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From microbial gene essentiality to novel antimicrobial drug targets.

Mobegi FM, van Hijum SA, Burghout P, Bootsma HJ, de Vries SP, van der Gaast-de Jongh CE, Simonetti E, Langereis JD, Hermans PW, de Jonge MI, Zomer A - BMC Genomics (2014)

Bottom Line: Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways.Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale.By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

View Article: PubMed Central - PubMed

Affiliation: Radboud Institute for Molecular Life Sciences, Laboratory of Paediatric Infectious Diseases, Radboud University Medical Centre, Nijmegen 6500 HB, The Netherlands. Sacha.vanHijum@radboudumc.nl.

ABSTRACT

Background: Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials.

Result: Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept.

Conclusion: Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

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Related in: MedlinePlus

Schematic overview of the drug target selection criteria. Genome annotations information for S. pneumoniae R6, S. pneumoniae TIGR4, H. influenzae 86 028NP, H. influenzae Rd KW20, and M. catarrhalis BBH18 were updated using RAST. The proteins with updated annotations were then clustered into putative orthologous groups using OrthoMCL, and their subcellular localizations predicted in various publicly available tools. ESSENTIALS was used to analyse various transposon mutant libraries and predict the essentiality metric for each ORF. Comparing the ensuing essential genes with the catalogue of human gut microbial genes, as well as with the human genome helped to eliminate genes with conserved orthologs, and subsequently prioritize potential drug targets.
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Fig1: Schematic overview of the drug target selection criteria. Genome annotations information for S. pneumoniae R6, S. pneumoniae TIGR4, H. influenzae 86 028NP, H. influenzae Rd KW20, and M. catarrhalis BBH18 were updated using RAST. The proteins with updated annotations were then clustered into putative orthologous groups using OrthoMCL, and their subcellular localizations predicted in various publicly available tools. ESSENTIALS was used to analyse various transposon mutant libraries and predict the essentiality metric for each ORF. Comparing the ensuing essential genes with the catalogue of human gut microbial genes, as well as with the human genome helped to eliminate genes with conserved orthologs, and subsequently prioritize potential drug targets.

Mentions: We sought to determine potential drug targets in S. pneumoniae, H. influenzae, and M. catarrhalis following the selection criteria outlined in Figure 1. For these species, five strains with the required Tn-seq data were available; S. pneumoniae strains R6 and TIGR4, H. influenzae strains Rd KW20 and 86 028NP, and M. catarrhalis strain BBH18. Altogether, genomes of these strains in their initial annotations constituted of 10,072 open reading frames (ORFs). These annotations were updated using RAST to ensure consistency and comparability among strains in subsequent analyses. This analysis resulted in putative annotations for about 50% of all ORFs originally annotated with a hypothetical function (Table 1; Additional file 1). Next, we clustered the updated protein sequences using OrthoMCL, producing 1,798 orthologous groups/clusters (OGs) with, and 2,729 without singletons respectively (Additional file 1). This clustering of orthologous proteins allowed for the determination of species and/or strain specific proteins, as well as determining the metabolic potential of the strains. For example, the “Gram-negative specific” periplasmic chaperones (SurA) were clustered in OG_756 (cluster 756), while the “Streptococci-specific” transcriptional regulators (LytR) were clustered in OG_2554. On the other hand, 300 OGs, including OG_184, OG_186, OG_216, and OG_224, among others, contained genes conserved in all the five strains. All protein in individual OGs constituted of similar or identical functional annotations. This consistency in grouping and annotation was observed across all OGs, suggesting a reliable clustering. Confirmatory clusters and respective annotations derived from the clusters of orthologous genes (COGs) database were consistent with our OrthoMCL clusters. Additionally, using the OG’s, we were able to curate annotations for the HI1586 locus in Haemophilus influenzae Rd KW20, which was possibly misannotated in the initial release, as an isoleucyl-tRNA synthetase instead of a NaP+P/HP+P antiporter.Figure 1


From microbial gene essentiality to novel antimicrobial drug targets.

