Experimental annotation of bovine respiratory disease pathogen genomes by proteogenomic mapping
NRI competitive grant no: 2006-35600-17688
Since 2000, the USDA has invested heavily in microbial genome sequencing, which has resulted in the completion of a large number of genome sequences from animal pathogens that impact U.S. agriculture. However, the ability to decipher the information content of sequenced genomes is currently limited and has seriously hindered the full experimental exploitation of these sequences. In particular, there is no experimental evidence for the existence of predicted protein products from the large majority of annotated genes in sequenced microbial genomes. Furthermore, standardized gene ontology (GO) to facilitate data retrieval is not consistently used.
Three animal pathogens whose genomes have been sequenced through USDA funding are the most important bacterial pathogens responsible for causing bovine respiratory disease, a syndrome costing more than $1 billion annually. These three pathogens are: Mannheimia haemolytica, Histophilus somni, and Pasteurella multocida. Accurate and accessible annotation of their genomes is needed to maximize the utility of the genome sequences to the U.S. agricultural research community. In this project, we will identify proteins from these three species and map the proteins onto their genome sequences to confirm predicted proteins and identify novel proteins. We will also assign standardized nomenclature and functional annotations for the identified proteins as well as build computational tools to use these annotations, which will be publicly accessible at our AgBase website.
A critical component of this proposal is a coordinated effort with the principal investigators of the genome sequencing projects for each of these pathogens. Sarah Highlander (Baylor College of Medicine) is the PI of the Mannheimia haemolytica genome sequencing project (http://www.hgsc.bcm.tmc.edu/projects/microbial/Mhaemolytica/), Tom Inzana (Virginia Polytechnic Institute and State University) is PI of the Histophilus somni genome sequencing project (http://micro-gen.ouhsc.edu/h_somnus/h_somnus_home.htm), and Allison Gillaspy (Oklahoma University Health Sciences Center) is PI of the Pasteurella multocida genome project (http://micro-gen.ouhsc.edu/p_mult/p_mult_home.htm). Information will be disseminated to these annotation projects through an established GO annotation website (AgBase; http://www.agbase.msstate.edu/), and GO annotation will be conducted in conjunction with these sequencing projects using a distributed annotation system.
Our rationale for conducting this research is that experimental annotation combined with standardized gene ontology, easily accessible databases, and new computational tools will achieve much more comprehensively annotated microbial genomes and will allow them to be more fully exploited for functional genomic and systems biology studies for agricultural research. Our expected outcomes are more comprehensively annotated genome sequences and tools for BRD pathogens that will be available to the research community in a single place and in an easily usable format. Specifically, we will: 1) improve structural annotation of the three genomes in terms of protein coding, 2) provide GO functional annotation for the three genomes, 3) provide computational tools for functional annotation, and 4) provide computational tools for conducting functional genomics in agricultural microbial genomes.
Our experimental approach will be to use multi-dimensional protein identification technology (MuDPIT) to produce a map of the coding sequences in these genomes by proteogenomic mapping. Proteogenomic maps of the M. haemolytica, H. somni, and P. multocida genomes will provide direct ex-vivo evidence for the existence of proteins that are predicted by homology, more accurately determine the boundaries and enumeration of functional ORFs, verify predicted proteins, and identify unknown ORFs that are not predicted by homology. In addition, because of the importance of BRD in cattle production, proteogenomic mapping will provide a highly relevant proteome dataset for the agricultural research community. Completion of the experimental work in this aim will result in improved databases for proteomics, a better understanding of the size and diversity of proteomes in Pasteurellaceae, and improved ability to model functional genomics datasets.
Our second aim is to provide standardized nomenclature and functional annotations for all expressed protein sequence tags (ePSTs) identified, to make all of this data publicly accessible, to link our data directly to Ensembl, UniProt and PRIDE, to keep current with changes in the UniProt and nomenclature databases, and to provide computational tools to facilitate comparative and functional genomics studies in microbial genomes. One of our major goals is that both the annotations and the tools be easily accessible to the research community. Our approach will be to do electronic and manual GO annotation from our experimental data, continue to provide computational tools for functional genomics, and continue to provide public access to these via AgBase. This approach will achieve more comprehensively annotated microbial genomes that are more easily accessible and have more user-friendly tools; in particular, it will enable the M. haemolytica, P. multocida, and H. somni genomes to be more fully exploited for functional genomic and systems biology studies. This expectation is based on the tools for functional genomics studies that we have already produced and that have decreased the time required for functional annotation and analyzing functional genomic data from months to hours.
Project director: Mark Lawrence
Project co-directors: Shane Burgess, Susan Bridges, Bindu Nanduri
Project personnel: Ranjit Kumar, Nan Wang, Michelle Banes
Publications:
Pending |
