Limits...
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 specific interaction between human LCN2 protein and c-di-GMP.(a) Cross-linking assay. STING was used as a positive control (lane 1). A competitive experiment was carried out by addition of unlabeled c-di-GMP at 67-, 134- and 333-fold excess to the reaction mixtures containing 15 μM rLCN2 and 1.5 μM c-di-[32P]GMP (lanes 2– 4). [32P]GTP was used as a negative control molecule. The reaction samples were subjected to a 12% w/v SDS–PAGE and radioactive gels were exposed to a storage phosphor screen (GE Healthcare). Radiolabelled nucleotides is indicated by an asterisk (*). (b) ITC assays for the specific interaction between rLCN2 and c-di-GMP. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. Open and filled rectangles indicate data for c-di-AMP and c-di-GMP, respectively. The solid line in the bottom panel represents the best fit to a one-site binding model of the interaction of rLCN2 with c-di-GMP. c-di-AMP was used as a control. (c) Schematic of the predicted contacts between c-di-GMP and LCN2 from docking results. Potential hydrogen bonds are indicated as green dashed lines. (d) Cross-linking assays for the binding of c-di-GMP with five recombinant mutant LCN2 proteins. Two mutant proteins, rLCN2-W79A (lane 3) and rLCN2-K125A (lane 4), did not exhibit c-di-GMP binding activities. (e) Assays for c-di-GMP secretion by two bacterial strains. The concentration of extracelluar release of c-di-GMP was obtained, 1.042 μM for E. coli BL21(DE3) and 0.568 μM for M. tuberculosis H37Ra. Error bars represent the variant range of the data derived from three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4595737&req=5

f2: Assays for specific interaction between human LCN2 protein and c-di-GMP.(a) Cross-linking assay. STING was used as a positive control (lane 1). A competitive experiment was carried out by addition of unlabeled c-di-GMP at 67-, 134- and 333-fold excess to the reaction mixtures containing 15 μM rLCN2 and 1.5 μM c-di-[32P]GMP (lanes 2– 4). [32P]GTP was used as a negative control molecule. The reaction samples were subjected to a 12% w/v SDS–PAGE and radioactive gels were exposed to a storage phosphor screen (GE Healthcare). Radiolabelled nucleotides is indicated by an asterisk (*). (b) ITC assays for the specific interaction between rLCN2 and c-di-GMP. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. Open and filled rectangles indicate data for c-di-AMP and c-di-GMP, respectively. The solid line in the bottom panel represents the best fit to a one-site binding model of the interaction of rLCN2 with c-di-GMP. c-di-AMP was used as a control. (c) Schematic of the predicted contacts between c-di-GMP and LCN2 from docking results. Potential hydrogen bonds are indicated as green dashed lines. (d) Cross-linking assays for the binding of c-di-GMP with five recombinant mutant LCN2 proteins. Two mutant proteins, rLCN2-W79A (lane 3) and rLCN2-K125A (lane 4), did not exhibit c-di-GMP binding activities. (e) Assays for c-di-GMP secretion by two bacterial strains. The concentration of extracelluar release of c-di-GMP was obtained, 1.042 μM for E. coli BL21(DE3) and 0.568 μM for M. tuberculosis H37Ra. Error bars represent the variant range of the data derived from three biological replicates.

Mentions: To determine whether c-di-GMP can directly bind LCN2 protein and whether this binding is specific, we first purified the recombinant human LCN2 (rLCN2) protein without a signal peptide fragment and identified the potentially direct interaction between rLCN2 and c-di-[32P]GMP by performing a cross-linking assay15. As expected, STING, as a positive control, could clearly bind to the second messenger as indicated by an autoradiograph signal on a polyacrylamide gel electrophoresis (PAGE) gel (Fig. 2a, lane 1). We detected a similar strong signal corresponding to rLCN2 bound to c-di-[32P]GMP under the same conditions, which indicates that rLCN2 can directly bind to c-di-GMP (Fig. 2a, lane 7). By contrast, rLCN2 did not bind to [32P]GTP (Fig. 2a, lane 5) and showed very weak binding to another bacterial second messenger molecule, 32P-labelled cyclic diadenosine monophosphate (c-di-[32P]AMP; Fig. 2a, lane 6). Further experiments on c-di-[32P]GMP cross-linking confirmed the binding specificity of c-di-GMP in the presence of unlabeled nucleotides. Addition of unlabelled c-di-GMP at 67-, 134- and 333-fold excess to the reaction mixtures competitively inhibited the binding of rLCN2 to c-di-[32P]GMP (Fig. 2a, lanes 2–4). Furthermore, isothermal titration calorimetry (ITC) assays confirmed the binding specificity of c-di-GMP. Figure 2b (top) shows the raw data for titration of c-di-GMP against rLCN2 and indicates that the interaction is exothermic. Furthermore, results showed that the binding stoichiometry between c-di-GMP against rLCN2 was 1:1 (n=1.05), and the binding affinity of the interaction (Kd) was 1.63±0.05 μM (data shown are mean±s.d. of three biological replicates) under our experimental conditions (Fig. 2b, bottom), which is comparable to the reported Kd values of several receptors451516. By contrast, no interaction between rLCN2 and c-di-AMP (Fig. 2b) or the eukaryotic cGAMP (Supplementary Fig. 1) was detected under similar experimental conditions. These results are consistent with those of cross-linking assays. Thus, rLCN2 can directly and specifically bind to c-di-GMP. Using the ITC assay, we further performed titration of c-di-GMP into rLCN2 in a series of temperatures and calculated the heat capacity change (ΔCp) as −2.383±1.4 kJ mol−1 K−1 (data shown are mean±s.d. of three biological replicates), indicating that there exists certain conformational change17 for LCN2 following c-di-GMP 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 specific interaction between human LCN2 protein and c-di-GMP.