Members of the genus Rickettsia are the etiologic agents of rocky mountain and other spotted fevers and endemic, scrub and epidemic typhus, diseases that pose a pernicious health threat worldwide. Rickettsia prowazekii, the etiologic agent of epidemic typhus is an obligate intracellular parasitic bacterium that can grow only within the cytoplasm of a eucaryotic host cell. The ability of 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 focuses on the development and application of genetic techniques to address questions regarding the pathogenic bacterium R. prowazekii and its obligate intracytoplasmic existence. It exploits the availability of the R. prowazekii genome sequence and the development of rickettsial genetic technologies to test hypotheses related to rickettsial gene function, DNA replication, and pathogenic mechanisms.
In Specific Aim 1 the PI's goal is to capitalize on a rickettsial transformation system and identification of a selectable antibiotic resistance gene that can be expressed in R. prowazekii to discriminate, via knockouts, essential function at the level of single genes. Specifically targeted genes include those that encode products with homology to known virulence genes of other bacteria, genes hypothesized to be expressed only in the arthropod vector, genes hypothesized to be non-functional and part of the process of rickettsial reductive evolution, and finally, genes with homologs within the R. prowazekii genome. In addition, a transposon-based approach will be used to generate random insertion mutants.
In Specific Aim 2, the PI's goal is to isolate the functional origin of replication. One approach will attempt to generate a rickettsial mini-chromosome by linking putative origin fragments with the selectable erythromycin-resistant gene, ereB. An alternate method will identify the origin by binding of rickettsial DnaA.
Specific Aim 3 will continue the PI's characterization of transcription termination and identification of rickettsial transcriptional changes that occur just prior to lysis of the host cell. Using ribonuclease protection studies, the PI will determine whether these changes reflect a general property of the rickettsiae by examining additional non-intrinsic termination sites and the effect of cell number on termination at these sites. Modulation of Rho and its correlation to these changes will be addressed.

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 #
2R37AI020384-17
Application #
6196835
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Baker, Phillip J
Project Start
1983-07-01
Project End
2005-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
17
Fiscal Year
2000
Total Cost
$317,938
Indirect Cost
Name
University of South Alabama
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
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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