Tissue-Specific Salmonella Typhimurium Gene Expression during Persistence in Pigs

et al. (2011) Tissue-Specific Salmonella Typhimurium Gene Expression during Persistence in Pigs. PLoS ONE 6(8): e24120. doi:10.1371/journal.pone.0024120 Tissue-Specific Salmonella Typhimurium Gene Expression during Persistence in Pigs Alexander Van Parys 0 Filip Boyen 0 Bregje Leyman 0 Elin Verbrugghe 0 Freddy Haesebrouck 0 Frank 0 Ben Adler, Monash University, Australia 0 Ghent University, Faculty of Veterinary Medicine, Department of Pathology , Bacteriology and Avian Diseases, Merelbeke , Belgium Salmonellosis caused by Salmonella Typhimurium is one of the most important bacterial zoonotic diseases. The bacterium persists in pigs resulting in asymptomatic 'carrier pigs', generating a major source for Salmonella contamination of pork. Until now, very little is known concerning the mechanisms used by Salmonella Typhimurium during persistence in pigs. Using in vivo expression technology (IVET), a promoter-trap method based on DpurA attenuation of the parent strain, we identified 37 Salmonella Typhimurium genes that were expressed 3 weeks post oral inoculation in the tonsils, ileum and ileocaecal lymph nodes of pigs. Several genes were expressed in all three analyzed organs, while other genes were only expressed in one or two organs. Subsequently, the identified IVET transformants were pooled and reintroduced in pigs to detect tissue-specific gene expression patterns. We found that efp and rpoZ were specifically expressed in the ileocaecal lymph nodes during Salmonella peristence in pigs. Furthermore, we compared the persistence ability of substitution mutants for the IVET-identified genes sifB and STM4067 to that of the wild type in a mixed infection model. The DSTM4067::kanR was significantly attenuated in the ileum contents, caecum and caecum contents and faeces of pigs 3 weeks post inoculation, while deletion of the SPI-2 effector gene sifB did not affect Salmonella Typhimurium persistence. Although our list of identified genes is not exhaustive, we found that efp and rpoZ were specifically expressed in the ileocaecal lymph nodes of pigs and we identified STM4067 as a factor involved in Salmonella persistence in pigs. To our knowledge, our study is the first to identify Salmonella Typhimurium genes expressed during persistence in pigs. - Funding: This work was supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT Vlaanderen; http://www.iwt.be), Brussels, Belgium (grant IWT Landbouw 040791) and the Research Foundation-Flanders (FWO; http://www.fwo.be). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Non-typhoidal salmonellosis is one of the most important bacterial zoonotic diseases, yearly resulting in an estimated 155,000 deaths worldwide [1]. In European countries, Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) is the serovar most frequently isolated from slaughter pigs [2]. Porcine carcass contamination with Salmonella Typhimurium can largely be attributed to persistently infected pigs [3]. Transmission of Salmonella Typhimurium between pigs occurs mainly via the faecal-oral route. After ingestion by the pig, the bacterium will preferentially colonize its tonsils and ileum, where it adheres to the intestinal epithelium. This is followed by invasion and subsequent migration of the Salmonella bacterium to the underlying lymphoid tissues, like the ileocaecal lymph nodes, resulting in so called carrier status pigs [4]. In the past, Salmonella interactions with hosts were largely examined in murine and avian models, while Salmonella Typhimurium behaviour in pigs is only poorly investigated. Due to the different and often hostile environments Salmonella must combat to successfully colonize one host, it is expected that the bacterium is equipped with a broad range of survival strategies, each one adapted to a certain biological niche. Therefore the bacterial genes involved in Salmonella survival in the tonsils might markedly differ from those essential for colonization of and persistence in for example the lymph nodes or the ileum in pigs [5]. The last decades, new techniques have been developed that allow high-throughput screening for genes important for microbial colonization and persistence in animal models [6,7]. Recently, using signature-tagged mutagenesis (STM), Salmonella genes were identified that were specifically expressed in the deleterious gastric environment of pigs [8]. Furthermore, genes involved in Salmonella virulence in pigs shortly after oral inoculation have been identified [9,10]. However, to date very little is known about the mechanisms employed by Salmonella Typhimurium during the persistent phase of infection of pigs. Because increasing evidence demonstrates that Salmonella Typhimurium behaves markedly different in various hosts, a thorough understanding of Salmonella pathogenesis in pigs is required to develop successful intervention strategies to cope with Salmonella infection of pigs. Using the genome-wide approach of IVET, our goal was to identify genes specifically induced in porcine tonsils, ileum and ileocaecal lymph nodes at 3 weeks post oral inoculation as an indication of genes involved in Salmonella persistence in these 3 different organs of pigs. Materials and Methods Ethics statement All animal experiments were approved by the ethical committee of the Faculty of Veterinary Medicine, Ghent University (EC2008/074; EC2009/021; EC2010/005 and EC2010/158 respectively). Bacterial strains and growth conditions Salmonella Typhimurium strain 112910a phage type 120/ad, isolated from a pig stool sample, was used as the wild type strain (WT). A spontaneous nalidixic acid resistant derivative of the wild type strain was used in the mixed inoculation in vivo experiments. Salmonella Typhimurium substitution mutants DpurA::kanR, DsifB::kanR and DSTM4067::kanR were constructed according to the one-step inactivation method described by [11] and slightly modified for use in Salmonella Typhimurium as described before [12]. Primers used to create the gene-specific linear PCR fragments are given as Supporting Information S1. For construction of the IVET pool, the DpurA deletion mutant was obtained from the DpurA::kanR substitution mutant, by excision of the kanamycin resistance cassette [11]. For oral inoculation of piglets, bacteria were cultured aerobically for 16 h at 37uC in 5 ml LuriaBertani broth (LB; SigmaAldrich Chemie Gmbh, Steinheim, Germany) with the appropriate antibiotic(s), if required, included at the following concentrations: 20 mg/ml for nalidixic acid (SigmaAldrich); 100 mg/ml for kanamycin (Sigma-Aldrich) and 50 mg/ml for ampicillin (Sigma-Aldrich). The DpurA strain was grown in LB enriched with 1.35% adenine and 0.337% thiamine. The bacterial 16 h cultures were then centri (...truncated)