Dietary trehalose enhances virulence of epidemic Clostridium difficile

Abstract

Clostridium difficile disease has recently increased to become a dominant nosocomial pathogen in North America and Europe, although little is known about what has driven this emergence. Here we show that two epidemic ribotypes (RT027 and RT078) have acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose. RT027 strains contain a single point mutation in the trehalose repressor that increases the sensitivity of this ribotype to trehalose by more than 500-fold. Furthermore, dietary trehalose increases the virulence of a RT027 strain in a mouse model of infection. RT078 strains acquired a cluster of four genes involved in trehalose metabolism, including a PTS permease that is both necessary and sufficient for growth on low concentrations of trehalose. We propose that the implementation of trehalose as a food additive into the human diet, shortly before the emergence of these two epidemic lineages, helped select for their emergence and contributed to hypervirulence.

Extended Data Fig. 1

Phylogenetic organization of C. difficile MLST profiles. Maximum likelihood tree based upon concatenated Multi Locus Sequence Typing genes of the 399 current profiles available at https://pubmlst.org/cdifficile/ . Stars indicate position of strains used in this study with red stars indicating sequence types possessing either the treR L172I (ST1, ST41) or 4 gene insertion (ST11). Tree constructed using MEGA7.

Extended Data Fig. 2. Growth of C. difficle strains

a, The majority of strains can grow on 50 mM trehalose. Dashed grey line and band indicate mean growth in DMM without a carbon source and s.d. Solid lines indicate mean growth yield (OD600) for groups: Non-RT027/078 (n=10), RT027 (n=8), and RT078 (n=3). b, Deletion of treA ablates the ability of both CD630 (RT12) and R20291 (RT027) to grow on trehalose. This phenotype can be restored by supplying treA on an inducible plasmid (n=3 for each strain/group). c, RT244 strains (DL3110 and DL3111) possessing the treR L172I mutation are capable of growth on 10 mM trehalose (n=3 for each strain/group). d, CD1015ΔptsT can metabolize 50 mM trehalose (n=4 for each strain/group). For panels a-d points represent biologically independent samples, solid bars are mean.

Extended Data Fig. 3. RT027 strains express treA at a significantly higher level than non-RT027 strains in the presence of 25 mM trehalose

Each data point (n=4 Ribotypes per group) represents gene expression from a different, biologically independent, strain and is an average from 2-5 independent experiments. P=0.029, Mann-Whitney-Wilcoxon Test (2 sided).

Extended Data Fig. 4. RT027 strains have a L172I mutation at a highly conserved site

a, treR genes from available C. difficile WGS files on NCBI (accessed 11-May-2017) were identified by tblastn and translated to protein sequences. Sequence fragments < 240 amino acids were discarded and the remaining 1010 sequences aligned with Clustal Omega. All 191 sequences containing the L172I SNP also contained the thyA gene, a marker for the RT027 lineage. ThyA was not found in any other genomes. Numbers indicate number of sequences with corresponding amino acid in that position. Multiple sequence alignment visualization generated with ProfileGrid. b, The TreR protein sequence from RT027 strain R20291 was blasted against non-C. difficile sequences in the NCBI database and the top 99 matches (along with R20291_treR) aligned with Clustal Omega. The Leucine at position 172 was found to be conserved in 93 of 99 non-C. difficile sequences. To confirm the importance of this residue, TreR was blasted against all non-Clostridial sequences in the NCBI database and the top 500 hits saved. Following removal of duplicate species 191 sequences were aligned with Clustal Omega. The Leucine at residue 172 was conserved in 83% of sequences (not shown).

Extended Data Fig. 5. A treA knockout strain has decreased toxin production 48 hours after infection

Mice were gavaged 10⁴ spores of either R20291 or R20291ΔtreA and provided 5 mM trehalose in drinking water. Points represent toxin levels from individual mice (R20291 n=10, R20291ΔtreA n=11) euthanized 48 hours after infection. Bars are mean. Mice gavaged with R20291ΔtreA had significantly lower toxin levels (p=.0268 Wilcoxon-Mann-Whitney test (2 sided), median 40960, IQR 23040-46080 Vs 92160, IQR 51200-102400).

