Progress 10/01/10 to 09/30/15
Outputs
Target Audience:Research scientists at universities and scientific institutions whi are itersted in tick biology, tick-borne pathogens, and in tick symbionts. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Under this project, one PhDstudent and one postdoctoral fellow were trained intick cellular biology and rickettsial genetics/genomics. The postdoctoral fellow submitted an R21 application that was funded in the first round of review. The student's research waspublished three peer-reviewed articles, including one first-author publication in PLoS One. How have the results been disseminated to communities of interest?Journal publications, meeting presentations What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We sequenced, assembled and annotated the genome of R. peacockii, and compared it to the genome of R. rickettsii isolate Sheila Smith. The R. peacockii genome consists of a 1.29 Mb chromosome and a 26.4 kb plasmid (pRPR). Rickettsia peacockii, also known as the East Side Agent, is a non-pathogenic obligate intracellular bacterium found as a symbiont in Dermacentor andersoni ticks in the western USA and Canada. Its presence in ticks on the eastern slopes of the bitterroot valley in Montana is correlated with reduced prevalence of R. rickettsii, the agent of Rocky Mountain Spotted Fever. The R. peacockii (Rustic) genome consists of a 1.29 Mb chromosome and a 26.4 kb plasmid (pRPR). There are 40 chromosomal copies of the ISRpe1 transposon, closely related to a transposon in a Cardinium species from ticks. The transposons are associated with numerous deletions via recombination and a lack of chromosomal synteny with R. rickettsii, and other spotted fever group rickettsiae. Genes deleted or mutated in R. peacockii which may relate to loss of virulence include those coding for an ankyrin repeat containing protein (effector molecule), DsbA (disulfide-bond formation during the folding of secreted proteins), RickA (actin-based motility), PtrB/protease II (host cell entry), OmpA and ScaI (interaction with host cells during adhesion and/or entry), and a putative phosphoethanolamine transferase (binding to host cells). The gene coding for the ankyrin repeat containing protein is especially implicated as it (RrIowa_1113) is mutated in R. rickettsii strain Iowa, which is avirulent. The plasmid contains two copies of the transposon and a number of genes from distantly related organisms, such as part of the glycosylation island of Pseudomonas aeruginosa. The presence of numerous copies of the ISRpe1 transposon, possibly acquired by lateral transfer from a Cardinium species, is associated with extensive genomic reorganization, the and mutation of genes involved with rickettsial pathogenicity resulting in numerous deletions via recombination, a lack of synteny between the two genomes and loss of virulence. We completed assembly and annotation of the genome sequence of our culture isolate of the rickettsial endosymbiont of Ixodes scapularis (Rickettsia buchneri) and compared it with Rickettsia monacensis and R. peacockii. The R. buchneri parent isolate has 4 plasmids as reported for its genome obtained from the Ixodes scapularis genome sequence. Two sub-clones of R. buchneri lacked the plasmid pREIS4 indicating that it is less prevalent and less stable in R. buchneri than the other 3 plasmids. Genomic analyses of the two symbionts, R. peacockii and R. buchneri, revealed several mutations in genes encoding proteins involved in rickettsial pathogenicity and motility. Neighbor joining analysis based on amino acid sequences of 10 concatenated genes showed that R. peacockii and R. rickettsii were closely related and that R. buchneri and R. monacensis were also closely related. Genes involved in rickettsial motility (rickA, sca2), outer membrane structure and function, and chaperonin function were mutated in the endosymbionts. We further obtained culture isolates of rickettsial symbionts of Ixodes pacificus (REIP) and of the bat tick, Carios capensis (RCCE3). The genomes of both were sequenced through a white paper to NIH (Sequencing Rickettsiales Genomes), along with a large number of other Rickettsiales species contributed by us (22 isolates, including 15 Rickettsia species) and collaborating laboratories (8 isolates), and 14 isolates were deposited with BEI (10 from our lab). Sequences have been deposited in GenBank by the Institute for Genome Science at the University of Maryland, Baltimore (Dr. Julie Dunning Hotopp, Project Lead). We designed new shuttle vectors containing rickettsial genes to evaluate heterologous gene expression in the nonpathogenic endosymbionts. R. bellii were successfully transformed with a shuttle vector that contained the R. monacensis rickA gene. We demonstrated that R. bellii transformed with R. monacensis rickA displayed altered motility compared to wild-type. Although velocity was enhanced, distance traveled was reduced due to greater path curvature in the transformant. These results provided the first direct evidence of the involvement of RickA in rickettsial motility. A high-resolution tiling microarray that covers all of the genomic sequence of R. rickettsii isolate Sheila Smith (RrSS) was used to compare its global transcriptome during infection of Vero (African green monkey kidney) and ISE6 (I. scapularis) cell lines. Unfractionated RNA (host cell plus RrSS) was used to generate Cy3-labeled cDNA for hybridization to the arrays. The results showed that differential gene expression of RrSS between ISE6 and Vero cells was greater than previously seen, with 101 genes in ISE6 cells transcribed between 3- and 34-fold more than in Vero cells, and 54 genes in Vero cells transcribed 3- to 20-fold more than in ISE6 cells. Many non-annotated sequences show transcription, suggesting the existence of novel genes and regulatory RNAs. To better track rickettsial endosymbiont movement in ticks we constructed a shuttle vector carrying the gene mKATE2. mKate2 is a far-red fluorescent protein that will be more easily seen through the tick cuticle. We examined the interaction of GFP-expressing R. buchneri, which contains mutated sca2 and rickA genes, with host cell actin using tick cells transformed to express a red fluorescent LifeAct protein that binds to f-actin, and visualized rickettsiae by confocal microscopy. In infected cells, actin distribution became compartmentalized and filopodia were reduced. In late-stage infection, filopodia were absent and actin was strongly