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Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli.

Turlin E, Heuck G, Simões Brandão MI, Szili N, Mellin JR, Lange N, Wandersman C - Microbiologyopen (2014)

Bottom Line: We also show that it is disrupted by iron chelation, which reduces the intracellular iron concentration necessary for loading iron into protoporphyrin IX (PPIX, the immediate heme precursor).The MacAB-TolC pump is required for Salmonella typhimurium survival in macrophages.We propose that PPIX is an endogenous substrate of the MacAB-TolC pump in E. coli and S. typhimurium and that this compound is produced inside bacteria when natural heme homeostasis is disrupted by iron shortage, as happens when bacteria invade the mammalian host.

View Article: PubMed Central - PubMed

Affiliation: Unité des Membranes Bactériennes, Département de Microbiologie, Institut Pasteur, 75724, Paris Cedex 15, France.

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Accumulation of Protoporphyrin IX (PPIX) in tolC and macAB mutants. (A) Immunodetection of YfeX amounts in various strains 10 indicated above the western blot. Protein extracts were obtained as described in Experimental Procedures (porphyrin extraction and HPLC analysis). Levels of 6His-YfeX were detected by immunoblotting using anti-His antibodies. Each lane was loaded with 10 lL of a cell extract of cultures grown to an OD600 of 1. Each extract contained the same total protein concentration of 2 mg mL−1 as measured by Bradford. (B) Visualization of the fluorescence of soluble fraction under near-UV light. The name of the corresponding culture strain is indicated above the western blot. (C) HPLC fluorescence chromatograms of porphyrins. The fluorescent soluble fraction of strains WT and mutants indicated in the figure were prepared as described in Experimental Procedures. The extracted porphyrin were separated by HPLC. The retention positions of control porphyrins (COPI, COP III, and PPIX) are indicated on the graph by arrows. There are not many endogenous fluorophors except prophyrins that can be excited at 405 nm and do fluorescence at 635 nm (chlorins and chlorophylls are fluorescing at about 670 nm when excited at 400 nm). The mass spectrometry data of the various fluorescent compounds were performed in Letoffe et al. 2009.
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fig01: Accumulation of Protoporphyrin IX (PPIX) in tolC and macAB mutants. (A) Immunodetection of YfeX amounts in various strains 10 indicated above the western blot. Protein extracts were obtained as described in Experimental Procedures (porphyrin extraction and HPLC analysis). Levels of 6His-YfeX were detected by immunoblotting using anti-His antibodies. Each lane was loaded with 10 lL of a cell extract of cultures grown to an OD600 of 1. Each extract contained the same total protein concentration of 2 mg mL−1 as measured by Bradford. (B) Visualization of the fluorescence of soluble fraction under near-UV light. The name of the corresponding culture strain is indicated above the western blot. (C) HPLC fluorescence chromatograms of porphyrins. The fluorescent soluble fraction of strains WT and mutants indicated in the figure were prepared as described in Experimental Procedures. The extracted porphyrin were separated by HPLC. The retention positions of control porphyrins (COPI, COP III, and PPIX) are indicated on the graph by arrows. There are not many endogenous fluorophors except prophyrins that can be excited at 405 nm and do fluorescence at 635 nm (chlorins and chlorophylls are fluorescing at about 670 nm when excited at 400 nm). The mass spectrometry data of the various fluorescent compounds were performed in Letoffe et al. 2009.

Mentions: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot were carried out according to standard protocols. Cultures were grown to an OD600 of 1. Cells were harvested by centrifugation for 20 min at 5000g at 4°C. The cell pellet was resuspended in 50 mmol/L Tris-HCl pH 8.0, 0.3 mol/L NaCl to OD600 of 50/mL and disrupted by sonication, then centrifuged at 20,000g for 60 min at 4°C to remove cellular debris. Protein concentration in cell extracts was determined by Bradford protein assay and is indicated in the Figure 1. Ten microliters of aliquots were loaded on SDS PAGE. Bound antibodies were detected with secondary anti-rabbit antibodies coupled with horseradish peroxidase and revealed by chemiluminescence.


Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli.

