title: Staphylococcus aureus Biofilms Induce Macrophage Dysfunction Through Leukocidin AB and Alpha-Toxin.
abstract: The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed by using hla and lukAB mutants, and synergy between the two toxins was demonstrated with a lukAB hla double mutant and verified by complementation. 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. The importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed by using a murine orthopedic implant biofilm infection model in which the lukAB hla double mutant displayed significantly lower bacterial burdens and more macrophage infiltrates than each single mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in promoting macrophage dysfunction and facilitating S. aureus biofilm development in vivo.
  
  
  OBJECTIVE
  Staphylococcus aureus has a propensity to form multicellular communities known as biofilms. While growing in a biofilm, S. aureus displays increased tolerance to nutrient deprivation, antibiotic insult, and even host immune challenge. Previous studies have shown that S. aureus biofilms thwart host immunity in part by preventing macrophage phagocytosis. It remained unclear whether this was influenced solely by the considerable size of biofilms or whether molecules were also actively secreted to circumvent macrophage-mediated phagocytosis. 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. Loss of leukocidin AB and alpha-toxin expression resulted in enhanced S. aureus biofilm clearance in a mouse model of orthopedic implant infection, suggesting that these toxins could be targeted therapeutically to facilitate biofilm clearance in humans.
authors: Departments of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.
authors: Tyler D
authors: TD
authors: Scherr
authors: Departments of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.
authors: Mark L
authors: ML
authors: Hanke
authors: Departments of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.
authors: Ouwen
authors: O
authors: Huang
authors: Department of Microbiology, New York University, New York, New York, USA.
authors: David B A
authors: DB
authors: James
authors: Departments of Microbiology and Immunology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA.
authors: Alexander R
authors: AR
authors: Horswill
authors: Departments of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.
authors: Kenneth W
authors: KW
authors: Bayles
authors: Departments of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.
authors: Paul D
authors: PD
authors: Fey
authors: Department of Microbiology, New York University, New York, New York, USA.
authors: Victor J
authors: VJ
authors: Torres
authors: Departments of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA tkielian@unmc.edu.
authors: Tammy
authors: T
authors: Kielian
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D018441:Biofilms; D017077:Culture Media, Conditioned; D004195:Disease Models, Animal; D006460:Hemolysin Proteins / Q000378:metabolism*; D006801:Humans; D007956:Leukocidins / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000276:immunology / Q000502:physiology*; D051379:Mice; D009154:Mutation; D010587:Phagocytosis; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017077:Culture Media, Conditioned; D006460:Hemolysin Proteins; D007956:Leukocidins; C586430:leukocidin AB, Staphylococcus aureus; C021091:staphylococcal alpha-toxin