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.

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Figure 1: Only RT027 and RT078 strains show enhanced growth on 10 mM trehalose.
Figure 2: The treA gene is responsible for trehalose metabolism.
Figure 3: Trehalose metabolism increases virulence.
Figure 4: The ptsT gene enables enhanced trehalose metabolism.
Figure 5: Trehalose can be detected in mouse caecum and human ileostomy fluid.
Figure 6: Timeline of trehalose adoption and spread of RT027 and RT078 lineages.

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Acknowledgements

This work was supported by the National Institutes of Health (U01AI124290-01 and 5U19AI09087202). We thank the members of the Ostomy Association of Greater Lansing for anonymously donating samples, and D. Lyras for the RT244 strains. We thank V. Young, D. Mills, J. Walter and M. Costa-Mattioli for comments on the manuscript.

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Concept and design of study: R.A.B., J.M.A., J.C. and C.R. Experiments: C. difficile growth, J.C. and C.R.; identification of L172I SNP and comparative analysis, C.R. and J.C.; treA RT–qPCR, H.D.; mouse infection model, J.C.; genetic manipulation of C. difficile strains, J.C. and C.R.; identification of RT078 trehalose insertion, C.W.K., H.C.L. and T.D.L.; faecal minibioreactor competitions, J.M.A.; spontaneous C. difficile mutant identification, H.D.; analysis, J.C., C.R., H.D., J.M.A. and R.B. The manuscript was drafted by J.C., J.M.A., and R.A.B., and revised by all authors.

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Correspondence to R. A. Britton.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks J. Ballard, E. Pamer and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 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/31. Stars indicate position of strains used in this study, with red stars indicating sequence types possessing either the TreR L172I amino acid substitution (ST1, ST41) or four-gene insertion (ST11). Tree constructed using MEGA7 (ref. 32).

Extended Data Figure 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 and s.d. in DMM without a carbon source. Solid lines indicate mean growth yield (A600nm) 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, CD1015∆ptsT can metabolize 50 mM trehalose (n = 4 for each strain/group). For ad, points represent biologically independent samples, solid bars are means. Source data

Extended Data Figure 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 two to five independent experiments. P = 0.029, Mann–Whitney–Wilcoxon test (two-sided). Bar indicates mean expression. Source data

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

a, The treR genes from available C. difficile whole-genome sequencing files on the NCBI database (accessed 11 May 2017) were identified by tblastn and translated to protein sequences. Sequence fragments shorter than 240 amino acids were discarded and the remaining 1,010 sequences aligned with Clustal Omega33. 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 the number of sequences with a corresponding amino acid in that position. Multiple sequence alignment visualization generated with ProfileGrid34. 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 R20291treR) 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. After removal of duplicate species, 191 sequences were aligned with Clustal Omega. The leucine at residue 172 was conserved in 83% of sequences (data not shown).

Extended Data Figure 5 A treA knockout strain decreased toxin production 48 h after infection.

Mice were gavaged with 104 spores of either R20291 or R20291∆treA and provided with 5 mM trehalose in drinking water. Points represent toxin levels from individual mice (R20291 n = 10, R20291∆treA n = 11) euthanized 48 h after infection. Bars are means. Mice gavaged with R20291∆treA had significantly lower toxin levels (P = 0.0268; Wilcoxon–Mann–Whitney test (two-sided), median 40,960, IQR 23,040–46,080 versus 92,160, IQR 51,200–102,400). Source data

Extended Data Figure 6 The four-gene trehalose insertion is only present in the RT078 lineage.

Artemis comparison tool 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 was absent in other ribotypes.

Extended Data Table 1 Compounds conferring at least 1.5-fold growth advantage in Biolog Phenotypic Microarray plates PM1 or PM2
Extended Data Table 2 Spontaneous C. difficile mutants able to utilize 10 mM trehalose
Extended Data Table 3 Strains, primers and plasmids

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Collins, J., Robinson, C., Danhof, H. et al. Dietary trehalose enhances virulence of epidemic Clostridium difficile. Nature 553, 291–294 (2018). https://doi.org/10.1038/nature25178

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