Turlin E, Heuck G, Simões Brandão MI, Szili N, Mellin JR, Lange N, Wandersman C - Microbiologyopen (2014)

Accumulation of Protoporphyrin IX (PPIX) in tolC and macAB mutants. (A) Immunodetection of YfeX amounts in various strains 10 indicated above the western blot. Protein extracts were obtained as described in Experimental Procedures (porphyrin extraction and HPLC analysis). Levels of 6His-YfeX were detected by immunoblotting using anti-His antibodies. Each lane was loaded with 10 lL of a cell extract of cultures grown to an OD600 of 1. Each extract contained the same total protein concentration of 2 mg mL−1 as measured by Bradford. (B) Visualization of the fluorescence of soluble fraction under near-UV light. The name of the corresponding culture strain is indicated above the western blot. (C) HPLC fluorescence chromatograms of porphyrins. The fluorescent soluble fraction of strains WT and mutants indicated in the figure were prepared as described in Experimental Procedures. The extracted porphyrin were separated by HPLC. The retention positions of control porphyrins (COPI, COP III, and PPIX) are indicated on the graph by arrows. There are not many endogenous fluorophors except prophyrins that can be excited at 405 nm and do fluorescence at 635 nm (chlorins and chlorophylls are fluorescing at about 670 nm when excited at 400 nm). The mass spectrometry data of the various fluorescent compounds were performed in Letoffe et al. 2009.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263509&req=5

fig01: Accumulation of Protoporphyrin IX (PPIX) in tolC and macAB mutants. (A) Immunodetection of YfeX amounts in various strains 10 indicated above the western blot. Protein extracts were obtained as described in Experimental Procedures (porphyrin extraction and HPLC analysis). Levels of 6His-YfeX were detected by immunoblotting using anti-His antibodies. Each lane was loaded with 10 lL of a cell extract of cultures grown to an OD600 of 1. Each extract contained the same total protein concentration of 2 mg mL−1 as measured by Bradford. (B) Visualization of the fluorescence of soluble fraction under near-UV light. The name of the corresponding culture strain is indicated above the western blot. (C) HPLC fluorescence chromatograms of porphyrins. The fluorescent soluble fraction of strains WT and mutants indicated in the figure were prepared as described in Experimental Procedures. The extracted porphyrin were separated by HPLC. The retention positions of control porphyrins (COPI, COP III, and PPIX) are indicated on the graph by arrows. There are not many endogenous fluorophors except prophyrins that can be excited at 405 nm and do fluorescence at 635 nm (chlorins and chlorophylls are fluorescing at about 670 nm when excited at 400 nm). The mass spectrometry data of the various fluorescent compounds were performed in Letoffe et al. 2009.
Mentions: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot were carried out according to standard protocols. Cultures were grown to an OD600 of 1. Cells were harvested by centrifugation for 20 min at 5000g at 4°C. The cell pellet was resuspended in 50 mmol/L Tris-HCl pH 8.0, 0.3 mol/L NaCl to OD600 of 50/mL and disrupted by sonication, then centrifuged at 20,000g for 60 min at 4°C to remove cellular debris. Protein concentration in cell extracts was determined by Bradford protein assay and is indicated in the Figure 1. Ten microliters of aliquots were loaded on SDS PAGE. Bound antibodies were detected with secondary anti-rabbit antibodies coupled with horseradish peroxidase and revealed by chemiluminescence.

Bottom Line: We also show that it is disrupted by iron chelation, which reduces the intracellular iron concentration necessary for loading iron into protoporphyrin IX (PPIX, the immediate heme precursor).The MacAB-TolC pump is required for Salmonella typhimurium survival in macrophages.We propose that PPIX is an endogenous substrate of the MacAB-TolC pump in E. coli and S. typhimurium and that this compound is produced inside bacteria when natural heme homeostasis is disrupted by iron shortage, as happens when bacteria invade the mammalian host.

View Article: PubMed Central - PubMed

Affiliation: Unité des Membranes Bactériennes, Département de Microbiologie, Institut Pasteur, 75724, Paris Cedex 15, France.

Show MeSH
Related in: MedlinePlus