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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 rLCN2-mediated inhibition of M. tuberculosis growth in the presence or absence of c-di-GMP.(a) ITC assays for the interaction between Fe-CMBs and rLCN2. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. (b) Cross-linking assay for the ability of c-di-[32P]GMP to bind to rLCN2 with Fe-CMBs. c-di-[32P]GMP retained >50% of the rLCN2 binding capacity even in the presence of 200-fold excess of Fe-CMBs (lane 3). The reaction samples were assayed on a 12% w/v SDS–PAGE. The c-di-[32P]GMP-rLCN2 complex was quantified, and the mean values of three independent labeling experiments along with error bars are shown. (c) Assays for changes in M. tuberculosis H37Ra growth mediated by the human rLCN2 protein in the presence or absence of c-di-GMP. FeCl3 and Fe-CMBs were used as controls for the growth inhibition assays. Bacterial counts were determined at two representative time points, 96 h and 120 h. and an asterisk (*) represents significant difference (P≤0.05, two-tailed Student's t-test) between two groups.
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f3: Assays for rLCN2-mediated inhibition of M. tuberculosis growth in the presence or absence of c-di-GMP.(a) ITC assays for the interaction between Fe-CMBs and rLCN2. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. (b) Cross-linking assay for the ability of c-di-[32P]GMP to bind to rLCN2 with Fe-CMBs. c-di-[32P]GMP retained >50% of the rLCN2 binding capacity even in the presence of 200-fold excess of Fe-CMBs (lane 3). The reaction samples were assayed on a 12% w/v SDS–PAGE. The c-di-[32P]GMP-rLCN2 complex was quantified, and the mean values of three independent labeling experiments along with error bars are shown. (c) Assays for changes in M. tuberculosis H37Ra growth mediated by the human rLCN2 protein in the presence or absence of c-di-GMP. FeCl3 and Fe-CMBs were used as controls for the growth inhibition assays. Bacterial counts were determined at two representative time points, 96 h and 120 h. and an asterisk (*) represents significant difference (P≤0.05, two-tailed Student's t-test) between two groups.

Mentions: In an ITC assay, we found that both rLCN2 (5.04±0.36) (Fig. 3a) and the mutant protein rLCN2-W79A (4.56±0.47 ) (data shown are mean±s.d. of three biological replicates; Supplementary Fig. 2a) could bind well to ferric carboxymycobactins (Fe-CMBs), although the mutant protein rLCN2-W79A almost lost c-di-GMP binding activity (Supplementary Fig. 2b). This observation indicated that some amino acid residues recognized by c-di-GMP may be different from those recognized by siderophores, even though the two molecules bind to the same pocket on LCN2. Using a similar c-di-[32P]GMP cross-linking assay (Fig. 2d), we further determined the competitive binding activity of c-di-[32P]GMP to the protein in the presence of Fe-CMBs. c-di-[32P]GMP retained a clear rLCN2 binding capacity even in the presence of 200-fold excess of Fe-CMBs (Fig. 3b, lane 3). This result indicated that c-di-[32P]GMP could easily compete with ferric carboxymycobactin 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 rLCN2-mediated inhibition of M. tuberculosis growth in the presence or absence of c-di-GMP.(a) ITC assays for the interaction between Fe-CMBs and rLCN2. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. (b) Cross-linking assay for the ability of c-di-[32P]GMP to bind to rLCN2 with Fe-CMBs. c-di-[32P]GMP retained >50% of the rLCN2 binding capacity even in the presence of 200-fold excess of Fe-CMBs (lane 3). The reaction samples were assayed on a 12% w/v SDS–PAGE. The c-di-[32P]GMP-rLCN2 complex was quantified, and the mean values of three independent labeling experiments along with error bars are shown. (c) Assays for changes in M. tuberculosis H37Ra growth mediated by the human rLCN2 protein in the presence or absence of c-di-GMP. FeCl3 and Fe-CMBs were used as controls for the growth inhibition assays. Bacterial counts were determined at two representative time points, 96 h and 120 h. and an asterisk (*) represents significant difference (P≤0.05, two-tailed Student's t-test) between two groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Assays for rLCN2-mediated inhibition of M. tuberculosis growth in the presence or absence of c-di-GMP.(a) ITC assays for the interaction between Fe-CMBs and rLCN2. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. (b) Cross-linking assay for the ability of c-di-[32P]GMP to bind to rLCN2 with Fe-CMBs. c-di-[32P]GMP retained >50% of the rLCN2 binding capacity even in the presence of 200-fold excess of Fe-CMBs (lane 3). The reaction samples were assayed on a 12% w/v SDS–PAGE. The c-di-[32P]GMP-rLCN2 complex was quantified, and the mean values of three independent labeling experiments along with error bars are shown. (c) Assays for changes in M. tuberculosis H37Ra growth mediated by the human rLCN2 protein in the presence or absence of c-di-GMP. FeCl3 and Fe-CMBs were used as controls for the growth inhibition assays. Bacterial counts were determined at two representative time points, 96 h and 120 h. and an asterisk (*) represents significant difference (P≤0.05, two-tailed Student's t-test) between two groups.
Mentions: In an ITC assay, we found that both rLCN2 (5.04±0.36) (Fig. 3a) and the mutant protein rLCN2-W79A (4.56±0.47 ) (data shown are mean±s.d. of three biological replicates; Supplementary Fig. 2a) could bind well to ferric carboxymycobactins (Fe-CMBs), although the mutant protein rLCN2-W79A almost lost c-di-GMP binding activity (Supplementary Fig. 2b). This observation indicated that some amino acid residues recognized by c-di-GMP may be different from those recognized by siderophores, even though the two molecules bind to the same pocket on LCN2. Using a similar c-di-[32P]GMP cross-linking assay (Fig. 2d), we further determined the competitive binding activity of c-di-[32P]GMP to the protein in the presence of Fe-CMBs. c-di-[32P]GMP retained a clear rLCN2 binding capacity even in the presence of 200-fold excess of Fe-CMBs (Fig. 3b, lane 3). This result indicated that c-di-[32P]GMP could easily compete with ferric carboxymycobactin 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