Progress 03/15/05 to 02/28/08
Outputs
OUTPUTS: The outputs of this project include the ongoing molecular characterization of tick-derived molecules required for rickettsial infection. The data have been presented at the annual meeting of the American Society for Tropical Medicine and Hygiene. We have also initiated studies to characterize rickettsial replication within the tick host. These data have been published this year in a peer reviewed journal. PARTICIPANTS: One postdoctoral fellow from Thailand has lead the tick receptor study - unpublished. One Master's of Science candidate, Zanetti, lead the rickettsial infection of tick study that lead to the publication. TARGET AUDIENCES: The data have been presented at regular rickettsiology meetings and we have published some findings to provide the first enumeration of rickettsiae within the arthropod host. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have identified and characterized tick-derived histone as a receptor for rickettsiae. The manuscript is in preparation. We have detailed a real-time quantitative polymerase chain reaction assay for the quantification of rickettsia in Lone Star ticks during feeding and at each life cycle stage. The assay will be transferable to any laboratory and the data are a component of the hypothesis of this project (rickettsial virulence is a component of rickettsial replication).
Publications
- Zanetti AS, Pornwiroon W, Kearney MT, KR Macaluso*. Characterization of rickettsial infection in Amblyomma americanum (Acari:Ixodidae) by quantitative real-time PCR. J Med Entomol 2008; 45:267-275.
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Progress 01/01/07 to 12/31/07
Outputs
Progress has continued in the characterization of putatively-identified tick-derived molecules. The full-length sequences for Dermacentor variabilis vATPase (Dv-vATPase) and D. variabilis α-catenin (Dv-α-catenin) were cloned and expressed in Drosophila and mosquito cell lines. We were able to generate specific antibodies. We have attempted chemical-knockdown of vATPase in tick cells and determined that this molecule is not a component of rickettsial entry, as treated and untreated cells were equally infected. We have also carried out functional characterization studies for catenin and demonstrated that this molecule may be present in some of our cell lines, but not all. This will allow use to assess function in cell lines that lack catenin. We have continued our studies on the identification of tick-derived molecules utilized by spotted fever group Rickettsia (SFGR) for attachment, entry, and spread through the tick cells. Progress includes the isolation of a
previously uncultivatable strain of Rickettsia felis in our tick cell line ISE6; and identification of the rickettsial receptor molecule in the ISE6 cells. We are working on the protocol for RNAi in the tick cell line assay as a way to prove function. We are excited by the identified tick-derived molecule bound by SFGR for which a similar molecule has recently been identified as a SFGR receptor in mammalian cells. Towards the quantification of SFGR in arthropods, we developed a quantitative real-time PCR assay using a single control plasmid that contains both rickettsial and tick housekeeping genes. We are able to quantify SFGR in tick and cell culture models and recently had a manuscript accepted for publication on these results
Impacts
IMPACT In the tick host, expression of a number of molecules is modulated in response to rickettsial infection. However, the importance of these molecules in rickettsial survival via host cell modification, or direct interaction with rickettsiae, has yet to be determined. Since the initiation of this award, significant progress has been made with both the molecular and functional characterization of tick molecules associated with rickettsial survival. We have put great effort into assembling the tools to identify and characterize molecules associated with rickettsial infection. Also the enumeration of rickettsiae within a vector allows us to use biologically relevant numbers of rickettsiae in our assay. In understanding the complex interaction between ticks and rickettsia, novel targets for rickettsial control will be elucidated.
Publications
- AS Zanetti, Pornwiroon W, Kearney MT, KR Macaluso. Characterization of rickettsial infection in Amblyomma americanum (Acari:Ixodidae) by quantitative real-time PCR. J Med Entomol. 2007. In Press.
