Abstract
Microbial pathogenesis studies are typically performed with reference strains, thereby overlooking within-species heterogeneity in microbial virulence. Here we integrated human epidemiological and clinical data with bacterial population genomics to harness the biodiversity of the model foodborne pathogen Listeria monocytogenes and decipher the basis of its neural and placental tropisms. Taking advantage of the clonal structure of this bacterial species, we identify clones epidemiologically associated either with food or with human central nervous system (CNS) or maternal-neonatal (MN) listeriosis. The latter clones are also most prevalent in patients without immunosuppressive comorbidities. Strikingly, CNS- and MN-associated clones are hypervirulent in a humanized mouse model of listeriosis. By integrating epidemiological data and comparative genomics, we have uncovered multiple new putative virulence factors and demonstrate experimentally the contribution of the first gene cluster mediating L. monocytogenes neural and placental tropisms. This study illustrates the exceptional power in harnessing microbial biodiversity to identify clinically relevant microbial virulence attributes.
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Change history
06 March 2017
In the version of this article initially published, in Figure 2b, in the panel called "CNS infections" the bar of CC3 should have been represented in red and the one of CC121 should have been represented in blue. The errors have been corrected in the HTML and PDF versions of the article.
06 March 2017
In the version of this article initially published, the titles of the x axes in Figure 5b and 5c should have been “Brain/blood CFU ratio” instead of "Blood/brain CFU ratio," and the title of the z axis in Figure 2c should have been "% of isolates" instead of "Number of isolates." The errors have been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank C. Soto Alvarez, G. Pontdeme, T. Cantinelli and L. Diancourt for their contributions to MLST data production and analysis, and S. Roche for providing low-virulence strains for genome sequencing. We also thank D. Mornico (Center of Bioinformatics, Biostatistics and Integrative Biology of the Institut Pasteur) for his help with the submission of genome reads and assemblies. This study was funded by the Institut Pasteur, INSERM, from the French government's Investissement d'Avenir program, Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases' (grant ANR-10-LABX-62-IBEID), the European Research Council (ERC), ERANET Proantilis, the Programme Hospitalier de Recherche Clinique MONALISA and the Programme de Recherche Translationnelle (PTR) ANSES–Institut Pasteur. Listeriosis surveillance in France is funded by the Institut de Veille Sanitaire (InVS) and the Institut Pasteur.
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Contributions
M.L. and S.B. conceived, supervised and directed the project. L. monocytogenes isolates were collected and characterized by A.L. in the context of French National Reference Center for Listeria activities, with the help of V.C.-F., as well as A.B. and S.R. Methods for clone identification were developed by M.M.M. and S.B. Epidemiological analyses were performed by M.M.M. and S.B. Clinical data collection and analysis was conducted by C.C. and M.L. Statistical analyses were performed by M.M.M. and E.P.C.R. Comparative genomics analyses were performed by M.M.M., M.T. and E.P.C.R. Phylogenetic analyses were performed by M.M.M., A.C. and M.T. Y.-H.T. generated mutant CC4 strains. In vivo experiments were performed by Y.-H.T., O.D. and C.G. M.M.M., S.B. and M.L. wrote the manuscript, with contributions from Y.-H.T., C.C., A.L., M.T. and E.P.C.R.
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Integrated supplementary information
Supplementary Figure 1 Flow chart of the study.
The strategy involving the four different approaches (epidemiological, clinical, genomics and experimental) used in this study is shown. C, clinical; F, food; A, animal infection; E, environmental.
Supplementary Figure 2 MLST diversity of lineages I and II represented by the genomes analyzed in the present study.
MLST profiles of the genomes used in this study (orange, newly sequenced genomes; dark blue, public genomes) were compared to published MLST data15–17,24,35 using the minimum spanning tree algorithm with the software tool BioNumerics v6.6 (Applied Maths). Each circle corresponds to a sequence type (ST). Gray zones surround STs that belong to the same clonal complex (CC). CC numbers are given next to the corresponding zones. The lines between STs are bold, plain, discontinuous and light discontinuous depending on the number of allelic mismatches between profiles (1, 2, 3, and 4 or more, respectively); note that links are only indicative, as alternative links with equal weight might exist. There were no common alleles between the two major lineages.
Supplementary Figure 3 Core and pan-genome size as a function of genome numbers.
The number of genes in common (core genome; in green) and total number of homologous gene families (pan-genome; in blue) for increasing numbers of genomes (in-house R script). Numbers of gene families were estimated by performing 1,000 random different input orders of genomes. Solid lines correspond to the average number of gene families obtained by taking into account all permutations. Dashed lines indicate the standard deviation of the mean. The upper and lower edges of the blue and green areas correspond to the maximum and minimum numbers of gene families, respectively. For 104 sequenced genomes, the pan-genome and core genome comprised 6,867 and 1,791 genes, respectively. The core genome represented 60% of the average number of genes per genome and 26% of the pan-genome.
Supplementary Figure 4 Phylogenetic distribution and size variation of gene products encoded in the LIPI-1, LIPI-3 and SSI-1 genomic islands.
