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Cyclic diguanylate monophosphate directly binds to human siderocalin and inhibits its antibacterial activity.

Li W, Cui T, Hu L, Wang Z, Li Z, He ZG - Nat Commun (2015)

Bottom Line: We demonstrate that c-di-GMP specifically binds to LCN2.In addition, c-di-GMP can compete with bacterial ferric siderophores to bind LCN2.Furthermore, c-di-GMP can significantly reduce LCN2-mediated inhibition on the in vitro growth of Escherichia coli.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.

ABSTRACT
Cyclic diguanylate monophosphate (c-di-GMP) is a well-conserved second messenger in bacteria. During infection, the innate immune system can also sense c-di-GMP; however, whether bacterial pathogens utilize c-di-GMP as a weapon to fight against host defense for survival and possible mechanisms underlying this process remain poorly understood. Siderocalin (LCN2) is a key antibacterial component of the innate immune system and sequesters bacterial siderophores to prevent acquisition of iron. Here we show that c-di-GMP can directly target the human LCN2 protein to inhibit its antibacterial activity. We demonstrate that c-di-GMP specifically binds to LCN2. In addition, c-di-GMP can compete with bacterial ferric siderophores to bind LCN2. Furthermore, c-di-GMP can significantly reduce LCN2-mediated inhibition on the in vitro growth of Escherichia coli. Thus, LCN2 acts as a c-di-GMP receptor. Our findings provide insight into the mechanism by which bacteria utilize c-di-GMP to interfere with the innate immune system for survival.

No MeSH data available.


Related in: MedlinePlus

Assays for LCN2-mediated inhibition of E. coli growth in the presence or absence of c-di-GMP.(a) ITC assays for the interaction between Fe-Ent and rLCN2 protein. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. (b) Assays for changes in E. coli growth mediated by the human rLCN2 protein in the presence or absence of c-di-GMP. c-di-GMP could rescue the growth inhibition by rLCN2, but hardly by rLCN2-W79A. Addition of FeCl3 or Fe-Ent could rescue rLCN2-mediated growth inhibition. The control nucleotide, c-di-AMP or cGAMP, could not rescue rLCN2-mediated growth inhibition. Bacterial counts were determined at two representative time points, 18 h (top panel) and 24 h (bottom panel). Double asterisks (**) represent significant difference between two groups (P≤0.01, two-tailed Student's t-test).
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f4: Assays for LCN2-mediated inhibition of E. coli growth in the presence or absence of c-di-GMP.(a) ITC assays for the interaction between Fe-Ent and rLCN2 protein. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. (b) Assays for changes in E. coli growth mediated by the human rLCN2 protein in the presence or absence of c-di-GMP. c-di-GMP could rescue the growth inhibition by rLCN2, but hardly by rLCN2-W79A. Addition of FeCl3 or Fe-Ent could rescue rLCN2-mediated growth inhibition. The control nucleotide, c-di-AMP or cGAMP, could not rescue rLCN2-mediated growth inhibition. Bacterial counts were determined at two representative time points, 18 h (top panel) and 24 h (bottom panel). Double asterisks (**) represent significant difference between two groups (P≤0.01, two-tailed Student's t-test).

Mentions: Next, we used E. coli, a fast-growing bacterium, as a model to determine whether rLCN2-mediated bacterial growth inhibition is rescued by c-di-GMP. rLCN2-mediated inhibition of growth has been previously described in E. coli78. In a similar ITC assay, we found that the rLCN2 protein could bind well to ferric enterobactin (Fe-Ent; Fig. 4a). The binding affinity of the interaction (Kd) was 0.24±0.03 μM (data shown are mean±s.d. of three biological replicates). The mutant rLCN2-W79A protein without the ability to bind c-di-GMP retained good binding activity for Fe-Ent (Kd 0.25±0.05 μM) (data shown are mean±s.d. of three biological replicates) (Supplementary Fig. 3b), which is comparable to the wild-type protein. Using a c-di-[32P]GMP cross-linking assay (Supplementary Fig. 4), we determined the competitive binding activity of c-di-[32P]GMP to the protein in the presence of Fe-Ent. Interestingly, binding of c-di-[32P]GMP to rLCN2 could be hardly detected in the presence of 100-fold excess Fe-Ent (Supplementary Fig. 4, lane 6). This result indicated that Fe-Ent could easily compete with c-di-[32P]GMP for protein binding.