compartmentalized. This indicated that REIS affected f-actin conformation and distribution in host cells using effector proteins other than RickA or Sca2. F-actin compartmentalization by REIS may prevent other rickettsiae from invading the ovaries of female I. scapularis, a tick species not known to carry pathogenic rickettsiae. It has long been accepted that f-actin-based motility is a means to enter adjacent host cells, however, despite hours of observation many species of Rickettsia rarely exit host cells by means of actin based motility. We performed live cell imaging of fluorescent protein transformed R. monacensis and R. parkeri in Vero, HMEC-1, and RF/6A cells expressing mCherry LifeAct and a variety of fluorescent fusion proteins that localize to endoplasmic reticulum (ER), mitochondria (MI), Golgi, or the nucleus. Motile R. monacensis and R. parkeri continuously penetrated ER, severed MI and fragmented Golgi cisternae. Rickettsiae that penetrated into the nucleus slowed and eventually stopped moving while their actin tails grew longer. Although destruction of mitochondria by Rickettsia parkeri was accompanied by release of cytochrome c, apoptosis was not observed, indicating that Rickettsia parkeri possesses the ability to interfere with that process. We hypothesize that the observed organelle destruction allows diversion of cellular resources to bacterial replication and defense.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Oliver JD, Lynn GE, Burkhardt NY, Price LD, Nelson CM, Kurtti TJ, Munderloh UG. Accepted. Infection of Immature Ixodes scapularis (Acari: Ixodidae) by Membrane Feeding. J. Med. Ent.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Oliver JD, Ch�vez AS, Felsheim RF, Kurtti TJ, Munderloh UG. 2015. An Ixodes scapularis cell line with a predominantly neuron-like phenotype. Exp Appl Acarol. 66(3):427-42.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Robinson S, Neitzel D, Moen R, Craft M, Hamilton K, Johnson L, Mulla D, Munderloh UG, Redig P, Smith K, Turner C, Umber J, Pelican K. 2015. Disease risk in a dynamic environment: the spread of tick-borne pathogens in Minnesota, USA. EcoHealth 12(1):152-63.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Kurtti TJ, Felsheim RF, Burkhardt NY, Oliver JD, Heu CC, Munderloh UG. 2015. Rickettsia buchneri sp. nov., a rickettsial endosymbiont of the blacklegged tick Ixodes scapularis. Int J Syst Evol Microbiol. 65(Pt 3):965-70.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Heu CC, Kurtti TJ, Nelson CM, Munderloh UG. 2015. Transcriptional Analysis of the Conjugal Transfer Genes of Rickettsia bellii RML 369-C. PLoS One. 2015 Sep 9;10(9):e0137214
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Research scientists at universities and scientific institutions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project supports one graduate student and a postdoctoral scientist through NIH funding How have the results been disseminated to communities of interest? Through publication in peer-reviewed-journals and presentation at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? We will continue using the shuttle vectors as a tool to probe rickettsial gene function in homologous and heterologous systems. We will further examine the effects of rickettsial motility on host cells using biological markers of organelle function.
Impacts
What was accomplished under these goals?
We have shown that black-legged tick (Ixodes scapularis) infestation of hamsters that have been immunized against the symbiont of these ticks (Rickettsia buchneri) significantly reduces symbiont numbers in the ticks, but does not eliminate them, and does not obviously reduce fitness and fecundity. We have developed a membrane feeding apparatus that supports engorgement of Ixodes nymphs and adults. Using this feeder, we have shown that ciprofloxacin antibiotic when added to the blood meal eliminates R. buchneri, and are now testing if ticks can be reconstituted with other rickettsiae. We have characterized the neuron-like phenotype of tick cell line ISE6. The I. scapularis embryonic cell line ISE6 is the most widely utilized tick-derived cell line due to its susceptibility to a wide variety of tick- and non-tick-vectored pathogens.Little is known about its tissue origin or biological background.Stimulation of ISE6 cells by in vivo exposure to compounds present in larval or molting ticks resulted in the development of neuron-like morphologic characteristics in the affected cells.Protein expression of ISE6 cells was compared with that of another I. scapularis-derived cell line, IDE12, and dissected tick synganglia.Results demonstrated the presence of the neuronal marker protein, type 3 β-tubulin, in all three samples, as well as of other shared and unique neuronal proteins. However, neuronal proteins were much more abundantly expressed in ISE6 cells, indicating this cell line could be a good tool for the development of novel acaricides. We have used live imaging and time-lapse microscopy to show that fluorescent marker expressing rickettsiae continuously collide with cellular organelles, resulting in physical damage to mitochondria, Golgi and endoplasmic reticulum. Surprisingly, damage to mitochondria did not induce apoptosis, as expected, suggesting that rickettsiae interfere with this process. These findings provide new knowledge about rickettsial pathogenesis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Oliver JD, Burkhardt NY, Felsheim RF, Kurtti TJ, Munderloh UG. 2014. Motility characteristics are altered for Rickettsia bellii transformed to overexpress a heterologous rickA. AEM 80:1170-1176
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2015
Citation:
The Structural Consequences of Bacterial Motility In Host Cell Cytoplasm. In preparation. Herron MJ, Kumagai Y, Burkhardt NY, Felsheim RF, Kurtti TJ, Munderloh UG. PLoS One
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Research scientists at universities and scientific institutions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project support one graduate student through NIH funding How have the results been disseminated to communities of interest? Through publication in peer-reviewed-journals and presentation at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? We will continue using the shuttle vectors as a tool to probe rickettsial gene function in homologous and heterologous systems. We will further examine the effects of rickettsial motility on host cells using biological markers of organelle function.
