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Integrating chemical and genetic silencing strategies to identify host kinase-phosphatase inhibitor networks that control bacterial infection.

Albers HM, Kuijl C, Bakker J, Hendrickx L, Wekker S, Farhou N, Liu N, Blasco-Moreno B, Scanu T, den Hertog J, Celie P, Ovaa H, Neefjes J - ACS Chem. Biol. (2013)

Bottom Line: Every year three million people die as a result of bacterial infections, and this number may further increase due to resistance to current antibiotics.We define host phosphatases inhibiting intracellular growth of Salmonella and identify corresponding inhibitors for the dual specificity phosphatases DUSP11 and 27.Inhibiting two enzyme classes with opposite activities-kinases and phosphatases-may be a new strategy to overcome infections by antibiotic-resistant bacteria.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, ‡Netherlands Proteomics Centre, and §Division of Biochemistry, The Netherlands Cancer Institute , Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Every year three million people die as a result of bacterial infections, and this number may further increase due to resistance to current antibiotics. These antibiotics target almost all essential bacterial processes, leaving only a few new targets for manipulation. The host proteome has many more potential targets for manipulation in order to control bacterial infection, as exemplified by the observation that inhibiting the host kinase Akt supports the elimination of different intracellular bacteria including Salmonella and M. tuberculosis. If host kinases are involved in the control of bacterial infections, phosphatases could be as well. Here we present an integrated small interference RNA and small molecule screen to identify host phosphatase-inhibitor combinations that control bacterial infection. We define host phosphatases inhibiting intracellular growth of Salmonella and identify corresponding inhibitors for the dual specificity phosphatases DUSP11 and 27. Pathway analysis places many kinases and phosphatases controlling bacterial infection in an integrated pathway centered around Akt. This network controls host cell metabolism, survival, and growth and bacterial survival and reflect a natural host cell response to bacterial infection. Inhibiting two enzyme classes with opposite activities-kinases and phosphatases-may be a new strategy to overcome infections by antibiotic-resistant bacteria.

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(A) Effect of identified S. typhimuriuminfection inhibitors on the activity of DUSP3, 11, and 27. DUSP activity(%) has been measured at an inhibitor concentration of 5 μM.Data are represented as average with standard deviation of triplicatemeasurements. (B) IC50 values (μM) for LH65.3 onDUSP3, 11, and 27 given as average with standard deviation of triplicatemeasurements. (C) Deconvolution of siRNAs for DUSP3, 11, and 27 andtheir effect on intracellular Salmonella growth.Shown is the Z-score for the four siRNAs tested pertarget. A negative Z-score indicates inhibition ofintracellular growth of Salmonella.
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fig4: (A) Effect of identified S. typhimuriuminfection inhibitors on the activity of DUSP3, 11, and 27. DUSP activity(%) has been measured at an inhibitor concentration of 5 μM.Data are represented as average with standard deviation of triplicatemeasurements. (B) IC50 values (μM) for LH65.3 onDUSP3, 11, and 27 given as average with standard deviation of triplicatemeasurements. (C) Deconvolution of siRNAs for DUSP3, 11, and 27 andtheir effect on intracellular Salmonella growth.Shown is the Z-score for the four siRNAs tested pertarget. A negative Z-score indicates inhibition ofintracellular growth of Salmonella.

Mentions: Only a limited number of phosphatases have been defined in the siRNAscreen that upon silencing showed a similar affect as the small moleculeinhibitors. We aimed at connecting the inhibitors with their respectivetargets, which can only be done in in vitro assayswith purified phosphatases. We assayed phosphatase activity using3-O-methylfluorescein phosphate (OMFP).18 The quenched OMFP substrate is hydrolyzed intofluorescent 3-O-methylfluorescein (OMF) and phosphate.We expressed and purified three DUSP family members identified inthe siRNA screen as inhibiting intracellular bacterial growth whensilenced: DUSP3, 11, and 27. DUSP3 (also called VHR) is reported tocontrol ERK1 (p44MAPK3) and 2 (p38MAPK1) kinases;19 DUSP11 targets RNA (although active on tyrosine phosphate-likesubstrates) and is involved in cell growth control;20 and DUSP27 may be a poorly defined phosphatase with a potentialrole in metabolism (that could include the mTOR pathway controlledby Akt).21 We tested whether the inhibitorLH65.3 affected ERK activity in response to Salmonella infection. No effect of the compound was observed (Supplemental Figure 3). This result urged us to control thedefined DUSPs again. These were identified with pools of four differentsiRNAs/target. Off target effects are highly unlikely when more than2 different siRNAs/target show inhibition of intracellular bacterialgrowth. We have therefore deconvoluted the pool and tested the foursiRNAs independently for their effect on Salmonella infection (Figure 4C). MCF7 cells were firsttransfected with the different siRNAs and infected 3 days later with Salmonella, and the rate of intracellular growth of Salmonella was determined 24 h later. The results are shownas a Z-score. Silencing of DUSP11 and DUSP27 with3 or 4 different siRNAs/target confirmed inhibition of intracellulargrowth of Salmonella, whereas only one siRNA couldbe confirmed for DUSP3. This suggests that DUSP11 and DUSP27, unlikeDUSP3, are involved in the control of intracellular infection by Salmonella.


