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===========================================

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37AI020384-22
Application #
6883772
Study Section
Special Emphasis Panel (NSS)
Program Officer
Perdue, Samuel S
Project Start
1983-07-01
Project End
2010-03-31
Budget Start
2005-07-01
Budget End
2006-03-31
Support Year
22
Fiscal Year
2005
Total Cost
$292,455
Indirect Cost
Name
University of South Alabama
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
172750234
City
Mobile
State
AL
Country
United States
Zip Code
36688
Wood, David O; Wood, Raphael R; Tucker, Aimee M (2014) Genetic systems for studying obligate intracellular pathogens: an update. Curr Opin Microbiol 17:11-6
Wood, David O; Hines, Andria; Tucker, Aimee M et al. (2012) Establishment of a replicating plasmid in Rickettsia prowazekii. PLoS One 7:e34715
Clark, Tina R; Lackey, Amanda M; Kleba, Betsy et al. (2011) Transformation frequency of a mariner-based transposon in Rickettsia rickettsii. J Bacteriol 193:4993-5
Woodard, Andrew; Wood, David O (2011) Analysis of convergent gene transcripts in the obligate intracellular bacterium Rickettsia prowazekii. PLoS One 6:e16537
Tucker, Aimee M; Driskell, Lonnie O; Pannell, Lewis K et al. (2011) Differential proteomic analysis of Rickettsia prowazekii propagated in diverse host backgrounds. Appl Environ Microbiol 77:4712-8
Liu, Zhi-Mei; Tucker, Aimee M; Driskell, Lonnie O et al. (2007) Mariner-based transposon mutagenesis of Rickettsia prowazekii. Appl Environ Microbiol 73:6644-9
Tucker, Aimee M; Pannell, Lewis K; Wood, David O (2005) Dissecting the Rickettsia prowazekii genome: genetic and proteomic approaches. Ann N Y Acad Sci 1063:35-46
Driskell, Lonnie O; Tucker, Aimee M; Winkler, Herbert H et al. (2005) Rickettsial metK-encoded methionine adenosyltransferase expression in an Escherichia coli metK deletion strain. J Bacteriol 187:5719-22
Qin, Aiping; Tucker, Aimee M; Hines, Andria et al. (2004) Transposon mutagenesis of the obligate intracellular pathogen Rickettsia prowazekii. Appl Environ Microbiol 70:2816-22
Rachek, L I; Hines, A; Tucker, A M et al. (2000) Transformation of Rickettsia prowazekii to erythromycin resistance encoded by the Escherichia coli ereB gene. J Bacteriol 182:3289-91

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