(a) Cross-linking assay. STING was used as a positive control (lane 1). A competitive experiment was carried out by addition of unlabeled c-di-GMP at 67-, 134- and 333-fold excess to the reaction mixtures containing 15 μM rLCN2 and 1.5 μM c-di-[32P]GMP (lanes 2– 4). [32P]GTP was used as a negative control molecule. The reaction samples were subjected to a 12% w/v SDS–PAGE and radioactive gels were exposed to a storage phosphor screen (GE Healthcare). Radiolabelled nucleotides is indicated by an asterisk (*). (b) ITC assays for the specific interaction between rLCN2 and c-di-GMP. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. Open and filled rectangles indicate data for c-di-AMP and c-di-GMP, respectively. The solid line in the bottom panel represents the best fit to a one-site binding model of the interaction of rLCN2 with c-di-GMP. c-di-AMP was used as a control. (c) Schematic of the predicted contacts between c-di-GMP and LCN2 from docking results. Potential hydrogen bonds are indicated as green dashed lines. (d) Cross-linking assays for the binding of c-di-GMP with five recombinant mutant LCN2 proteins. Two mutant proteins, rLCN2-W79A (lane 3) and rLCN2-K125A (lane 4), did not exhibit c-di-GMP binding activities. (e) Assays for c-di-GMP secretion by two bacterial strains. The concentration of extracelluar release of c-di-GMP was obtained, 1.042 μM for E. coli BL21(DE3) and 0.568 μM for M. tuberculosis H37Ra. Error bars represent the variant range of the data derived from three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Assays for specific interaction between human LCN2 protein and c-di-GMP.(a) Cross-linking assay. STING was used as a positive control (lane 1). A competitive experiment was carried out by addition of unlabeled c-di-GMP at 67-, 134- and 333-fold excess to the reaction mixtures containing 15 μM rLCN2 and 1.5 μM c-di-[32P]GMP (lanes 2– 4). [32P]GTP was used as a negative control molecule. The reaction samples were subjected to a 12% w/v SDS–PAGE and radioactive gels were exposed to a storage phosphor screen (GE Healthcare). Radiolabelled nucleotides is indicated by an asterisk (*). (b) ITC assays for the specific interaction between rLCN2 and c-di-GMP. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. Open and filled rectangles indicate data for c-di-AMP and c-di-GMP, respectively. The solid line in the bottom panel represents the best fit to a one-site binding model of the interaction of rLCN2 with c-di-GMP. c-di-AMP was used as a control. (c) Schematic of the predicted contacts between c-di-GMP and LCN2 from docking results. Potential hydrogen bonds are indicated as green dashed lines. (d) Cross-linking assays for the binding of c-di-GMP with five recombinant mutant LCN2 proteins. Two mutant proteins, rLCN2-W79A (lane 3) and rLCN2-K125A (lane 4), did not exhibit c-di-GMP binding activities. (e) Assays for c-di-GMP secretion by two bacterial strains. The concentration of extracelluar release of c-di-GMP was obtained, 1.042 μM for E. coli BL21(DE3) and 0.568 μM for M. tuberculosis H37Ra. Error bars represent the variant range of the data derived from three biological replicates.
Mentions: To determine whether c-di-GMP can directly bind LCN2 protein and whether this binding is specific, we first purified the recombinant human LCN2 (rLCN2) protein without a signal peptide fragment and identified the potentially direct interaction between rLCN2 and c-di-[32P]GMP by performing a cross-linking assay15. As expected, STING, as a positive control, could clearly bind to the second messenger as indicated by an autoradiograph signal on a polyacrylamide gel electrophoresis (PAGE) gel (Fig. 2a, lane 1). We detected a similar strong signal corresponding to rLCN2 bound to c-di-[32P]GMP under the same conditions, which indicates that rLCN2 can directly bind to c-di-GMP (Fig. 2a, lane 7). By contrast, rLCN2 did not bind to [32P]GTP (Fig. 2a, lane 5) and showed very weak binding to another bacterial second messenger molecule, 32P-labelled cyclic diadenosine monophosphate (c-di-[32P]AMP; Fig. 2a, lane 6). Further experiments on c-di-[32P]GMP cross-linking confirmed the binding specificity of c-di-GMP in the presence of unlabeled nucleotides. Addition of unlabelled c-di-GMP at 67-, 134- and 333-fold excess to the reaction mixtures competitively inhibited the binding of rLCN2 to c-di-[32P]GMP (Fig. 2a, lanes 2–4). Furthermore, isothermal titration calorimetry (ITC) assays confirmed the binding specificity of c-di-GMP. Figure 2b (top) shows the raw data for titration of c-di-GMP against rLCN2 and indicates that the interaction is exothermic. Furthermore, results showed that the binding stoichiometry between c-di-GMP against rLCN2 was 1:1 (n=1.05), and the binding affinity of the interaction (Kd) was 1.63±0.05 μM (data shown are mean±s.d. of three biological replicates) under our experimental conditions (Fig. 2b, bottom), which is comparable to the reported Kd values of several receptors451516. By contrast, no interaction between rLCN2 and c-di-AMP (Fig. 2b) or the eukaryotic cGAMP (Supplementary Fig. 1) was detected under similar experimental conditions. These results are consistent with those of cross-linking assays. Thus, rLCN2 can directly and specifically bind to c-di-GMP. Using the ITC assay, we further performed titration of c-di-GMP into rLCN2 in a series of temperatures and calculated the heat capacity change (ΔCp) as −2.383±1.4 kJ mol−1 K−1 (data shown are mean±s.d. of three biological replicates), indicating that there exists certain conformational change17 for LCN2 following c-di-GMP 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