Cyclic diguanylate monophosphate directly binds to human siderocalin and inhibits its antibacterial activity.

Li W, Cui T, Hu L, Wang Z, Li Z, He ZG - Nat Commun (2015)

Assays for LCN2-mediated inhibition of E. coli growth in the presence or absence of c-di-GMP.(a) ITC assays for the interaction between Fe-Ent and rLCN2 protein. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. (b) Assays for changes in E. coli growth mediated by the human rLCN2 protein in the presence or absence of c-di-GMP. c-di-GMP could rescue the growth inhibition by rLCN2, but hardly by rLCN2-W79A. Addition of FeCl3 or Fe-Ent could rescue rLCN2-mediated growth inhibition. The control nucleotide, c-di-AMP or cGAMP, could not rescue rLCN2-mediated growth inhibition. Bacterial counts were determined at two representative time points, 18 h (top panel) and 24 h (bottom panel). Double asterisks (**) represent significant difference between two groups (P≤0.01, two-tailed Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4595737&req=5

f4: Assays for LCN2-mediated inhibition of E. coli growth in the presence or absence of c-di-GMP.(a) ITC assays for the interaction between Fe-Ent and rLCN2 protein. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. (b) Assays for changes in E. coli growth mediated by the human rLCN2 protein in the presence or absence of c-di-GMP. c-di-GMP could rescue the growth inhibition by rLCN2, but hardly by rLCN2-W79A. Addition of FeCl3 or Fe-Ent could rescue rLCN2-mediated growth inhibition. The control nucleotide, c-di-AMP or cGAMP, could not rescue rLCN2-mediated growth inhibition. Bacterial counts were determined at two representative time points, 18 h (top panel) and 24 h (bottom panel). Double asterisks (**) represent significant difference between two groups (P≤0.01, two-tailed Student's t-test).
Mentions: Next, we used E. coli, a fast-growing bacterium, as a model to determine whether rLCN2-mediated bacterial growth inhibition is rescued by c-di-GMP. rLCN2-mediated inhibition of growth has been previously described in E. coli78. In a similar ITC assay, we found that the rLCN2 protein could bind well to ferric enterobactin (Fe-Ent; Fig. 4a). The binding affinity of the interaction (Kd) was 0.24±0.03 μM (data shown are mean±s.d. of three biological replicates). The mutant rLCN2-W79A protein without the ability to bind c-di-GMP retained good binding activity for Fe-Ent (Kd 0.25±0.05 μM) (data shown are mean±s.d. of three biological replicates) (Supplementary Fig. 3b), which is comparable to the wild-type protein. Using a c-di-[32P]GMP cross-linking assay (Supplementary Fig. 4), we determined the competitive binding activity of c-di-[32P]GMP to the protein in the presence of Fe-Ent. Interestingly, binding of c-di-[32P]GMP to rLCN2 could be hardly detected in the presence of 100-fold excess Fe-Ent (Supplementary Fig. 4, lane 6). This result indicated that Fe-Ent could easily compete with c-di-[32P]GMP for protein binding.

Bottom Line: We demonstrate that c-di-GMP specifically binds to LCN2.In addition, c-di-GMP can compete with bacterial ferric siderophores to bind LCN2.Furthermore, c-di-GMP can significantly reduce LCN2-mediated inhibition on the in vitro growth of Escherichia coli.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.

ABSTRACT
Cyclic diguanylate monophosphate (c-di-GMP) is a well-conserved second messenger in bacteria. During infection, the innate immune system can also sense c-di-GMP; however, whether bacterial pathogens utilize c-di-GMP as a weapon to fight against host defense for survival and possible mechanisms underlying this process remain poorly understood. Siderocalin (LCN2) is a key antibacterial component of the innate immune system and sequesters bacterial siderophores to prevent acquisition of iron. Here we show that c-di-GMP can directly target the human LCN2 protein to inhibit its antibacterial activity. We demonstrate that c-di-GMP specifically binds to LCN2. In addition, c-di-GMP can compete with bacterial ferric siderophores to bind LCN2. Furthermore, c-di-GMP can significantly reduce LCN2-mediated inhibition on the in vitro growth of Escherichia coli. Thus, LCN2 acts as a c-di-GMP receptor. Our findings provide insight into the mechanism by which bacteria utilize c-di-GMP to interfere with the innate immune system for survival.

No MeSH data available.


Related in: MedlinePlus