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Model-driven discovery of synergistic inhibitors against E. coli and S. enterica serovar Typhimurium targeting a novel synthetic lethal pair, aldA and prpC.

Aziz RK, Khaw VL, Monk JM, Brunk E, Lewis R, Loh SI, Mishra A, Nagle AA, Satyanarayana C, Dhakshinamoorthy S, Luche M, Kitchen DB, Andrews KA, Palsson BØ, Charusanti P - Front Microbiol (2015)

Bottom Line: Here, we reconcile this disparity by providing evidence that aldA and prpC form a synthetic lethal pair, as the double knockout could only be created through complementation with a plasmid-borne copy of aldA.Moreover, virtual and biological screening against the two proteins led to a set of compounds that inhibited the growth of E. coli and Salmonella enterica serovar Typhimurium synergistically at 100-200 μM individual concentrations.These results highlight the power of metabolic models to drive basic biological discovery and their potential use to discover new combination antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University Cairo, Egypt ; Department of Bioengineering, University of California, San Diego La Jolla, CA, USA.

ABSTRACT
Mathematical models of biochemical networks form a cornerstone of bacterial systems biology. Inconsistencies between simulation output and experimental data point to gaps in knowledge about the fundamental biology of the organism. One such inconsistency centers on the gene aldA in Escherichia coli: it is essential in a computational model of E. coli metabolism, but experimentally it is not. Here, we reconcile this disparity by providing evidence that aldA and prpC form a synthetic lethal pair, as the double knockout could only be created through complementation with a plasmid-borne copy of aldA. Moreover, virtual and biological screening against the two proteins led to a set of compounds that inhibited the growth of E. coli and Salmonella enterica serovar Typhimurium synergistically at 100-200 μM individual concentrations. These results highlight the power of metabolic models to drive basic biological discovery and their potential use to discover new combination antibiotics.

No MeSH data available.


Related in: MedlinePlus

Confirmation of the ΔprpC ΔaldA deletions at their annotated chromosomal positions, and the continued presence of a copy of aldA at an undefined location. Colonies were isolated in which the chromosomal copies of prpC and aldA had been successfully deleted (lanes 3 and 5 versus lanes 2 and 4, respectively), but PCR amplification using primers that targeted a region wholly within aldA revealed that a copy of this gene was still present in the double mutant (lane 6). The identity of this amplicon was also confirmed by Sanger sequencing. Abbrevations: WT, wild-type; DKO, ΔprpC ΔaldA double knockout; ext, external; int, internal. Numbers on the far left indicate DNA band sizes in kb.
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Figure 2: Confirmation of the ΔprpC ΔaldA deletions at their annotated chromosomal positions, and the continued presence of a copy of aldA at an undefined location. Colonies were isolated in which the chromosomal copies of prpC and aldA had been successfully deleted (lanes 3 and 5 versus lanes 2 and 4, respectively), but PCR amplification using primers that targeted a region wholly within aldA revealed that a copy of this gene was still present in the double mutant (lane 6). The identity of this amplicon was also confirmed by Sanger sequencing. Abbrevations: WT, wild-type; DKO, ΔprpC ΔaldA double knockout; ext, external; int, internal. Numbers on the far left indicate DNA band sizes in kb.

Mentions: Based on this outcome, we hypothesized that aldA and prpC might form a SL gene pair in both LB and glucose M9. To further investigate this possibility, we cloned aldA into the pASK1988 overexpression vector (Fong et al., 2013), transformed it into a ΔprpC mutant, and re-attempted to create the double knockout. With this complementation, we could successfully delete the chromosomal copy of aldA. We next removed the kanamycin selection marker and attempted to cure the overexpression plasmid from the double mutant. All colonies regained sensitivity to the selection marker present on the complementation plasmid (chloramphenicol resistance), but the presence of aldA could still be detected within the double mutant (Figure 2 and Supplementary Figure S2). These data support the hypothesis that aldA and prpC are synthetically lethal.


