Chlamydia is an obligate intracellular bacterial pathogen that is the cause of a wide spectrum of human diseases, including sexually transmitted diseases, blinding trachoma and possibly coronary artery disease. The singular host cell hallmark of infection is the vacuole that contains chlamydiae, called the inclusion. Chlamydiae infect mammalian cells and differentiate and grow only within the inclusion. The inclusion is a unique cellular compartment that physically sequesters chlamydiae from the host cytosol. This compartment is modified by chlamydial proteins and host lipids. Interactions with the host and acquisition of required host nutrients must be translated across the inclusion membrane. This host cell compartment represents the keystone to chlamydial intracellular biology and parasitism, although little is known about its composition, structure or function. Our hypothesis is that the inclusion defines elements of chlamydial biology that are essential for chlamydial growth and differentiation, and interactions with other host cell compartments. The specific aims of this application target the development of experimental methods for the purification of inclusions that enable defining their composition and biological functions. This research will promote advancement of methodologies across disciplines of basic and applied chlamydial research. The long-term objectives are to understand the requirements for chlamydial growth and differentiation, and determine the molecular bases and roles for interaction with other host cell compartments. This will yield important fundamental information for the development of new approaches for intervention. The significance of these studies is to expand the capacity for research and to advance the understanding of fundamental mechanisms of chlamydial pathogenesis and virulence as an active interplay between chlamydiae and their hosts. The specific aims are: 1) Isolate and purify chlamydial inclusions. 2) Characterize inclusion membrane composition. 3) Evaluate functional capacity of isolated inclusions. PUBLIC HEALTH RELEVANCE: Chlamydia organisms cause bacterial infections with major public health and medical significance. Like a virus, Chlamydia must infect human cells to grow and cause disease;yet, this process is not understood. This research will establish the mechanism of microbial survival in cells that will serve as targets for prevention and control of chlamydial infection.