Fructose-asparagine is a primary nutrient during growth of Salmonella in the inflamed intestine

Abstract

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.

Figure 1. Protection of mice against Salmonella serovar Typhimurium strain 14028 by Enterobacter cloacae strain JLD400.

Germ-free C57BL/6 mice were divided into two groups. One group was colonized with 10⁷ cfu of Enterobacter cloacae via the intragastric route (i.g.) and one group was not. One day later both groups were challenged i.g. with 10⁷ cfu of Salmonella. After 24 hours, the cecum and spleen were homogenized and plated to enumerate Salmonella. Each point represents the CFU/g recovered from one mouse with the geometric mean shown by a horizontal line. Statistical significance between select groups was determined by using an unpaired two-tailed Student t test. ** = P value<0.01, *** = P value<0.001.

Figure 2. Competitive index (CI) measurements of a sirA mutant in mouse models.

A) 10⁷ wild-type MA43 and sirA mutant MA45 in germ-free mice, via the intragastric route (i.g.) and recovered from the cecum after 24 hours. B) 10⁷ wild-type MA43 vs sirA mutant MA45 in germ-free mice mono-associated with Enterobacter cloacae, via the i.g. route and recovered from the cecum after 24 hours. Each point represents the CI from one mouse with the median shown by a horizontal line. Statistical significance of each group being different than 1 was determined by using a one sample Student's t test. Statistical significance between groups was determined using a Mann-Whitney test. * = P value<0.05, *** = P value<0.001.

Figure 3. Map of the fra locus of Salmonella enterica.

The five genes of the fra locus are shown as grey arrows. The gor and treF genes are shown as black arrows and are conserved throughout the Enterobacteriaceae while the fra locus is not, suggesting that the fra locus was horizontally acquired. The proposed functions and names of each gene are shown below and above the arrows, respectively. The names are based upon the distantly related frl locus of E. coli. For example, the deglycase enzyme of the frl locus is encoded by frlB so we have named the putative deglycase of the fra locus, fraB. The fra locus has no frlC homolog, while the frl locus does not have an asparaginase. Therefore, the name fraC was not used and the asparaginase was named fraE. The locus tags using the Salmonella nomenclature for strains 14028 (STM14 numbers) and LT2 (STM numbers) are shown above the gene names.

Figure 4. Fitness defect of a fraB1::kan mutant as measured by competitive index (CI) in various genetic backgrounds and mouse models.

A) 10⁷ wild-type MA43 and fraB1::kan mutant MA59 in germ-free (GF) C57BL/6 mice, via the intragastric route (i.g.) and recovered from the cecum after 24 hours. B) 10⁷ wild-type MA43 and fraB1::kan mutant MA59 in germ-free C57BL/6 mice mono-associated with Enterobacter cloacae, via the i.g. route and recovered from the cecum after 24 hours. C) 10⁹ wild-type MA43 and fraB1::kan mutant MA59 in C57BL/6 conventional mice, via the i.g. route and recovered from the cecum after 24 hours. D) 10⁷ wild-type IR715 and fraB1::kan mutant MA59 in streptomycin-treated (ST) C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. E) 10⁷ wild-type IR715 and fraB1::kan mutant MA59 in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. F) Complementation of the fraB1::kan mutation with a plasmid encoding the entire fra island, pASD5006. 10⁷ ASD6090 and ASD6000 in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. G) 10⁷ wild-type IR715 and fraB4::kan mutant CS1032 in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. H) 10⁷ wild-type IR715 and fraB4::kan mutant CS1032 in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. I) Complementation of the fraB4::kan mutation with a plasmid encoding the entire fra island, pASD5006.10⁷ wild-type ASD6090 and fraB4::kan mutant ASD6040 in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. J) 10⁷ fra ⁺ MA4301 and fraB1::cam mutant MA5900, both strains are a SPI1⁻ SPI2⁻ background, in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. K) 10⁷ fra ⁺ MA4301 and fraB1::cam mutant MA5900, both strains are a SPI1⁻ SPI2⁻ background, in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. L) 10⁷ fra ⁺ MA4301 vs fraB1::cam mutant MA5900, both strains in a SPI1⁻ SPI2⁻ background, in germ-free C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. M) 10⁷ fra ⁺ MA4310 vs fraB1::kan mutant MA5910, both strains are a ttrA ⁻ background, in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. N) 10⁷ fra ⁺ MA4310 vs fraB1::kan mutant MA5910, both strains are a ttrA ⁻ background, in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. O) 10⁷ fra ⁺ MA4310 vs fraB1::kan mutant MA5910, both strains are a ttrA ⁻ background, in germ-free C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. P) 10⁴ wild-type MA43 and fraB1::kan mutant MA59 in conventional C57BL/6 mice, via the intraperitoneal route (i.p.) and recovered from the spleen after 24 hours. Q) 10⁴ wild-type MA43 and fraB1::kan mutant MA59 in streptomycin-treated C57BL/6 mice, via the i.p. route and recovered from the spleen after 24 hours. Each data point represents the CI from one mouse with the median shown by a horizontal line. Statistical significance of each group being different than 1 was determined by using a one sample Student's t test. Statistical significance between select groups was determined using a Mann-Whitney test. * = P value<0.05, ** = P value<0.01, *** = P value<0.001.