Impacts
What was accomplished under these goals?
1) Comparative microarray analysis of global gene expression in R. rickettsii and R. peacockii during replication in a Rocky Mountain wood tick (Dermacentor andersoni) cell line A high-resolution tiling microarray that covers all of the genomic sequence of R. rickettsii isolate Sheila Smith (RrSS) was used to compare its global transcriptome during infection of Vero (African green monkey kidney), DAE100 (Dermacentor andersoni),and ISE6 (Ixodes scapularis) cell lines. Unfractionated RNA (host cell plus RrSS) was directly labeled for hybridization to the arrays. The results, viewed using the genome browser Artemis, showed specific transcription without confounding background. Differential gene expression of RrSS between ISE6 and Vero cells was greater than previously seen, with 101 genes in ISE6 cells transcibed between 3- and 34-fold more than in Vero cells, and 54 genes in Vero cells transcribed 3- to 20-fold more than in ISE6 cells. Many non-annotated sequences show transcription, suggesting the existence of novel genes and regulatory RNAs. Results from RNA harvested from DAE100 cells are still in progress. 2) Sequence of rickettsial plasmids Recent work has focused on the whole genomes and plasmid sequences of poorly characterized isolates of Rickettsia hoogstraalii, Rickettsia bellii, and Rickettsia amblyommii. An interesting result has been the high amount of tick and/or eukaryote DNA being sequenced in genomes run on the Illumina platform (up to 57% of total reads) but low amounts of eukaryote sequences (~4%) for reads from PacBio, indicating that PacBio excels for de novo assembly due to fewer contaminating reads as well as its greater read length. This is particularly important for analysis of plasmids where high amounts of lateral gene transfer are expected. Despite high coverage of plasmids in our reads, reference based assembly to any of the already sequenced plasmids is typically poor due to lack of synteny and conservation of genes within this genus. Nonetheless, substantial plasmid coverage is available for all isolates. 3) Role of plasmids in rickettsial adaptation to environmental changes To improve our understanding of transcriptional control in rickettsiae, we focused on non-pathogenic R. bellii RML-369C as it grows in tick cell culture. We used qRT-PCR to screen 10 housekeeping genes: atpB, dnaK, gltA, gyrA, infB, metG, nrdF, rpoB, 16s rRNA, and tlc5 for use as reference genes. Out of the 5 genes assessed using Normfinder, we identifiedmetG as the most stably expressed gene throughout a 72 hr post infection growth period in tick cell culture. A combination of atpB and 16s rRNA was identified as being best suited for transcriptional analysis. A unique feature of the REIS-plasmid pREIS2 is that it encodes a biotin operon, likely acquired by lateral gene transfer, which possibly enhances its ability to serve as a symbiont by providing a key factor to its tick host that is missing from the blood meal. To investigate functionality of this symbiont operon, we cloned the entire operon encoding all 6 genes into the multiple cloning site of our shuttle vector, yielding pRAM18dSGA[biotin].
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Oliver JD, Burkhardt NY, Felsheim RF, Kurtti TJ, Munderloh UG. 2013. Motility characteristics are altered for Rickettsia bellii transformed to overexpress a heterologous rickA. AEM Dec. 2, Epub ahead of print
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Ticks transmit a great variety of pathogens, many of which cause emerging diseases, and include pathogens classified as potential bioterrorism agents, e.g., Rickettsia rickettsii, the agent of Rocky Mountain spotted fever. Globally, the incidence of tick-borne diseases affecting humans and animals is rising as tick vectors spread aided by climate change that enables them to expand their habitat into areas that used to be unsuitable for their survival. Due to toxicity of acaricides and increasing levels of resistance in tick populations, chemical control of ticks may be undesirable, is often impractical, and increasingly ineffective. We have developed methods for genetic manipulation of tick-borne rickettsiae using transposon mutagenesis to knock out genes randomly, and for insertion of shuttle vectors carrying genes of interest into rickettsiae. We are collaborating with five laboratories at the University of Florida (Gainesville), Kansas State University (Manhattan), the University of South Alabama (Mobile), the University of California (Riverside), and Washington State University. Our results (since last year) have been published in peer-reviewed articles and an invited book chapter. We have shared materials such as tick cell lines, rickettsial pathogen isolates and mutants, and plasmid constructs with these collaborators, and many more laboratories in the USA, UK, and Germany using MTAs. PARTICIPANTS: Ulrike Munderloh is the lead scientist of this project, which involves collaboration with Tim Kurtti. Lab staff include Rod Felsheim, Curt Nelson, Nicole Burkhardt, Lisa Price and Dr. Jon Oliver. Two Ph.D. students are supported by this project, Adela Oliva Chavez and Chan Heu. Outside collaborators were Dr. Jeffrey Foster, University of Northern Arizona, Flagstaff; Dr. Anthony F. Barbet, University of Florida, Gainesville; Dr. Roman Ganta, Kansas State University, Manhattan; Dr. Joao Pedra, University of California, Riverside; Dr. Kelly Brayton, Washington Sate University, Pullman; Dr. David Wood, University of South Alabama, Mobile. TARGET AUDIENCES: Research scientists at universities and scientific institutions. