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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) Results from thephosphatase siRNA screen. The effect of silencing the different phosphatases(for details see Supplementary Table 1)on intracellular S. typhimurium growth is quantifiedby flow cytometry, and the results are expressed as a Z-score. The gray lines depict the variation in the triplicate datapoints. (B) The 13 phosphatases that most strongly reduced or inducedintracellular growth of S. typhimurium are shownwith standard deviation of triplicate measurements.
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fig2: (A) Results from thephosphatase siRNA screen. The effect of silencing the different phosphatases(for details see Supplementary Table 1)on intracellular S. typhimurium growth is quantifiedby flow cytometry, and the results are expressed as a Z-score. The gray lines depict the variation in the triplicate datapoints. (B) The 13 phosphatases that most strongly reduced or inducedintracellular growth of S. typhimurium are shownwith standard deviation of triplicate measurements.

Mentions: We aimed to identify phosphatases controllingintracellular bacterial infections since we already defined the opposingclass of enzymes, kinases.6 Around 190phosphatase and phosphatase-like genes encoded in the human genomewere silenced with siRNAs. (Supplementary TableS1). After transfection with siRNA, the cells were grown forthree days before infection with fluorescent DsRed-expressing Salmonella typhimurium17 andcultured for another 18 h before the cells were analyzed for intracellular(fluorescent Ds-Red expressing) bacteria by flow cytometry. This experimentvisualized the involvement of host phosphatases in the control ofintracellular growth of S. typhimurium. We identifiedphosphatases that when silenced accelerated or inhibited intracellulargrowth of S. typhimurium (Figure 2A,B). The phosphatases accelerating intracellular growth includefive subunits of PPP2, a phosphatase complex that controls the activityof kinase Akt, which we had shown in a previous study limits intracellulargrowth of Salmonella and other intracellular bacteria.6−9 This illustrates the complementarities of phosphatase-kinase reactionsin one biological process and supports our approach to control bacterialinfection by targeting host proteins. Various phosphatases that stronglyinhibit intracellular infection when silenced were members of thedual specificity phosphatase (DUSP) family.


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) Results from thephosphatase siRNA screen. The effect of silencing the different phosphatases(for details see Supplementary Table 1)on intracellular S. typhimurium growth is quantifiedby flow cytometry, and the results are expressed as a Z-score. The gray lines depict the variation in the triplicate datapoints. (B) The 13 phosphatases that most strongly reduced or inducedintracellular growth of S. typhimurium are shownwith standard deviation of triplicate measurements.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: (A) Results from thephosphatase siRNA screen. The effect of silencing the different phosphatases(for details see Supplementary Table 1)on intracellular S. typhimurium growth is quantifiedby flow cytometry, and the results are expressed as a Z-score. The gray lines depict the variation in the triplicate datapoints. (B) The 13 phosphatases that most strongly reduced or inducedintracellular growth of S. typhimurium are shownwith standard deviation of triplicate measurements.
Mentions: We aimed to identify phosphatases controllingintracellular bacterial infections since we already defined the opposingclass of enzymes, kinases.6 Around 190phosphatase and phosphatase-like genes encoded in the human genomewere silenced with siRNAs. (Supplementary TableS1). After transfection with siRNA, the cells were grown forthree days before infection with fluorescent DsRed-expressing Salmonella typhimurium17 andcultured for another 18 h before the cells were analyzed for intracellular(fluorescent Ds-Red expressing) bacteria by flow cytometry. This experimentvisualized the involvement of host phosphatases in the control ofintracellular growth of S. typhimurium. We identifiedphosphatases that when silenced accelerated or inhibited intracellulargrowth of S. typhimurium (Figure 2A,B). The phosphatases accelerating intracellular growth includefive subunits of PPP2, a phosphatase complex that controls the activityof kinase Akt, which we had shown in a previous study limits intracellulargrowth of Salmonella and other intracellular bacteria.6−9 This illustrates the complementarities of phosphatase-kinase reactionsin one biological process and supports our approach to control bacterialinfection by targeting host proteins. Various phosphatases that stronglyinhibit intracellular infection when silenced were members of thedual specificity phosphatase (DUSP) family.

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