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Zinc piracy as a mechanism of Neisseria meningitidis for evasion of nutritional immunity.

Stork M, Grijpstra J, Bos MP, Mañas Torres C, Devos N, Poolman JT, Chazin WJ, Tommassen J - PLoS Pathog. (2013)

Bottom Line: Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source.Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process.Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Utrecht, Netherlands.

ABSTRACT
The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as "nutritional immunity." The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.

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Related in: MedlinePlus

CbpA is a cell-surface-exposed outer membrane protein.(A) OMVs were isolated from cells of strain CE1523 containing pEN-cbpA grown in the presence or absence of isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by SDS-PAGE. The gel was stained with Coomassie brilliant blue. The position of CbpA is indicated. (B) Intact cells were treated with proteinase K (Prot K) at the concentrations indicated at the top and analyzed by SDS-PAGE followed by staining with Coomassie brilliant blue (left) or immunoblotting (right) with antibodies directed against the proteins indicated. In all panels, the positions of molecular weight marker proteins (MW) are indicated at the left (in kDa).
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ppat-1003733-g002: CbpA is a cell-surface-exposed outer membrane protein.(A) OMVs were isolated from cells of strain CE1523 containing pEN-cbpA grown in the presence or absence of isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by SDS-PAGE. The gel was stained with Coomassie brilliant blue. The position of CbpA is indicated. (B) Intact cells were treated with proteinase K (Prot K) at the concentrations indicated at the top and analyzed by SDS-PAGE followed by staining with Coomassie brilliant blue (left) or immunoblotting (right) with antibodies directed against the proteins indicated. In all panels, the positions of molecular weight marker proteins (MW) are indicated at the left (in kDa).

Mentions: As a Tdf family member, CbpA is expected to be embedded in the outer membrane as a 22-stranded β-barrel with an N-terminal plug domain that closes the pore in the barrel [1]. The outer membrane localization of CbpA was confirmed by isolating outer membrane vesicles (OMVs) from strain CE1523 containing cbpA under lac-promoter control on plasmid pEN11-cbpA by extracting the cells with deoxycholic acid (DOC). Like the outer membrane marker protein, the porin PorB, CbpA was present in the insoluble OMV fraction (Figure 2A). The cell-surface exposure of CbpA was confirmed in protease-accessibility experiments. Like the cell-surface-exposed lipoprotein fHbp (factor H-binding protein), CbpA was degraded when intact cells were treated with proteinase K, while the periplasmic iron-binding protein FbpA was inaccessible (Figure 2B). Thus, CbpA is a surface-exposed outer membrane protein that is expected to bind a ligand from the environment.


Zinc piracy as a mechanism of Neisseria meningitidis for evasion of nutritional immunity.

Stork M, Grijpstra J, Bos MP, Mañas Torres C, Devos N, Poolman JT, Chazin WJ, Tommassen J - PLoS Pathog. (2013)

CbpA is a cell-surface-exposed outer membrane protein.(A) OMVs were isolated from cells of strain CE1523 containing pEN-cbpA grown in the presence or absence of isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by SDS-PAGE. The gel was stained with Coomassie brilliant blue. The position of CbpA is indicated. (B) Intact cells were treated with proteinase K (Prot K) at the concentrations indicated at the top and analyzed by SDS-PAGE followed by staining with Coomassie brilliant blue (left) or immunoblotting (right) with antibodies directed against the proteins indicated. In all panels, the positions of molecular weight marker proteins (MW) are indicated at the left (in kDa).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814407&req=5

ppat-1003733-g002: CbpA is a cell-surface-exposed outer membrane protein.(A) OMVs were isolated from cells of strain CE1523 containing pEN-cbpA grown in the presence or absence of isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by SDS-PAGE. The gel was stained with Coomassie brilliant blue. The position of CbpA is indicated. (B) Intact cells were treated with proteinase K (Prot K) at the concentrations indicated at the top and analyzed by SDS-PAGE followed by staining with Coomassie brilliant blue (left) or immunoblotting (right) with antibodies directed against the proteins indicated. In all panels, the positions of molecular weight marker proteins (MW) are indicated at the left (in kDa).
Mentions: As a Tdf family member, CbpA is expected to be embedded in the outer membrane as a 22-stranded β-barrel with an N-terminal plug domain that closes the pore in the barrel [1]. The outer membrane localization of CbpA was confirmed by isolating outer membrane vesicles (OMVs) from strain CE1523 containing cbpA under lac-promoter control on plasmid pEN11-cbpA by extracting the cells with deoxycholic acid (DOC). Like the outer membrane marker protein, the porin PorB, CbpA was present in the insoluble OMV fraction (Figure 2A). The cell-surface exposure of CbpA was confirmed in protease-accessibility experiments. Like the cell-surface-exposed lipoprotein fHbp (factor H-binding protein), CbpA was degraded when intact cells were treated with proteinase K, while the periplasmic iron-binding protein FbpA was inaccessible (Figure 2B). Thus, CbpA is a surface-exposed outer membrane protein that is expected to bind a ligand from the environment.

Bottom Line: Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source.Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process.Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Utrecht, Netherlands.

ABSTRACT
The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as "nutritional immunity." The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.

Show MeSH
Related in: MedlinePlus