Recipient Organization
MIDWESTERN UNIVERSITY
19555 N. 59TH AVENUE
GLENDALE,AZ 85308-6813
Performing Department
College of Veterinary Medicine
Non Technical Summary
Streptococcus suis causes a wide range of diseases such as fever, meningitis, septicemia, arthritis, polyserocitis, anorexia, endocarditis, permanent hearing loss and sudden death in animals. In humans, it can cause toxic shock, permanent hearing loss, meningitis and colon carcinoma. The economic impact in veterinary medicine and in the swine industry in particular is substantial (estimated at over 300 million dollars in the swine industry in the United States alone). Recently, we isolated and reported for the first time a highly virulent strain of S. suis from cattle. Although the isolate caused typical S. suis diseases including death, preliminary data showed differences in the pathogenic properties and growth characteristics compared with swine and human isolates. We believe that the observed differences might be due to evolution and adaptation to environment. To test our reasoning, we aim to sequence the entire genome of the cattle S. suis and compare the results with those obtained from swine and human S. suis isolates because it will address the critical need for an improved understanding of how cattle S. suis causes disease, protective antigens, evolutionary biology and ecology. We will focus on two specific objectives. In the first objective, we will complete the genome-sequencing project of the S. suis isolate from cattle which is currently in progress in my laboratory. In the second objective, we will compare the complete genome sequences of the isolate against the complete genome sequences of swine and human S. suis isolates, including the genome sequences of other disease causing bacteria in the whole genome sequence database to explore regions that are involved in ability of the cattle isolate to cause disease. We will also search for proteins that can serve as vaccine candidate and involved in environmental adaptation. It is our believe that the fundamental data on overall genome content information that our study will generate, will open up novel therapeutic and preventative (vaccine) strategies, provide more information on molecular mechanism of S. suis pathogenicity, and insight on evolutionary adaptation. This may lead to the development of improved control strategies to reduce the incidence of S. suis infection in animals and humans.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The long-term goal of the project is to understand in detail the genetic content of a highly virulent strain of Streptococcus suis isolated from cattle. Availability of this data will open up novel therapeutic and preventative (vaccine) strategies, provide more information on molecular mechanism of S. suis pathogenicity, and provide insight on evolutionary adaptation and niche specificity. The economic impact of S. suis in veterinary medicine is substantial (estimated at over 300 million dollars a year in the swine industry in the United States alone). This is due in part to the lack of thorough and comprehensive understanding of the factors that contribute to pathogenicity and virulence, effective vaccines, rapid diagnostic reagents, and knowledge gap on evolution and host specificity. Although the incidence of S. suis in the cattle industry has increased annually, there is minimal research effort in addressing the economic impact in the cattle industry. Recently, we isolated for the first time a highly virulent strain of S. suis from cattle (4). The isolate caused typical S. suis diseases including death, and exhibited differences in the pathogenic properties and growth characteristics compared with swine and human isolates. Based on our preliminary data, we hypothesize that the observed differences might be due to genomic content differences, which in turn may influence the development of effective control strategies, adaptive molecular evolution as well as niche specificity. To test this hypothesis, we propose to sequence the whole genome of the cattle S. suis to completion and perform comparative genomics analysis against the sequences of swine and human isolates as well as other bacterial pathogens at the nucleotide database using bioinformatics tools as it addresses the critical need for an improved understanding of S. suis virulence associated factors, evolutionary biology, ecology, niche specificity and protective antigens.The specific objectives are(1) To sequence to completion and annotate the whole genome of a virulent S. suis isolate from cattle.(2) To compare the genome sequences with the genome sequences of Swine and human S. suis in the genome sequence database including the genome sequences of other bacterial pathogens in order to identify regions that contribute to immunogenicity, pathogenicity, adaptive molecular evolution as well as niche specificity.