The columns show the presence/absence and truncation/deletion status of virulence gene products. The maximum-likelihood phylogeny was obtained on the basis of the core genome of 104 L. monocytogenes isolates (Supplementary Note). Genes located in the same syntenic blocks are indicated with black lines above the corresponding genes. Gene products encoded by LIPI-3 are named with the terminal part of the locus tags (LMOf2365_1113 to LMOf2365_1119) in the F2365 genome. InlA truncation was the main feature correlated with hypovirulent clones. Strain LM13656 (CC2) was initially selected because it was non-hemolytic. Consistently, it showed a truncation in the hly gene encoding LLO (listeriolysin O), the virulence factor listeriolysin involved in hemolysis.
Supplementary Figure 5 Distribution and variability of virulence gene products.
All 69 variable reported virulence genes28 are shown except those already shown in Supplementary Figure 4. The colored rounded squares show the distribution and size variations of virulence gene products. The numbers above the figure correspond to genes listed in Supplementary Table 7. Genes present in all the genomes with invariable size are not shown. Genes located in the same syntenic blocks are indicated with black solid lines above the corresponding genes.
Supplementary Figure 6 Description of the CC4-associated PTS cluster LIPI-4.
(a) The gene content of the PTS cluster and flanking core genes in the CC4 strain LM09-00558 (below) is shown in comparison to the genome of CC1 strain LL195 (above). Putative functions are indicated. Identity percentages between sequences were determined by nucleotide BLAST and are represented using Easyfig 2.1. (b) The genomic region of the PTS in the CC4 strain LM09-00558 in which the PTS cluster was deleted (CC4∆PTS) is shown in comparison to the isogenic wild-type strain. Orange, genes of the PTS cluster; blue, flanking core genes.
Supplementary Figure 7 Implication of LIPI-4, the hypervirulent clone CC4-associated PTS, in CNS and placental infection.
The data shown here are supplementary to Figure 5. (a,b) Humanized mice were inoculated orally at a dose of 3 × 108 CFUs (a) or intravenously at a dose of 5 × 105 CFUs (b) with reference strain EGDe (n = 5 in a), representative CC4 strain LM09-00558 (CC4; n = 9 in a and n = 6 in b) or a whole-PTS-cluster deletion mutant derived from LM09-00558 (CC4ΔPTS; n = 7 in a and n = 6 in b). (c) Humanized mice were intravenously infected at a dose of 5 × 105 CFUs by CC4ΔPTS containing either a single copy of pIMC (n = 9) or pIMC with the PTS cluster under its native promoter on the chromosome (n = 9). (d) The competition index of WT EGDe (n = 4) or WT CC4 (n = 4) was tested against chloramphenicol-resistant EGDe (EGDe containing pIMC) in pregnant humanized mice. (e) The competition index of WT CC4 was tested against chloramphenicol-resistant CC4ΔPTS (pIMC) (n = 3) or CC4ΔPTS (pIMC-PTS) (n = 4) in pregnant humanized mice. Pregnant humanized mice at day 14/21 of gestation were intravenously infected with a 1:1 mixture of the two strains as indicated at a total dose of 2 × 105 CFUs. Mice were sacrificed on day 5 after infection when bacteria were orally inoculated (a) or day 2 after infection when intravenously infected (b–e). Results are shown as medians with interquartile range. Each dot represents the bacterial count from a whole organ. Statistical analyses were carried out by a Dunn’s multiple-comparison test (a), Mann-Whitney U test (b,c) or Wilcoxon matched-pairs signed-rank test (d,e): *P < 0.05, **P < 0.01, ***P < 0.001.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 and Supplementary Note. (PDF 1905 kb)
Supplementary Table 1
Distribution of genotypic categories in food and clinical sources. (XLSX 18 kb)
Supplementary Table 2
Distribution of genotypic categories in bacteremia, CNS and MN infections. (XLSX 16 kb)
Supplementary Table 3
Bacterial strains used for in vivo tests. (XLSX 11 kb)
Supplementary Table 4
Stepwise multiple regression of the parameters recorded during the in vivo experiments on the clinical frequencies of clones. (XLSX 13 kb)
Supplementary Table 5
Genomes used in this study. (XLSX 26 kb)
Supplementary Table 6
Gene families of the core genome. (XLSX 1923 kb)
Supplementary Table 7
Virulence gene products shown in Supplementary Figure 5. (XLSX 13 kb)
Supplementary Table 8
Pan-genome of the 104 genomes. (XLSX 12318 kb)
Supplementary Table 9
Correlation of the pattern of presence/absence of gene families of the pan-genome with the clinical frequency of clones. (XLSX 364 kb)
Supplementary Table 10
Primers used for CC4-specific PTS mutagenesis. (XLSX 11 kb)
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Maury, M., Tsai, YH., Charlier, C. et al. Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity. Nat Genet 48, 308–313 (2016). https://doi.org/10.1038/ng.3501
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DOI: https://doi.org/10.1038/ng.3501
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