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The plasmids of symbionts of Ixodes scapularis (REIS) and Ixodes ricinus (Rickettsia monacensis) were sequenced. REIS carries four plasmids, and R. monacensis one. Pulsed-field gel electrophoresis and Southern blot analysis confirmed the presence of all 4 plasmids in uncloned REIS, and the absence of pREIS4 in cloned REIS, suggesting that it is present in only a small subpopulation of rickettsiae. We generated multiple versions of recombinant plasmids incorporating a spectinomycin resistance cassette to test the utility of the multiple cloning site in pRAM18dRGA for introduction of different genes into rickettsial species. We successfully transformed R. montanensis with a shuttle vector encoding rickA in the multiple cloning site. RT-PCR and rescue cloning demonstrated that the transgene was expressed from the recombinant plasmid, but functional assays using time-lapse imaging did not reveal any differences in the motility of R. montanensis carrying the extra rickA gene. rickA and sca2 were cloned into the MCS of shuttle vectors, and tested in R. peacockii, R. montanensis and R. bellii. R. peacockii was successfully transformed only with the basic shuttle vector, whereas R. montanensis and R. bellii were successfully transformed with the shuttle vector carrying rickA. We were not able to transform sca2 into these species. A collaborator employed complimentary next-generation sequencing technologies using Illumina GAIIX 100 bp paired-end reads, PacBio Continuous Long Reads generating up to 10,000 bp reads, and the Ion Torrent PGM on a 318 chip with 200 bp reads for whole genome analysis of Rickettsia spp., taking advantage of each of their strengths. Using 50 whole genome sequences representing 24 species, including R. amblyommii An13 and AaRSc, R. hoogstraalii RCee3, and R. bellii, the lab carried out a single-nucleotide polymorphism (SNP) based phylogenomic analysis of rickettsiae to infer genetic relationships among closely related Rickettsia spp. by making use of the entire genome including intergenic regions. Along with the rickettsial chromosomes, plasmid sequences were also acquired for these species. Attempts to demonstrate exchange of rickettsial plasmid DNA have thus far been unsuccessful. Since R. bellii RML-369C encodes a complete set of tra genes putatively involved in the transfer of genetic material, we demonstrated active transcription of the tra cluster in R. bellii in tick and Vero cells as a single operon, including intergenic regions. We have for the first time been able to express an outer surface transgene in a Rickettsia species, but is not clear why this was only possible in R. belli and R. montanensis, and not R. peacockii. Additionally, the reasons for the failure to transfect rickettisae with sca2 need to be investigated. Although a number of Rickttsia spp. are known to possess tra genes, their transcriptional activity has not been analyzed. The research summarized here has demonstrated that the R. belli tra cluster is actively transcribed in both tick and mammalian host cells. Because the R. belli isolate used does not carry plasmids, we conclude that they are not exclusively linked to plasmid transfer.
Publications
- Oliva Chavez AS, Felsheim RF, Kurtti TJ, Brayton KA, Ku P-S, Munderloh UG. 2012. Expression Patterns of Anaplasma marginale msp2 Variants Change in Response to Growth in Cattle, and Tick Cells versus Mammalian Cells. PloSOne. 7(4):e36012
- Al-Khedery B, Lundgren AM, Stuen S, Granquist EG, Munderloh UG, Nelson CM, Alleman RA, Mahan SM, Barbet AF. 2012. Structure of the Type IV Secretion System in Different Strains of Anaplasma phagocytophilum. BMC Genomics.
- Cheng C, Nair ADS, Indukuri VV, S, Felsheim RF, Jaworski D, Munderloh UG, Ganta RR. 2012. Targeted and Random Mutagenesis of Ehrlichia chaffeensis for the Identification of Genes Required for In vivo Infection. PLoS Pathogens.
- Chen G, Severo MS, Sakhon OS, Choy A, MJ, Felsheim RF, Wiryawan H, Liao J, Johns JL, Munderloh UG, Sutterwala FS, Kotsyfakis M, Pedra JHF. 2012. Anaplasma phagocytophilum LPDA1 Affects Host-Derived Immunopathology During Microbial Colonization. Infect Immun 80(9):3194-205.
