EXCEED THE SPACE PROVIDED. Members of the genus Rickettsia are the etiologic agents of sonde of the most severe of human diseases caused by bacteria. These include rocky mountain and other spotted fevers, and endemic, scrub, and epidemic typhus, diseases that pose a pernicious health threat worldwide. Rickettsia prowazekii, categorized as a select agent and potential bioterrorist agent, is the causative agent of epidemic typhus. This pathogen is an obligate, intracellular, parasitic bacterium that can grow only with the cytoplasm of a eukaryotic host cell. The ability of the rickettsiae to exploit this intracellular niche in animals as diverse as arthropods and humans and to subsequently cause serious human disease provides the impetus for this study. This proposal will focus on applying genetic techniques to address questions concerning rickettsial pathogenicity and intracellular growth. It will capitalize on the availability of rickettsial genome sequences and protein expression databases, as well as recent breakthroughs in the genetic manipulation of K prowazekiL to test hypotheses related to rickettsial gene function and pathogenic mechanisms.
In Specific Aim 1 we will continue our groundbreaking studies on the generation of rickettsial knockout mutants and the categorization of rickettsial genes as essential or non-essential under specific growth conditions. Random mutagenesis, as well as mutagenesis targeted to specific genes (e.g. genes exhibiting homology to known virulence genes of other bacteria) will be employed. Mutants isolated by these techniques will be characterized for detectable phenotypes, including the ability to survive in an arthropod vector system.
In Specific Aim 2 we will examine the role of a putative virulence system, the Type IV secretion system, and a possible transported substrate, Sec7/RalF, on K prowazekii intraeellular growth and host membrane trafficking. RalF export by the rickettsiae, the generation of rickettsial mutants, the localization of RalF in the host cell, and the effect of RalF expression from a eukaryotic expression vector will be scrutinized.
In Specific Aim 3 we will investigate DNA replication in K prowazekii by examining the role of the global regulator CzeR in controlling the initiation of DNA replication and the timing of rickettsial DNA replication following host cell infection.
In Specific Aim 4 we will continue our characterization of rickettsial gene transcription at different stages of rickettsial growth, especially during lysis of the host cell. In addition, we will characterize an unusual aspect of rickettsial transcription that was recently identified; the presence of rickettsial antisense RNA. PERFORMANCE SITE ========================================Section End===========================================
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