ALL Metrics
-
Views
-
Downloads
Get PDF
Get XML
Cite
Export
Track
Brief Report
Revised

Large scale enterohemorrhagic E coli population genomic analysis using whole genome typing reveals recombination clusters and potential drug target

[version 3; peer review: 2 approved]
Previously titled: Pangenome guided pharmacophore modelling of enterohemorrhagic Escherichia coli sdiA
PUBLISHED 01 Sep 2020
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

Abstract

Enterohemorrhagic Escherichia coli continues to be a significant public health risk. With the onset of next generation sequencing, whole genome sequences require a new paradigm of analysis relevant for epidemiology and drug discovery. A large-scale bacterial population genomic analysis was applied to 702 isolates of serotypes associated with EHEC resulting in five pangenome clusters. Serotype incongruence with pangenome types suggests recombination clusters. Core genome analysis was performed to determine the population wide distribution of sdiA as potential drug target. Protein modelling revealed nonsynonymous variants are notably absent in the ligand binding site for quorum sensing, indicating that population wide conservation of the sdiA ligand site can be targeted for potential prophylactic purposes. Applying pathotype-wide pangenomics as a guide for determining evolution of pharmacophore sites is a potential approach in drug discovery.

Keywords

pangenome,pharmacophore,EHEC, Escherichia coli

Revised Amendments from Version 2

Version 3 contains the McDonald-Kreitman test calculations to determine the nonsynonymous to synonymous mutations ratios.

See the author's detailed response to the review by Kerry K. Cooper
See the author's detailed response to the review by Olivier Tenaillon

Introduction

One of the more prominent strains of Escherichia coli is the enterohemorrhagic E. coli (EHEC) pathotype associated with global outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) usually by consumption of undercooked beef1. Within the cattle reservoir, sdiA gene is required by E. coli to survive within the acidic rumen environment. SdiA is used by E. coli to sense acyl homoserine in a quorum sensing system2. However, it is considered as an orphan as the cognate acyl homoserine synthase is absent, and hence sdiA is considered an environmental sensor to sense the nearby microbial community. SdiA is stabilized by acyl homoserine lactone and acts as transcription factor glutamate decarboxylase needed for survival in the acidic environment. Hence blocking the ability of EHEC to survive the acidic ruminal environment is a proposed mechanism to control shedding in the cattle reservoir.

Whole genome sequencing of bacterial pathogens, particularly EHEC, is quickly transforming the workflows of epidemiological investigations. However, most bioinformatic pipelines used in clinical investigation perform data reduction of genomes and artificially reduce diversity due to comparison of a limited number of housekeeping genes3. While wgMLST attempts to increase the number of genes for analysis, the assignment of a single reference genome appears to be inadequate in light of the pangenome. Various studies have shown that a significant number of genes that are present to the entire universe of genes within a species are missed for variant calling if only a single reference gene is used4. In this study, a multi-scale approach was applied to generate genome wide clustering using the entire pangenome, composed of the core genome and the accessory genome via variable k-mers5. This approach allows differentiation between clusters as well as within serotypes, which is a limitation of using low resolution techniques like MLST.

The concept of the pangenome, which represents the entirety of the genes that are present within a species, which can also be adjusted to the pathotype level, was applied in this particular study. The EHEC pangenome represents the combination of genes seen in the EHEC pathotype. While a prior pangenome of E. coli contained 17 genomes, I generated and updated EHEC pangenome with 702 genomes, representing the largest population wide whole genome comparison to date6. The pangenome enables clustering of isolates using gene presence and absence. Targetting the core genome, represented in this study by sdiA, enables integration of population genomics with drug discovery target identification. This strategy enables to capture the pangenome wide variation and ensures all conserved variants are targeted by the drug discovery pipeline coupling the pangenome to pharmacophore modelling.

