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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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After being phagocytosed by hMDM S. aureus strain Newman decreasingly persists intracellularly for several days until a burst of growth on day 6.A. CFU of S. aureus in cell lysates and culture medium on six consecutive days post-infection. Macrophages were allowed to engulf S. aureus at a MOI of 25 for 2 h, washed, and extracellular bacteria killed by gentamycin. Macrophages were cultured in media without antibiotics. At consecutive days post-phagocytosis media was aspired and hMDMs were lysed. Both conditioned media and cell lysates were plated onto TSA for CFU enumeration. The data shown details of one representative experiment (means±SD) of the 76 we performed in triplicate. B. CFU of S. aureus and LDH activity levels (A490 nm) in the conditioned culture medium on six consecutive days post infection (left panel); and transmission (upper row) and fluorescent (propidium iodide staining, lower row) light micrographs (x40) of S. aureus-infected cells maintained for the indicated time interval post infection. C. Confocal fluorescence microscopy images of hMDMs after 1, 3, and 5 days post-infection at MOI of 25. Cells were fixed, treated with RNase and stained with acridine orange (see Experimental Procedures section). Internalized bacteria are observed as green spots. All scale bars = 10 µm. D. Confocal fluorescence microscopy images of viable bacteria in hMDMs on the 5th day post-phagocytosis. Infected hMDMs were collected, permeabilized with 0.2 % Triton X-100 and double- labeled with propidium iodide and Syto9 (LIVE/DEAD BacLight Kit). Viable S. aureus cells are stained green while red signals represent dead bacteria and the host cell's nuclear DNA stained mainly with propidium. Scale bar = 7.5 µm. E. Specific PCR amplification of the S. aureus 16S rRNA gene (left panel) from cell lysates and the MLVF pattern (right panel) of bacteria infecting cells on consecutive days post-phagocytosis (numbers above lanes). Lane 0, sample collected immediately upon completion of phagocytosis (2h). Lane N, the MLVF pattern of S. aureus Newman before infection.
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pone-0001409-g003: After being phagocytosed by hMDM S. aureus strain Newman decreasingly persists intracellularly for several days until a burst of growth on day 6.A. CFU of S. aureus in cell lysates and culture medium on six consecutive days post-infection. Macrophages were allowed to engulf S. aureus at a MOI of 25 for 2 h, washed, and extracellular bacteria killed by gentamycin. Macrophages were cultured in media without antibiotics. At consecutive days post-phagocytosis media was aspired and hMDMs were lysed. Both conditioned media and cell lysates were plated onto TSA for CFU enumeration. The data shown details of one representative experiment (means±SD) of the 76 we performed in triplicate. B. CFU of S. aureus and LDH activity levels (A490 nm) in the conditioned culture medium on six consecutive days post infection (left panel); and transmission (upper row) and fluorescent (propidium iodide staining, lower row) light micrographs (x40) of S. aureus-infected cells maintained for the indicated time interval post infection. C. Confocal fluorescence microscopy images of hMDMs after 1, 3, and 5 days post-infection at MOI of 25. Cells were fixed, treated with RNase and stained with acridine orange (see Experimental Procedures section). Internalized bacteria are observed as green spots. All scale bars = 10 µm. D. Confocal fluorescence microscopy images of viable bacteria in hMDMs on the 5th day post-phagocytosis. Infected hMDMs were collected, permeabilized with 0.2 % Triton X-100 and double- labeled with propidium iodide and Syto9 (LIVE/DEAD BacLight Kit). Viable S. aureus cells are stained green while red signals represent dead bacteria and the host cell's nuclear DNA stained mainly with propidium. Scale bar = 7.5 µm. E. Specific PCR amplification of the S. aureus 16S rRNA gene (left panel) from cell lysates and the MLVF pattern (right panel) of bacteria infecting cells on consecutive days post-phagocytosis (numbers above lanes). Lane 0, sample collected immediately upon completion of phagocytosis (2h). Lane N, the MLVF pattern of S. aureus Newman before infection.

Mentions: Cultured hMDMs engulfed S. aureus strain Newman efficiently, with the majority of bacteria being internalized by 2 h. To determine the intracellular survival/killing rate of S. aureus cells the infected hMDMs were lysed at specific time points post-phagocytosis and plated onto TSA. The intracellular survival of S. aureus was dependent on the MOI. At MOI = 5 or 10 all of the intracellular bacteria were killed by 3 days postphagocytosis (data not shown). Conversely, at MOI = 25 the number of viable intracellular staphylococci decreased gradually between day 1 and day 5 by approximately five orders of magnitude compared to day 0 (Fig. 3A). Significantly, the conditioned medium of the infected cultures was sterile throughout. This suggests that hMDM effectively killed the internalized S. aureus, and the surviving bacteria did not escape intracellular confinement. Nevertheless, at higher MOI macrophages could not completely eradicate the bacteria, since an explosion of bacterial growth occurred on day 6, with similarly large numbers of S. aureus free in the medium and apparently adherent to cell corpses. At this time all macrophages that had appeared healthy on the preceding day had lost their plasma membrane integrity. Significantly, the same kinetic of S. aureus intracellular survival was observed in hMDMs cultivated for both 1 and 2 weeks, eliminating the possibility of macrophage aging triggering bacterial release (data not shown).


