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Membrane damage elicits an immunomodulatory program in Staphylococcus aureus.

Attia AS, Benson MA, Stauff DL, Torres VJ, Skaar EP - PLoS Pathog. (2010)

Bottom Line: This result indicates that the physical absence of HrtA is responsible for the increased expression of immunomodulatory factors, whereas deficiencies in the ATPase activity of HrtA do not contribute to this process.Furthermore, HrtB expression in strains lacking hrtA decreases membrane integrity consistent with dysregulated permease function.These results suggest that S. aureus senses membrane damage and elaborates a gene expression program that protects the organism from the innate immune response of the host.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
The Staphylococcus aureus HrtAB system is a hemin-regulated ABC transporter composed of an ATPase (HrtA) and a permease (HrtB) that protect S. aureus against hemin toxicity. S. aureus strains lacking hrtA exhibit liver-specific hyper-virulence and upon hemin exposure over-express and secrete immunomodulatory factors that interfere with neutrophil recruitment to the site of infection. It has been proposed that heme accumulation in strains lacking hrtAB is the signal which triggers S. aureus to elaborate this anti-neutrophil response. However, we report here that S. aureus strains expressing catalytically inactive HrtA do not elaborate the same secreted protein profile. This result indicates that the physical absence of HrtA is responsible for the increased expression of immunomodulatory factors, whereas deficiencies in the ATPase activity of HrtA do not contribute to this process. Furthermore, HrtB expression in strains lacking hrtA decreases membrane integrity consistent with dysregulated permease function. Based on these findings, we propose a model whereby hemin-mediated over-expression of HrtB in the absence of HrtA damages the staphylococcal membrane through pore formation. In turn, S. aureus senses this membrane damage, triggering the increased expression of immunomodulatory factors. In support of this model, wildtype S. aureus treated with anti-staphylococcal channel-forming peptides produce a secreted protein profile that mimics the effect of treating DeltahrtA with hemin. These results suggest that S. aureus senses membrane damage and elaborates a gene expression program that protects the organism from the innate immune response of the host.

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Up-regulation of immunomodulatory proteins is caused by loss of expression of HrtA.(A) Bacterial strains were grown in (0, 0.5 and 2 µM) hemin in RPMI/CAS for 18 hours and proteins in culture supernatants were precipitated using 10% TCA, separated using 15% SDS-PAGE, and stained with Coomassie blue. (B) Exoprotein profiles of wildtype transformed with control plasmid pOS1 and ΔhrtA transformed with control plasmid or plasmids expressing WT HrtA, catalytically inactive HrtA variants (K45A, G145A, G145T, and E167Q) or partially catalytically active HrtA (R76A) [14]. All strains were grown in the presence of 1 µM hemin. Proteins up-regulated under the indicated condition that have been identified previously using mass spectrometry are marked with a #. The predicted identities of the proteins in these bands are as follows; #1 (Exotoxin 8, SACOL0472), #2 (Exotoxin, SACOL0478), #3 (Exotoxin 3 and 5, SACOL0468/0473), #4 (Efb, SACOL1168), and #5 (FLIPr, SACOL1166). Positions of protein molecular mass markers in kilodaltons (kDa) are indicated on the left side of panels A and B. (C) Immunoblot analysis of protoplast lysates of strains analyzed in (B). Proteins were resolved using 15% SDS-PAGE, transferred to nitrocellulose membrane, probed with 9E10 anti-C-Myc monoclonal primary and AlexaFluor-680-conjugated anti-mouse secondary antibodies. Membranes were then dried and scanned using an Odyssey Infrared Imaging System.
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ppat-1000802-g001: Up-regulation of immunomodulatory proteins is caused by loss of expression of HrtA.(A) Bacterial strains were grown in (0, 0.5 and 2 µM) hemin in RPMI/CAS for 18 hours and proteins in culture supernatants were precipitated using 10% TCA, separated using 15% SDS-PAGE, and stained with Coomassie blue. (B) Exoprotein profiles of wildtype transformed with control plasmid pOS1 and ΔhrtA transformed with control plasmid or plasmids expressing WT HrtA, catalytically inactive HrtA variants (K45A, G145A, G145T, and E167Q) or partially catalytically active HrtA (R76A) [14]. All strains were grown in the presence of 1 µM hemin. Proteins up-regulated under the indicated condition that have been identified previously using mass spectrometry are marked with a #. The predicted identities of the proteins in these bands are as follows; #1 (Exotoxin 8, SACOL0472), #2 (Exotoxin, SACOL0478), #3 (Exotoxin 3 and 5, SACOL0468/0473), #4 (Efb, SACOL1168), and #5 (FLIPr, SACOL1166). Positions of protein molecular mass markers in kilodaltons (kDa) are indicated on the left side of panels A and B. (C) Immunoblot analysis of protoplast lysates of strains analyzed in (B). Proteins were resolved using 15% SDS-PAGE, transferred to nitrocellulose membrane, probed with 9E10 anti-C-Myc monoclonal primary and AlexaFluor-680-conjugated anti-mouse secondary antibodies. Membranes were then dried and scanned using an Odyssey Infrared Imaging System.

