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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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Mcl-1 expression is induced specifically by S. aureus phagocytosis and is dependent on bacterial dose and viability. (a) The effect of phagocytosis of S. aureus (Sa), E. coli (Ec), and latex beads (LTX) on Mcl-1 protein levels was assessed by immunoblot. After phagocytosis, cells were cultured for 8 h and then protein fractions were prepared as described in Section 2. Representative immunoblot from three separate experiments performed on hMDMs derived from different donors is shown. Mcl-1 was visualised by immunoblot using anti-Mcl-1-specific antibodies. (b) The effect of S. aureus phagocytosis on Mcl-1 levels is proportional to an infection dose. A representative immunoblot from three separate experiments performed on hMDMs derived from different donors is shown. (c) The effect of phagocytosis of live (Sa) and dead, heat-killed S. aureus (Sa HI) on MCL1 expression was determined by qRT-PCR. Data are from independent reactions using hMDMs derived from different donors. Paired points represent the response of hMDMs obtained from the same donors to both live and dead bacteria. Bars represent relative means ± SD. ***P < 0.001; NS: not significant. (d) The comparison of expression levels of proapoptotic MCL1S (white bars) versus antiapoptotic MCL1 (filled bars). Results were obtained by qRT-PCR from three separate experiments. **P < 0.01.
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fig1: Mcl-1 expression is induced specifically by S. aureus phagocytosis and is dependent on bacterial dose and viability. (a) The effect of phagocytosis of S. aureus (Sa), E. coli (Ec), and latex beads (LTX) on Mcl-1 protein levels was assessed by immunoblot. After phagocytosis, cells were cultured for 8 h and then protein fractions were prepared as described in Section 2. Representative immunoblot from three separate experiments performed on hMDMs derived from different donors is shown. Mcl-1 was visualised by immunoblot using anti-Mcl-1-specific antibodies. (b) The effect of S. aureus phagocytosis on Mcl-1 levels is proportional to an infection dose. A representative immunoblot from three separate experiments performed on hMDMs derived from different donors is shown. (c) The effect of phagocytosis of live (Sa) and dead, heat-killed S. aureus (Sa HI) on MCL1 expression was determined by qRT-PCR. Data are from independent reactions using hMDMs derived from different donors. Paired points represent the response of hMDMs obtained from the same donors to both live and dead bacteria. Bars represent relative means ± SD. ***P < 0.001; NS: not significant. (d) The comparison of expression levels of proapoptotic MCL1S (white bars) versus antiapoptotic MCL1 (filled bars). Results were obtained by qRT-PCR from three separate experiments. **P < 0.01.

Mentions: We previously showed that S. aureus can protect infected macrophages against apoptosis through upregulation of expression of antiapoptotic genes [7]. Among these genes, MCL1 plays a key role in macrophage survival [13, 14]. Here, we investigated potential mechanisms of Mcl-1 regulation, as well as its role in cytoprotection induced by S. aureus. The Mcl-1 induction in response to phagocytosis of different bacteria and particles was examined by incubating macrophages with S. aureus, E. coli, or latex beads for 8 h. S. aureus induced the highest level of Mcl-1, about five-times more (4.78 ± 0.96-fold above the control level) than that seen in mock-infected cells (Figure 1(a)). By contrast, no change in Mcl-1 levels was observed after incubation with latex beads (1.06 ± 0.06) and only a slight upshift after E. coli (1.78 ± 0.63) (Figure 1(a)). The enhanced Mcl-1 production in response to S. aureus was proportional to infection rate (Figure 1(b)) and was exerted only by viable bacterial cells (Figure 1(c)). The latter was clearly apparent from a comparison of relative levels of MCL1 mRNA induced in response to live or dead bacteria in macrophages derived from different blood donors (8.87 versus 2.75, 4.36 versus 2.35, and 2.24 versus 0.4, resp.). An intriguing feature of S. aureus-induced Mcl-1 expression was the synthesis of an alternatively spliced MCL1 gene product (MCL1S proapoptotic isoform), which was observed at the mRNA level early after infection (Figure 1(d)). Significantly, however, the expression of MCL1S was at much lower level compared to the full-length, antiapoptotic MCL1 form (Figure 1(d)).


