136. CA-MRSA Activate Calpain/Caspase-1 Signaling to Invade through Human Keratinocytes
Session: Oral Abstract Session: Bacterial Pathogenesis and Virulence
Friday, October 21, 2011: 9:45 AM
Room: 151AB
Background: USA300 CA-MRSA are the most common cause of skin infection in the US, associated with over 11 million ER visits/year.  Multiple staphylococcal virulence factors are associated with these strains, identified primarily in murine and rabbit models of infection, which lack many of the key features of human skin. 

Methods: We used the human keratinocyte HaCat cell line grown in a polarized fashion to determine which of the major USA300 virulence factors are required for invasion. 

Results: The α hemolysin (Hla) but not PVL or SpA (protein A) was associated with invasion through the keratinocyte barrier at 24 hours, quantified by both increased permeability to 3000 MW fluorescent dextran and by enumerating cfus in the basal chamber of Transwells (P<0.0001 for both assays). Staphylococci were not internalized by the keratinocytes and cytochalasin D treatment increased S. aureus transmigration by 2-fold.  Keratinocytes were shown to constitutively express pro-IL-1β and were readily activated by Hla+ strains to release IL-1β (P<0.001 as compared with Hla-). Caspase-1 activity and pyroptosis were induced by Hla+ but not Hla- mutants. We monitored apoptosis using YO-PRO-1 staining, imaged by confocal microscopy and confirmed by flow cytometry.  HMGB1 production was immunoblotted as an indicator of pyroptosis.  Pretreatment of the keratinocytes with Z-VAD-FMK, an inhibitor of caspase-1 or blocking calpain activity (an intracellular protease that participates in programmed cell death) with calpeptin or BAPTA decreased IL-1β production, prevented USA300 invasion (P< 0.05 for each) and inhibited pyroptosis. 

Conclusion: These studies using human keratinocytes suggest that activation of the inflammasome via the pore forming toxin Hla initiates apoptosis then pyroptosis causing local areas of cell death that provide a nidus for the invasion of these non-motile organisms through the keratinocyte barrier.


Subject Category: B. Bacterial pathogenesis, studies in animal models, molecular pathogenicity

Grace Soong, BS, Jarin Chun, PhD, Bryan Harfenist, BA and Alice Prince, MD, FIDSA, Pediatrics, Columbia University, New York, NY

Disclosures:

G. Soong, None

J. Chun, None

B. Harfenist, None

A. Prince, None

Findings in the abstracts are embargoed until 12:01 a.m. EST Thursday, Oct. 20 with the exception of research findings presented at IDSA press conferences.