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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

LukSF-PV inhibits LukED cytotoxic and haemolytic activity.(a) Primary murine bone marrow cells were incubated with LukED (1 μg ml−1) and either LukS-PV or LukF-PV (50 μg ml−1) for 1 h and cell metabolism measured using the CellTiter reagent. (b) Assay plate containing primary murine bone marrow cells in the presence or absence of indicated toxins, post centrifugation. Picture highlights red blood cell pellets in samples treated with LukED (E+D), LukE (E) or LukD (D) in the presence of increasing concentrations of LukSF-PV. (c) Primary murine (left panel) and primary human (right panel) RBCs were incubated with or without LukED in the presence of increasing concentrations of LukSF-PV or LukAB and haemolysis of the RBCs monitored. (d) Wildtype CA-MRSA strains USA400-MW2 and USA300-LAC (WT LukSF-PV+), and their isogenic ΔlukSF-PV counterparts were transformed with a plasmid overexpressing LukED (pOS-lukED), or a control plasmid (pOS), followed by incubation with human red blood cells at a MOI of 100. RBC lysis was measured at 405 nm after 1 h. 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 (a,d) or two-way analysis of variance (c).
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f2: LukSF-PV inhibits LukED cytotoxic and haemolytic activity.(a) Primary murine bone marrow cells were incubated with LukED (1 μg ml−1) and either LukS-PV or LukF-PV (50 μg ml−1) for 1 h and cell metabolism measured using the CellTiter reagent. (b) Assay plate containing primary murine bone marrow cells in the presence or absence of indicated toxins, post centrifugation. Picture highlights red blood cell pellets in samples treated with LukED (E+D), LukE (E) or LukD (D) in the presence of increasing concentrations of LukSF-PV. (c) Primary murine (left panel) and primary human (right panel) RBCs were incubated with or without LukED in the presence of increasing concentrations of LukSF-PV or LukAB and haemolysis of the RBCs monitored. (d) Wildtype CA-MRSA strains USA400-MW2 and USA300-LAC (WT LukSF-PV+), and their isogenic ΔlukSF-PV counterparts were transformed with a plasmid overexpressing LukED (pOS-lukED), or a control plasmid (pOS), followed by incubation with human red blood cells at a MOI of 100. RBC lysis was measured at 405 nm after 1 h. 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 (a,d) or two-way analysis of variance (c).

Mentions: To determine the potential contribution of LukED/LukSF-PV antagonism to S. aureus pathogenesis in vivo, we first tested if this phenomenon was observed using murine leukocytes. LukED is highly active against murine leukocytes, which has been shown to be critical for the lethality observed in mice infected systemically with S. aureus91024. In contrast, LukSF-PV is unable to kill murine leukocytes (Table 2 and Supplementary Fig. 2F)25, a finding consistent with the specific tropism exhibited by this toxin towards the human C5aR receptor11. Thus, we evaluated the potential for LukSF-PV to inhibit LukED leukocidal activity. Primary murine leukocytes isolated from bone marrow, which consisted mostly of neutrophils, monocytes and macrophages, were incubated with LukED alone, or LukED with a 25-fold molar excess of LukS-PV, or LukF-PV. As expected, exposure to LukED resulted in the death of ∼20% murine leukocytes (Fig. 2a). LukED-mediated killing of these cells was found to be unaffected by the presence of excess LukS-PV, while LukF-PV protected the cells reducing cell death to <5% (Fig. 2a). Consistent with the data from PMN-HL60 cells and primary human neutrophils, it is the inert ‘F' subunit of LukSF-PV that antagonizes the cytotoxic activity of LukED.


Counter inhibition between leukotoxins attenuates Staphylococcus aureus virulence.

Yoong P, Torres VJ - Nat Commun (2015)

LukSF-PV inhibits LukED cytotoxic and haemolytic activity.(a) Primary murine bone marrow cells were incubated with LukED (1 μg ml−1) and either LukS-PV or LukF-PV (50 μg ml−1) for 1 h and cell metabolism measured using the CellTiter reagent. (b) Assay plate containing primary murine bone marrow cells in the presence or absence of indicated toxins, post centrifugation. Picture highlights red blood cell pellets in samples treated with LukED (E+D), LukE (E) or LukD (D) in the presence of increasing concentrations of LukSF-PV. (c) Primary murine (left panel) and primary human (right panel) RBCs were incubated with or without LukED in the presence of increasing concentrations of LukSF-PV or LukAB and haemolysis of the RBCs monitored. (d) Wildtype CA-MRSA strains USA400-MW2 and USA300-LAC (WT LukSF-PV+), and their isogenic ΔlukSF-PV counterparts were transformed with a plasmid overexpressing LukED (pOS-lukED), or a control plasmid (pOS), followed by incubation with human red blood cells at a MOI of 100. RBC lysis was measured at 405 nm after 1 h. 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 (a,d) or two-way analysis of variance (c).
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Related In: Results  -  Collection

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f2: LukSF-PV inhibits LukED cytotoxic and haemolytic activity.(a) Primary murine bone marrow cells were incubated with LukED (1 μg ml−1) and either LukS-PV or LukF-PV (50 μg ml−1) for 1 h and cell metabolism measured using the CellTiter reagent. (b) Assay plate containing primary murine bone marrow cells in the presence or absence of indicated toxins, post centrifugation. Picture highlights red blood cell pellets in samples treated with LukED (E+D), LukE (E) or LukD (D) in the presence of increasing concentrations of LukSF-PV. (c) Primary murine (left panel) and primary human (right panel) RBCs were incubated with or without LukED in the presence of increasing concentrations of LukSF-PV or LukAB and haemolysis of the RBCs monitored. (d) Wildtype CA-MRSA strains USA400-MW2 and USA300-LAC (WT LukSF-PV+), and their isogenic ΔlukSF-PV counterparts were transformed with a plasmid overexpressing LukED (pOS-lukED), or a control plasmid (pOS), followed by incubation with human red blood cells at a MOI of 100. RBC lysis was measured at 405 nm after 1 h. 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 (a,d) or two-way analysis of variance (c).
Mentions: To determine the potential contribution of LukED/LukSF-PV antagonism to S. aureus pathogenesis in vivo, we first tested if this phenomenon was observed using murine leukocytes. LukED is highly active against murine leukocytes, which has been shown to be critical for the lethality observed in mice infected systemically with S. aureus91024. In contrast, LukSF-PV is unable to kill murine leukocytes (Table 2 and Supplementary Fig. 2F)25, a finding consistent with the specific tropism exhibited by this toxin towards the human C5aR receptor11. Thus, we evaluated the potential for LukSF-PV to inhibit LukED leukocidal activity. Primary murine leukocytes isolated from bone marrow, which consisted mostly of neutrophils, monocytes and macrophages, were incubated with LukED alone, or LukED with a 25-fold molar excess of LukS-PV, or LukF-PV. As expected, exposure to LukED resulted in the death of ∼20% murine leukocytes (Fig. 2a). LukED-mediated killing of these cells was found to be unaffected by the presence of excess LukS-PV, while LukF-PV protected the cells reducing cell death to <5% (Fig. 2a). Consistent with the data from PMN-HL60 cells and primary human neutrophils, it is the inert ‘F' subunit of LukSF-PV that antagonizes the cytotoxic activity of LukED.

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