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A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages.

Kubica M, Guzik K, Koziel J, Zarebski M, Richter W, Gajkowska B, Golda A, Maciag-Gudowska A, Brix K, Shaw L, Foster T, Potempa J - PLoS ONE (2008)

Bottom Line: In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages.Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types.S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. staphaureus@gmail.com

ABSTRACT
Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-gamma at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in alpha-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.

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Internalization of S. aureus strain Newman does not affect hMDM viability until the plasma membrane is permeabilized.A. Transmission light (upper two rows) and fluorescence (lower two rows) micrographs (x 40) of control (upper panel) and S. aureus-infected cells (bottom panel) maintained in culture for 2h (A) and 1 day (B), 4- (C), 5- (D), and 6-days (E). Propidium iodide-positive cells represent infected host cells with leaky plasma membranes. B. BODIPY495/503 staining of lipid droplets of control and S. aureus infected hMDM cultures on five consecutive days post-phagocytosis. Cells were permeabilized with 0.2 % Triton X-100 and stained as described in the Materials and Methods section. All scale bars = 10 µm. C. Transmission electron micrographs of control (panel A) and infected cells (panels B and C) one day post-phagocytosis. Black arrowheads point to lipid droplets. Magnification: x10,000 (A and C) and ×7,500 (B). The photographs presented are representative of a minimum of 20 fields observed. D. Cytotoxicity (%) of S. aureus infection. Plasma membrane permeabilization or cell lysis induced in macrophage cultures by S. aureus infection at different MOI was determined as LDH activity levels. Cytotoxicity was calculated according to the formula: % cytotoxicity = [(experimental value–low control)/(high control–low control)] × 100, where a low control is the LDH activity in the conditioned medium of the control non-infected culture, while the LDH activity in the whole cell culture with cells lysed with detergent (2% Triton X-100) constitutes a high control. An experimental value was the activity in the conditioned medium from the culture infected with S. aureus. According to this calculation the control non-infected culture was assumed to show 0% cytotoxicity. All assays were performed in triplicate.
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pone-0001409-g001: Internalization of S. aureus strain Newman does not affect hMDM viability until the plasma membrane is permeabilized.A. Transmission light (upper two rows) and fluorescence (lower two rows) micrographs (x 40) of control (upper panel) and S. aureus-infected cells (bottom panel) maintained in culture for 2h (A) and 1 day (B), 4- (C), 5- (D), and 6-days (E). Propidium iodide-positive cells represent infected host cells with leaky plasma membranes. B. BODIPY495/503 staining of lipid droplets of control and S. aureus infected hMDM cultures on five consecutive days post-phagocytosis. Cells were permeabilized with 0.2 % Triton X-100 and stained as described in the Materials and Methods section. All scale bars = 10 µm. C. Transmission electron micrographs of control (panel A) and infected cells (panels B and C) one day post-phagocytosis. Black arrowheads point to lipid droplets. Magnification: x10,000 (A and C) and ×7,500 (B). The photographs presented are representative of a minimum of 20 fields observed. D. Cytotoxicity (%) of S. aureus infection. Plasma membrane permeabilization or cell lysis induced in macrophage cultures by S. aureus infection at different MOI was determined as LDH activity levels. Cytotoxicity was calculated according to the formula: % cytotoxicity = [(experimental value–low control)/(high control–low control)] × 100, where a low control is the LDH activity in the conditioned medium of the control non-infected culture, while the LDH activity in the whole cell culture with cells lysed with detergent (2% Triton X-100) constitutes a high control. An experimental value was the activity in the conditioned medium from the culture infected with S. aureus. According to this calculation the control non-infected culture was assumed to show 0% cytotoxicity. All assays were performed in triplicate.

Mentions: Previous reports have shown that hMDMs and murine macrophages that have ingested virulent bacteria such as Streptococcus pneumoniae, Shigella flexnerii, Burkholderia pseudomallei, Yersinia ssp., Salmonella typhimurium, die by cytolytic processes that involve cell swelling, plasma membrane disintegration and karyolysis or by apoptosis [50]–[55]. Contrary to this we have found that the phagocytosis of opsonised S. aureus strain Newman by hMDMs in ratios bacteria to macrophages of up to 50:1 (multiplicity of infection, MOI = 50), led to a prolonged, survival of bacteria in phagocytes (see below) that did not affect the viability of the host cells. Indeed hMDMs maintained an intact plasma membrane up to 5 days post-phagocytosis as discerned by a lack of staining with propidium iodide (Fig. 1A), an impermeable fluorophore which can only enter cells with compromised plasma membranes. The infected cells were metabolically active for up to 5 days after uptake of S. aureus as indicated by a MTT cell proliferation and cytotoxicity assay which measures the reduction of tetrazolium (MTT) into an insoluble formazan product in viable cells (data not shown). Furthermore, the intracellular staphylococci apparently stimulated lipid metabolism since infected cells were stained with Bodipy 493/503, a marker of neutral lipids and lipid droplets, more vividly than control cells. This difference was visible at 1 day post-uptake (Fig. 1B). At this time point the enhanced accumulation of lipid inside infected cells was also apparent by TEM analysis (Fig. 1C).


A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages.