Integrating chemical and genetic silencing strategies to identify host kinase-phosphatase inhibitor networks that control bacterial infection.

Albers HM, Kuijl C, Bakker J, Hendrickx L, Wekker S, Farhou N, Liu N, Blasco-Moreno B, Scanu T, den Hertog J, Celie P, Ovaa H, Neefjes J - ACS Chem. Biol. (2013)

(A) Effect of identified S. typhimuriuminfection inhibitors on the activity of DUSP3, 11, and 27. DUSP activity(%) has been measured at an inhibitor concentration of 5 μM.Data are represented as average with standard deviation of triplicatemeasurements. (B) IC50 values (μM) for LH65.3 onDUSP3, 11, and 27 given as average with standard deviation of triplicatemeasurements. (C) Deconvolution of siRNAs for DUSP3, 11, and 27 andtheir effect on intracellular Salmonella growth.Shown is the Z-score for the four siRNAs tested pertarget. A negative Z-score indicates inhibition ofintracellular growth of Salmonella.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3934374&req=5

fig4: (A) Effect of identified S. typhimuriuminfection inhibitors on the activity of DUSP3, 11, and 27. DUSP activity(%) has been measured at an inhibitor concentration of 5 μM.Data are represented as average with standard deviation of triplicatemeasurements. (B) IC50 values (μM) for LH65.3 onDUSP3, 11, and 27 given as average with standard deviation of triplicatemeasurements. (C) Deconvolution of siRNAs for DUSP3, 11, and 27 andtheir effect on intracellular Salmonella growth.Shown is the Z-score for the four siRNAs tested pertarget. A negative Z-score indicates inhibition ofintracellular growth of Salmonella.
Mentions: Only a limited number of phosphatases have been defined in the siRNAscreen that upon silencing showed a similar affect as the small moleculeinhibitors. We aimed at connecting the inhibitors with their respectivetargets, which can only be done in in vitro assayswith purified phosphatases. We assayed phosphatase activity using3-O-methylfluorescein phosphate (OMFP).18 The quenched OMFP substrate is hydrolyzed intofluorescent 3-O-methylfluorescein (OMF) and phosphate.We expressed and purified three DUSP family members identified inthe siRNA screen as inhibiting intracellular bacterial growth whensilenced: DUSP3, 11, and 27. DUSP3 (also called VHR) is reported tocontrol ERK1 (p44MAPK3) and 2 (p38MAPK1) kinases;19 DUSP11 targets RNA (although active on tyrosine phosphate-likesubstrates) and is involved in cell growth control;20 and DUSP27 may be a poorly defined phosphatase with a potentialrole in metabolism (that could include the mTOR pathway controlledby Akt).21 We tested whether the inhibitorLH65.3 affected ERK activity in response to Salmonella infection. No effect of the compound was observed (Supplemental Figure 3). This result urged us to control thedefined DUSPs again. These were identified with pools of four differentsiRNAs/target. Off target effects are highly unlikely when more than2 different siRNAs/target show inhibition of intracellular bacterialgrowth. We have therefore deconvoluted the pool and tested the foursiRNAs independently for their effect on Salmonella infection (Figure 4C). MCF7 cells were firsttransfected with the different siRNAs and infected 3 days later with Salmonella, and the rate of intracellular growth of Salmonella was determined 24 h later. The results are shownas a Z-score. Silencing of DUSP11 and DUSP27 with3 or 4 different siRNAs/target confirmed inhibition of intracellulargrowth of Salmonella, whereas only one siRNA couldbe confirmed for DUSP3. This suggests that DUSP11 and DUSP27, unlikeDUSP3, are involved in the control of intracellular infection by Salmonella.

Bottom Line: Every year three million people die as a result of bacterial infections, and this number may further increase due to resistance to current antibiotics.We define host phosphatases inhibiting intracellular growth of Salmonella and identify corresponding inhibitors for the dual specificity phosphatases DUSP11 and 27.Inhibiting two enzyme classes with opposite activities-kinases and phosphatases-may be a new strategy to overcome infections by antibiotic-resistant bacteria.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, ‡Netherlands Proteomics Centre, and §Division of Biochemistry, The Netherlands Cancer Institute , Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Every year three million people die as a result of bacterial infections, and this number may further increase due to resistance to current antibiotics. These antibiotics target almost all essential bacterial processes, leaving only a few new targets for manipulation. The host proteome has many more potential targets for manipulation in order to control bacterial infection, as exemplified by the observation that inhibiting the host kinase Akt supports the elimination of different intracellular bacteria including Salmonella and M. tuberculosis. If host kinases are involved in the control of bacterial infections, phosphatases could be as well. Here we present an integrated small interference RNA and small molecule screen to identify host phosphatase-inhibitor combinations that control bacterial infection. We define host phosphatases inhibiting intracellular growth of Salmonella and identify corresponding inhibitors for the dual specificity phosphatases DUSP11 and 27. Pathway analysis places many kinases and phosphatases controlling bacterial infection in an integrated pathway centered around Akt. This network controls host cell metabolism, survival, and growth and bacterial survival and reflect a natural host cell response to bacterial infection. Inhibiting two enzyme classes with opposite activities-kinases and phosphatases-may be a new strategy to overcome infections by antibiotic-resistant bacteria.

Show MeSH
Related in: MedlinePlus