Performing Department
VETERINARY MEDICINE
Non Technical Summary
Despite the economic importance of avian colibacillosis, the mechanisms of virulence employed by avian pathogenic Escherichia coli (APEC) have long remained a puzzle, confounding efforts to control the disease. However, in recent years, a widespread attribute of these organisms has been recognized that provides a focus for their study. It is the presence of large plasmids. We propose the sequencing of a large APEC resistance plasmid, and subsequent use of the data generated to produce an E. coli plasmid microarray that will be used to scan APEC isolated over the past 35 years for plasmid-associated sequences. The results obtained in the proposed project will allow us to determine if plasmid-containing strains of APEC have emerged in the poultry environment. It will also offer us an opportunity to explore the evolution of plasmid-mediated virulence and antimicrobial resistance among E. coli.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Despite the economic importance of avian colibacillosis, the mechanisms of virulence employed by avian pathogenic Escherichia coli (APEC) have long remained a puzzle, confounding efforts to control the disease. However, in recent years, a widespread attribute of these organisms has been recognized that provides a focus for their study. It is the presence of large plasmids. These plasmids encode antimicrobial resistance and virulence, and they are significantly more likely to be found in APEC than in commensal E. coli of healthy birds. Interestingly, plasmid-associated virulence factors, and the large plasmids that encode them, appear to have recently emerged as a defining feature of APEC. In the present study, we propose testing the hypothesis that plasmid-containing APEC have recently emerged via comparison of plasmid content and constitution among APEC isolated over the past 35 years. To do this, we will use a comprehensive E. coli "plasmid chip", created with DNA
microarray technology. To support this study, we have generated the first APEC plasmid sequences, including two large virulence plasmids (the first ColV and ColBM plasmids sequenced), as well as two large R plasmids. Here, we propose the sequencing of an additional large APEC IncN plasmid, as it is representative of a type of plasmid that occurs commonly in present day APEC isolates and it is the only APEC plasmid type yet to be sequenced. We can then use comparative genomics to discern similarities and differences among APEC plasmids. The sequence data obtained will also be used to produce an E. coli plasmid chip that will be used to scan APEC, isolated over the past 35 years, for plasmid-associated sequences. From these results, we will be able to determine if plasmid-containing strains of APEC have emerged in the poultry environment. It will also offer us an opportunity to explore the evolution of plasmid-mediated virulence and antimicrobial resistance among E. coli.
Project Methods
SPECIFIC OBJECTIVE 1. Completely sequence a 100-kb IncN plasmid from an APEC strain. Methods. Shotgun sequencing, assembly, and finishing will be performed on the 100-kb IncN APEC plasmid. Plasmid DNA will be prepared according to the methods of Wang and Rossman, sheared, concentrated and desalted using standard protocols. One thousand clones will be picked and arrayed in an effort to obtain eight-fold sequencing coverage. Bi-directional shotgun sequencing will be performed by MWG Biotech, Inc. The data will be collected with ABI 3700 and ABI 3730xl capillary sequencers and assembled using the SeqMan program from DNASTAR. Assembly and finishing will be perfomed by the PI, Tim Johnson, and will involve an optimized multiplex PCR procedure. Any remaining gaps in the plasmid sequences will be resolved using high-stringency PCR. SPECIFIC OBJECTIVE 2. Analyze and compare the sequences of five APEC plasmids. Methods. Open reading frames (ORFs) in the plasmid sequences will
be identified using GeneQuest from DNASTAR, followed by manual inspection. Translated ORFs will then be compared to known protein sequences using the BLAST algorithm. Physical genetic maps will be created for each plasmid using Visual Cloning software. Plasmid G+C content of individual ORFs will be analyzed using GeneQuest (DNASTAR). Insertion sequences and repetitive elements will be identified using IS FINDER (http://www-is.biotoul.fr/). Genomic comparisons of the five APEC plasmid sequences obtained in our laboratory will be performed using MAUVE alignments and manual inspection. Amino acid sequence alignments will also be performed using MegAlign (DNASTAR). The codon bias of each plasmid will be analyzed using the General Codon Usage Analysis software and compared to E. coli K-12. SPECIFIC OBJECTIVE 3. Construct the comprehensive plasmid chip. Methods. Unique oligonucleotides from each of the five plasmids' genes plus others identified in the literature will be designed using
Primer3 software, modified to eliminate false priming and potential cross hybridization of genome sequences. Each probe will be printed in duplicate for each array with four complete arrays printed to each glass substrate, UltraGAPS (Corning). Besides the probes being printed in non-adjacent, well-spaced duplicate locations, control spots, consisting of non-homologous DNA sequences of the same size as oligonucleotide probes, will be added. Additionally, negative control probes will be included. SPECIFIC OBJECTIVE 4. Using the chip, scan APEC isolated over the past 35 years will be scanned for plasmid genes. Methods. Total DNA will be isolated from the APEC isolates, fragmented to <800 bp by mechanical shearing and then labeled using a commercially available kit. At least 40 APEC isolates per decade (1970s, 80s, 90s, and 00s) will be used.