Project Methods
Objective 1: To sequence to completion and annotate the whole genome of a virulent S. suis isolate from cattle.Rationale and Approach: Determining the whole genome sequence of S. suis from cattle is important to determine its genome content and facilitate comparative genomic studies in order to obtain insight on the biological properties of the organism. Over 95% of available studies on S. suis were performed in Asia, Canada and Europe using isolates recovered from swine in those regions. To use such information as sole source to predict what happens in the US agriculture with regard to the pathogen will be unwise due to geographical and host differences. To develop effective control strategies across the board, and have a better picture of the pathogenic process and host adaptability attention must be paid to isolates from different hosts and geographical locations. We intend to take a systematic approach in addressing this knowledge gap. We have chosen to work with cattle S. suis because no information is available on its mechanism of pathogenicity and protective antigens and if not properly addressed it may pose a major veterinary and economic concern considering the importance of cattle in the industry and society. Whole genome sequencing is a very useful tool that has successfully been used by several investigators to aid the search for new therapies and diagnostic reagents.Methods:We have sequenced greater than 68% of the genome of cattle S. suis and will now bring it to completion with full annotation. To obtain more DNA, the isolate will be grown at 37oC overnight in Todd-Hewitt broth containing 0.6% yeast extract. DNA will be extracted using the DNeasy blood and tissue kit (Qiagen) following the manufactures recommendation. DNA concentration, purity and quality will be checked using the Nanodrop spectrometry (Thermo Fisher Scientific, Waltham, MA) and agarose gel electrophoresis. Sequencing will be performed at the University of Wisconsin-Madison biotechnology Center using illumina HiSeq 2000 platform (Illimina, Inc. San Diego, CA). Reads will be assembled using the Newbler assembler (version 2.6 ) into scaffolds. Auto-annotation of the contigs will be performed using the CLC Genomics Workbench version 4.6 (CLCBio), and the MacVector software version 16.0.8 or at NCBI using the Prokaryotic Genome Annotation Pipeline (PGAP). Open reading frames (ORFs) will be predicted on the genome using Prodigal software and translated into proteins. The sequences will be mapped against complete genome sequences of S suis deposited at NCBI). To fill the intra scaffolds gaps, we will use paired-end information to retrieve read pairs that has one read that aligns to the contigs and another read that is located in the gap region. With this information, we will perform local assembly for the collected reads. Then, the scaffolds will be ordered relative to the genome of S. suis strain P1/7 using MUMmer3. We will close gaps by primer walking and sequencing of PCR products. Possible misassemblies will be corrected using PCR amplification and direct sequencing.Objective 2: To compare the genome sequences with the genome sequences of Swine and human S. suis in the genome sequence database including the genome sequences of other bacterial pathogens in order to identify regions that contribute to immunogenicity, pathogenicity, adaptive molecular evolution as well as niche specificity.Rationale and Approach: The evolutionary interplay between microbial pathogens and their hosts is a continual process of adaptation, manifested by genomic variation of host adaptation factors, and by the gain and loss of genes via horizontal gene transfer (HGT). Comparative sequence analysis will provide a better understanding of the host association, evolution of S. suis strains, protective antigens, and could help in deciphering the differences observed among strains of similar and different niche. We believe that acquisition of novel genes has enabled S. suis to adopt distinct host-specific lifestyles. Genome sequence data for swine and human S. suis isolates are publicly available. We will compare sequences of the swine and human strains to the cattle strain and against sequences of other bacterial pathogens to explore the molecular details of factors involved in immunogenicity, pathogenicity, adaptive molecular evolution and niche specificity. The results will provide insight on the molecular details involved in virulence and explain the strikingly different host tropism and clinical manifestations. It will also provide information on antigens that may be good vaccine candidates. Methods:The assembled and annotated sequences from the cattle S. suis will be aligned to the previously sequenced Swine (strain P1/7, GenBank accession number AM946016.1) and human (strain 05ZYH33; GenBank accession number CP000407.1) S. suis by MUMmer). Alignment will be performed with CLC work bench and the MacVector software using ClusterW. We will construct a phylogenetic tree from the alignment using the Macvector software.