Methods

EHEC population

EHEC associated serotypes are defined based on a previous study7. This study defined EHEC strains as subgroup of Shiga-toxin producing E. coli and are belonging to the following serotypes (O26:H11,O45:H2,O103:H2,O111:H8,O121: H19, O145:H28, and O157:H7). Whole genome sequences with the associated EHEC metadata was downloaded from Enterobase 1.1.2 using the keyword search of the respective serotypes within the E. coli species8. This search yielded 702 genomes from environmental, animal and clinical samples. (Underlying data: Metadata from Enterobase 1.1.2 of EHEC pangenome9). As this genomes are different from version 1 of this paper, previous Figure 1 was deleted and new Figure 1A was generated reflecting the expanded genomes used in the analysis.

EHEC pangenome

Whole genome typing in the context of the pangenome was performed using PopPUNK (POPulation Partitioning Using Nucleotide Kmers) 1.1.6.5. The genomes were annotated with Prokka 1.13.3 as per published protocol10. Gff files were extracted as input for the pangenome pipeline Roary 3.11.2 using the following parameters for not splitting paralogs (roary -s -p 32 *.gff) and the resulting presence absence matrix together with the accessory genome phylogeny visualized in Phandango 1.3.0 and is represented as Figure 1B11. Each blue bar represents an individual gene and solid blue blocks represent gene clusters. Previous Figure 1B was deleted and new version of Figure 1B was regenerated integrating the new genomes.

bdd9b3da-cd4d-469c-aa72-ae64a7f3dc29_figure1a.gif

Figure 1A. Pangenome wide clustering using k-mers.

There are three clusters within the 0157 serotype, 026 is clustered with O111 as well as 103 with O45. Previous Figure 1A was replaced to reflect the increase in genomes analyzed.

bdd9b3da-cd4d-469c-aa72-ae64a7f3dc29_figure1b.gif

Figure 1B. EHEC pangenome showing genomic diveristy with the gene presence absence variation matrix.

Previous Figure 1B was replaced to reflect the increase in genomes analyzed.

Allelic variant calling

Snippy variant calling pipeline 4.3.5 was used to determine the synonymous and nonsynonymous protein mutations using sdiA of Escherichia coli O157:H7 str. Sakai as reference. The –contigs option was added to the standard commandline (snippy –outdir –ref sdiA_sakai.gbk). The resulting individual variants of sdiA was merged into EHEC E. coli sdiA variant calling data (Underlying data9). Previous Figure 3 in version was removed as the new data was better represented by a new Table 2. McDonald-Kreitman test was done using the Snippy output containing data on synonymous and nonsynonymous mutations12.

In silico sdiA protein modelling

SdiA genes were extracted from the pangenome output of Roary and protein in silico modelling performed using SWISS-MODEL1317. SdiA protein sequences were used as targets to search for protein templates within the SWISS-MODEL library. Model selection was based on the template with the highest quality prediction by the target-template alignment.

Results and discussion

Pangenome based clustering integrated the core and accessory elements was applied on 702 whole genomes sequences from serotypes associated with EHEC from diverse sources in the environment as well as animal and human hosts capture the evolutionary space. The majority of the available sequences are from O157 H7 representing 68.5% (481 out of 702) and the rest from the other major non-O157 serotype designated as the “big six”, with O45 H2 1.9% (13 out of 702), O103 H2 10.7% (77 out of 702), O26 H11 1.3% (9 out of 702), O111 H8 6.0% (42 out of 702), O121 H19 8.1% (57 out of 702) and O145 H28 3.2% (23 out of 702). The variable-length k-mer analysis and comparison software (PopPUNK) enables scalable, annotation and alignment free approach to large scale population genomics5. The accessory genome details the recent acquisition of mobile elements via horizontal gene transfer conveying metabolic, virulence and antibiotic resistance properties which cannot be captured by classical approaches. Eliminating an integral property of recombigenic organism underestimates the diversity and artificially creates similarity and relatedness. The analysis yielded five major pangenomic clusters of EHEC associated isolates. Cluster I is represented by O157 with three genomic subclusters, cluster two contains serotypes O103 and O45, cluster III contains serotype O121, cluster IV contains serotypes O26 and O111 and cluster V contains serotype O145 (Figure 1A). This updated analysis expanded the genomes from version 1 of this paper with 152 genomes into 702 which necessitates the regeneration of Figure 1. A better visualization of the pangenome cluster was also utilized. Clusters containing several serotypes like cluster II and IV indicate that recombination events blur the genomic boundary resulting to being meshed together in a gradient of dots visually. This novel genome wide framework allows a greater resolution of comparison, as it is now possible to compare similar organisms within the same serotype and determine specific lineages integrating the accessory genome. The acquisition of genomic islands unique to individual isolates are well defined in the pangenome gene presence absence matrix (Figure 1B). The core genome is 2966 (Table 1) and total gene count within the EHEC pangenome is 27774, exceeding previous estimates of total E. coli pangenome 22,000. This enormous difference between the core gene and total gene highlights the variation between the different isolates, which can be strain specific and individual isolate specific as indicated by the pangenome data. However, further analysis is limited due to the incompleteness of the metadata entry with regards to the pertinent parameters such specific geolocation, organ of isolation, severity of clinical signs and others.