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)

Mcl-1 expression is induced specifically by S. aureus phagocytosis and is dependent on bacterial dose and viability. (a) The effect of phagocytosis of S. aureus (Sa), E. coli (Ec), and latex beads (LTX) on Mcl-1 protein levels was assessed by immunoblot. After phagocytosis, cells were cultured for 8 h and then protein fractions were prepared as described in Section 2. Representative immunoblot from three separate experiments performed on hMDMs derived from different donors is shown. Mcl-1 was visualised by immunoblot using anti-Mcl-1-specific antibodies. (b) The effect of S. aureus phagocytosis on Mcl-1 levels is proportional to an infection dose. A representative immunoblot from three separate experiments performed on hMDMs derived from different donors is shown. (c) The effect of phagocytosis of live (Sa) and dead, heat-killed S. aureus (Sa HI) on MCL1 expression was determined by qRT-PCR. Data are from independent reactions using hMDMs derived from different donors. Paired points represent the response of hMDMs obtained from the same donors to both live and dead bacteria. Bars represent relative means ± SD. ***P < 0.001; NS: not significant. (d) The comparison of expression levels of proapoptotic MCL1S (white bars) versus antiapoptotic MCL1 (filled bars). Results were obtained by qRT-PCR from three separate experiments. **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569898&req=5

fig1: Mcl-1 expression is induced specifically by S. aureus phagocytosis and is dependent on bacterial dose and viability. (a) The effect of phagocytosis of S. aureus (Sa), E. coli (Ec), and latex beads (LTX) on Mcl-1 protein levels was assessed by immunoblot. After phagocytosis, cells were cultured for 8 h and then protein fractions were prepared as described in Section 2. Representative immunoblot from three separate experiments performed on hMDMs derived from different donors is shown. Mcl-1 was visualised by immunoblot using anti-Mcl-1-specific antibodies. (b) The effect of S. aureus phagocytosis on Mcl-1 levels is proportional to an infection dose. A representative immunoblot from three separate experiments performed on hMDMs derived from different donors is shown. (c) The effect of phagocytosis of live (Sa) and dead, heat-killed S. aureus (Sa HI) on MCL1 expression was determined by qRT-PCR. Data are from independent reactions using hMDMs derived from different donors. Paired points represent the response of hMDMs obtained from the same donors to both live and dead bacteria. Bars represent relative means ± SD. ***P < 0.001; NS: not significant. (d) The comparison of expression levels of proapoptotic MCL1S (white bars) versus antiapoptotic MCL1 (filled bars). Results were obtained by qRT-PCR from three separate experiments. **P < 0.01.
Mentions: We previously showed that S. aureus can protect infected macrophages against apoptosis through upregulation of expression of antiapoptotic genes [7]. Among these genes, MCL1 plays a key role in macrophage survival [13, 14]. Here, we investigated potential mechanisms of Mcl-1 regulation, as well as its role in cytoprotection induced by S. aureus. The Mcl-1 induction in response to phagocytosis of different bacteria and particles was examined by incubating macrophages with S. aureus, E. coli, or latex beads for 8 h. S. aureus induced the highest level of Mcl-1, about five-times more (4.78 ± 0.96-fold above the control level) than that seen in mock-infected cells (Figure 1(a)). By contrast, no change in Mcl-1 levels was observed after incubation with latex beads (1.06 ± 0.06) and only a slight upshift after E. coli (1.78 ± 0.63) (Figure 1(a)). The enhanced Mcl-1 production in response to S. aureus was proportional to infection rate (Figure 1(b)) and was exerted only by viable bacterial cells (Figure 1(c)). The latter was clearly apparent from a comparison of relative levels of MCL1 mRNA induced in response to live or dead bacteria in macrophages derived from different blood donors (8.87 versus 2.75, 4.36 versus 2.35, and 2.24 versus 0.4, resp.). An intriguing feature of S. aureus-induced Mcl-1 expression was the synthesis of an alternatively spliced MCL1 gene product (MCL1S proapoptotic isoform), which was observed at the mRNA level early after infection (Figure 1(d)). Significantly, however, the expression of MCL1S was at much lower level compared to the full-length, antiapoptotic MCL1 form (Figure 1(d)).

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