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The role of Mcl-1 in S. aureus-induced cytoprotection of infected macrophages.

Koziel J, Kmiecik K, Chmiest D, Maresz K, Mizgalska D, Maciag-Gudowska A, Mydel P, Potempa J - Mediators Inflamm. (2013)

Bottom Line: Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis.Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection.Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Ul. Gronostajowa 7, 30-387 Kraków, Poland. joanna.koziel@uj.edu.pl

ABSTRACT
As a facultative intracellular pathogen, Staphylococcus aureus invades macrophages and then promotes the cytoprotection of infected cells thus stabilizing safe niche for silent persistence. This process occurs through the upregulation of crucial antiapoptotic genes, in particular, myeloid cell leukemia-1 (MCL-1). Here, we investigated the underlying mechanism and signal transduction pathways leading to increased MCL-1 expression in infected macrophages. Live S. aureus not only stimulated de novo synthesis of Mcl-1, but also prolonged the stability of this antiapoptotic protein. Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis. Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection. Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression. Therefore, we propose that S. aureus is hijacking the Mcl-1-dependent inhibition of apoptosis to prevent the elimination of infected host cells, thus allowing the intracellular persistence of the pathogen, its dissemination by infected macrophages, and the progression of staphylococci diseases.

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S. aureus increases both de novo Mcl-1 synthesis and stability. (a) Time course of MCL1 expression in control and S. aureus-infected macrophages (hours after-infection; p.i.) was monitored by qRT-PCR, as described in Section 2. Data represent the mean values calculated from the results of three independent experiments using hMDMs derived from different donors. Bars represent mean relative expression ± SD. *P < 0.05; ***P < 0.001. (b, c) Time course of Mcl-1 protein synthesis following S. aureus infection. Mcl-1 levels were measured at different time points between 0.5 and 20 h p.i. by immunoblot. (b) Representative immunoblot from three separate experiments performed on macrophages derived from different donors. (c) Relative Mcl-1 levels obtained by densitometric analyses of western blots. Results from three separate experiments. Data represent means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. (d) Mcl-1 stability in macrophages incubated in the presence of cycloheximide (CHX) (10 μg/mL) in the absence (circles) or presence (squares) of S. aureus (MOI 1 : 50). At time periods of up to 3 h, Mcl-1 levels were detected by immunoblot in cell lysates. Data represents Mcl-1 levels relative to time 0, which was arbitrarily set as 100%, obtained by densitometric analyses of western blots. Data represent means ± SD of three separate experiments. *P < 0.05.
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fig2: S. aureus increases both de novo Mcl-1 synthesis and stability. (a) Time course of MCL1 expression in control and S. aureus-infected macrophages (hours after-infection; p.i.) was monitored by qRT-PCR, as described in Section 2. Data represent the mean values calculated from the results of three independent experiments using hMDMs derived from different donors. Bars represent mean relative expression ± SD. *P < 0.05; ***P < 0.001. (b, c) Time course of Mcl-1 protein synthesis following S. aureus infection. Mcl-1 levels were measured at different time points between 0.5 and 20 h p.i. by immunoblot. (b) Representative immunoblot from three separate experiments performed on macrophages derived from different donors. (c) Relative Mcl-1 levels obtained by densitometric analyses of western blots. Results from three separate experiments. Data represent means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. (d) Mcl-1 stability in macrophages incubated in the presence of cycloheximide (CHX) (10 μg/mL) in the absence (circles) or presence (squares) of S. aureus (MOI 1 : 50). At time periods of up to 3 h, Mcl-1 levels were detected by immunoblot in cell lysates. Data represents Mcl-1 levels relative to time 0, which was arbitrarily set as 100%, obtained by densitometric analyses of western blots. Data represent means ± SD of three separate experiments. *P < 0.05.

Mentions: To correlate S. aureus-induced cytoprotection with the expression of MCL1, we determined the time dependence of the induction of specific mRNA after macrophage challenge with bacteria. A 4-fold increase of MCL1 mRNA was observed 1 h after-infection, with sustained upregulation observed for up to 6 h (Figure 2(a)). At the protein level, Mcl-1 levels were significantly increased 2 h after-infection, reached a maximum at 8 h, and remained at 3-fold higher levels compared to mock-infected cells for at least 20 h (Figures 2(b) and 2(c)).