Table 1. Pangenome metrics.

Percentage Occurence
Core genes(99% <= strains <= 100%)2966
Soft core genes(95% <= strains < 99%)301
Shell genes(15% <= strains < 95%)2889
Cloud genes(0% <= strains < 15%)21618
Total genes(0% <= strains <= 100%)27774

SdiA is a core gene found across the EHEC pangenome clusters based on the genome wide pangenome analysis, indicating that it can be a suitable interventional target. Considering the huge diversity between pangenome clusters, sdiA homology was analyzed and compared. Remarkably, pangenome cluster I showed highly conserved sdiA structure across global spatial and temporal range (30 years), in spite of cluster I diverging to three separate subsclusters. Divergence from the canonical sdiA structure is more prominent in other genomic clusters. Pangenome cluster II yielded the most number of nonsynonymous mutations (50%) in sdiA gene (Table 2). The percentage distribution for the rest of the pangenome clusters are as follows: 22% for cluster IV, 21% for cluster III and 4% for cluster V. The topological relevance of the predominant mutations was further contextualized by protein modelling.

Table 2. Nonsynonymous mutations summary integrating the pangenome clusters.

EHEC Pangenome
Cluster
SerotypeNonsynonymous mutation
position
101_240140_240189_240
IIO103H2777777
IVO111H8404040
IIIO121H195555
VO145H2823
IO157H7221
IIO45H2131313
Total210187131

The impact of the most prevalent nonsynonymous mutations were analyzed with protein modelling using sdiA of Escherichia coli O157:H7 str. Sakai as template. The most ranked nonsynonymous mutation is asparagine to serine at amino acid position 101 with 39.1% (210/536 located adjacent to η-4 phenylalanine which is associated with the ligand docking (Figure 2B). This is followed by 24.4% (131/536) of the nonsynonymous mutation is due to conversion of arginine to lysine at position 189 of sdiA (Figure 2A). This amino acid is located with the α-6 domain, adjacent to the amino acid clusters associated with sdiA dimerization. Previous protein modelling determined the role of guanidinium group of arginine which enables interactions in three different directions enabling a more complex electrostatic interaction versus lysine as well as the higher pKa value in arginine that can yield a more stable ionic interaction compared to lysine18. β-5 domain alanine to threonine change at amino acid position 140 is the third ranked nonsynonymous mutation with 34.9% (187/536) (Figure 2C). None of the highly ranked nonsynonymous mutations impact the ligand interaction, indicating the conservation of the sdiA motif across the population in geographic and temporal distribution, which suggests the possibility of targeting sdiA for quorum sensing inhibition. Mutational analysis using McDonald-Kreitman test indicate differential selection pressures between serotypes. Serotypes O103:H2,O45:H2 and O111:H8 have slightly higher between group nonsynonymous/synonymous ratios (0.42,0.45,0.43 respectively) than within species nonsynonymous/synonymous ratios (0.375 using O157:H7 as within species group). Serotypes O145:H28, O121:H19, O26:H11 have lower values compared to the within species values (0.33, 0.22,0 respectively).

bdd9b3da-cd4d-469c-aa72-ae64a7f3dc29_figure2a.gif

Figure 2A. Protein model of the nonsynonymous variant at amino acid position 189.

bdd9b3da-cd4d-469c-aa72-ae64a7f3dc29_figure2b.gif

Figure 2B. Protein model of the nonsynonymous variant at amino acid position 101.

bdd9b3da-cd4d-469c-aa72-ae64a7f3dc29_figure2c.gif

Figure 2C. Protein model of the nonsynonymous variant at amino acid position 140.

