Limits...
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

Validation of growth inhibition using disk diffusion essays. Cell culture plate cross-sectional images showing the area of growth inhibition for: a. M. catarrhalis in 5′deoxyadenosine, and S. pneumoniae in; b. (R)-6-fluoromevalonate diphosphate, 1-methyluric acid, d. 5, 5′-dithiobis (2-nitrobenzoate), and e. 5′deoxyadenosine respectively.
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Fig3: Validation of growth inhibition using disk diffusion essays. Cell culture plate cross-sectional images showing the area of growth inhibition for: a. M. catarrhalis in 5′deoxyadenosine, and S. pneumoniae in; b. (R)-6-fluoromevalonate diphosphate, 1-methyluric acid, d. 5, 5′-dithiobis (2-nitrobenzoate), and e. 5′deoxyadenosine respectively.

Mentions: As an essential amino acid, methionine is not synthesized de novo in humans, who must rely on dietary intake. Enzymes involved in microbial methionine biosynthesis therefore offer highly specific and selective drug targets. We used 1-methyluric acid (CAS: 708-79-2) to target S-adenosylmethionine synthetase (EC: 2.5.1.6); a key enzyme in methionine biosynthesis, whose drug target potential has been explored in various pathogens [53, 54]. Contrary to expectations, no growth inhibition was observed in Gram-negative strains (Table 3; Figure 3): growth inhibition was only observed in S. pneumoniae. Since 1-methyluric acid formed a precipitate in concentrations above 312 μg /ml, no MIC values could be calculated. This lack of growth inhibition in Gram-negative strains may possibly be due to their double layered cell walls which are less penetrable [55], or the bacteria have expanded their resistance mechanisms to evade killing by antimicrobials [55, 56]. It is also possible that the two Gram-negative species have alternative mechanisms for methionine biosynthesis, further complicating screening for effective drugs.Figure 3


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)

Validation of growth inhibition using disk diffusion essays. Cell culture plate cross-sectional images showing the area of growth inhibition for: a. M. catarrhalis in 5′deoxyadenosine, and S. pneumoniae in; b. (R)-6-fluoromevalonate diphosphate, 1-methyluric acid, d. 5, 5′-dithiobis (2-nitrobenzoate), and e. 5′deoxyadenosine respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Validation of growth inhibition using disk diffusion essays. Cell culture plate cross-sectional images showing the area of growth inhibition for: a. M. catarrhalis in 5′deoxyadenosine, and S. pneumoniae in; b. (R)-6-fluoromevalonate diphosphate, 1-methyluric acid, d. 5, 5′-dithiobis (2-nitrobenzoate), and e. 5′deoxyadenosine respectively.
Mentions: As an essential amino acid, methionine is not synthesized de novo in humans, who must rely on dietary intake. Enzymes involved in microbial methionine biosynthesis therefore offer highly specific and selective drug targets. We used 1-methyluric acid (CAS: 708-79-2) to target S-adenosylmethionine synthetase (EC: 2.5.1.6); a key enzyme in methionine biosynthesis, whose drug target potential has been explored in various pathogens [53, 54]. Contrary to expectations, no growth inhibition was observed in Gram-negative strains (Table 3; Figure 3): growth inhibition was only observed in S. pneumoniae. Since 1-methyluric acid formed a precipitate in concentrations above 312 μg /ml, no MIC values could be calculated. This lack of growth inhibition in Gram-negative strains may possibly be due to their double layered cell walls which are less penetrable [55], or the bacteria have expanded their resistance mechanisms to evade killing by antimicrobials [55, 56]. It is also possible that the two Gram-negative species have alternative mechanisms for methionine biosynthesis, further complicating screening for effective drugs.Figure 3

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