Kubica M, Guzik K, Koziel J, Zarebski M, Richter W, Gajkowska B, Golda A, Maciag-Gudowska A, Brix K, Shaw L, Foster T, Potempa J - PLoS ONE (2008)

Internalization of S. aureus strain Newman does not affect hMDM viability until the plasma membrane is permeabilized.A. Transmission light (upper two rows) and fluorescence (lower two rows) micrographs (x 40) of control (upper panel) and S. aureus-infected cells (bottom panel) maintained in culture for 2h (A) and 1 day (B), 4- (C), 5- (D), and 6-days (E). Propidium iodide-positive cells represent infected host cells with leaky plasma membranes. B. BODIPY495/503 staining of lipid droplets of control and S. aureus infected hMDM cultures on five consecutive days post-phagocytosis. Cells were permeabilized with 0.2 % Triton X-100 and stained as described in the Materials and Methods section. All scale bars = 10 µm. C. Transmission electron micrographs of control (panel A) and infected cells (panels B and C) one day post-phagocytosis. Black arrowheads point to lipid droplets. Magnification: x10,000 (A and C) and ×7,500 (B). The photographs presented are representative of a minimum of 20 fields observed. D. Cytotoxicity (%) of S. aureus infection. Plasma membrane permeabilization or cell lysis induced in macrophage cultures by S. aureus infection at different MOI was determined as LDH activity levels. Cytotoxicity was calculated according to the formula: % cytotoxicity = [(experimental value–low control)/(high control–low control)] × 100, where a low control is the LDH activity in the conditioned medium of the control non-infected culture, while the LDH activity in the whole cell culture with cells lysed with detergent (2% Triton X-100) constitutes a high control. An experimental value was the activity in the conditioned medium from the culture infected with S. aureus. According to this calculation the control non-infected culture was assumed to show 0% cytotoxicity. All assays were performed in triplicate.
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Related In: Results  -  Collection

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pone-0001409-g001: Internalization of S. aureus strain Newman does not affect hMDM viability until the plasma membrane is permeabilized.A. Transmission light (upper two rows) and fluorescence (lower two rows) micrographs (x 40) of control (upper panel) and S. aureus-infected cells (bottom panel) maintained in culture for 2h (A) and 1 day (B), 4- (C), 5- (D), and 6-days (E). Propidium iodide-positive cells represent infected host cells with leaky plasma membranes. B. BODIPY495/503 staining of lipid droplets of control and S. aureus infected hMDM cultures on five consecutive days post-phagocytosis. Cells were permeabilized with 0.2 % Triton X-100 and stained as described in the Materials and Methods section. All scale bars = 10 µm. C. Transmission electron micrographs of control (panel A) and infected cells (panels B and C) one day post-phagocytosis. Black arrowheads point to lipid droplets. Magnification: x10,000 (A and C) and ×7,500 (B). The photographs presented are representative of a minimum of 20 fields observed. D. Cytotoxicity (%) of S. aureus infection. Plasma membrane permeabilization or cell lysis induced in macrophage cultures by S. aureus infection at different MOI was determined as LDH activity levels. Cytotoxicity was calculated according to the formula: % cytotoxicity = [(experimental value–low control)/(high control–low control)] × 100, where a low control is the LDH activity in the conditioned medium of the control non-infected culture, while the LDH activity in the whole cell culture with cells lysed with detergent (2% Triton X-100) constitutes a high control. An experimental value was the activity in the conditioned medium from the culture infected with S. aureus. According to this calculation the control non-infected culture was assumed to show 0% cytotoxicity. All assays were performed in triplicate.
Mentions: Previous reports have shown that hMDMs and murine macrophages that have ingested virulent bacteria such as Streptococcus pneumoniae, Shigella flexnerii, Burkholderia pseudomallei, Yersinia ssp., Salmonella typhimurium, die by cytolytic processes that involve cell swelling, plasma membrane disintegration and karyolysis or by apoptosis [50]–[55]. Contrary to this we have found that the phagocytosis of opsonised S. aureus strain Newman by hMDMs in ratios bacteria to macrophages of up to 50:1 (multiplicity of infection, MOI = 50), led to a prolonged, survival of bacteria in phagocytes (see below) that did not affect the viability of the host cells. Indeed hMDMs maintained an intact plasma membrane up to 5 days post-phagocytosis as discerned by a lack of staining with propidium iodide (Fig. 1A), an impermeable fluorophore which can only enter cells with compromised plasma membranes. The infected cells were metabolically active for up to 5 days after uptake of S. aureus as indicated by a MTT cell proliferation and cytotoxicity assay which measures the reduction of tetrazolium (MTT) into an insoluble formazan product in viable cells (data not shown). Furthermore, the intracellular staphylococci apparently stimulated lipid metabolism since infected cells were stained with Bodipy 493/503, a marker of neutral lipids and lipid droplets, more vividly than control cells. This difference was visible at 1 day post-uptake (Fig. 1B). At this time point the enhanced accumulation of lipid inside infected cells was also apparent by TEM analysis (Fig. 1C).

Bottom Line: In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages.Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types.S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. staphaureus@gmail.com

ABSTRACT
Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-gamma at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in alpha-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.

Show MeSH
Related in: MedlinePlus