Disulfiram Enhances the Activity of Polymyxin B Against Klebsiella pneumoniae by Inhibiting Lipid A Modification

Figures & data

Table 1 Potency of Molecules in Dehydrogenase Inhibitor Libraries Alone or in Combination with Polymyxin B Against K. pneumoniae ATCC13883

Structure	Function	MIC (μM)	ƩFIC
	Noncompetitive, selective and reversible inosine monophosphate dehydrogenase I/II	>100	≤0.75
	Phosphoglycerate dehydrogenase (PHGDH) inhibitor	>100	≤0.38
	Aldehyde dehydrogenase (ALDH1) inhibitor used to treat chronic alcoholism	>100	≤0.25
	Highly selective, effective mIDH1 inhibitor with oral bioavailability	>100	≤0.38
	Dihydrodehydrogenase inhibitor that inhibits early homologous recombination in double-strand break repair	>100	≤0.75
	Unique product from licorice root with very high sweetness	>100	≤1
	Inhibitor of tetrahydrofolate dehydrogenase and antitumor antimetabolites with immunosuppressive activity	>100	≤0.75
	Potent and selective IDH2 R140Q mutant inhibitor	>100	≤0.75
	Selective inhibitor of 15-hydroxyprostaglandin dehydrogenase (15-PGDH)	>100	≤0.28
	New, potent and selective reversible inhibitor of IDH2 mutant enzymes	>100	≤0.75
	Human dihydroorotate dehydrogenase (DHODH) inhibitor.	>100	≤0.50

Notes: ∑FIC, sum of the fractional inhibitory concentration; ∑FIC ≤ 0.5 indicates synergism; 4 ≥ ∑FIC > 0.5 indicates indifference; and ƩFIC > 4 indicates antagonism.

Abbreviations: MIC, minimal inhibitory concentration; ƩFIC, fractional inhibitory concentration index.

Table 2 Potency of DSF Alone or in Combination with Polymyxin B Against Different Species

	Species	Strains	MIC (μM)	ƩFIC
DSF	Klebsiella pneumoniae	P2445-4	>100	≤0.5
	Klebsiella pneumoniae	AH1313-1	>100	≤0.5
	Klebsiella pneumoniae	P2418-1	>100	≤0.38
	Klebsiella pneumoniae	P1291-19	>100	≤0.25
DTC	Klebsiella pneumoniae	ATCC13883	>100	>1

Notes: ∑FIC, sum of the fractional inhibitory concentration; ∑FIC ≤ 0.5 indicates synergism; 4 ≥ ∑FIC > 0.5 indicates indifference; and ƩFIC > 4 indicates antagonism.

Abbreviations: MIC, minimal inhibitory concentration; ƩFIC, fractional inhibitory concentration index; DSF, disulfiram; DTC, diethyldithiocarbamate.

Figure 1 Inhibitory effect of DSF on the transcription level of genes in the arn operon and its inhibition of lipid A modification. (A) Effect of DSF on the UDP-GlcA-specific dehydrogenase activity of the recombinant ArnA protein. (B) Transcription levels of genes in the arn operon after treatment with PB alone or with the combination of PB and DSF. (C) Protein- and cell-based effects of DSF on the UDP-GlcA-specific dehydrogenase activity of total protein. A schematic of the sample addition operation is shown. DSF was added to the bacterial culture (cell-based) or total protein sample (protein-based). (D) The percent of the L-Ara4N-lipid A conjugate was determined based on LC–MS/MS analysis. The reported structures of K. pneumoniae lipid A isolated from the strains included in this study are shown in Table S2. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The means are shown, and the error bars represent the means ± standard errors of the mean (SEMs). Three biologically independent experiments were performed.

Abbreviation: ns, no significance.

Figure 2 Effect of DSF on membrane integrity. (A) The integrity of the K. pneumoniae ATCC13883 membrane was not affected by treatment with DSF alone (0–237.6 μg/mL). DSF enhanced the disruption of the membrane integrity induced by PB. The membrane was probed with 10 nmol/L PI. ***P < 0.001. The means are shown, and the error bars represent the SEMs. The experiments were performed in triplicate. (B) Confocal images of K. pneumoniae ATCC13883 after treatment with PB (2 μg/mL) and/or DSF (100 μg/mL). The viable bacterial cells were stained green by SYTO9, and the dead cells were stained red by PI.

Abbreviations: a.u., arbitrary unit; ns, no significance.

Figure 3 DSF enhances the activity of PB in a mouse infection model. The survival rates of mice with pulmonary infection (n = 10) are shown. Specifically, the survival rates of BALB/c mice 7 d after a single dose of 1.0×10⁷ colony-forming units (CFUs) of PB-resistant K. pneumoniae (P2418-1, MIC = 16 μg/mL) were determined. The survival rates increased after treatment with PB alone (0.2 mg/kg) or in combination with DSF (1, 5 and 10 mg/kg). *P < 0.05, **P < 0.01.