Mobegi FM, van Hijum SA, Burghout P, Bootsma HJ, de Vries SP, van der Gaast-de Jongh CE, Simonetti E, Langereis JD, Hermans PW, de Jonge MI, Zomer A - BMC Genomics (2014)

Schematic overview of the drug target selection criteria. Genome annotations information for S. pneumoniae R6, S. pneumoniae TIGR4, H. influenzae 86 028NP, H. influenzae Rd KW20, and M. catarrhalis BBH18 were updated using RAST. The proteins with updated annotations were then clustered into putative orthologous groups using OrthoMCL, and their subcellular localizations predicted in various publicly available tools. ESSENTIALS was used to analyse various transposon mutant libraries and predict the essentiality metric for each ORF. Comparing the ensuing essential genes with the catalogue of human gut microbial genes, as well as with the human genome helped to eliminate genes with conserved orthologs, and subsequently prioritize potential drug targets.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4233050&req=5

Fig1: Schematic overview of the drug target selection criteria. Genome annotations information for S. pneumoniae R6, S. pneumoniae TIGR4, H. influenzae 86 028NP, H. influenzae Rd KW20, and M. catarrhalis BBH18 were updated using RAST. The proteins with updated annotations were then clustered into putative orthologous groups using OrthoMCL, and their subcellular localizations predicted in various publicly available tools. ESSENTIALS was used to analyse various transposon mutant libraries and predict the essentiality metric for each ORF. Comparing the ensuing essential genes with the catalogue of human gut microbial genes, as well as with the human genome helped to eliminate genes with conserved orthologs, and subsequently prioritize potential drug targets.
Mentions: We sought to determine potential drug targets in S. pneumoniae, H. influenzae, and M. catarrhalis following the selection criteria outlined in Figure 1. For these species, five strains with the required Tn-seq data were available; S. pneumoniae strains R6 and TIGR4, H. influenzae strains Rd KW20 and 86 028NP, and M. catarrhalis strain BBH18. Altogether, genomes of these strains in their initial annotations constituted of 10,072 open reading frames (ORFs). These annotations were updated using RAST to ensure consistency and comparability among strains in subsequent analyses. This analysis resulted in putative annotations for about 50% of all ORFs originally annotated with a hypothetical function (Table 1; Additional file 1). Next, we clustered the updated protein sequences using OrthoMCL, producing 1,798 orthologous groups/clusters (OGs) with, and 2,729 without singletons respectively (Additional file 1). This clustering of orthologous proteins allowed for the determination of species and/or strain specific proteins, as well as determining the metabolic potential of the strains. For example, the “Gram-negative specific” periplasmic chaperones (SurA) were clustered in OG_756 (cluster 756), while the “Streptococci-specific” transcriptional regulators (LytR) were clustered in OG_2554. On the other hand, 300 OGs, including OG_184, OG_186, OG_216, and OG_224, among others, contained genes conserved in all the five strains. All protein in individual OGs constituted of similar or identical functional annotations. This consistency in grouping and annotation was observed across all OGs, suggesting a reliable clustering. Confirmatory clusters and respective annotations derived from the clusters of orthologous genes (COGs) database were consistent with our OrthoMCL clusters. Additionally, using the OG’s, we were able to curate annotations for the HI1586 locus in Haemophilus influenzae Rd KW20, which was possibly misannotated in the initial release, as an isoleucyl-tRNA synthetase instead of a NaP+P/HP+P antiporter.Figure 1

Bottom Line: Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways.Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale.By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

View Article: PubMed Central - PubMed

Affiliation: Radboud Institute for Molecular Life Sciences, Laboratory of Paediatric Infectious Diseases, Radboud University Medical Centre, Nijmegen 6500 HB, The Netherlands. Sacha.vanHijum@radboudumc.nl.

ABSTRACT

Background: Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials.

Result: Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept.

Conclusion: Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

Show MeSH
Related in: MedlinePlus