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Counter inhibition between leukotoxins attenuates Staphylococcus aureus virulence.

Yoong P, Torres VJ - Nat Commun (2015)

Bottom Line: Despite extensive sequence conservation, each leukotoxin has unique properties, including disparate cellular receptors and species specificities.Using in vivo murine models of infection, we show that LukSF-PV negatively influences S. aureus virulence and colonization by inhibiting LukED.Thus, while S. aureus leukotoxins can certainly injure immune cells, the discovery of leukotoxin antagonism suggests that they may also play a role in reducing S. aureus virulence and maintaining infection without killing the host.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, New York University School of Medicine, New York, New York 10016, USA.

ABSTRACT
Staphylococcus aureus subverts host defences by producing a collection of virulence factors including bi-component pore-forming leukotoxins. Despite extensive sequence conservation, each leukotoxin has unique properties, including disparate cellular receptors and species specificities. How these toxins collectively influence S. aureus pathogenesis is unknown. Here we demonstrate that the leukotoxins LukSF-PV and LukED antagonize each other's cytolytic activities on leukocytes and erythrocytes by forming inactive hybrid complexes. Remarkably, LukSF-PV inhibition of LukED haemolytic activity on both human and murine erythrocytes prevents the release of nutrients required for in vitro bacterial growth. Using in vivo murine models of infection, we show that LukSF-PV negatively influences S. aureus virulence and colonization by inhibiting LukED. Thus, while S. aureus leukotoxins can certainly injure immune cells, the discovery of leukotoxin antagonism suggests that they may also play a role in reducing S. aureus virulence and maintaining infection without killing the host.

No MeSH data available.


Related in: MedlinePlus

LukED inhibition of LukSF-PV-mediated toxicity on PMN-HL60 cells and primary human neutrophils.(a) PMN-HL60s were incubated with 1 μg ml−1 LukSF-PV or PBS in the presence of increasing concentrations of LukED. After 1 h incubation, cell lysis was monitored by measuring LDH release. (b) Culture supernatants from WT MW2 (WT LukSF-PV+) and an isogenic MW2 strain lacking lukSF-PV (ΔlukSF-PV) transformed with a plasmid overexpressing LukED (pOS-lukED), or a control plasmid (pOS) were incubated with PMN-HL60 cells at a final concentration of 10% (v/v). The formation of toxin-mediated pores in the plasma membrane of PMN-HL60s was monitored by ethidium bromide incorporation. (c) PMN-HL60 cells were first incubated with 1 μg ml−1 LukS-PV, any unbound LukS-PV washed away followed by addition of LukE and/or LukD, and finally, 1 μg ml−1 of LukF-PV. After 1 h incubation, cell lysis was monitored by measuring LDH release. (d) Binding of fluorescently labelled LukD680 nm to PMN-HL60 cells, in the presence or absence of 0.25 μg ml−1 LukS-PV monitored by fluorescence-activated cell sorting. (e) Primary human neutrophils were incubated with 50 ng ml−1 LukSF-PV with increasing concentrations of LukE or LukD. After 1 h incubation, cell metabolism was monitored using the CellTiter reagent. Results represent the averages from three or more independent experiments±s.e.m. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 using one-way (b) or two-way analysis of variance (a,c,e). Results in part D are representative of three independent experiments.
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f1: LukED inhibition of LukSF-PV-mediated toxicity on PMN-HL60 cells and primary human neutrophils.(a) PMN-HL60s were incubated with 1 μg ml−1 LukSF-PV or PBS in the presence of increasing concentrations of LukED. After 1 h incubation, cell lysis was monitored by measuring LDH release. (b) Culture supernatants from WT MW2 (WT LukSF-PV+) and an isogenic MW2 strain lacking lukSF-PV (ΔlukSF-PV) transformed with a plasmid overexpressing LukED (pOS-lukED), or a control plasmid (pOS) were incubated with PMN-HL60 cells at a final concentration of 10% (v/v). The formation of toxin-mediated pores in the plasma membrane of PMN-HL60s was monitored by ethidium bromide incorporation. (c) PMN-HL60 cells were first incubated with 1 μg ml−1 LukS-PV, any unbound LukS-PV washed away followed by addition of LukE and/or LukD, and finally, 1 μg ml−1 of LukF-PV. After 1 h incubation, cell lysis was monitored by measuring LDH release. (d) Binding of fluorescently labelled LukD680 nm to PMN-HL60 cells, in the presence or absence of 0.25 μg ml−1 LukS-PV monitored by fluorescence-activated cell sorting. (e) Primary human neutrophils were incubated with 50 ng ml−1 LukSF-PV with increasing concentrations of LukE or LukD. After 1 h incubation, cell metabolism was monitored using the CellTiter reagent. Results represent the averages from three or more independent experiments±s.e.m. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 using one-way (b) or two-way analysis of variance (a,c,e). Results in part D are representative of three independent experiments.

Mentions: The observation that PMN-HL60s are resistant to the hybrid leukotoxins, but not to LukSF-PV, prompted us to investigate if LukED and LukSF-PV could inhibit each other by forming inactive complexes. To this end, PMN-HL60 cells were incubated with LukSF-PV, in the presence or absence of increasing concentrations of LukED. As expected, in the absence of LukED, LukSF-PV resulted in complete lysis of PMN-HL60s. However, inclusion of LukED in 10–20 fold excess protected ∼50% of the cells (Fig. 1a).


