Chlamydia trachomatis(CT) is the leading cause of sexually transmitted diseases (STD) in the developed world. CT infections and their sequelae of pelvic inflammatory disease, ectopic pregnancy, and infertility are responsible for ~80% of the estimated $2.5 billion annual cost of these infections in the United States. Further, up to 50% of women become reinfected and are at increased risk for these sequelae. Many reinfections reflect persistence which likely plays an important role in pathogenesis. The major outer membrane protein is considered to be the immunodominant protein of CT. However, the discovery of open reading frames predicted to encode a nine-member polymorphic membrane protein (Prop) gene (pmp) family in the recently published genome sequence of CT serovar D suggest that these Props may also be important in clalamydial biology. Further, CT contains a partial tryptophan biosynthesis operon (trpR, trpA, trpB) not found in a CT mouse strain (MoPn) or other species of Chlamydia. Tryptophan is essential for chlamydial replication, and tryptophan depletion in vitro results in cblamydial persistence. Our hypothesis is that the prop and tryptophan genes may undergo selection that results in differential expression or activity of these proteins that: 1) consequently determine active or persistent infection; and 2) are significantly involved in pathogenesis as an outcome of persistence or outcome of other factors. By analyzing the genetic profile of prototype and serial recurrent and persistent CT STD patient strains and by correlating these data with epidemiologic and clinical findings, we hope to identify the genes, genetic/protein variation and evolution of this variation in the organism, and how these are linked to persistence and patbogenesis. Thus, this grant will answer broad questions about the genetic and protein basis for persistence and for pathogenesis, and provide important research tools including a Database and DNA microarray that will be of long term benefit to investigators in the field of Chlamydia. The Specific Aims for this grant are to: 1) Sequence the nine pmps, and trpR, trpA, and trpB genes for the 19 prototype serovars of CT and create a DNA microarray for these genes and ompA to differentiate strains of CT, and for use in Aim 2; and 2) Identify polymorphisms in and protein expression of the nine props, specific tryptophan operon genes, and other constitutively expressed genes among serial cervical samples from patients with persistent versus non-persistent CT STDs; correlate the genetic and protein expression profiles of these serial samples with epidemiologic and clinical findings. )ERFORMANCE SiTE(S) (organization, city, state) Children's Hospital Oakland Research Institute, Oakland, California The Institute for Genome Research, Rockville, Maryland University of Maryland, Baltimore, Maryland KEY PERSONNEL. See instructions on Page 11. Use continuation pages as needed to provide the required Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name Organization Dean, Deborah Children's Hospital Oakland Research Institute, Oakland, California Cheng, Lihai Children's Hospital Oakland Research Institute, Oakland, California Hsia, Ru-ching University of Maryland Baltimore, Maryland Millman, Kim Children's Hospital Oakland Research Institute, Oakland, California Powers, Virginia Children's Hospital Oakland Research Institute, Oakland, California Read, Timothy The Institute for Genome Research Rockville, Maryland PHS 398 (Rev. 05/01 ) Page 2 information in the format shown below. Role on Project Principal Investigator Postdoctoral Fellow Co-Investigator Graduate Student Staff Research Associate III Co-Investigator Form page z Principal Investigator/Program Director (Last, first, middle): Dean, Deborah Anne The nameof the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page ......................................................................................................................................................... 1 Description,