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)

After being phagocytosed by hMDM S. aureus strain Newman decreasingly persists intracellularly for several days until a burst of growth on day 6.A. CFU of S. aureus in cell lysates and culture medium on six consecutive days post-infection. Macrophages were allowed to engulf S. aureus at a MOI of 25 for 2 h, washed, and extracellular bacteria killed by gentamycin. Macrophages were cultured in media without antibiotics. At consecutive days post-phagocytosis media was aspired and hMDMs were lysed. Both conditioned media and cell lysates were plated onto TSA for CFU enumeration. The data shown details of one representative experiment (means±SD) of the 76 we performed in triplicate. B. CFU of S. aureus and LDH activity levels (A490 nm) in the conditioned culture medium on six consecutive days post infection (left panel); and transmission (upper row) and fluorescent (propidium iodide staining, lower row) light micrographs (x40) of S. aureus-infected cells maintained for the indicated time interval post infection. C. Confocal fluorescence microscopy images of hMDMs after 1, 3, and 5 days post-infection at MOI of 25. Cells were fixed, treated with RNase and stained with acridine orange (see Experimental Procedures section). Internalized bacteria are observed as green spots. All scale bars = 10 µm. D. Confocal fluorescence microscopy images of viable bacteria in hMDMs on the 5th day post-phagocytosis. Infected hMDMs were collected, permeabilized with 0.2 % Triton X-100 and double- labeled with propidium iodide and Syto9 (LIVE/DEAD BacLight Kit). Viable S. aureus cells are stained green while red signals represent dead bacteria and the host cell's nuclear DNA stained mainly with propidium. Scale bar = 7.5 µm. E. Specific PCR amplification of the S. aureus 16S rRNA gene (left panel) from cell lysates and the MLVF pattern (right panel) of bacteria infecting cells on consecutive days post-phagocytosis (numbers above lanes). Lane 0, sample collected immediately upon completion of phagocytosis (2h). Lane N, the MLVF pattern of S. aureus Newman before infection.
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Related In: Results  -  Collection

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pone-0001409-g003: After being phagocytosed by hMDM S. aureus strain Newman decreasingly persists intracellularly for several days until a burst of growth on day 6.A. CFU of S. aureus in cell lysates and culture medium on six consecutive days post-infection. Macrophages were allowed to engulf S. aureus at a MOI of 25 for 2 h, washed, and extracellular bacteria killed by gentamycin. Macrophages were cultured in media without antibiotics. At consecutive days post-phagocytosis media was aspired and hMDMs were lysed. Both conditioned media and cell lysates were plated onto TSA for CFU enumeration. The data shown details of one representative experiment (means±SD) of the 76 we performed in triplicate. B. CFU of S. aureus and LDH activity levels (A490 nm) in the conditioned culture medium on six consecutive days post infection (left panel); and transmission (upper row) and fluorescent (propidium iodide staining, lower row) light micrographs (x40) of S. aureus-infected cells maintained for the indicated time interval post infection. C. Confocal fluorescence microscopy images of hMDMs after 1, 3, and 5 days post-infection at MOI of 25. Cells were fixed, treated with RNase and stained with acridine orange (see Experimental Procedures section). Internalized bacteria are observed as green spots. All scale bars = 10 µm. D. Confocal fluorescence microscopy images of viable bacteria in hMDMs on the 5th day post-phagocytosis. Infected hMDMs were collected, permeabilized with 0.2 % Triton X-100 and double- labeled with propidium iodide and Syto9 (LIVE/DEAD BacLight Kit). Viable S. aureus cells are stained green while red signals represent dead bacteria and the host cell's nuclear DNA stained mainly with propidium. Scale bar = 7.5 µm. E. Specific PCR amplification of the S. aureus 16S rRNA gene (left panel) from cell lysates and the MLVF pattern (right panel) of bacteria infecting cells on consecutive days post-phagocytosis (numbers above lanes). Lane 0, sample collected immediately upon completion of phagocytosis (2h). Lane N, the MLVF pattern of S. aureus Newman before infection.
Mentions: Cultured hMDMs engulfed S. aureus strain Newman efficiently, with the majority of bacteria being internalized by 2 h. To determine the intracellular survival/killing rate of S. aureus cells the infected hMDMs were lysed at specific time points post-phagocytosis and plated onto TSA. The intracellular survival of S. aureus was dependent on the MOI. At MOI = 5 or 10 all of the intracellular bacteria were killed by 3 days postphagocytosis (data not shown). Conversely, at MOI = 25 the number of viable intracellular staphylococci decreased gradually between day 1 and day 5 by approximately five orders of magnitude compared to day 0 (Fig. 3A). Significantly, the conditioned medium of the infected cultures was sterile throughout. This suggests that hMDM effectively killed the internalized S. aureus, and the surviving bacteria did not escape intracellular confinement. Nevertheless, at higher MOI macrophages could not completely eradicate the bacteria, since an explosion of bacterial growth occurred on day 6, with similarly large numbers of S. aureus free in the medium and apparently adherent to cell corpses. At this time all macrophages that had appeared healthy on the preceding day had lost their plasma membrane integrity. Significantly, the same kinetic of S. aureus intracellular survival was observed in hMDMs cultivated for both 1 and 2 weeks, eliminating the possibility of macrophage aging triggering bacterial release (data not shown).

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