Salmonella Persistence in Tomatoes Requires a Distinct Set of Metabolic Functions Identified by Transposon Insertion Sequencing

Abstract

Human enteric pathogens, such as <i>Salmonella</i> spp. and verotoxigenic <i>Escherichia coli</i>, are increasingly recognized as causes of gastroenteritis outbreaks associated with the consumption of fruits and vegetables. Persistence in plants represents an important part of the life cycle of these pathogens. The identification of the full complement of <i>Salmonella</i> genes involved in the colonization of the model plant (tomato) was carried out using transposon insertion sequencing analysis. With this approach, 230,000 transposon insertions were screened in tomato pericarps to identify loci with reduction in fitness, followed by validation of the screen results using competition assays of the isogenic mutants against the wild type. A comparison with studies in animals revealed a distinct plant-associated set of genes, which only partially overlaps with the genes required to elicit disease in animals. <i>De novo</i> biosynthesis of amino acids was critical to persistence within tomatoes, while amino acid scavenging was prevalent in animal infections. Fitness reduction of the <i>Salmonella</i> amino acid synthesis mutants was generally more severe in the tomato <i>rin</i> mutant, which hyperaccumulates certain amino acids, suggesting that these nutrients remain unavailable to <i>Salmonella</i> spp. within plants. <i>Salmonella</i> lipopolysaccharide (LPS) was required for persistence in both animals and plants, exemplifying some shared pathogenesis-related mechanisms in animal and plant hosts. Similarly to phytopathogens, <i>Salmonella</i> spp. required biosynthesis of amino acids, LPS, and nucleotides to colonize tomatoes. Overall, however, it appears that while <i>Salmonella</i> shares some strategies with phytopathogens and taps into its animal virulence-related functions, colonization of tomatoes represents a distinct strategy, highlighting this pathogen's flexible metabolism.<b>IMPORTANCE</b> Outbreaks of gastroenteritis caused by human pathogens have been increasingly associated with foods of plant origin, with tomatoes being one of the common culprits. Recent studies also suggest that these human pathogens can use plants as alternate hosts as a part of their life cycle. While dual (animal/plant) lifestyles of other members of the <i>Enterobacteriaceae</i> family are well known, the strategies with which <i>Salmonella</i> colonizes plants are only partially understood. Therefore, we undertook a high-throughput characterization of the functions required for <i>Salmonella</i> persistence within tomatoes. The results of this study were compared with what is known about genes required for <i>Salmonella</i> virulence in animals and interactions of plant pathogens with their hosts to determine whether <i>Salmonella</i> repurposes its virulence repertoire inside plants or whether it behaves more as a phytopathogen during plant colonization. Even though <i>Salmonella</i> utilized some of its virulence-related genes in tomatoes, plant colonization required a distinct set of functions.