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

Abstract

Human enteric pathogens, such as Salmonella spp. and verotoxigenic Escherichia coli, 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 Salmonella 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. De novo biosynthesis of amino acids was critical to persistence within tomatoes, while amino acid scavenging was prevalent in animal infections. Fitness reduction of the Salmonella amino acid synthesis mutants was generally more severe in the tomato rin mutant, which hyperaccumulates certain amino acids, suggesting that these nutrients remain unavailable to Salmonella spp. within plants. Salmonella 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, Salmonella spp. required biosynthesis of amino acids, LPS, and nucleotides to colonize tomatoes. Overall, however, it appears that while Salmonella 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.IMPORTANCE 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 Enterobacteriaceae family are well known, the strategies with which Salmonella colonizes plants are only partially understood. Therefore, we undertook a high-throughput characterization of the functions required for Salmonella persistence within tomatoes. The results of this study were compared with what is known about genes required for Salmonella virulence in animals and interactions of plant pathogens with their hosts to determine whether Salmonella repurposes its virulence repertoire inside plants or whether it behaves more as a phytopathogen during plant colonization. Even though Salmonella utilized some of its virulence-related genes in tomatoes, plant colonization required a distinct set of functions.KEYWORDS Salmonella, foodborne pathogens, plant-microbe interactions

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens

“…We used two global approaches to discover Salmonella genes that affect the P22 lytic life cycle. First, a library of random integrations of a transposon (Tn) into bacterial genomes was employed, a technique that can identify genes under selection in various growth conditions (Sassetti et al, 2001;Canals et al, 2012;de Moraes et al, 2017). We used a high complexity Tn5 library of S. enterica serovar Typhimurium strain 14028s to identify either genes whose products enable or promote phage infection (mutation resulted in blocked or delayed infection by P22) or genes that increased bacterial host resistance to P22 infection (mutation resulted in faster loss of host DNA, likely as a result of increased phage propagation).…”

“…We used a high complexity Tn5 library of S. enterica serovar Typhimurium strain 14028s to identify either genes whose products enable or promote phage infection (mutation resulted in blocked or delayed infection by P22) or genes that increased bacterial host resistance to P22 infection (mutation resulted in faster loss of host DNA, likely as a result of increased phage propagation). This 14028s Tn5 library contained 240,000 mutants, and insertions were present at an average density of about one every 25 bp and an average of 42.5 per gene (de Moraes et al, 2017). Almost all non-essential genes were represented by multiple independent insertions at different locations.…”

“…We compared the genetic profile of mutants with no infection at 'time zero' (T 0 ), and after 90 min of further growth (T 90-control ), to the genetic profile of mutants present after 90 min of infection (T 90-infected ). The quantitative pattern of Tn5 insertions was measured by PCR of a region that contains the barcode unique to each Tn insertion, followed by Illumina sequencing of the amplified DNA, as previously described (de Moraes et al, 2017). See Experimental procedures for details.…”

Attachment and colonization of Salmonella on ‘Rayada’, ‘Golden Delicious’, and ‘Red Delicious’ apples

“…Some similar metabolic functions associated with Salmonella colonization in animals (mouse) and plants (tomato) have been identified, such as biosynthesis of fatty acids, lipopolysaccharides (LPSs) and nucleotides. Lipopolysaccharides have a protective role in the immunity mechanisms of animals and plants . Other researchers have found that Salmonella fimbriae are required for successful attachment in a plant cell wall model …”

“…Lipopolysaccharides have a protective role in the immunity mechanisms of animals and plants. 14 Other researchers have found that Salmonella fimbriae are required for successful attachment in a plant cell wall model. 15 The attachment percentages found in our study are similar to those reported in tomato (0.8%), green tomato (1.3%), small green tomato (0.2%), and peach (3.0%).…”