Model-driven discovery of synergistic inhibitors against E. coli and S. enterica serovar Typhimurium targeting a novel synthetic lethal pair, aldA and prpC.

Aziz RK, Khaw VL, Monk JM, Brunk E, Lewis R, Loh SI, Mishra A, Nagle AA, Satyanarayana C, Dhakshinamoorthy S, Luche M, Kitchen DB, Andrews KA, Palsson BØ, Charusanti P - Front Microbiol (2015)

Confirmation of the ΔprpC ΔaldA deletions at their annotated chromosomal positions, and the continued presence of a copy of aldA at an undefined location. Colonies were isolated in which the chromosomal copies of prpC and aldA had been successfully deleted (lanes 3 and 5 versus lanes 2 and 4, respectively), but PCR amplification using primers that targeted a region wholly within aldA revealed that a copy of this gene was still present in the double mutant (lane 6). The identity of this amplicon was also confirmed by Sanger sequencing. Abbrevations: WT, wild-type; DKO, ΔprpC ΔaldA double knockout; ext, external; int, internal. Numbers on the far left indicate DNA band sizes in kb.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585216&req=5

Figure 2: Confirmation of the ΔprpC ΔaldA deletions at their annotated chromosomal positions, and the continued presence of a copy of aldA at an undefined location. Colonies were isolated in which the chromosomal copies of prpC and aldA had been successfully deleted (lanes 3 and 5 versus lanes 2 and 4, respectively), but PCR amplification using primers that targeted a region wholly within aldA revealed that a copy of this gene was still present in the double mutant (lane 6). The identity of this amplicon was also confirmed by Sanger sequencing. Abbrevations: WT, wild-type; DKO, ΔprpC ΔaldA double knockout; ext, external; int, internal. Numbers on the far left indicate DNA band sizes in kb.
Mentions: Based on this outcome, we hypothesized that aldA and prpC might form a SL gene pair in both LB and glucose M9. To further investigate this possibility, we cloned aldA into the pASK1988 overexpression vector (Fong et al., 2013), transformed it into a ΔprpC mutant, and re-attempted to create the double knockout. With this complementation, we could successfully delete the chromosomal copy of aldA. We next removed the kanamycin selection marker and attempted to cure the overexpression plasmid from the double mutant. All colonies regained sensitivity to the selection marker present on the complementation plasmid (chloramphenicol resistance), but the presence of aldA could still be detected within the double mutant (Figure 2 and Supplementary Figure S2). These data support the hypothesis that aldA and prpC are synthetically lethal.

Bottom Line: Here, we reconcile this disparity by providing evidence that aldA and prpC form a synthetic lethal pair, as the double knockout could only be created through complementation with a plasmid-borne copy of aldA.Moreover, virtual and biological screening against the two proteins led to a set of compounds that inhibited the growth of E. coli and Salmonella enterica serovar Typhimurium synergistically at 100-200 μM individual concentrations.These results highlight the power of metabolic models to drive basic biological discovery and their potential use to discover new combination antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University Cairo, Egypt ; Department of Bioengineering, University of California, San Diego La Jolla, CA, USA.

ABSTRACT
Mathematical models of biochemical networks form a cornerstone of bacterial systems biology. Inconsistencies between simulation output and experimental data point to gaps in knowledge about the fundamental biology of the organism. One such inconsistency centers on the gene aldA in Escherichia coli: it is essential in a computational model of E. coli metabolism, but experimentally it is not. Here, we reconcile this disparity by providing evidence that aldA and prpC form a synthetic lethal pair, as the double knockout could only be created through complementation with a plasmid-borne copy of aldA. Moreover, virtual and biological screening against the two proteins led to a set of compounds that inhibited the growth of E. coli and Salmonella enterica serovar Typhimurium synergistically at 100-200 μM individual concentrations. These results highlight the power of metabolic models to drive basic biological discovery and their potential use to discover new combination antibiotics.

No MeSH data available.


Related in: MedlinePlus