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Staphylococcus aureus Biofilms Induce Macrophage Dysfunction Through Leukocidin AB and Alpha-Toxin.

Scherr TD, Hanke ML, Huang O, James DB, Horswill AR, Bayles KW, Fey PD, Torres VJ, Kielian T - MBio (2015)

Bottom Line: Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent.Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide.This is the first report to demonstrate that S. aureus biofilms inhibit macrophage phagocytosis and induce macrophage death through the combined action of leukocidin AB and alpha-toxin.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

No MeSH data available.


Related in: MedlinePlus

S. aureus Hla and LukAB act in concert to promote macrophage dysfunction. Bone marrow-derived macrophages were exposed to fresh or conditioned medium from S. aureus WT or isogenic ΔlukA mutant, ΔlukB mutant, and ΔlukAB hla double mutant biofilms plus Hla Ab (α-Hla). After a 3-h treatment period, phagocytosis of fluorescent microspheres (A) and viable macrophages (B) were quantitated by confocal microscopy. Significant differences are denoted by asterisks (***, P < 0.001; unpaired two-tailed Student t test). Results are representative of at least three independent experiments.
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fig6: S. aureus Hla and LukAB act in concert to promote macrophage dysfunction. Bone marrow-derived macrophages were exposed to fresh or conditioned medium from S. aureus WT or isogenic ΔlukA mutant, ΔlukB mutant, and ΔlukAB hla double mutant biofilms plus Hla Ab (α-Hla). After a 3-h treatment period, phagocytosis of fluorescent microspheres (A) and viable macrophages (B) were quantitated by confocal microscopy. Significant differences are denoted by asterisks (***, P < 0.001; unpaired two-tailed Student t test). Results are representative of at least three independent experiments.

Mentions: The expression of both Hla and LukAB was markedly greater in conditioned medium from WT biofilms than in conditioned medium from planktonic bacteria (Fig. 4). Therefore, to assess whether LukAB and Hla act cooperatively to effect macrophage activity, ΔlukA and ΔlukB mutant biofilm-conditioned media were treated with an Hla-neutralizing Ab (Fig. 6). Interestingly, negating the action of Hla in ΔlukA and ΔlukB mutant biofilm-conditioned media significantly improved macrophage phagocytosis over that in supernatants where Hla was active (Fig. 6A). Similar findings were obtained with a ΔlukAB Δhla double mutant (Fig. 6A). Hla blockade in ΔlukA and ΔlukB mutant biofilm-conditioned media had no additional effect on macrophage survival, which was not unexpected since viability had nearly been restored with each of the single mutants to levels observed in fresh medium (Fig. 6B). When macrophages were treated with ΔlukAB mutant biofilm-conditioned medium (which still produces Hla) in combination with purified LukAB or a point mutant that lacks lytic activity (LukABE323A) (56), only bioactive LukAB returned both phagocytic inhibition and cytotoxicity to levels observed in WT biofilm-conditioned medium (see Fig. S4 in the supplemental material). Collectively, these results demonstrate that LukAB acts in concert with Hla to induce macrophage dysfunction.


Staphylococcus aureus Biofilms Induce Macrophage Dysfunction Through Leukocidin AB and Alpha-Toxin.

Scherr TD, Hanke ML, Huang O, James DB, Horswill AR, Bayles KW, Fey PD, Torres VJ, Kielian T - MBio (2015)

S. aureus Hla and LukAB act in concert to promote macrophage dysfunction. Bone marrow-derived macrophages were exposed to fresh or conditioned medium from S. aureus WT or isogenic ΔlukA mutant, ΔlukB mutant, and ΔlukAB hla double mutant biofilms plus Hla Ab (α-Hla). After a 3-h treatment period, phagocytosis of fluorescent microspheres (A) and viable macrophages (B) were quantitated by confocal microscopy. Significant differences are denoted by asterisks (***, P < 0.001; unpaired two-tailed Student t test). Results are representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4550693&req=5

fig6: S. aureus Hla and LukAB act in concert to promote macrophage dysfunction. Bone marrow-derived macrophages were exposed to fresh or conditioned medium from S. aureus WT or isogenic ΔlukA mutant, ΔlukB mutant, and ΔlukAB hla double mutant biofilms plus Hla Ab (α-Hla). After a 3-h treatment period, phagocytosis of fluorescent microspheres (A) and viable macrophages (B) were quantitated by confocal microscopy. Significant differences are denoted by asterisks (***, P < 0.001; unpaired two-tailed Student t test). Results are representative of at least three independent experiments.
Mentions: The expression of both Hla and LukAB was markedly greater in conditioned medium from WT biofilms than in conditioned medium from planktonic bacteria (Fig. 4). Therefore, to assess whether LukAB and Hla act cooperatively to effect macrophage activity, ΔlukA and ΔlukB mutant biofilm-conditioned media were treated with an Hla-neutralizing Ab (Fig. 6). Interestingly, negating the action of Hla in ΔlukA and ΔlukB mutant biofilm-conditioned media significantly improved macrophage phagocytosis over that in supernatants where Hla was active (Fig. 6A). Similar findings were obtained with a ΔlukAB Δhla double mutant (Fig. 6A). Hla blockade in ΔlukA and ΔlukB mutant biofilm-conditioned media had no additional effect on macrophage survival, which was not unexpected since viability had nearly been restored with each of the single mutants to levels observed in fresh medium (Fig. 6B). When macrophages were treated with ΔlukAB mutant biofilm-conditioned medium (which still produces Hla) in combination with purified LukAB or a point mutant that lacks lytic activity (LukABE323A) (56), only bioactive LukAB returned both phagocytic inhibition and cytotoxicity to levels observed in WT biofilm-conditioned medium (see Fig. S4 in the supplemental material). Collectively, these results demonstrate that LukAB acts in concert with Hla to induce macrophage dysfunction.

Bottom Line: Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent.Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide.This is the first report to demonstrate that S. aureus biofilms inhibit macrophage phagocytosis and induce macrophage death through the combined action of leukocidin AB and alpha-toxin.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

No MeSH data available.


Related in: MedlinePlus