- Wood DO, Hines A, Tucker AM, Woodard A, Driskell LO, Burkhardt NY, Kurtti TJ, Baldridge GD, Munderloh UG. 2012. Establishment of a Replicating Plasmid in Rickettsia prowazekii. PLoSOne 7(4):e34715. Epub Apr 17.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Among arthropods, ticks transmit the greatest variety of pathogens: paralysis toxins, viruses, bacteria and protozoa. Moreover, many of these cause emerging diseases, and include pathogens classified as potential bioterrorism agents, e.g., Rickettsia rickettsii, the agent of Rocky Mountain spotted fever. Globally, the incidence of tick-borne diseases affecting humans and animals is on the rise as tick vectors spread, aided by climate change that enables them to expand their habitat into areas that used to be too cold or too dry to permit survival. Due to the broad toxicity of acaricides and increasing levels of resistance in tick populations, chemical control of ticks may be undesirable, is often impractical, and increasingly ineffective. At the same time, manipulation of tick-borne rickettsiae has been difficult or impossible, because of their obligate intracellular nature. Our research aims to create tools suitable for genetic manipulation of rickettsiae in order to identify genes and gene families that encode pathogenicity factors, or traits that govern tick transmissibility. Of particular interest are transposons that we have harnessed to carry selected genes into the rickettsial genome (transposon mutagenesis), or torandomly disrupt genes, as well as the rickettsial plasmids that we are developing as genetically mobile elements to facilitate transfer of much larger sets of genes into rickettsiae. We are generating a family of molecular tools to eventually enable genetically based methods to prevent tick-borne rickettsial diseases. PARTICIPANTS: Dr. Phil Williamson at the University of North Texas Health Sciences Center TARGET AUDIENCES: Research scientists at universities and scientific institutions. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We sequenced 3 Rickettsia amblyommii plasmids and used them to produce shuttle vectors using backbones from commercial vectors. Function of constructs was confirmed by transformation of 5 Rickettsia spp. We added a multiple cloning site (MCS) into which we inserted the rickA gene from R. monacensis, and are working to transform R. montanensis. To extend applicability, we replaced the selectable marker with spectinomycin resistance, an antibiotic without clinical relevance in rickettsioses, and used only rarely. We sequenced the genome of the rickettsial endosymbiont of Ixodes scapularis (REIS) and now have two symbiont genomes from different tick vectors of important pathogens. We are focusing on REIS because it carries four different plasmids that we are interested in adding to our suite of plasmid vectors. We initiated studies on gene expression in Rickettsia rickettsii Sheila Smith in 3 cell lines (tick, human, and Vero cells). RNA was purified from cultures grown at 34 C, 37 C and 40 C, and will be used to examine gene expression. We performed live cell imaging of several rickettsial species moving within Vero cells expressing an mCherry-f-actin or EGFP-f-actin fusion construct. We revealed species specific patterns of motility that included spinning in place, reversal, and differences in velocity, suggesting that actin based motility serves differing roles in Rickettsia spp.. The absence of host cell escape behaviors in most rickettsiae indicates that this is not the primary purpose of actin-based motility. Using fluorescent probes to label intracellular organelles, we observed destruction of endoplasmic reticulum, mitochondria, and Golgi cisternae by rickettsiae colliding with them. We infected I. scapularis (natural host) and Dermacentor variabilis (non-host) with GFP-expressing REIS by capillary-feeding and intrahemocoelic injection. One week post feeding, REIS had extensively colonized the gut wall, and was retained cell-free in the hemolymph of adult I. scapularis for >5 days. When injected onto non-vector D. variabilis, rickettsial numbers declined, and none could be detected by day 3. These results indicate that plasmid-transformed REIS avoid elimination by the immune response of the susceptible tick, I. scapularis, but are cleared from the hemolymph of refractory D. variabilis. We plan to analyze candidate rickettsial motility genes to complement symbiotic R. peacockii and REIS, both of which are naturally non-motile. We want to test parA and dnaA genes of other rickettsial plasmid for their ability to mediate maintenance of shuttle plasmids in rickettsiae, so as to expand the reservoir of genetic tools. Availability of virulent R. rickettsii RNA from tick and human host cell cultures will allow us to complete gene expression studies using custom tiling arrays. Towards the ultimate goal of tick paratransgenesis, we will attempt to experimentally replace wild-type REIS with transformants in laboratory ticks. We will seek the required approvals from relevant authorities beforehand. This would pave the way towards using REIS as vehicles to introduce iRNA or other gene products that could block pathogen acquisition and transmission.
Publications
- Munderloh UG, Kurtti TJ. 2011. Emerging and Re-emerging Tick-borne Diseases: New Challenges at the Interface of Human and Animal Health. In: Critical Needs and Gaps in UnderstandingWorkshop Report. Committee on Lyme Disease and Other Tick-Borne Diseases: The State of the Science. Washington (DC): National Academies Press (US).
- Munderloh UG, Burkhardt NY, Felsheim RF, Baldridge GD, Wood DO, Hines A, Tucker AM, Woodward A, Driskell O, Kurtti TJ. 2011. Rickettsial plasmids what good are they Invited talk, 6th International Meeting on Rickettsiae and Rickettsial Diseases; Heraklion, Crete, June.
- Burkhardt NY, Heu C, Williamson PC, Billingsley PM, Felsheim RF, Kurtti TJ, Munderloh UG. 2011. Rickettsia amblyommii plasmids as shuttle vectors to examine rickettsial gene function. Poster, 6th International Meeting on Rickettsiae and Rickettsial Diseases; Heraklion, Crete, June.
