Date Published: June 26, 2014
Publisher: Public Library of Science
Author(s): Mohamed M. Ali, David L. Newsom, Juan F. González, Anice Sabag-Daigle, Christopher Stahl, Brandi Steidley, Judith Dubena, Jessica L. Dyszel, Jenee N. Smith, Yakhya Dieye, Razvan Arsenescu, Prosper N. Boyaka, Steven Krakowka, Tony Romeo, Edward J. Behrman, Peter White, Brian M. M. Ahmer, Renée M. Tsolis.
Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that Salmonella encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn), which is essential for Salmonella fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10−/− mice). The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1− SPI2− or ttrA mutants, respectively). The severe fitness defect of a Salmonella fra mutant suggests that F-Asn is the primary nutrient utilized by Salmonella in the inflamed intestine and that this system provides a valuable target for novel therapies.
Salmonella is a foodborne pathogen that causes significant morbidity and mortality in both developing and developed countries , . It is widely believed that there are no undiscovered drug targets in Salmonella enterica, largely due to the high number of nutrients available during infection and redundancy in metabolic pathways , . To acquire nutrients in the intestine, Salmonella initiates inflammation, which disrupts the microbiota and causes an oxidative burst that leads to the formation of tetrathionate –, –. Tetrathionate is used as a terminal electron acceptor for the anaerobic respiration of carbon compounds that otherwise would not be metabolized . One of these carbon sources is ethanolamine, which is derived from host phospholipids. Ethanolamine can be respired by Salmonella, but not fermented . Salmonella actively initiates inflammation using two Type 3 Secretion Systems (T3SS), each encoded within a distinct, horizontally acquired pathogenicity island. SPI1 (Salmonella Pathogenicity Island 1) contributes to invasion of host cells and elicitation of inflammation in the host. SPI2 is required for survival within macrophages and contributes to intestinal inflammation. Salmonella strains lacking SPI1 and SPI2 cause very little intestinal inflammation , , , . Here, we have identified fructose-asparagine (F-Asn) as another carbon source that is consumed by Salmonella using tetrathionate respiration during the host inflammatory response. The phenotypes of mutants lacking this utilization system are quite severe, suggesting that this is the primary nutrient utilized during Salmonella-mediated gastroenteritis. No other organism is known to synthesize or utilize F-Asn.
The fructose-asparagine (F-Asn) utilization system was discovered during a genetic screen designed to identify novel microbial interactions between Salmonella and the normal microbiota. Transposon site hybridization (TraSH) was used to measure and compare the relative fitness of Salmonella transposon insertion mutants after oral inoculation and recovery from the cecum of two types of gnotobiotic mice, differing from each other by a single intestinal microbial species –. The two types of mice were germ-free and ex-germ-free colonized by a single member of the normal microbiota, Enterobacter cloacae. E. cloacae was chosen because it is a commensal isolate from our laboratory mice, easily cultured, genetically tractable, and it protects mice against Salmonella infection (Figure 1). In total, five genes conferred a greater fitness defect in the mice containing Enterobacter than in the germ-free mice (Table 1).
The mechanisms by which microbes interact with each other in the gastrointestinal tract are largely unknown. Screening large libraries of bacterial mutants for fitness defects in animals with defined microbiota can be used to identify those genes that are only required in the presence of specific members of the microbiota . In this report, we took a highly reductionist approach and screened for genes that were differentially required in germ-free mice versus ex-germ-free mice colonized with a single commensal Enterobacter cloacae isolate. Only five genes were differentially required, a two component response regulatory pair, barA/sirA, and three genes within the fra locus (Table 1). Individual sirA and fraB mutants were used to confirm the findings. The sirA gene was required for fitness in the presence of E. cloacae but not in its absence (Figure 2). The fra locus was required for fitness in both situations, but the phenotype was more severe in the presence of E. cloacae (Figure 4A, B). Thus, the differential screening strategy was successful in identifying genes that are more important in the presence of other bacteria within the gastrointestinal tract. The reason(s) that sirA is required in the presence, but not the absence, of E. cloacae is not known. It is thought that BarA detects short chain fatty acids produced by the normal microbiota and then phosphorylates SirA –. SirA then activates the transcription of two small RNAs, csrB and csrC, which antagonize the activity of the CsrA protein , –. The CsrA protein is an RNA-binding protein that regulates the stability and translation of hundreds of mRNAs involved with metabolism and virulence , , . One possible reason that sirA differentially affects fitness in the two mouse models may be that the Enterobacter-colonized mouse offers an environment richer in carboxylic acids that act as stimuli for BarA-SirA signaling with resulting effects on metabolism and growth –. The fitness effects could also be due to the regulation of genes involved in the induction of inflammation and/or anareobic metabolism including SPI1, SPI2, ethanolamine utilization, and vitamin B12 biosynthesis by CsrA , , –. Finally, SirA or CsrA may regulate the fra locus itself.