The role of Mcl-1 in S. aureus-induced cytoprotection of infected macrophages.

Koziel J, Kmiecik K, Chmiest D, Maresz K, Mizgalska D, Maciag-Gudowska A, Mydel P, Potempa J - Mediators Inflamm. (2013)

S. aureus increases both de novo Mcl-1 synthesis and stability. (a) Time course of MCL1 expression in control and S. aureus-infected macrophages (hours after-infection; p.i.) was monitored by qRT-PCR, as described in Section 2. Data represent the mean values calculated from the results of three independent experiments using hMDMs derived from different donors. Bars represent mean relative expression ± SD. *P < 0.05; ***P < 0.001. (b, c) Time course of Mcl-1 protein synthesis following S. aureus infection. Mcl-1 levels were measured at different time points between 0.5 and 20 h p.i. by immunoblot. (b) Representative immunoblot from three separate experiments performed on macrophages derived from different donors. (c) Relative Mcl-1 levels obtained by densitometric analyses of western blots. Results from three separate experiments. Data represent means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. (d) Mcl-1 stability in macrophages incubated in the presence of cycloheximide (CHX) (10 μg/mL) in the absence (circles) or presence (squares) of S. aureus (MOI 1 : 50). At time periods of up to 3 h, Mcl-1 levels were detected by immunoblot in cell lysates. Data represents Mcl-1 levels relative to time 0, which was arbitrarily set as 100%, obtained by densitometric analyses of western blots. Data represent means ± SD of three separate experiments. *P < 0.05.
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Related In: Results  -  Collection

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fig2: S. aureus increases both de novo Mcl-1 synthesis and stability. (a) Time course of MCL1 expression in control and S. aureus-infected macrophages (hours after-infection; p.i.) was monitored by qRT-PCR, as described in Section 2. Data represent the mean values calculated from the results of three independent experiments using hMDMs derived from different donors. Bars represent mean relative expression ± SD. *P < 0.05; ***P < 0.001. (b, c) Time course of Mcl-1 protein synthesis following S. aureus infection. Mcl-1 levels were measured at different time points between 0.5 and 20 h p.i. by immunoblot. (b) Representative immunoblot from three separate experiments performed on macrophages derived from different donors. (c) Relative Mcl-1 levels obtained by densitometric analyses of western blots. Results from three separate experiments. Data represent means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. (d) Mcl-1 stability in macrophages incubated in the presence of cycloheximide (CHX) (10 μg/mL) in the absence (circles) or presence (squares) of S. aureus (MOI 1 : 50). At time periods of up to 3 h, Mcl-1 levels were detected by immunoblot in cell lysates. Data represents Mcl-1 levels relative to time 0, which was arbitrarily set as 100%, obtained by densitometric analyses of western blots. Data represent means ± SD of three separate experiments. *P < 0.05.
Mentions: To correlate S. aureus-induced cytoprotection with the expression of MCL1, we determined the time dependence of the induction of specific mRNA after macrophage challenge with bacteria. A 4-fold increase of MCL1 mRNA was observed 1 h after-infection, with sustained upregulation observed for up to 6 h (Figure 2(a)). At the protein level, Mcl-1 levels were significantly increased 2 h after-infection, reached a maximum at 8 h, and remained at 3-fold higher levels compared to mock-infected cells for at least 20 h (Figures 2(b) and 2(c)).

Bottom Line: Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis.Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection.Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Ul. Gronostajowa 7, 30-387 Kraków, Poland. joanna.koziel@uj.edu.pl

ABSTRACT
As a facultative intracellular pathogen, Staphylococcus aureus invades macrophages and then promotes the cytoprotection of infected cells thus stabilizing safe niche for silent persistence. This process occurs through the upregulation of crucial antiapoptotic genes, in particular, myeloid cell leukemia-1 (MCL-1). Here, we investigated the underlying mechanism and signal transduction pathways leading to increased MCL-1 expression in infected macrophages. Live S. aureus not only stimulated de novo synthesis of Mcl-1, but also prolonged the stability of this antiapoptotic protein. Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis. Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection. Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression. Therefore, we propose that S. aureus is hijacking the Mcl-1-dependent inhibition of apoptosis to prevent the elimination of infected host cells, thus allowing the intracellular persistence of the pathogen, its dissemination by infected macrophages, and the progression of staphylococci diseases.

Show MeSH
Related in: MedlinePlus