysis of Chlamydia-specific genes in a yeast expression system.
Our understanding of how obligate intracellular pathogens co-opt eukaryotic cellular functions has been limited by their intractability to genetic manipulation and by the abundance of pathogen-specific genes with no known functional homologues. In this report we describe a gene expression system to characterize proteins of unknown function from the obligate intracellular bacterial pathogen Chlamydia trachomatis. We have devised a homologous recombination-based cloning strategy to construct an ordered array of Saccharomyces cerevisiae strains expressing all Chlamydia-specific genes. These strains were screened to identify chlamydial proteins that impaired various yeast cellular functions or that displayed tropism towards eukaryotic organelles. In addition, to identify bacterial factors that are secreted into the host cell, recombinant chlamydial proteins were screened for reactivity towards antisera raised against vacuolar membranes purified from infected mammalian cells. We report the identification of 34 C. trachomatis proteins that impact yeast cellular functions or are tropic for a range of eukaryotic organelles including mitochondria, nucleus and cytoplasmic lipid droplets, and a new family of Chlamydia-specific proteins that are exported from the parasitopherous vacuole. The versatility of molecular manipulations and protein expression in yeast allows for the rapid construction of comprehensive protein expression arrays to explore the function of pathogen-specific gene products from microorganisms that are difficult to genetically manipulate, grow in culture or too dangerous for routine analysis in the laboratory.
Sisko
Jennifer L
JL
Department of Molecular Genetics and Microbiology and Center for Microbial Pathogenesis, Duke University Medical Center, Durham, NC 27710, USA.
Spaeth
Kris
K
Kumar
Yadunanda
Y
Valdivia
Raphael H
RH
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000648:ultrastructure; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D005821:Genetic Techniques; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000145:classification / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure
D001426:Bacterial Proteins; D011994:Recombinant Proteins