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Progress 01/01/06 to 12/31/06
Outputs
Progress has continued in the characterization of putatively-identified tick-derived molecules. The full-length sequences for Dermacentor variabilis vATPase (Dv-vATPase) and D. variabilis α-catenin (Dv-α-catenin), were cloned into the Escherichia coli pET32(a+) expression vector and recombinant protein (rDv-vATPase) was generated. rDv-vATPase and rDv-α-catenin were used to generate polyclonal antibodies in BALB/C mice. Peptide identification via mass spectrometry identified that polyclonal antibodies were non-specific. Subsequently we have now expressed both molecules in Drosophila and mosquito cell lines. The utilization of insect cell lines will facilitate the proper folding of recombinant protein and generation of more specific antibodies. We have continued our studies on the identification of tick-derived molecules utilized by spotted fever group Rickettsia (SFGR) for attachment, entry, and spread through the tick cells. Progress includes the
isolation of a previously uncultivatable strain of Rickettsia felis in our tick cell line ISE6, the results of which were recently published. We also identified tick-derived molecule bound by SFGR for which a similar molecule has recently been identified as a SFGR receptor in mammalian cells. A vATPase inhibitor was assessed in rickettsial invasion assays and we observe less intracellular rickettsiae in treated cells, compared to untreated controls. However, one major limitation to our functional assays was the quantification of SFGR in treatments. To address this limitation we developed a quantitative real-time PCR assay using a single control plasmid that contains both rickettsial and tick housekeeping genes. To date we are able to quantify SFGR in tick and cell culture models. The development of the necessary tools to quantify SFGR combined with the ISE6 tick cell system will facilitate in vitro studies to assess tick and Rickettsia interactions.
Impacts
In the tick host, expression of a number of molecules is modulated in response to rickettsial infection. However, the importance of these molecules in rickettsial survival via host cell modification, or direct interaction with rickettsiae, has yet to be determined. Since the initiation of this award, significant progress has been made with both the molecular and functional characterization of tick molecules associated with rickettsial survival. In understanding the complex interaction between ticks and rickettsia, novel targets for rickettsial control will be elucidated.
Publications
- Macaluso KR, Mulenga A, Simser JA, Azad AF. 2006. Characterization of Dermacentor variabilis molecules associated with rickettsial infection. Ann N Y Acad Sci 2006; 1078:384-8.
- Pornwiroon W, Pourciau SS, Foil LD, Macaluso KR. Rickettsia felis from cat fleas: isolation and culture in a tick-derived cell line. Appl Environ Microbiol. 2006; 72:5589-5595.
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Progress 01/01/05 to 12/31/05
Outputs
Progress has been made in the characterization of putatively-identified tick-derived molecules. The full-length sequence for Dermacentor variabilis vATPase (Dv-vATPase) was cloned into the Escherichia coli pET32(a+) expression vector and recombinant protein (rDv-vATPase) was generated. rDv-vATPase was used to generate polyclonal antibodies in BALB/C mice. Antibodies were used to confirm expression of vATPase in tick tissues by western blotting and for preliminary localization studies using confocal microscopy. Attempts to functionally characterize Dv-vATPase include using a vATPase inhibitor in rickettsial invasion assays. Many of the planned experiments, and those currently underway, require unique tools or assays to be developed for the tick model system. Specific assays developed or adapted to facilitate experiments under this aim include a quantitative real-time PCR assay using a single control plasmid that contains both rickettsial and tick housekeeping genes. We
have invested in the Gateway expression system which allows for high-level protein expression and easy movement of the gene of interest between different vectors. We have utilized this system with the lumino vector to visualize rDv-vATPase/dsRed fused protein expression in transfected Vero cell culture. We have also established a tick cell line, ISE6, in the laboratory to facilitate in vitro studies to assess tick and rickettsia interactions.
Impacts
In the tick host, expression of a number of molecules is modulated in response to rickettsial infection. However, the importance of these molecules in rickettsial survival via host cell modification, or direct interaction with rickettsiae, has yet to be determined. Since the initiation of this award, significant progress has been made with both the molecular and functional characterization of tick molecules associated with rickettsial survival. In understanding the complex interaction between ticks and rickettsia, novel targets for rickettsial control will be elucidated.
Publications
- No publications reported this period
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