A novel signature-tagged mutagenesis system for Streptococcus suis serotype 2
Introduction
Streptococcus suis is an important and world-wide pathogen that causes meningitis, endocarditis, septicemia, arthritis, polyserositis and pneumonia in swine. S. suis has become a major problem in swine-producing countries due to intensive management practices. On rare occasions S. suis can infect humans, mainly associated with exposure to infected pigs or tissues. In fact, the largest outbreak in humans was recently reported, along with a newly described toxic shock syndrome clinical manifestation (Yu et al., 2006). The magnitude and severity of the outbreak has sparked new interest in the zoonotic potential of this organism. There are 35 known serotypes of S. suis with serotype 2 being the most prevalent in North America and Europe (Gottschalk and Segura, 2000). Important S. suis virulence associated factors include the capsular polysaccharide (CPS), muramidase-released protein (MRP), extracellular protein factor (EF), suilysin, and adhesins (reviewed in Gottschalk and Segura, 2000). However, many virulent isolates lacking these factors have also been isolated from clinical cases (Gottschalk et al., 1998).
Experimental vaccines in mice have been shown to induce significant protection against strains of homologous serotype, but protection was serotype specific (Kebede et al., 1990). In addition, currently available commercial vaccines have been formulated with multiple serotypes, presumably to increase the spectrum of protection. Successful comprehensive coverage of vaccines may be improved by a more thorough knowledge of virulence factors. The infection process can be directly influenced by factors, such as toxins, adhesins, and various other proteins, or it can be indirectly influenced by factors that control cellular metabolism and regulatory processes in response to the host environment.
Comprehensive screens for virulence related genes, such as in vivo expression technology (IVET), signature-tagged mutagenesis (STM), subtractive hybridization, and differential display have not been reported to date for S. suis. Limited screening by in vivo complementation (Smith et al., 2001) has been employed, and the preliminary development of a transposon system for S. suis has been reported (Slater et al., 2003). STM is a negative selection procedure that is used to identify attenuated bacteria from pools of mutants, thus allowing subsequent identification of genes critical for in vivo survival (Hensel et al., 1995). Since its inception, the STM process has been utilized for screens of numerous pathogenic bacteria (reviewed in Autret and Charbit, 2005). Several STM screens have been performed in Streptococcus organisms (Jones et al., 2000, Miller and Neely, 2005, Paik et al., 2005, Polissi et al., 1998).
Over the past decade, technical advances have been developed to enhance the STM process, including microarray and PCR detection systems (reviewed in Saenz and Dehio, 2005). The Luminex xMAP® system (http://www.luminexcorp.com/) utilizes microfluidics and microsphere based technology for analysis of up to 100 different analytes simultaneously and quantitatively. The capabilities of the technology are well suited to improving the throughput of STM screening. In this paper, we report development of a novel STM analysis method using the Luminex xMAP® system, a modified protocol for direct genomic sequencing of an ISS1 insertion site, the successful screening of approximately 2600 S. suis serotype 2 mutants in both the natural host and a laboratory model, and the identification of genes critical for the virulence of S. suis in both mice and pigs.
Section snippets
Construction of a S. suis library containing transposon-tagged mutants
S. suis serotype 2 S735 cells were kindly provided by Marcelo Gottschalk (University of Montreal, Faculty of Veterinary Medicine, St. Hyacinthe, Quebec, Canada) under USDA Import permit 49703. Cells were made electrocompetent essentially as described by Takamatsu et al. (2001) except cells were grown without dl-threonine. A library of S. suis serotype 2 S735 signature-tagged mutants was constructed using the suicide vector pGh9:ISS1 (Maguin et al., 1996) modified by incorporation of a
Preparation of STM mutant pools
A library of 30 pools containing approximately 2600 S. suis serotype 2 S735 signature-tagged mutants was constructed using signature-tagged pGh9:ISS1 plasmids. The ISS1 element on the vector allowed integration of the plasmid into the chromosome when thermosensitive replication was inhibited at 37 °C (Maguin et al., 1996). The Luminex screening protocol in Section 2 was developed for screening of STM tags after evaluation of several parameters including oligo tag length, hybridization
Discussion
In order to identify virulence genes necessary for in vivo survival, a signature-tagged mutagenesis (STM) system for S. suis was developed. Approximately 2600 S. suis serotype 2 S735 pGh9:ISS1 STM mutants were successfully constructed, assembled into 30 pools, and screened through a septicemic mouse model. The Luminex xMAP® system was adapted for use with the STM process, allowing a higher throughput analysis of samples. A total of 174 insertion mutants were initially identified as possible
Acknowledgements
The authors thank Stephen X. Behan, Heather R. Francis, James A. Jackson, Rika Jolie, Rob Keich, Vickie L. King, Albert MacKenzie, Paul Runnels, Gary J. Sibert, Lucas P. Taylor, Janet F. Teel, Sandra S. Walters, and the Animal Technicians at Pfizer Animal Health, Richland, MI.
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