- Munderloh UG, Burkhardt NY, Felsheim RF, Baldridge GD, Kurtti TJ. 2011. Rickettsial Plasmids what good are they. Invited talk, ASM General Meeting, New Orleans, May.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Among arthropods, ticks transmit the greatest variety of pathogens: paralysis toxins, viruses, bacteria and protozoa. Moreover, many of these cause emerging diseases, and include pathogens classified as potential bioterrorism agents, e.g., Rickettsia rickettsii, the agent of Rocky Mountain spotted fever. Globally, the incidence of tick-borne diseases affecting humans and animals is on the rise as tick vectors spread, aided by climate change that enables them to expand their habitat into areas that used to be too cold or too dry to permit survival. Due to the broad toxicity of acaricides and increasing levels of resistance in tick populations, chemical control of ticks may be undesirable, is often impractical, and increasingly ineffective. At the same time, manipulation of tick-borne rickettsiae has been difficult, or impossible, because of their obligate intracellular nature. Our research aims to create tools suitable for genetic manipulation of rickettsiae in order to identify genes and gene families that encode pathogenicity factors, or traits that govern tick transmissibility. Of particular interest are transposons that may be harnessed to carry selected genes into the rickettsial genome, as well as the newly discovered rickettsial plasmids as genetically mobile elements and carriers of horizontally acquired genes. A family of molecular tools will eventually enable genetically based methods to prevent tick-borne rickettsial diseases. PARTICIPANTS: Dr. Phil Williamson at the University of North Texas Health Sciences Center TARGET AUDIENCES: Microbiologists working with rickettsiae in university- and government labs PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We transformed non-pathogenic Rickettsia montanensis by electroporation with TN5 transposon pMOD700 containing GFPuv and chloramphenicol acetyltransferase genes under regulation of the R. rickettsii ompB promoters, yielding transformant Rmontanensis700. Northern blots showed that GFPuv and CAT mRNAs were larger and smaller than predicted, whereas immunoblots showed that the GFPuv protein was the predicted size. The ompB promoter may be the most problematic of the four promoters tested so far. Plasmids may be significant in rickettsial evolution by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria. We detected plasmids in rickettsiae from humans and ticks, including R. akari, R. amblyommii, R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis, but not in R. parkeri. We confirmed existence of multiple plasmids in some species by cloning plasmids with different sequences from two R. amblyommii isolates. Phylogenetic analysis of rickettsial ParA proteins indicated multiple origins and plasmid incompatibility groups; rickettsial small heat shock protein genes (hsp2) were plasmid specific and hsp1 genes, found only on plasmids of R. amblyommii, R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers ranged from 2.4 to 9.2 per chromosome, as determined by real-time qPCR. In collaboration with Dr. Williamson at the University of North Texas Health Sciences Center, two plasmids from R. amblyommii from the Lone Star tick, were sequenced and annotated, and a third was partially sequenced. Importantly, parA- and dnaA-like regions were identified which control plasmid replication and partitioning in bacteria. Subclones were transferred to University of Minnesota and utilized to investigate the minimum requirement for plasmid maintenance in plasmid-less Rickettsiae as well as those carrying their own plasmids. This information is needed for the design of broad host-range shuttle vectors for rickettsial transformation. A 27.6kb pRAM18 construct encoding rifampicin resistance and GFPuv (pRAM18/Rif/GFPuv) was used to transform R. bellii. To create a more efficient construct we transferred the parA and dnaA portion of pRAM18 into pGEM/Rif/GFPuv and used this 10.3kb version to stably transform R. montanensis, R. monacensis and R. bellii. A 8.87kb subclone containing parA and dnaA from plasmid pRAM30 was cloned into a pUC19 backbone and used successfully in the same species, with maintenance of up to 15 copies. However, attempts to transform R. amblyommii with pRAM18 constructs have failed. Transformation of R. monacensis, R. montanensis, R. felis, R. peacockii and R. amblyommii strains Ac/Pa & WB82 with partial pRM constructs containing parA and dnaA was also unsuccessful. We are constructing a pRAM18 vector with a multiple cloning site and have inserted AmTr/Cherry to test its stability. The pGEM and pUC shuttle plasmids have been modified to encode spectinomycin resistance, and are candidates for use in pathogenic rickettsiae.
Publications
- Burkhardt NY, Baldridge GD, Williamson PC, Billingsley PM, Felsheim RF, Kurtti TJ, Munderloh UG. 2010. Development of a Shuttle Vector for Transformation of Diverse Rickettsia Species. Poster, 24th Meeting of the ASR, Stevenson, WA
- Felsheim, RF, Kurtti, TJ, and Munderloh, UG. 2010. One way to become an endosymbiont; the case of Rickettsia peacockii. Poster, Sixth International Wolbachia Conference, Asilomar, CA. June 9-14.
- Baldridge GD, Burkhardt NY, Labruna MB, Pacheco RC, Paddock CD, Williamson PC, Billingsley PM, Felsheim RF, Kurtti TJ, Munderloh UG. 2010. Low Copy Number Plasmids are Widely Dispersed in Rickettsia Species Associated with Blood-feeding Arthropods and may have Multiple Origins. Mar;76(6):1718-31
- Baldridge GD, Burkhardt NY, Oliva AS, Kurtti TJ, Munderloh UG. 2010. Rickettsial ompB Promoter Regulated Expression of GFPuv in Transformed Rickettsia montanensis. PLoS One Jan. 2
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Tick-borne diseases have increased in incidence globally, including in the Midwestern United States and Minnesota (e.g. a fatal case of Rocky Mountain Spotted fever in a child from Dakota County was reported this summer for the first time). Ticks carry a vast array of microbes that cause emerging or reemerging diseases in mammals, such as spotted fever rickettsioses. Additionally, non-pathogenic rickettsiae are symbiotically associated with ticks and have been implicated in regulating spotted fever epidemiology by a phenomenon known as interference. The molecular biology of either rickettsial group is poorly defined, but offers promise for control of ticks as well as for control of rickettsial diseases in humans. Research with tick-borne rickettsiae has been neglected for decades because antibiotics discovered last century provided effective treatment, and research with BSL3 level spotted fever rickettsiae is cumbersome. Recently, interest in rickettsia research is rising due to discovery and increasing incidence of new and previously known rickettsioses, and potential abuse of rickettsiae as bioterrorism agents. Using nonpathogenic rickettsiae, we have developed the first effective tools for molecular manipulation of these fastidious, obligate intracellular bacteria. Our research results were published in both open access and traditional journals. PARTICIPANTS: Uli Munderloh is the Principal Investigator for this CRIS project. Professor Tim Kurtti, Dr. Gerald Baldridge, Nicole Burkhardt, Rod Felsheim and Adela S. O. Chavez assisted with the design and execution of the research. Dr. Chris Paddock (CDC, Atlanta, Ga) and Dr. Marcelo LaBruna (Sao Paulo, Brasil) provided several additional rickettsia isolates for plasmid analysis. Collaborations also included projects on tick functional genomics (Department of Primary Industries and Fisheries, Emerging Technologies, Moorooka, Queensland, Australia), cat scratch fever/Bartonellosis (Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC), and characterization of novel ehrlichiosis agents (School of Forestry and Natural Resources, University of Georgia, Athens, GA; New York State Department of Health, Albany, NY). TARGET AUDIENCES: The research is relevant to other scientists in public health, medicine and agriculture working with rickettsial disease agents of humans and animals. Moreover, rickettsiae are being detected in insects that are plant pests, or suspected of directly causing plant diseases, and this research will impact those areas of investigation as well. The molecular tools we are developing will provide ways to genetically manipulate obligate intracellular bacteria to create vaccines and diagnostics for medical and agricultural applications. PROJECT MODIFICATIONS: No changes were made.
