Genetic and chemical knockdown: a complementary strategy for evaluating an anti-infective target

Figures & data

Figure 1 Network diagram of metabolic pathway of shikimic acid production in Escherichia coli.

Notes: Key enzymes and genes of the pathway: 2-dehydro-3-deoxyphosphoheptonate aldolase (aroH), 3-dehydroquinate synthase (aroB), 3-dehydroquinate dehydratase (aroD), shikimate dehydrogenase (aroE), shikimate kinase I (aroK), 3-phosphoshikimate-1-carboxyvinyltransferase (aroA), chorismate synthase (aroC).
Abbreviations: PEP, phosphoenol pyruvate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehyroshikimate; SKM, shikimate; S3P, shikimate-3-phosphate; EPSP, 5-enolpyruvylshikimate-3-phosphate; CHA, chorismate.

Table 1 Classification of genes as bacteriostatic (growth arrest) and bactericidal (growth arrest and increase in redox ratio)

Bactericidal	Bacteristatic
Essential genes
aroA, aroB, aroC, aroD, folA, folD, folE, folK, glnA, glyA, gmk, guaA, guaB, nadE, prs, purA, purB, purC, purD, purF, purH, purK, purL, purM, pyrF, pyrG	cdsA, dut, folC, gpsA, kdsA, kdsB, rlpB, lpxA, lpxB, lpxC, lpxH, lpxK, msbA, murA, murB, murC, murD, murE, murF, murG, murI, nadA, nadD, pgsA, plsB, plsC, psd, pssA, pyrC, pyrD, pyrE, pyrH, thyA, tmk, waaA, waaB, waaC, waaF, waaG, waaO, waaP, waaQ, waaR, waaU, waaY

Figure 2 Effect of different concentrations (250 μM, 500 μM, 1000 μM) of drug (uncompetitive inhibitor) against aroA on biomass in glucose media was estimated through in silico simulation.

Notes: Up to 250 μM of the inhibitor, the biomass levels were found to be similar to that of untreated. At 500 μM and beyond, the biomass levels indicated growth arrest. All the inhibitors that were used in simulation had the same molecular weight as glyphosate (169 Da).

Figure 3 Effect of different concentrations (250 μM, 500 μM, 1000 μM) of drug (uncompetitive inhibitor) against aroA on redox ratio (nicotinamide adenine dinucleotide [NAD]/reduced NAD [NADH]) in glucose media.

Notes: At 500 μM and 1000 μM, the NAD/NADH ratio showed a sharp increase, indicating cidality. The mode of action and molecular weight is equivalent to that of glyphosate.

Table 2 Graded knockdown data of shikimate pathway genes in glucose media and impact on growth

Gene	Knockdown
	70%	90%	99%	100%
aroA	+	+	+	−
aroB	+	+	+	−
aroC	+	+	−	−
aroD	+	+	+	−
aroE	+	+	+	−
aroF	+	+	+	+
aroG	+	+	+	+
aroH	+	+	+	+
aroK	+	+	+	+
aroL	+	+	+	+
ydiB	+	+	+	+

Notes:

+, growth;

−, growth arrest.

Figure 4 Effect of various inhibitors (uncompetitive, noncompetitive, competitive) on growth rate of Escherichia coli; uncompetitive inhibitor arrest growth at lower concentrations followed by noncompetitive and competitive inhibitors.

Note: All the inhibitors that were used in simulation had the same molecular weight as glyphosate (169 Da).

Table 3 Targets from shikimate pathway: relationship between MIC, flux, and the type of inhibition

Gene	Concentration for MIC
	Competitive		Noncompetitive		Uncompetitive
	Drug concentration (μM)	Flux (μmol/second)	Drug concentration (μM)	Flux (μmol/second)	Drug concentration (μM)	Flux (μmol/second)
Control	0	0.147	0	0.147	0	0.147
aroA	80,000	0.0002	30,000	0.0019	500	5E-04
aroB	120,000	0.0539	5000	0.0024	1000	0.012
aroC	80,000	0.0598	5000	0.0008	250	0.015
aroD	400,000	0.0522	5000	0.0043	3000	0.007

Note: The concentrations that brought in growth arrest (MIC) were different amongst the various targets, inhibitor type, and flux through the pathway.

Abbreviation: MIC, minimum inhibitory concentration.

Figure 5 Determination of Km of shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) for Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Kinetic parameters of Mtu EPSPS determined at a concentration of one substrate and variable concentrations of the other. (A) Apparent Km for PEP was determined to be 104 μM (○–○); (B) apparent Km for S3P was determined to be 16 μM (•–•).

Note: Data points indicate mean reaction rates with standard deviation from a single experiment with each of the sample run as triplicates.

