Identification of Bacterial Species That Can Utilize Fructose-Asparagine

Abstract

Salmonella enterica serovar Typhimurium is the only organism demonstrated to utilize fructose-asparagine (F-Asn) as a source of carbon and nitrogen. In this report, we first used a bioinformatics approach to identify other microorganisms that encode homologs of the Salmonella F-Asn utilization enzymes FraB (deglycase), FraD (kinase), and FraE (asparaginase). These candidate organisms were then tested with up to four different methods to confirm their ability to utilize F-Asn. The easiest and most broadly applicable method utilized a biological toxicity assay, which is based on the observation that F-Asn is toxic to a Salmonella fraB mutant. Candidate organisms were grown in a rich medium containing F-Asn, and depletion of F-Asn from the medium was inferred by the growth of a Salmonella fraB mutant in that same medium. For select organisms, the toxicity assay was cross-validated by direct mass spectrometry-aided measurement of F-Asn in the spent-culture media and through demonstration of FraB and FraD enzyme activity in cellular extracts. For prototrophs, F-Asn utilization was additionally confirmed by growth in a minimal medium containing F-Asn as the sole carbon source. Collectively, these studies established that Clostridiumbolteae, Clostridium acetobutylicum, and Clostridium clostridioforme can utilize F-Asn, but Clostridium difficile cannot; Klebsiella oxytoca and some Klebsiella pneumoniae subspecies can utilize F-Asn; and some Citrobacter rodentium and Citrobacter freundii strains can also utilize F-Asn. Within Salmonella enterica, the host-adapted serovars Typhi and Paratyphi A have lost the ability to utilize F-Asn.IMPORTANCE Fructose-asparagine (F-Asn) is a precursor to acrylamide that is found in human foods, and it is also a nutrient source for Salmonella enterica, a foodborne pathogen. Here, we determined that among the normal intestinal microbiota, there are species of Clostridium that encode the enzymes required for F-Asn utilization. Using complementary experimental approaches, we have confirmed that three members of Clostridium, two members of Klebsiella, and two members of Citrobacter can indeed utilize F-Asn. The Clostridium spp. likely compete with Salmonella for F-Asn in the gut and contribute to competitive exclusion. FraB, one of the enzymes in the F-Asn utilization pathway, is a potential drug target because inhibition of this enzyme leads to the accumulation of a toxic metabolite that inhibits the growth of Salmonella species. This study identifies the potential off-target organisms that need to be considered when developing therapeutics directed at FraB.

Keywords: Amadori products; Citrobacter; Clostridium; FraB; Klebsiella; Salmonella; fructosamines; fructose-asparagine; pathovar; phylogeny; typhoid.

FIG 1

Maximum likelihood trees of concatenated FraBD (A) and FraE (B) amino acid sequences that were recovered from a BLAST search of HMRGD isolates and CBA/J mouse fecal metagenomes are shown. All putative homologs found with BLAST search of the HMRGD and mouse metagenomes can be found in File S1 in the supplemental material. Bootstrap values are based on 100 resamplings, with values of >80 shown. The metagenome and scaffold associated with each FraBD from mouse metagenomes (black text) are reported with the metagenome number (1 to 3), followed by an underscore and scaffold number. E. coli K-12 strain W3110 was used as an outgroup that does not contain fra genes.

FIG 1

Maximum likelihood trees of concatenated FraBD (A) and FraE (B) amino acid sequences that were recovered from a BLAST search of HMRGD isolates and CBA/J mouse fecal metagenomes are shown. All putative homologs found with BLAST search of the HMRGD and mouse metagenomes can be found in File S1 in the supplemental material. Bootstrap values are based on 100 resamplings, with values of >80 shown. The metagenome and scaffold associated with each FraBD from mouse metagenomes (black text) are reported with the metagenome number (1 to 3), followed by an underscore and scaffold number. E. coli K-12 strain W3110 was used as an outgroup that does not contain fra genes.

FIG 2

Synteny map of the fra locus from select bacterial species. Putative homologs are indicated by the same color.

FIG 3

(A) A Salmonella toxicity assay used to determine if Clostridium species can deplete F-Asn from growth medium. The log competitive index (logCI) is plotted, with a horizontal bar indicating the mean of the results from three independent experiments that include a total of eight technical replicates. Competitions in which the result was closer to neutral than the “medium with F-Asn” competition were tested for statistical significance. Asterisks represent differences in an unpaired t test (parametric) compared to medium with F-Asn (****, P < 0.0001). (B) MS measurements to determine if Clostridium species can remove F-Asn from growth medium. The horizontal bar in the MS data indicates the mean value of three measurements; the dotted line represents the limit of detection (LOD) of 0.055 mM.

FIG 4

Identification of Klebsiella and Citrobacter species that can utilize F-Asn. The Salmonella toxicity assay was used to determine which Klebsiella (A) or Citrobacter (B) strains could deplete F-Asn from their medium. The log competitive index (logCI) is plotted, with a horizontal bar indicating the mean of independent replicates. Competitions in which the result was closer to neutral than the “medium with F-Asn” competition were tested for statistical significance. Asterisks indicate difference from medium with F-Asn (unpaired t test, parametric: *, P < 0.05; **, P < 0.01; ****, P < 0.0001). (C) MS measurements to determine if Klebsiella species can deplete F-Asn from growth medium. The horizontal bar in the MS data indicates mean value of three independent measurements; the dotted line represents the limit of detection (LOD) of 0.055 mM. Growth at 37°C was tested in minimal medium containing either glucose (open symbols) or F-Asn (closed symbols) as the sole carbon source for Klebsiella (D) or Citrobacter (E) strains. Growth curves represent two independent experiments with six total replicates for each point. Error bars indicate the standard deviation. OD₆₀₀, optical density at 600 nm.

FIG 5

Some Salmonella serovars have lost the ability to utilize F-Asn. The log competitive index (logCI) is plotted, with a horizontal bar indicating the mean of the results from two independent experiments that include a total of four technical replicates. Competitions in which the result was closer to neutral than the “medium with F-Asn” competition were tested for statistical significance. Asterisks indicate difference from medium with F-Asn (unpaired t test, parametric: ***, P < 0.001).