Conclusion

While EHEC pangenome is remarkably diverse, the allelic variants of sdiA, particularly nonsynonymous mutants, indicate the conservation of quorum sensing domain, indicating that targeting this structure can be effective across the different lineages of EHEC pathotype.

Data availability

All underlying and extended data available from Open Science Framework: Supplemental Data for Pangenome guided pharmacophore modelling of enterohemorrhagic Escherichia coli sdiA, https://doi.org/10.17605/OSF.IO/BNZ859

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Underlying data

Table 1 Metadata from Patric Database of EHEC E. coli pangenome, version 1 replaced with the updated 702 genomes

Table 2 EHEC E. coli pangenome presence absence matrix, version 1 replaced with the updated 702 genomes

Table 3 EHEC E. coli sdiA variant calling data, version 1 replaced with the updated 702 genomes

Extended data

SWISS-MODEL Homology Modelling Report available at osf.io/bnz85.

Comments on this article Comments (0)

Version 3
VERSION 3 PUBLISHED 09 Jan 2019
Comment
Author details Author details
Competing interests
Grant information
Copyright
Download
 
Export To
metrics
Views Downloads
F1000Research - -
PubMed Central
Data from PMC are received and updated monthly.
- -
Citations
CITE
how to cite this article
Bandoy DD. Large scale enterohemorrhagic E coli population genomic analysis using whole genome typing reveals recombination clusters and potential drug target [version 3; peer review: 2 approved]. F1000Research 2020, 8:33 (https://doi.org/10.12688/f1000research.17620.3)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
track
receive updates on this article
Track an article to receive email alerts on any updates to this article.

Open Peer Review

Current Reviewer Status: ?
Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 3
VERSION 3
PUBLISHED 01 Sep 2020
Revised
Views
9
Cite
Reviewer Report 03 Nov 2020
Olivier Tenaillon, IAME (Infection Antimicrobials Modelling Evolution), UMR 1137, French Institute of Health and Medical Research (INSERM), Paris, France 
Approved
VIEWS 9
Table 2 is not properly labelled. Positions should be presented without the _240 and eventually the precise mutation named. It should be mentionned that the numbers refers to number of strains carrying the mutation.

Results from the ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Tenaillon O. Reviewer Report For: Large scale enterohemorrhagic E coli population genomic analysis using whole genome typing reveals recombination clusters and potential drug target [version 3; peer review: 2 approved]. F1000Research 2020, 8:33 (https://doi.org/10.5256/f1000research.29079.r70557)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Version 2
VERSION 2
PUBLISHED 01 Oct 2019
Revised
Views
15
Cite
Reviewer Report 06 Nov 2019
Kerry K. Cooper, School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, USA 
Approved
VIEWS 15
While I disagree with the author that "The inclusion of non-EHEC is necessary as outgroup comparison groups and does not debunk the validity of the analysis pipeline". As including non-EHEC genomes in an EHEC core genome analysis does alter the results ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Cooper KK. Reviewer Report For: Large scale enterohemorrhagic E coli population genomic analysis using whole genome typing reveals recombination clusters and potential drug target [version 3; peer review: 2 approved]. F1000Research 2020, 8:33 (https://doi.org/10.5256/f1000research.22447.r54557)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Views
18
Cite
Reviewer Report 29 Oct 2019
Olivier Tenaillon, IAME (Infection Antimicrobials Modelling Evolution), UMR 1137, French Institute of Health and Medical Research (INSERM), Paris, France 
Approved with Reservations
VIEWS 18
I think this version is better than the previous one, but I still think the connection to sidA could be made stronger and the relevance of the focus on that gene and this subgroup also. Any conserved gene could be ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Tenaillon O. Reviewer Report For: Large scale enterohemorrhagic E coli population genomic analysis using whole genome typing reveals recombination clusters and potential drug target [version 3; peer review: 2 approved]. F1000Research 2020, 8:33 (https://doi.org/10.5256/f1000research.22447.r54556)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Version 1
VERSION 1
PUBLISHED 09 Jan 2019
Views
28
Cite
Reviewer Report 15 May 2019
Olivier Tenaillon, IAME (Infection Antimicrobials Modelling Evolution), UMR 1137, French Institute of Health and Medical Research (INSERM), Paris, France 
Approved with Reservations
VIEWS 28
The present manuscript presents a state-of-the-art pan genome analysis of EHEC strains and a subsequent analysis of the variation in sdiA.