Figure 5. Histopathology scores of C57BL/6 mice after i.g. inoculation with Salmonella.

All groups received 10⁷ cfu except conventional mice (D), which received 10⁹ cfu. A) Germ-free (GF) mice 24 hours post-infection with wild-type MA43 and fraB1::kan mutant MA59; B) GF mice 24 hours post-infection with SPI1⁻ SPI2⁻ Salmonella (fra ⁺ MA4301 vs fraB1::cam mutant MA5900); C) GF mice 24 hours post-infection with ttrA ⁻ Salmonella (fra⁺ MA4310 vs fraB1::kan mutant MA5910); D) Conventional mice 24 hours post-infection with wild-type MA43 and fraB1::kan mutant MA59. E) Strep-treated (ST) mice 4 days post-infection with wild-type IR715 and fraB1::kan mutant MA59; F) ST mice 4 days post-infection with SPI1⁻ SPI2⁻ Salmonella (fra ⁺ MA4301 and fraB1::cam mutant MA5900; G) ST mice 4 days post-infection with ttrA ⁻ Salmonella (fra ⁺ MA4310 vs fraB1::kan mutant MA5910). Error bars represent mean+SD. Statistical significance between select groups was determined using a Mann-Whitney test. * = P value<0.05, ** = P value<0.01.

Figure 6. Phenotype of a fraB1::kan mutant in the cecum of “humanized” and IL10 knockout mice.

10⁹ wild-type IR715 vs fraB1::kan mutant MA59 in “humanized” Swiss Webster mice (germ free mice inoculated orally with a human fecal sample), or C57BL/6 IL10 knockout mice, as indicated, via the i.g. route and recovered from cecum on day 3 post-infection. A) Each data point represents the CI from one mouse with the median shown by a horizontal line. Statistical significance of each group being different than 1 was determined by using a one sample Student's t test. *** = P value<0.001. B) Histopathology scores of mice from panel A. Error bars represent mean+SD.

Figure 7. Quantitation of Salmonella in feces on days 1 through 4, and cecum on day 4, post-infection.

Groups of five C57BL/6 mice heterozygous for Nramp1 were orally inoculated with 10⁷ CFU of IR715 (wild-type), MA59 (fraB1::kan mutant), or ASD6000 (fraB1::kan mutant with complementation plasmid pASD5006). The geometric mean+SE is shown. Statistical significance between select groups was determined by using an unpaired two-tailed Student t test. * = P value<0.05, ** = P value<0.01.

Figure 8. Growth of wild-type and fraB1::kan mutant Salmonella on Amadori products.

