Rickettsial diseases, such as epidemic typhus, a louse-vectored disease of humans, are a world-wide scourge for which fundamental understanding of the etiologic agent, better diagnostic tools, and vaccines are needed. The etiologic agent, R. prowazekii, is an obligate intracellular bacterial parasite;rickettsiae only grow directly in the cytoplasm of hosts and vectors. Rickettsiae are marvelous organisms to unravel the biological mysteries of obligate intracellular parasitism and living probes for the investigation of the eukaryotic host cell cytoplasm. R. prowazekii has only 834 genes because many metabolic intermediates need not be synthesized, but are transported from the cytoplasm of the host cell - often by unusual transport systems that have no counterparts in free-living bacteria. The ATP/ADP translocase (Tlc1) of R. prowazekii transports energy via the obligatory exchange of rickettsial ADP for host cell ATP. We now have an excellent two-dimensional topology for this protein and evidence for which transmembrane domains (TMs) form the water-filled channel through which ATP/ADP crosses the membrane. Our current Aim 1 is to elucidate the structure of its 12 TMs, the Tlc1 helix packing.
In Aim 2 the substrate-binding site of Tlc1 will be determined through DNA shuffling and formation of chimeric proteins. We will do 'evolution in the test-tube'by shuffling the tlc1 genes of Caedibacter caryophilus and R. prowazekii. Sequence analysis of shuffled fragments within these chimeric proteins will associate conserved sequence motifs with substrate specificity.
In Aim 3 we will identify and characterize the transport system of NAD, a principle metabolite in R. prowazekii, and in Aim 4 we will investigate the phospholipase-like proteins, patatin and ExoU, in R. prowazekii. We have demonstrated that interaction of rickettsiae with host cells results in the biochemical products of phospholipase activity. This is involved in one of the most fundamental questions in rickettsial biology: its entry. The hypothesis is that activation and/or translocation is required for enzyme activity and rickettsial entry and we will determine if ExoU and patatin play a role in this process.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
3R01AI015035-32S1
Application #
8053160
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Perdue, Samuel S
Project Start
2010-04-12
Project End
2010-12-31
Budget Start
2010-04-12
Budget End
2010-12-31
Support Year
32
Fiscal Year
2010
Total Cost
$355,059
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
Housley, Nicole A; Winkler, Herbert H; Audia, Jonathon P (2011) The Rickettsia prowazekii ExoU homologue possesses phospholipase A1 (PLA1), PLA2, and lyso-PLA2 activities and can function in the absence of any eukaryotic cofactors in vitro. J Bacteriol 193:4634-42
Audia, Jonathon P; Winkler, Herbert H (2006) Study of the five Rickettsia prowazekii proteins annotated as ATP/ADP translocases (Tlc): Only Tlc1 transports ATP/ADP, while Tlc4 and Tlc5 transport other ribonucleotides. J Bacteriol 188:6261-8
Audia, Jonathon P; Roberts, Rosemary A W; Winkler, Herbert H (2006) Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: characterization of TMs IV-VII and IX-XII and their accessibility to the aqueous translocation pathway. Biochemistry 45:2648-56
Alexeyev, Mikhail F; Roberts, Rosemary A W; Daugherty, Robin M et al. (2004) Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: evidence that transmembrane regions I and II, but not III, are structural components of the aqueous translocation channel. Biochemistry 43:6995-7002
Daugherty, Robin M; Linka, Nicole; Audia, Jonathon P et al. (2004) The nucleotide transporter of Caedibacter caryophilus exhibits an extended substrate spectrum compared to the analogous ATP/ADP translocase of Rickettsia prowazekii. J Bacteriol 186:3262-5
Winkler, Herbert H; Daugherty, Robin M; Audia, Jonathon P (2003) Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: evidence that TM VIII faces an aqueous channel. Biochemistry 42:12562-9
Linka, Nicole; Hurka, Herbert; Lang, B Franz et al. (2003) Phylogenetic relationships of non-mitochondrial nucleotide transport proteins in bacteria and eukaryotes. Gene 306:27-35
Schwoppe, Christian; Winkler, Herbert H; Neuhaus, H Ekkehard (2003) Connection of transport and sensing by UhpC, the sensor for external glucose-6-phosphate in Escherichia coli. Eur J Biochem 270:1450-7
Alexeyev, Mikhail F; Winkler, Herbert H (2002) Complete replacement of basic amino acid residues with cysteines in Rickettsia prowazekii ATP/ADP translocase. Biochim Biophys Acta 1565:136
Schwoppe, Christian; Winkler, Herbert H; Neuhaus, H Ekkehard (2002) Properties of the glucose-6-phosphate transporter from Chlamydia pneumoniae (HPTcp) and the glucose-6-phosphate sensor from Escherichia coli (UhpC). J Bacteriol 184:2108-15

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