Impacts
Objective 1. A Shuttle Vector for Transformation of Rickettsiae. We have achieved transformation of R. bellii by electroporation with a recombinant plasmid cloned from R. amblyommii. pRAM18 carries Rifampicin resistance and GFPuv markers, and is designated as pRAM18/GFPuv/Rif. Resistant rickettsial populations expressing GFPuv were detected in tick cell cultures within 2 weeks. Pulsed field gel electrophoresis (PFGE) and Southern blotting of pRAM18 transformants resolved a plasmid band that hybridized to a GFPuv probe. Therefore, pRAM18/GFPuv/Rif DNA in the transformants was maintained as an intact plasmid and was not integrated into the chromosome. The R. bellii pRAM18/GFPuv/Rif transformants are the most intensely fluorescent rickettsiae that we have isolated. This is likely due to the multiple plasmid copies present, significantly more than the native plasmids in Rickettsia species. More recently, we have been able to produce a significantly smaller shuttle vector likewise cloned from the native R. amblyommii plasmid, but with reduced content. This construct is currently under evaluation, and showing promise. Objective 2. We found that Himar1 transposase constructs in "cis" configuration with spectinomycin resistance (aadA) and fluorophore expression driven by the A. marginale tr promoter were active in rickettsiae and yielded R. peacockii transformants with single chromosomal integrations. We then designed both "cis" and "trans" Himar1 transposons in which tr was replaced by the rickettsial ompA promoter, and the chloramphenicol acetyl transferase (CAT) gene replaced aadA. In addition, we spliced the pMOD cassette that we have previously used with Epicentre's Tn5 system into pHimar, between Himar inverted repeats, likewise in a "trans" configuration where the transposase is encoded on a second plasmid. We are evaluating the transformation efficiency of these transpon plasmids using R. bellii, R. parkeri and R. peacockii. We determined that the Himar1 transposon integration sites in our R. felis LSU and R. bellii Mogi transformants are chromosomal, and are now evaluating an R. bellii Himar1 transformant population.Both new systems will be extraordinarily valuable for genetic manipulation and analysis of tick-and insect-borne rickettsiae important in medicine and agriculture.
Publications
- Felsheim RF, Kurtti TJ, Munderloh UG. 2009. Genome sequence of the endosymbiont Rickettsia peacockii and comparison with virulent Rickettsia rickettsii: Identification of Virulence Factors. PLoS One 4(12): e8361.
- Felsheim RF, Oliva Chavez AS, Palmer GH, Crosby L, Barbet AF, Kurtti TJ, Munderloh UG. 2009. Transformation of Anaplasma marginale. Vet. Parasitol. Sept. 20. Epub ahead of print.
- Billeter SA, V.P. Diniz PP, Battisti JM, Munderloh UG, Breitschwerdt EB, Levy MG. 2009. Infection and Replication of Bartonella Species Within A Tick Cell Line. Experimental and Applied Acarology. 49(3):193-208. Epub Feb. 26.
- Baldridge GD, Scoles GA, Burkhardt NY, Schroeder B, Kurtti TJ, Munderloh UG. 2009. Transovarial transmission of Francisella-like Endosymbionts and Anaplasma phagocytophilum in Dermacentor albipictus (Acari:Ixodidae). J Med Ent 46:625-32.
- Kurscheid S, Lew-Tabor AE, Rodriguez Valle M, Bruyeres AG, Doogan VJ, Munderloh UG, Guerrero FD, Barrero RA, Bellgard MI. 2009. Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila. BMC Molecular Biology 10:26.
- Yabsley MJ, Murphy SM, Luttrell MP, Wilcox BR, Howerth EW, Munderloh UG. 2009. Characterization of Candidatus Neoehrlichia lotoris (Family Anaplasmataceae) from raccoons (Procyon lotor). IJSEM 58: 2794-2798.