Mentions: In an effort to elucidate the mechanism responsible for the hypervirulence of S. aureus ΔhrtA, we analyzed the secreted protein profiles of staphylococcal strains with mutations in the Hss and Hrt systems. Upon exposure of ΔhrtA to hemin in concentrations of either 0.5 or 2 µM, up-regulation of the production and secretion of at least five proteins was observed (Fig. 1A). These proteins have been previously identified through mass spectrometry to be immunomodulatory proteins that interfere with neutrophil recruitment to the site of infection [13],[19],[20],[21]. This finding suggests that strains unable to alleviate hemin toxicity through HrtAB increase the expression of immunomodulatory proteins. However, a hemin-dependent increase in protein expression was not observed in either wildtype S. aureus or a strain lacking functional HssRS (ΔhssR), the regulatory system which controls hemin-dependent hrtAB expression (Fig. 1A) [12]. In addition, the secreted protein profile of a hemin-exposed S. aureus strain containing a transposon insertion that inactivates the permease HrtB (thrtB) closely resembled that of wildtype and did not show changes similar to those observed in ΔhrtA exposed to hemin (Fig. 1A). These data indicate that hemin-exposed S. aureus ΔhrtA increases the secretion of immunomodulatory proteins, but this immunomodulatory response does not occur upon loss of HrtB.


Membrane damage elicits an immunomodulatory program in Staphylococcus aureus.

Attia AS, Benson MA, Stauff DL, Torres VJ, Skaar EP - PLoS Pathog. (2010)

Up-regulation of immunomodulatory proteins is caused by loss of expression of HrtA.(A) Bacterial strains were grown in (0, 0.5 and 2 µM) hemin in RPMI/CAS for 18 hours and proteins in culture supernatants were precipitated using 10% TCA, separated using 15% SDS-PAGE, and stained with Coomassie blue. (B) Exoprotein profiles of wildtype transformed with control plasmid pOS1 and ΔhrtA transformed with control plasmid or plasmids expressing WT HrtA, catalytically inactive HrtA variants (K45A, G145A, G145T, and E167Q) or partially catalytically active HrtA (R76A) [14]. All strains were grown in the presence of 1 µM hemin. Proteins up-regulated under the indicated condition that have been identified previously using mass spectrometry are marked with a #. The predicted identities of the proteins in these bands are as follows; #1 (Exotoxin 8, SACOL0472), #2 (Exotoxin, SACOL0478), #3 (Exotoxin 3 and 5, SACOL0468/0473), #4 (Efb, SACOL1168), and #5 (FLIPr, SACOL1166). Positions of protein molecular mass markers in kilodaltons (kDa) are indicated on the left side of panels A and B. (C) Immunoblot analysis of protoplast lysates of strains analyzed in (B). Proteins were resolved using 15% SDS-PAGE, transferred to nitrocellulose membrane, probed with 9E10 anti-C-Myc monoclonal primary and AlexaFluor-680-conjugated anti-mouse secondary antibodies. Membranes were then dried and scanned using an Odyssey Infrared Imaging System.
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Related In: Results  -  Collection

