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Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane.

Jabado N, Jankowski A, Dougaparsad S, Picard V, Grinstein S, Gros P - J. Exp. Med. (2000)

Bottom Line: The resulting conjugate, zymosan-FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging.The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H(+) dependent.Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal H3G-1Y6, Quebec, Canada.

ABSTRACT
Mutations at the natural resistance-associated macrophage protein 1 (Nramp1) locus cause susceptibility to infection with antigenically unrelated intracellular pathogens. Nramp1 codes for an integral membrane protein expressed in the lysosomal compartment of macrophages, and is recruited to the membrane of phagosomes soon after the completion of phagocytosis. To define whether Nramp1 functions as a transporter at the phagosomal membrane, a divalent cation-sensitive fluorescent probe was designed and covalently attached to a porous particle. The resulting conjugate, zymosan-FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging. Quenching of the probe by Mn(2+) was used to monitor the flux of divalent cations across the phagosomal membrane in peritoneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficient (-/-) macrophages. Phagosomes from Nramp1(+/+) mice extrude Mn(2+) faster than their Nramp(-/-) counterparts. The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H(+) dependent. These studies suggest that Nramp1 contributes to defense against infection by extrusion of divalent cations from the phagosomal space. Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.

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Related in: MedlinePlus

Synthesis of the fluorescent probe zymosan–FF6. Step a, activation of COOH-FF6(COOEt)4 using dicyclohexylcarbodiimide and N-hydroxysuccinimide. Step b, coupling of the activated intermediate to zymosan. Step c, deprotection of the conjugate by silanolate-induced ester hydrolysis.
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Figure 1: Synthesis of the fluorescent probe zymosan–FF6. Step a, activation of COOH-FF6(COOEt)4 using dicyclohexylcarbodiimide and N-hydroxysuccinimide. Step b, coupling of the activated intermediate to zymosan. Step c, deprotection of the conjugate by silanolate-induced ester hydrolysis.

Mentions: The strategy used to synthesize the particulate fluorescent probe zymosan–FF6 is shown in Fig. 1. The precursor, Fura-6-FF-tetraethylester (COOH-FF6[COOEt]4), was purchased from TeFLabs. COOH-FF6 (COOEt)4 has four cation-sensitive carboxyl groups that are protected by esterification, with a fifth unprotected carboxylate that is used to covalently couple the precursor to amino groups on zymosan by carbodiimide-mediated activation using a succinimidyl ester. After attachment to the particles, the protecting ethyl ester moieties were removed by treatment with trimethylsilanoate.


Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane.

Jabado N, Jankowski A, Dougaparsad S, Picard V, Grinstein S, Gros P - J. Exp. Med. (2000)

Synthesis of the fluorescent probe zymosan–FF6. Step a, activation of COOH-FF6(COOEt)4 using dicyclohexylcarbodiimide and N-hydroxysuccinimide. Step b, coupling of the activated intermediate to zymosan. Step c, deprotection of the conjugate by silanolate-induced ester hydrolysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193348&req=5

Figure 1: Synthesis of the fluorescent probe zymosan–FF6. Step a, activation of COOH-FF6(COOEt)4 using dicyclohexylcarbodiimide and N-hydroxysuccinimide. Step b, coupling of the activated intermediate to zymosan. Step c, deprotection of the conjugate by silanolate-induced ester hydrolysis.
Mentions: The strategy used to synthesize the particulate fluorescent probe zymosan–FF6 is shown in Fig. 1. The precursor, Fura-6-FF-tetraethylester (COOH-FF6[COOEt]4), was purchased from TeFLabs. COOH-FF6 (COOEt)4 has four cation-sensitive carboxyl groups that are protected by esterification, with a fifth unprotected carboxylate that is used to covalently couple the precursor to amino groups on zymosan by carbodiimide-mediated activation using a succinimidyl ester. After attachment to the particles, the protecting ethyl ester moieties were removed by treatment with trimethylsilanoate.

Bottom Line: The resulting conjugate, zymosan-FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging.The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H(+) dependent.Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal H3G-1Y6, Quebec, Canada.

ABSTRACT
Mutations at the natural resistance-associated macrophage protein 1 (Nramp1) locus cause susceptibility to infection with antigenically unrelated intracellular pathogens. Nramp1 codes for an integral membrane protein expressed in the lysosomal compartment of macrophages, and is recruited to the membrane of phagosomes soon after the completion of phagocytosis. To define whether Nramp1 functions as a transporter at the phagosomal membrane, a divalent cation-sensitive fluorescent probe was designed and covalently attached to a porous particle. The resulting conjugate, zymosan-FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging. Quenching of the probe by Mn(2+) was used to monitor the flux of divalent cations across the phagosomal membrane in peritoneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficient (-/-) macrophages. Phagosomes from Nramp1(+/+) mice extrude Mn(2+) faster than their Nramp(-/-) counterparts. The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H(+) dependent. These studies suggest that Nramp1 contributes to defense against infection by extrusion of divalent cations from the phagosomal space. Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.

Show MeSH
Related in: MedlinePlus