The analysis of sdiA could have been completed with simple KA/Ks analysis and compared to that ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Tenaillon O. Reviewer Report For: Large scale enterohemorrhagic E coli population genomic analysis using whole genome typing reveals recombination clusters and potential drug target [version 3; peer review: 2 approved]. F1000Research 2020, 8:33 (https://doi.org/10.5256/f1000research.19267.r46991)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 16 May 2019
    DJ Darwin Bandoy, Department of Veterinary Paraclinical Sciences, University of the Philippines Los Baños, Los Baños, 4031, Philippines
    16 May 2019
    Author Response
    I thank the reviewer for the effort in doing the review. I accept all the suggestions and will add the population genetic analysis in the next version of the paper.
    Competing Interests: No competing interests were disclosed.
COMMENTS ON THIS REPORT
  • Author Response 16 May 2019
    DJ Darwin Bandoy, Department of Veterinary Paraclinical Sciences, University of the Philippines Los Baños, Los Baños, 4031, Philippines
    16 May 2019
    Author Response
    I thank the reviewer for the effort in doing the review. I accept all the suggestions and will add the population genetic analysis in the next version of the paper.
    Competing Interests: No competing interests were disclosed.
Views
29
Cite
Reviewer Report 24 Apr 2019
Kerry K. Cooper, School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, USA 
Not Approved
VIEWS 29
I would state the biggest issue with the manuscript is the work is not technically sound, because upon examining the metadata file from Patric, numerous strains included in the EHEC pangenome were in fact not EHEC strains. Many of the isolates ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Cooper KK. Reviewer Report For: Large scale enterohemorrhagic E coli population genomic analysis using whole genome typing reveals recombination clusters and potential drug target [version 3; peer review: 2 approved]. F1000Research 2020, 8:33 (https://doi.org/10.5256/f1000research.19267.r47169)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 16 May 2019
    DJ Darwin Bandoy, Department of Veterinary Paraclinical Sciences, University of the Philippines Los Baños, Los Baños, 4031, Philippines
    16 May 2019
    Author Response
    I appreciate the work of the reviewer for going through the metadata. The inclusion of non-EHEC is necessary as outgroup comparison group and does not debunk the validity of the ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 16 May 2019
    DJ Darwin Bandoy, Department of Veterinary Paraclinical Sciences, University of the Philippines Los Baños, Los Baños, 4031, Philippines
    16 May 2019
    Author Response
    I appreciate the work of the reviewer for going through the metadata. The inclusion of non-EHEC is necessary as outgroup comparison group and does not debunk the validity of the ... Continue reading

Comments on this article Comments (0)

Version 3
VERSION 3 PUBLISHED 09 Jan 2019
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
Sign In
If you've forgotten your password, please enter your email address below and we'll send you instructions on how to reset your password.

The email address should be the one you originally registered with F1000.

Email address not valid, please try again

You registered with F1000 via Google, so we cannot reset your password.

To sign in, please click here.

If you still need help with your Google account password, please click here.

You registered with F1000 via Facebook, so we cannot reset your password.

To sign in, please click here.

If you still need help with your Facebook account password, please click here.

Code not correct, please try again
Email us for further assistance.
Server error, please try again.