Anaplasma marginale Genome Sequencing Projects
Anaplasma marginale is the most prevalent tick-borne pathogen of cattle worldwide. The disease results in significant morbidity and mortality in U.S. cattle and is a constraint to export. Critically, there is no federally licensed vaccine available and the live, blood-based vaccines widely used in tropical countries cannot be licensed in the U.S. due to the risk of transmitting both known and unknown pathogens. A vaccine for anaplasmosis is a priority for the USDA National Cattlemen’s Beef Association and many other research groups worldwide.
A. marginale is the type species for the genus Anaplasma which contains both animal and human pathogens.
The first strain selected for sequencing was the St. Maries strain as it represents a virulent tick transmissible strain. The second strain selected for sequencing was the virulent non-tick-transmissible Florida strain. These projects were completed using a targeted BAC-based clone-by clone approach. More recent genome projects have employed pyrosequencing to provide >96% genome coverage of the Virginia, Puerto Rico and Mississippi strains.
Two strains of A. marginale from Australia were sequenced revealing that there is limited diversity between these strains. The Dawn strain is avirulent, while the Gypsy Plains strain is a virulent field isolate.
The A. marginale centrale subspecies (strain Israel) genome was sequenced using a traditional shotgun based approach. This strain is employed as a live vaccine in a number of countries.
The A. marginale genome sequences were generated with funding from USDA/ARS CRIS, USDA CSREES and the Wellcome Trust.
Genome maps of the St. Maries, Florida, Gypsy Plains, Dawn and Israel strains. The inner most circle shows GC skew, the next circle show positions of tRNA (purple) and rRNA (orange) genes. The positions of CDSs are indicated as bars with genes on the reverse strand shown within the circle depicting genes on the forward strand. The St. Maries strain then shows the positions of the BAC clones (blue and pink arcs) or gap spanning PCR fragments (green and yellow arcs) that were used to obtain the complete sequence. The next ring shows the locations of the msp1, 2, 3 genes, while the outermost ring indicates the genome size coordinates (in 100 kb increments). The Florida strain map shows genome size (in bp) below the black circle. The outer four series of bars indicate repetitive genes (black), and regions missing from pyrosequenced genomes relative to the FL sequence: Virginia (blue), Puerto Rico (red) and Mississippi (green).
Brayton, K. A., D. P. Knowles, T. C. McGuire, and G. H. Palmer. Efficient use of a small genome to generate antigenic diversity in tick-borne ehrlichial pathogens. Proceedings of the National Academy of Sciences. 98: 4130-4135, 2001.
Brayton, K. A., L. S. Kappmeyer, D. R. Herndon, M. J. Dark, D. L. Tibbals, G. H. Palmer, T. C. McGuire and D. P. Knowles Jr. Complete genome sequencing of Anaplasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins. Proceedings of the National Academy of Sciences. 102: 844-849, 2005.
Dark, M. J., D. R. Herndon, L. S. Kappmeyer, M. P. Gonzales, E. Nordeen, G. H. Palmer, D. P. Knowles, Jr., and K. A. Brayton. Conservation in the face of diversity: Multistrain analysis of an intracellular bacterium. BMC Genomics. 10: 16. 2009.
Herndon, D. R., G. H. Palmer, V. Shkap, D. P. Knowles, Jr., and K. A. Brayton. Complete Genome Sequence of Anaplasma marginale ss. centrale. Journal of Bacteriology. 192: 379-380. 2010.
Aguilar-Pierlé, S., I. Imaz-Rosshandler, A. Akim Kerudin, J. Sambono, A. Lew-Tabor, P. J. Rolls, C. Rangel-Escareño, and K. A. Brayton. Genetic diversity of tick-borne rickettsial pathogens, insights gained from distant strains. Invited for a special issue entitled: Bacterial Pathogenomics: From Technology to Application. Pathogens. 3: 57-72. 2014.
BLAST the Anaplasma marginale genomes
BLAST on local server
NCBI BLAST (Basic local alignment search tool) BLAST with Microbial Genomes
Anaplasma marginale Transcriptome Sequencing Projects
Obtaining pure nucleic acids from obligate intracellular pathogens remains a challenge due to contamination with host material. However, the cost of high through-put sequencing has reduced to a level where it becomes feasible to sequence cDNA from obligate intracellular organisms. We have obtained sequence data from 1) A. marginale St. Maries strain during peak infection in the bovine host, 2) from the Florida strain at similar infection levels in the host, 3) St. Maries grown in ISE6 tick cell culture and 4) from a GFP insertional mutant created from the St. Maries strain and grown in ISE6 cell culture.
These experiments have highlighted genes that are involved in tick transmission, and have revealed pathways that respond to the transition from the bovine host to the tick vector. The GFP mutant was observed to grow more slowly than wild type, and analysis of the differences in transcription reveals pathways involved in protein synthesis and nucleotide synthesis are altered in this mutant.
Aguilar Pierlé, S., M. J. Dark, D. Dahmen, G. H. Palmer, and K. A. Brayton. Comparative genomics and transcriptomics of vector borne transmission. BMC Genomics. 13: 669. 2012.
Aguilar Pierlé, S., G. K. Hammac, G. H. Palmer, and K. A. Brayton. Transcriptional pathways associated with the slow growth phenotype of transformed Anaplasma marginale. BMC Genomics. 14: 272. 2013.
Hammac, G. K., S. Aguilar Pierlé, X. Cheng, G. A. Scoles, and K. A. Brayton. Global transcriptional analysis reveals surface remodeling of Anaplasma marginale in the tick vector. Parasites and Vectors. 7: 193. 2014.
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