Growth of wild-type MA43 and fraB1::kan mutant MA59 on F-Asn (A), F-Arg (B), F-Lys (C), asparagine, arginine, lysine, or glucose (D). Bacteria were grown overnight in LB at 37°C shaking, centrifuged, resuspended in water, and subcultured 1∶1000 into NCE medium containing the indicated carbon source at 5 mM. The optical density at 600 nm was then read at time points during growth at 37°C with shaking. Controls included NCE with no carbon source, and NCE with glucose that was not inoculated, as a sterility control (D). E) Complementation of a fraB1::kan mutation with plasmid pASD5006 encoding the fra island (ASD6000) or the vector control, pWSK29 (ASD6010). Each point in (A)–(E) represents the mean of three cultures with error bars indicating standard deviation. F) The structure of F-Asn (CAS#34393-27-6).

Figure 9. Growth of Salmonella on F-Asn as sole nitrogen source.

Growth of wild-type MA43 and fraB1::kan mutant MA59 on F-Asn. Bacteria were grown overnight in LB at 37°C shaking, centrifuged, resuspended in water, and subcultured 1∶1000 into NCE medium lacking a nitrogen source (NCE-N) but containing the indicated carbon source at 5 mM. The optical density at 600 nm was then read at time points during growth at 37°C with shaking. Controls included NCE-N with no carbon source, NCE-N with 5 mM glucose, and NCE-N with glucose that was not inoculated, as a sterility control. Each point represents the mean of four cultures and error bars represent standard deviation.

Figure 10. Growth of Salmonella on F-Asn in the presence or absence of tetrathionate or oxygen.

Growth of wild-type MA43 and fraB1::kan mutant MA59 on 5 mM F-Asn or 5 mM glucose anaerobically (A and B) or aerobically (C and D) in the presence (A and C) or absence (B and D) of 40 mM tetrathionate (S₄0₆ ²⁻). Bacteria were grown overnight in LB at 37°C shaking, centrifuged, resuspended in water, and subcultured 1∶1000 into NCE medium containing the indicated carbon source. The optical density at 600 nm was then read at time points during growth at 37°C with shaking. Each point represents the mean of four cultures with error bars indicating standard deviation.

Figure 11. Competitive index measurements of a fraB1::kan mutant during in vitro growth.

Cultures were grown overnight in LB, pelleted and washed in water, subcultured 1∶10,000 and grown for 24 hours at 37°C in NCE minimal medium containing 5 mM F-Asn, aerobically or anaerobically, in the presence or absence of tetrathionate (S₄O₆ ²⁻), as indicated. A) Anaerobic growth in the presence of tetrathionate, B) anaerobic growth in the absence of tetrathionate, C) aerobic growth in the presence of tetrathionate, D) aerobic growth in the absence of tetrathionate. Each data point represents the CI from one culture with the median shown by a horizontal line. Statistical significance of each group being different than 1 was determined by using a one sample Student's t test. Statistical significance between select groups was determined using a Mann-Whitney test. ** = P value<0.01, *** = P value<0.001.

Figure 12. A proposed model of Fra protein localization and functions.

A proteomic survey of subcellular fractions of Salmonella previously identified FraB (the deglycase) as cytoplasmic and FraE (the asparaginase) as periplasmic . Therefore, it is possible that F-Asn is converted to F-Asp in the periplasm by the asparaginase and that the transporter and kinase actually use F-Asp as substrate rather than F-Asn. The FraD kinase of Salmonella shares 30% amino acid identity with the FrlD kinase of E. coli. FrlD phosphorylates F-Lys to form F-Lys-6-P . Therefore, we hypothesize that FraD phosphorylates F-Asp to form F-Asp-6-P. The FrlB deglycase of E. coli shares 28% amino acid identity with FraB of Salmonella. The FrlB deglycase converts F-Lys-6-P to lysine and glucose-6-P , so we hypothesize that FraB of Salmonella converts F-Asp-6-P to aspartate and glucose-6-P.