Virulence factors of Salmonella enterica serovar Enteritidis

Abstract

Salmonella enterica serovar Enteritidis is one of the major etiologic agents of human food-borne gastrointestinal infections. Efforts to control the number of serovar Enteritidis infections have had a limited success, in part because of the lack of knowledge of the molecular mechanisms that contribute to the spread and development of the infection. In this thesis, novel Salmonella virulence determinants were identified through screening of a mutant library of the serovar Enteritidis strain CVI-1 for bacteria that were impaired in their ability to infect chicken macrophages. The approach followed in this thesis consisted of a systematic search for Salmonella virulence genes, followed by a detailed in vitro and in vivo analysis of the most interesting virulence determinants. In Chapter 2, it is described how a mutant library of the serovar Enteritidis genome was constructed by random insertional mutagenesis using Transposon mini-Tn10 (KanR). The genome of serovar Enteritidis is 4.5 mb in size, and contains about 4000 genes. To cover most genes, a library containing nearly 8000 mutants was constructed. The library was used to search for mutants that were impaired in their ability to infect macrophages, which are target cells of Salmonella in chicken. Forty-four mutants were identified that were impaired in their ability to enter and/or to survive in the HD-11 cells. Genetic analysis of the mutants showed that most of the affected genes have previously been associated with bacterial virulence in the related serovar Typhimurium. This indicates that both serovars share a large part of their virulence repertoire. Five mutants were found to carry the transposon in genes not previously reported to be involved in virulence. The defects in three of the mutants (the pbpA2, yegQ and cat2 mutant) could be complemented and were further studied in detail. During these studies, a novel Salmonella pathogenicity island (designated as SPI-6) was discovered as well as a novel carbon source-dependent type of regulation of the bacterial growth. The latter may be important for the ability of the bacterium to resist oxidative challenge and killing by cationic antimicrobial peptides in an intracellular environment.