Figure 6 (A and B) IC₅₀ determination. (A) Inhibition of Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) by glyphosate. M. tuberculosis EPSPS was assayed in the presence of increasing concentrations of glyphosate. Data points (○–○) indicate mean of triplicate measurements. Analysis was done using GraphPad Prism software. The IC₅₀ of glyphosate for the Mtu EPSPS was found to be 260 μM. (B) Inhibition of Escherichia coli EPSPS by glyphosate. E. coli EPSPS was assayed in the presence of increasing concentration of glyphosate. Data points (•–•) indicate mean of triplicate measurements. Analysis was done using GraphPad Prism software. The IC₅₀ of glyphosate for E. coli EPSPS was found to be 3 μM.

Table 4 MIC of glyphosate in Escherichia coli K-12

Sample no.	Compound name	Medium used	MIC (μg/mL)
1	Glyphosate	M9 with sorbitol	300
2	Glyphosate	M9 with glucose	600
3	Glyphosate	M9 with sorbitol with (20 μg/mL) aromatic amino acids	>2500
4	Glyphosate	M9 with glucose with (20 μg/mL) aromatic amino acids	2500
5	Tetracycline	M9 with sorbitol	32
6	Tetracycline	M9 with glucose	16
7	Tetracycline	M9 with sorbitol with (20 μg/mL) aromatic amino acids	32
8	Tetracycline	M9 with glucose with (20 μg/mL) aromatic amino acids	16
9	Novobiocin	M9 with sorbitol	1
10	Novobiocin	M9 with glucose	1
11	Novobiocin	M9 with sorbitol with (20 μg/mL) aromatic amino acids	1
12	Novobiocin	M9 with glucose with (20 μg/mL) aromatic amino acids	1

Notes: MIC of glyphosate for Escherichia coli K-12 estimated under different growth conditions. MIC was determined using broth dilution method. The growth medium was varied with respect to the carbon source and the supplements.

Abbreviation: MIC, minimum inhibitory concentration.

Table 5 MBC of glyphosate for Escherichia coli K-12 estimated under different growth conditions

Sample no.	Compound name	Medium used	Start concentration (μg/mL)	MIC (μg/mL)	MBC (μg/mL)	MBC/MIC ratio
1	Glyphosate	M9 with sorbitol	2500	300	>2500	>8.3
2	Glyphosate	M9 with glucose	2500	600	600	1

Notes: The growth medium was varied with respect to the carbon source and the supplements. Colony-forming units were enumerated after samples were plated on M9 agar, and the lowest concentration that resulted in >99.9% reduction in counts was considered as MBC.

Abbreviations: MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration.

Table 6 Relationship between inhibition type and kinetic parameters

Inhibition	Vmax	Km
KD/noncompetitive	Lower	Unchanged
Competitive	Unchanged	Increases
Uncompetitive	Lower	Lower

Abbreviations: V_max, maximum reaction rate; Km, substrate concentration at half V_max; KD, knockdown.

Table S1 Comparison of predicted flux distribution trends for pgi gene knockout mutant with reported experimental trends obtained using glucose as the sole carbon source

Reactions	Trend of reaction as per literature	Trend of reaction as per in silico system
Glucose 6-phosphate to fructose 6 phosphate (catalyzed by PGI enzyme)	Decreases	Decreases
Glucose 6-phosphate to glyceraldehyde 3-phosphate (via pentose phosphate pathway)	Increases	Increases
Glyceraldehyde 3-phosphate to PEP (in glycolysis)	Decreases	Decreases
Phospho 6-gluconate to pyruvate (ED pathway)	Increases	Increases
Malate to pyruvate (catalyzed by maeB enzyme)	Decreases	Decreases
Oxaloacetate to phosphoenol pyruvate (catalyzed by pck enzyme)	Increases	Increases

Table S2 Comparison of experimental and in silico result in case of ppc knockout mutant

Reaction	Trend of reaction as per literature	Trend of reaction as per in silico system
Pyruvate to acetyl coenzyme A	Increases	Increases
Malate to oxaloacetate	Increases	Increases
Oxaloacetate to phosphoenol pyruvate^***	Decreases	Decreases
Malate to pyruvate	Increases	Increases

Note:

*** Data taken from Fong et al.²⁹

Table S3 Growth rate in wild-type and pgi–udhA double knockout³⁰

Strain	Growth rate
WT	0.67 per hour
pgi–udhA	0

Abbreviations: WT, wild-type strain of E. coli; pgi–udhA, pgi–udhA double mutant.

Figure S1 Effect of double knockout (pgi–udhA) on biomass levels and growth rates.

Notes: From the plot, growth rates (slope of the lines) of wild-type E. coli strain and pgi–udhA double-mutant strain can be compared. Simulation results show steady-state attainment of biomass in pgi–udhA double-mutant strain, which is indicative of growth arrest.

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