Tissue-Specific Salmonella Typhimurium Gene Expression during Persistence in Pigs

Alexander Van Parys,Bregje Leyman,Elin Verbrugghe,Filip Boyen,Freddy Haesebrouck,Frank Pasmans,Ben Adler

doi:10.1371/journal.pone.0024120

Abstract

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 ΔpurA 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 ΔSTM4067::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.

Highlights

Non-typhoidal salmonellosis is one of the most important bacterial zoonotic diseases, yearly resulting in an estimated 155,000 deaths worldwide [1]
The wild type Salmonella Typhimurium was recovered from all organs and organ contents from all piglets, except for the ileum contents and faeces from 1 animal, while the purA::kanR substitution mutant was found in the tonsils of only 1 piglet and the faeces of another, eight days post oral inoculation (Table 1)
in vivo expression technology (IVET) screening for in vivo induced genes To screen for Salmonella Typhimurium genes that are induced in porcine tissues during the persistent phase of infection, we constructed an IVET transformants pool covering the major part of the Salmonella Typhimurium genome [14]

Summary

Introduction

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]

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