- Munderloh UG, Silverman DJ, MacNamara KC, Ahlstrand GG, Chatterjee M, Winslow GM. 2009. Ixodes ovatus ehrlichia exhibits unique ultrastructural characteristics in mammalian endothelial and tick-derived cells. Annals NY Acad. Sci. 1166:112-9.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Tick-borne diseases have increased in incidence globally, including in the midwestern United States and Minnesota. Ticks carry a vast array of microbes that cause emerging or reemerging diseases in mammals, such as spotted fever rickettsioses. Additionally, non-pathogenic rickettsiae are symbiotically associated with ticks and have been implicated in regulating spotted fever epidemiology by a phenomenon known as interference. The molecular biology of either rickettsial group is poorly defined, but offers promise for control of ticks as well as for control of rickettsial diseases in humans. Research with tick-borne rickettsiae has been neglected for decades because antibiotics discovered last century provided effective treatment, and research with BSL3 level spotted fever rickettsiae is cumbersome. Recently, interest in rickettsia research is rising due to discovery and increasing incidence of new and previously known rickettsioses, and potential abuse of rickettsiae as bioterrorism agents. Using nonpathogenic rickettsiae, we have developed the first effective tools for molecular manipulation of these fastidious, obligate intracellular bacteria. Our research results should be equally applicable to studying rickettsiae associated with insects that are plant pests. PARTICIPANTS: Uli Munderloh is the Principal Investigator for this CRIS project. Professor Tim Kurtti, Dr. Gerald Baldridge, Nicole Burkhardt, Rod Felsheim and Adela S. O. Chavez assisted with the design and execution of the research. Dr. Chris Paddock (CDC, Atlanta, Ga) and Dr. Marcelo LaBruna (Sao Paulo, Brasil) provided several additional rickettsia isolates for plasmid analysis. TARGET AUDIENCES: The research is relevant to other scientists working with rickettsial disease agents of humans and animals. Moreover, rickettsiae are being detected in insects that are plant pests, or suspected of directly causing plant diseases, and this research will impact those areas of investigation as well. The molecular tools we are developing will provide ways to genetically manipulate obligate intracellular bacteria to create vaccines and diagnostics for medical and agricultural applications. PROJECT MODIFICATIONS: No major changes
Impacts
Objective 1. A Shuttle Vector for Transformation of Rickettsiae. We have completed sequence analysis of the pRM plasmid from Rickettsia monacensis, and have begun testing them for their ability to replicate in recipient rickettsiae. Constructs were designed to contain selectable markers, as well as the putative origin of replication. Although there was evidence for transient replication, stable transformants were not obtained. We discovered that rickettsial plasmids were lost during in vitro passage, and surveyed isolates in our lab and from collaborators at the CDC and in Brasil. They included: R. montanensis, R. peacockii, R. helvetica, R. hoogstraalii, R. massiliae, and several R. bellii, R. parkeri, and R. amblyommii isolates. All except R. parkeri contained plasmids, and there were differences in plasmid complement among isolates of the same species depending on geographic origin. We developed a method to clone the higher copy number plasmids in order to define common sequences necessary for plasmid replication. We prepared pJAZZ shotgun libraries of R. amblyommii DNA digested with HpaI and SwaI, whose recognition sites should occur at low frequency in AT-rich rickettsial plasmids. Colony lifts of the libraries were hybridized with a pool of probes for genes common to both R. amblyommii and pRM plasmids. We obtained clones containing plasmid inserts of approximately 6.5 - 20 kbp, including clone L19. PFGE analysis showed that clone L19 contains an insert with end terminal sequences that respectively begin within 20 bp of each other in an R. massiliae pRMa gene homolog on the pRa plasmid and proceed in opposite directions toward the termini of that gene. Clone L19 likely contains most or all of pRA and we will clone it into the CopyRight v2 BAC vector. Objective 2. A Himar1 transposase system optimized for SFG rickettsiae. We recently used Himar1 transposase constructs similar to those that have yielded our Anaplasma transformants to generate transformed R. peacockii. Himar1 has also been successfully adapted to transformation of typhus group rickettsiae in the laboratory of Dr. David Wood, and we have adapted his constructs to isolate several transformants of R. amblyommii and R. bellii. Using cis constructs in which the Himar1 transposase, a fluorescent marker, and a selectable marker conferring spectinomycin resistance are encoded on the same plasmid (configured as a non-autonomous transposable element), we have generated two R. peacockii transformants, one expressing the red-fluorescent mCherry, and the second GFPuv under regulation of the Anaplasma marginale transcriptional promoter Amtr1. Furthermore, an mCherry-transformant of rickettsial symbionts from the Lone Star tick, R. amblyommii strain MOAa, was obtained using the same construct. This important result demonstrates that promoters that are not native to rickettsiae are nevertheless functional in rickettsiae, increasing the choices of constructs available for transformation. Both new systems will be extraordinarily valuable for genetic manipulation and analysis of tick-and insect-borne rickettsiae important in medicine and agriculture.
Publications
- Baldridge GD, Burkhardt NY, Felsheim RF, Kurtti TJ, Munderloh UG. 2008. Plasmids of the pRM/pRF Family Occur in Diverse Rickettsia species. Appl Environ. Microbiol. 74:645-52.
- Baldridge GD, Burkhardt NY, Felsheim RF, Kurtti TJ, Munderloh UG. Transposon Insertion Reveals pRM, a Plasmid of Rickettsia monacensis. Appl Environ Microbiol. 2007 73:4984-95.
- Baldridge GD, Kurtti TJ, Burkhardt NY, Baldridge AS, Nelson CM, Oliva AS, Munderloh UG. 2007. Infection of Ixodes scapularis ticks with Rickettsia monacensis expressing green fluorescent protein: A model system. Journal of Invertebrate Pathology 94:163-74.
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