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Rickettsia Prowazekii Angela Mcgaugh1, David Wood2, and Aimee Tucker2 1Department of Biomedical Sciences, University of South Alabama, Mobile, AL 36688

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Rickettsia Prowazekii Angela Mcgaugh1, David Wood2, and Aimee Tucker2 1Department of Biomedical Sciences, University of South Alabama, Mobile, AL 36688 Identification of Putative Secretion Effectors in Rickettsia prowazekii Angela McGaugh1, David Wood2, and Aimee Tucker2 1Department of Biomedical Sciences, University of South Alabama, Mobile, AL 36688. 2Department of Microbiology and Immunology, University of South Alabama, Mobile, AL 36688. Abstract . Fusion Cassette The obligate intracellular, gram-negative bacterium Rickettsia prowazekii, the louse-borne, causative agent of epidemic typhus, is a historically significant pathogen that has caused millions of deaths during periods of war and famine. It is also identified as a potential bioterrorism weapon. Because R. prowazekii only grows within the cytosol of host cells, it has evolved mechanisms that aid its infection, intracellular growth, and ability to evade host cell defense. The rickettsial genome contains several secretion systems that may support the Results delivery of secreted effectors that would interact with its host. To identify Methodology Targets have been successfully amplified and ligated with the putative effectors, a shuttle vector was constructed containing an expression plasmid vector. Currently, E. coli transformants are being cassette that incorporates a tandem FLAG and glycogen synthase kinase (GSK) Selection of Secreted Effectors screened using antibiotic selection and the presence of “pink” tag that can be fused to a protein of interest. The FLAG-GSK tag allows the colonies. Selected transformants are digested with specific proteins to be identified within the eukaryotic cytosol, confirming protein • Signal sequences Potential Secretion Proposed Function Effector restriction enzymes unique to each target to confirm the proper secretion from the pathogen and supporting its role as an effector molecule. orientation of inserts. Once the proper orientation of an insert Finally, incorporation of an RpCherry-Arr2 fusion protein into the plasmid allows • Increased G/C content RP016 Possible Cell Surface is confirmed for a target, transformants will be grown overnight for fluorescence detection and selection of the desired transformants using the Antigen in LB, diluted, and allowed to grow for 4 hours. Cultures will antibiotic rifampin. This system permits the screening of hypothesized secreted • Eukaryotic domains RP290 Type IV Sec Pathway then be analyzed using Western Blotting (probe for anti-FLAG). effectors in Escherichia coli, a surrogate host. Targets secreted can then be RP621 Zinc Uptake Mechanism Once the expression of FLAG is confirmed, targets will then be identified prior to validating the secretion of the effectors in BSL-3 agent R. • Homologous to effectors RP807 Penicillin Binding analyzed in R. montanesis, a Biosafety Level-2 rickettsiae. prowazekii. The identification of potential secreted effectors that allow in other pathogens Protein Confirming that RP016, RP290, RP621, and RP807 as secreted rickettsiae to enter, manipulate, and exit its host will reveal crucial information Selected Genes and their Proposed Functions effectors will allow us to then identify specific protein targets to regarding rickettsial obligate intracellular growth and ability to cause human be analyzed in R. prowazekii. disease. Verified Insert Orientation Background Fusion Plasmid Construction M 1 Secretion of effector proteins may be involved in several aspects of rickettsial growth, including invasion, intracellular replication, and ability to evade host cell Plasmid Elements defense mechanisms. Investigating potential secreted effectors of R. prowazekii is crucial to understanding how rickettsiae manipulate its host and cause • FLAG tag disease. As many as 40 proteins are hypothesized to be putative secreted • GSK tag effectors based on bioinformatics. Based on their G/C content, signal sequences, presence of eukaryotic domains, and homology to effectors in other pathogens, • RpCherry-Arr2 fusion five genes (RP016, RP290, RP621, RP787, and RP807) were selected for this initial study. To investigate putative effectors, a shuttle vector was constructed containing an expression cassette that contains a FLAG-GSK tag and RpCherry- Restriction Enzyme Digestion Arr2 fusion protein. The FLAG tag was selected because cytoplasmic expression 1) RP807, Marker (M) and secretion of recombinant fusion proteins can be easily detected using antibody recognition in cellular localization studies and immunofluorescence. Future Directions The incorporation of the GSK tag is imperative because only a eukaryotic kinase, •Identify specific rickettsial proteins that are secreted based on located in the cytoplasm, can phosphorylate GSK, validating the secretion of localization studies in the E.coli and R. monacensis surrogate proteins into the cytosol of the host. The plasmid construct also contains Engineered Plasmid systems using Western blotting and fluorescence microscopy. RpCherry-Arr2 fusion protein, permitting fluorescence detection and selection Pink Cultures from Expression of the desired transformants using the antibiotic rifampin and presence of of RpCherry-Arr2 fusion •Specific protein targets in R. prowazekii will be validated in the “pink” colonies. These putative effectors are initially expressed in Escherichia University of South Alabama Laboratory of Infectious Diseases’ coli and Biosafety Level-2 Rickettsia sp. as surrogates to identify the potential Biosafety Level-3 laboratories using secretion assays and a secreted effectors. Protein targets will then be analyzed in secretion assays using transposon system. R. prowazekii, a Biosafety Level-3 pathogen. 1 M 2 M 3 1. PCR Amplify Targets •This project will further our knowledge regarding intracellular Rickettsial Secretion Systems interactions with R. prowazekii and its host, increasing our overall R. prowazekii systems: Type I, Type II, Type IV and Type V. 2. Ligate to Fusion Plasmid understanding of intracellular growth and pathogenicity of R. prowazekii. 3. Transform Competent E. coli Referencences 1 Azad A. F. and Radulovic, S. (2003), Pathogenic Rickettsiae as 4. Isolate Plasmid DNA from Bioterrorism Agents. Annals of the New York Academy of Sciences, Colonies Type I Type IV 990: 734–738. doi: 10.1111/j.1749-6632.2003.tb07452.x TolC RP224 HlyD RP314/RP243 VirB1 RP400/RP457 2Andersson SG, et al. (1998) The genome sequence of Rickettsia HlyB RP315/RP157 VirB2 RP192 5. Digestion with Restriction VirB3 RRP0100 prowazekii and the origin of mitochondria. Nature 396:133–140. Type II VirB4 RP103/RP784 Enzymes Sec-SRP VirB5 absent . 3 Pang, H., & Winkler, H. H. (1996). Transcriptional analysis of the 16s SecA RP575 SecB RP070 VirB6 RP104-RP108 SecE RP134 SecD/F RP586/RP114 VirB7 RP288 PCR Amplification of Genes 1) RP016, rRNA gene in Rickettsia prowazekii. Journal of Bacteriology, 178(6), SecG RP079 SecY RP639 6. Verify Insert Orientation on Gel VirB8 RP287/RP289 2) RP290, and 3) RP621 Marker (M) Ffh RP173 FtsY RP775YidC RP048 VirB9 RP286/RP290 1750–1755. YajC RP585 VirB10 RP291 4 VirB11 RP292 Qin, A., Tucker, A. M., Hines, A., & Wood, D. O. (2004). Transposon Twin Arginine Transport (TAT) VirD4 RP293 Mutagenesis of the Obligate Intracellular Pathogen Rickettsia TatA RP749 TatC RP782 prowazekii.Applied and Environmental Microbiology, 70(5), 2816– Acknowledgements to Dr. Ryan Wood, Lonnie Driskell, and Robin Daughtery for their 2822. doi:10.1128/AEM.70.5.2816-2822.2004. technical expertise. This work was supported by NIH Grant AI020384..
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