Extended Data Fig. 6. The 4 gene trehalose insertion is only present in the RT078 lineage

Artemis comparison tool (ACT) displaying pairwise comparisons between C. difficile RT078 genome (M120) sequence and genome sequences from other C. difficile ribotypes (ribotypes indicated on the left). Numbers between grey bars indicate the genomic region where the trehalose four gene insert is located (3231169..3237057). Regions of sequence homology are displayed in red. The trehalose four gene insert of RT078 (indicated by the arrow on the top) was observed in RT078, but absent in other ribotypes.

Figure 1. Only RT027 and 078 strains show enhanced growth on 10 mM trehalose

Dashed grey line and band indicate mean growth in DMM without a carbon source and s.d. for all samples (n=21). Solid lines are mean growth yield (OD600) for groups: Non-RT027/078 (n=10), RT027 (n=8), and RT078 (n=3). All points represent biologically independent samples.

Figure 2. treA is responsible for trehalose metabolism

a, Trehalose metabolism operon found in all C. difficile strains; consisting of a phosphotrehalase (treA) and its transcriptional regulator (treR). b, RT027 strains strongly induce treA at 50 μM trehalose and at a significantly higher level than non-RT027 strains (n=4 biologically independent samples per trehalose concentration/strain). Bars are average fold increase, error bars are s.d.; p values derived from t-test (2-tailed) and Holm corrected for multiple comparisons. c, Structure of TreR monomer highlighting proximity of L172I mutation to trehalose-6-P binding pocket.

Figure 3. Trehalose metabolism increases virulence

a, Mice Infected with R20291ΔtreA (n=27 animals) have significantly attenuated risk of mortality when compared to mice infected with R20291 (n=28 animals) (78% lower risk with ΔtreA mutant; hazard ratio, 0.22; 95% CI, 0.09 to 0.59; P=0.003). b, Mice infected with R20291 (RT027) have a significantly higher risk of mortality when trehalose is supplemented in the diet (n=28 animals) than those with no trehalose supplementation (n=27 animals) (3-fold increased risk with trehalose; hazard ratio, 3.20; 95% CI, 1.09 to 9.42; P=0.035). All statistical tests were 2 sided.

Figure 4. ptsT enables enhanced trehalose metabolism

a, Structure of horizontally acquired trehalose metabolism module found in RT078 and closely related strains. b, Deletion of the trehalose transporter from a clinical RT078 strain (CD1015) ablates its ability to grow on 10 mM trehalose. Expression of ptsT from an inducible plasmid restores growth of CD1015ΔptsT on 10 mM trehalose, (CD1015, n=4; CD1015ΔptsT, n=4; CD1015ΔptsT::ptsT, n=5). c, Expressing ptsT from an inducible plasmid enables enhanced growth of CD630 (RT012) on 10 mM trehalose (CD630 n=3; CD630::ptsT n=3). d, The ptsT provides a competitive advantage in complex microbial communities. Dashed grey line (CI = 1) indicates equal fitness of the competing strains, points above this line represent out-competition by CD1015. All points (Fig 4b-d) represent biologically independent samples, bars are mean, p values derived from t-test (2-tailed) and Holm corrected for multiple comparisons where appropriate.

Figure 5. Trehalose can be detected in murine cecum and human ileostomy fluid

a, Twenty minutes post gavage, trehalose reaches high enough levels in the murine cecum to turn on expression of treA in RT027 but not non-RT027 in both non-antibiotic and antibiotic treated mice (n=3 animals per trehalose concentration/strain). b, trehalose can be detected by RT027 but not non-RT027 in the cecum of antibiotic treated mice gavaged with just 100 μl 5 mM trehalose (n=3 animals per group). c, RT027 strains can detect trehalose in 2/3 human ileostomy fluid samples tested from patients eating a normal (no deliberate trehalose addition) diet. Points represent biologically independent replicates, bars are average fold increase, error bars are s.d.

Figure 6. Timeline of trehalose adoption and spread of RT027 & RT078 lineages

Flags indicate reported outbreaks or first reports of ribotype 027 (top) or 078 (bottom) in PubMed. _† Stoke Mandeville outbreaks.