Counter inhibition between leukotoxins attenuates Staphylococcus aureus virulence.

Yoong P, Torres VJ - Nat Commun (2015)

LukED inhibition of LukSF-PV-mediated toxicity on PMN-HL60 cells and primary human neutrophils.(a) PMN-HL60s were incubated with 1 μg ml−1 LukSF-PV or PBS in the presence of increasing concentrations of LukED. After 1 h incubation, cell lysis was monitored by measuring LDH release. (b) Culture supernatants from WT MW2 (WT LukSF-PV+) and an isogenic MW2 strain lacking lukSF-PV (ΔlukSF-PV) transformed with a plasmid overexpressing LukED (pOS-lukED), or a control plasmid (pOS) were incubated with PMN-HL60 cells at a final concentration of 10% (v/v). The formation of toxin-mediated pores in the plasma membrane of PMN-HL60s was monitored by ethidium bromide incorporation. (c) PMN-HL60 cells were first incubated with 1 μg ml−1 LukS-PV, any unbound LukS-PV washed away followed by addition of LukE and/or LukD, and finally, 1 μg ml−1 of LukF-PV. After 1 h incubation, cell lysis was monitored by measuring LDH release. (d) Binding of fluorescently labelled LukD680 nm to PMN-HL60 cells, in the presence or absence of 0.25 μg ml−1 LukS-PV monitored by fluorescence-activated cell sorting. (e) Primary human neutrophils were incubated with 50 ng ml−1 LukSF-PV with increasing concentrations of LukE or LukD. After 1 h incubation, cell metabolism was monitored using the CellTiter reagent. Results represent the averages from three or more independent experiments±s.e.m. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 using one-way (b) or two-way analysis of variance (a,c,e). Results in part D are representative of three independent experiments.
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f1: LukED inhibition of LukSF-PV-mediated toxicity on PMN-HL60 cells and primary human neutrophils.(a) PMN-HL60s were incubated with 1 μg ml−1 LukSF-PV or PBS in the presence of increasing concentrations of LukED. After 1 h incubation, cell lysis was monitored by measuring LDH release. (b) Culture supernatants from WT MW2 (WT LukSF-PV+) and an isogenic MW2 strain lacking lukSF-PV (ΔlukSF-PV) transformed with a plasmid overexpressing LukED (pOS-lukED), or a control plasmid (pOS) were incubated with PMN-HL60 cells at a final concentration of 10% (v/v). The formation of toxin-mediated pores in the plasma membrane of PMN-HL60s was monitored by ethidium bromide incorporation. (c) PMN-HL60 cells were first incubated with 1 μg ml−1 LukS-PV, any unbound LukS-PV washed away followed by addition of LukE and/or LukD, and finally, 1 μg ml−1 of LukF-PV. After 1 h incubation, cell lysis was monitored by measuring LDH release. (d) Binding of fluorescently labelled LukD680 nm to PMN-HL60 cells, in the presence or absence of 0.25 μg ml−1 LukS-PV monitored by fluorescence-activated cell sorting. (e) Primary human neutrophils were incubated with 50 ng ml−1 LukSF-PV with increasing concentrations of LukE or LukD. After 1 h incubation, cell metabolism was monitored using the CellTiter reagent. Results represent the averages from three or more independent experiments±s.e.m. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 using one-way (b) or two-way analysis of variance (a,c,e). Results in part D are representative of three independent experiments.
Mentions: The observation that PMN-HL60s are resistant to the hybrid leukotoxins, but not to LukSF-PV, prompted us to investigate if LukED and LukSF-PV could inhibit each other by forming inactive complexes. To this end, PMN-HL60 cells were incubated with LukSF-PV, in the presence or absence of increasing concentrations of LukED. As expected, in the absence of LukED, LukSF-PV resulted in complete lysis of PMN-HL60s. However, inclusion of LukED in 10–20 fold excess protected ∼50% of the cells (Fig. 1a).

Bottom Line: Despite extensive sequence conservation, each leukotoxin has unique properties, including disparate cellular receptors and species specificities.Using in vivo murine models of infection, we show that LukSF-PV negatively influences S. aureus virulence and colonization by inhibiting LukED.Thus, while S. aureus leukotoxins can certainly injure immune cells, the discovery of leukotoxin antagonism suggests that they may also play a role in reducing S. aureus virulence and maintaining infection without killing the host.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, New York University School of Medicine, New York, New York 10016, USA.

ABSTRACT
Staphylococcus aureus subverts host defences by producing a collection of virulence factors including bi-component pore-forming leukotoxins. Despite extensive sequence conservation, each leukotoxin has unique properties, including disparate cellular receptors and species specificities. How these toxins collectively influence S. aureus pathogenesis is unknown. Here we demonstrate that the leukotoxins LukSF-PV and LukED antagonize each other's cytolytic activities on leukocytes and erythrocytes by forming inactive hybrid complexes. Remarkably, LukSF-PV inhibition of LukED haemolytic activity on both human and murine erythrocytes prevents the release of nutrients required for in vitro bacterial growth. Using in vivo murine models of infection, we show that LukSF-PV negatively influences S. aureus virulence and colonization by inhibiting LukED. Thus, while S. aureus leukotoxins can certainly injure immune cells, the discovery of leukotoxin antagonism suggests that they may also play a role in reducing S. aureus virulence and maintaining infection without killing the host.

No MeSH data available.


Related in: MedlinePlus