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ppat-1000802-g001: Up-regulation of immunomodulatory proteins is caused by loss of expression of HrtA.(A) Bacterial strains were grown in (0, 0.5 and 2 µM) hemin in RPMI/CAS for 18 hours and proteins in culture supernatants were precipitated using 10% TCA, separated using 15% SDS-PAGE, and stained with Coomassie blue. (B) Exoprotein profiles of wildtype transformed with control plasmid pOS1 and ΔhrtA transformed with control plasmid or plasmids expressing WT HrtA, catalytically inactive HrtA variants (K45A, G145A, G145T, and E167Q) or partially catalytically active HrtA (R76A) [14]. All strains were grown in the presence of 1 µM hemin. Proteins up-regulated under the indicated condition that have been identified previously using mass spectrometry are marked with a #. The predicted identities of the proteins in these bands are as follows; #1 (Exotoxin 8, SACOL0472), #2 (Exotoxin, SACOL0478), #3 (Exotoxin 3 and 5, SACOL0468/0473), #4 (Efb, SACOL1168), and #5 (FLIPr, SACOL1166). Positions of protein molecular mass markers in kilodaltons (kDa) are indicated on the left side of panels A and B. (C) Immunoblot analysis of protoplast lysates of strains analyzed in (B). Proteins were resolved using 15% SDS-PAGE, transferred to nitrocellulose membrane, probed with 9E10 anti-C-Myc monoclonal primary and AlexaFluor-680-conjugated anti-mouse secondary antibodies. Membranes were then dried and scanned using an Odyssey Infrared Imaging System.
Mentions: In an effort to elucidate the mechanism responsible for the hypervirulence of S. aureus ΔhrtA, we analyzed the secreted protein profiles of staphylococcal strains with mutations in the Hss and Hrt systems. Upon exposure of ΔhrtA to hemin in concentrations of either 0.5 or 2 µM, up-regulation of the production and secretion of at least five proteins was observed (Fig. 1A). These proteins have been previously identified through mass spectrometry to be immunomodulatory proteins that interfere with neutrophil recruitment to the site of infection [13],[19],[20],[21]. This finding suggests that strains unable to alleviate hemin toxicity through HrtAB increase the expression of immunomodulatory proteins. However, a hemin-dependent increase in protein expression was not observed in either wildtype S. aureus or a strain lacking functional HssRS (ΔhssR), the regulatory system which controls hemin-dependent hrtAB expression (Fig. 1A) [12]. In addition, the secreted protein profile of a hemin-exposed S. aureus strain containing a transposon insertion that inactivates the permease HrtB (thrtB) closely resembled that of wildtype and did not show changes similar to those observed in ΔhrtA exposed to hemin (Fig. 1A). These data indicate that hemin-exposed S. aureus ΔhrtA increases the secretion of immunomodulatory proteins, but this immunomodulatory response does not occur upon loss of HrtB.

Bottom Line: This result indicates that the physical absence of HrtA is responsible for the increased expression of immunomodulatory factors, whereas deficiencies in the ATPase activity of HrtA do not contribute to this process.Furthermore, HrtB expression in strains lacking hrtA decreases membrane integrity consistent with dysregulated permease function.These results suggest that S. aureus senses membrane damage and elaborates a gene expression program that protects the organism from the innate immune response of the host.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
The Staphylococcus aureus HrtAB system is a hemin-regulated ABC transporter composed of an ATPase (HrtA) and a permease (HrtB) that protect S. aureus against hemin toxicity. S. aureus strains lacking hrtA exhibit liver-specific hyper-virulence and upon hemin exposure over-express and secrete immunomodulatory factors that interfere with neutrophil recruitment to the site of infection. It has been proposed that heme accumulation in strains lacking hrtAB is the signal which triggers S. aureus to elaborate this anti-neutrophil response. However, we report here that S. aureus strains expressing catalytically inactive HrtA do not elaborate the same secreted protein profile. This result indicates that the physical absence of HrtA is responsible for the increased expression of immunomodulatory factors, whereas deficiencies in the ATPase activity of HrtA do not contribute to this process. Furthermore, HrtB expression in strains lacking hrtA decreases membrane integrity consistent with dysregulated permease function. Based on these findings, we propose a model whereby hemin-mediated over-expression of HrtB in the absence of HrtA damages the staphylococcal membrane through pore formation. In turn, S. aureus senses this membrane damage, triggering the increased expression of immunomodulatory factors. In support of this model, wildtype S. aureus treated with anti-staphylococcal channel-forming peptides produce a secreted protein profile that mimics the effect of treating DeltahrtA with hemin. These results suggest that S. aureus senses membrane damage and elaborates a gene expression program that protects the organism from the innate immune response of the host.

Show MeSH
Related in: MedlinePlus