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.2017 Sep 5;8(5):e01224-17.
doi: 10.1128/mBio.01224-17.

Dehydrosqualene Desaturase as a Novel Target for Anti-Virulence Therapy againstStaphylococcus aureus

Affiliations

Dehydrosqualene Desaturase as a Novel Target for Anti-Virulence Therapy againstStaphylococcus aureus

Peng Gao et al. mBio..

Abstract

Staphylococcus aureus, especially methicillin-resistantS. aureus (MRSA), is a life-threatening pathogen in hospital- and community-acquired infections. The golden-colored carotenoid pigment ofS. aureus, staphyloxanthin, contributes to the resistance to reactive oxygen species (ROS) and host neutrophil-based killing. Here, we describe a novel inhibitor (NP16) ofS. aureus pigment production that reduces the survival ofS. aureus under oxidative stress conditions. Carotenoid components analysis, enzyme inhibition, andcrtN mutational studies indicated that the molecular target of NP16 is dehydrosqualene desaturase (CrtN).S. aureus treated with NP16 showed increased susceptibility to human neutrophil killing and to innate immune clearance in a mouse infection model. Our study validates CrtN as a novel druggable target inS. aureus and presents a potent and effective lead compound for the development of virulence factor-based therapy againstS. aureusIMPORTANCES. aureus staphyloxanthin contributes substantially to pathogenesis by interfering with host immune clearance mechanisms, but it has little impact onex vivo survival of the bacterium. Agents blocking staphyloxanthin production may discourage the establishment and maintenance of bacterial infection without exerting selective pressure for antimicrobial resistance. Our newly discovered CrtN inhibitor, NP16, may offer an effective strategy for combatingS. aureus infections.

Keywords: MRSA; anti-virulence; bacterial infection; staphyloxanthin.

Copyright © 2017 Gao et al.

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Figures

FIG 1
FIG 1
Biosynthesis pathway of staphyloxanthin. First, in staphyloxanthin biosynthesis, two molecules of farnesyl diphosphate are condensed head-to-head to form dehydrosqualene (4,4′-diapophytoene); this reaction is catalyzed by the dehydrosqualene synthase CrtM. Second, dehydrosqualene is dehydrogenated by the dehydrosqualene desaturase CrtN to form the yellow intermediate 4,4′-diaponeurosporene. Third, oxidation of the terminal methyl group of 4,4′-diaponeurosporene is catalyzed by a mixed-function oxidase, CrtP, to form 4,4′-diaponeurosporenic acid. Then, glycosyl 4,4′-diaponeurosporenoate is formed by esterification of glucose at the C-1″ position of 4,4′-diaponeurosporenic acid via CrtQ, a glycosyltransferase. Finally, glucose at the C-6″ position is esterified with the carboxyl group of 12-methyltetradecanoic acid by the acyltransferase CrtO to yield staphyloxanthin (2).
FIG 2
FIG 2
In vitro pigment inhibition by compound NP16. (A) Inhibition of wild-type (WT)S. aureus pigmentation in the presence of increasing concentrations of NP16. (B) Pigment inhibition by NP16; the IC50 for pigment formation is ∼300 nM. (C) The chemical structure of compound NP16. (D) Growth curve ofS. aureus COL in the presence of different concentrations of NP16. All data represent mean values ± SD.
FIG 3
FIG 3
Effects of compound NP16 on CrtM and CrtN enzyme activity. (A) The inhibition of CrtM with NP16 and inhibitor BPH-652; the reaction mixture or the reaction mixture without enzyme showed activity ratios of 1 and 0, respectively. (B) An assay of CrtN enzyme activity was conducted with different concentrations of NP16, which were monitored by LC-MS. All data represent mean values ± SD.
FIG 4
FIG 4
4,4′-Diapophytoene accumulation after NP16 treatment. (A) Comparison of carotenoid components among strain COL, COL-ΔcrtN, COL-ΔcrtN-pOS1hrtAB-crtN, or COL-ΔcrtN-pOS1hrtAB-crtN plus 10 μM hemin (inducer), or COL treated with NP16. Plasmid pOShrtAB-crtN has a hemin-inducible promoter. (B) Comparison of the pigmentation of the wild-type COL strain,crtN deletion strain, andcrtN complemented strain.
FIG 5
FIG 5
Homologous expression and intracellular inhibition of CrtN by NP16. (A) Homologous expression ofcrtN in the wild-type COL strain, treated with NP16, and with BPH652 combined with different concentrations of hemin to confirm the reduced inhibition ratio whencrtN was overexpressed. (B) LC-MS analysis of the effects of different concentrations of NP16 on thecrtN-overexpressing strain and the parent strain. All data represent mean values ± SD.
FIG 6
FIG 6
NP16 treatment leads to increased sensitivity to oxidation and neutrophil killing. (A) Cytotoxic activity of compound NP16 on different cell lines. (B) UV spectrum of carotenoids extracted from different strains, with or without NP16 treatment. (C) Increased susceptibility of the NP16-treatedS. aureus COL strain to killing by hydrogen peroxide. (D) Increased susceptibility of the NP16-treatedS. aureus COL strain to killing by neutrophils. All data represent mean values ± SD. ns, not significant; *,P < 0.05; **,P < 0.01; ***,P < 0.001; ****,P < 0.0001.P values were determined using GraphPad Prism with an unpaired parametrict test and Welch’s correction.
FIG 7
FIG 7
In vivo effect of CrtN and its inhibition by NP16. (A and B) Bacteria recovered from the spleens and livers of mice infected with the wild-type COL or COL-ΔcrtN strains. (C and D) Bacteria recovered from the spleens and livers of mice infected with the COL strain, with or without compound NP16 treatment. (E) Bacteria recovered from the kidneys of mice infected with clinical isolate strain AE052 or strain AE052-ΔcrtN. (F) Bacteria recovered from the kidneys of mice infected with strain AE052, with or without compound NP16 treatment. All data represent mean values ± the standard errors of the means. *,P < 0.05; **,P < 0.01; ***,P < 0.001.P values were determined using GraphPad Prism with an unpaired parametrict test and Welch’s correction.
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References

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