Document Type

Dissertation

Date of Degree

Summer 2013

Degree Name

PhD (Doctor of Philosophy)

Degree In

Microbiology

First Advisor

Alexander Horswill

Abstract

Staphylococcus aureus is a known cause of chronic biofilm infections. Recent studies have demonstrated the importance of proteinaceous material in the biofilm matrix. S. aureus secretes at least ten proteases and there is growing evidence that these enzymes have self-cleavage roles that alter biofilm integrity. The goal of this dissertation is to characterize the function of secreted proteases with respect to S. aureus biofilms. In addition, we aimed to determine whether a known regulator of S. aureus proteases, the Regulator of toxins (Rot), is also a regulator of biofilms.

Studies presented in Chapter II utilize a mutation in Sigma factor B (SigB) to analyze the role of secreted proteases in biofilm formation. S. aureus strains with sigB mutations have enhanced protease activity and a biofilm negative phenotype, and this biofilm phenotype was conserved on human plasma coated surfaces. To identify the protease(s) responsible for the phenotype, inhibitor studies revealed that the addition of the cysteine protease inhibitor E-64, or Staphostatin inhibitors that specifically target the S. aureus cysteine proteases SspB or ScpA (Staphopains), could restore biofilm formation. Using gene deletion mutants, we identified that the combined mutation of sspB and scpA also restored ΔsigB biofilm formation. Together these findings indicated that Staphopain enzymes are responsible for the ΔsigB biofilm negative phenotype. To address the Staphopain inhibitory role in biofilms further, regulatory studies indicated that enzymes levels were maintained at a low level during biofilm formation and exogenous addition of purified Staphopains inhibited biofilm formation or disassembled established biofilms across multiple S. aureus strain lineages. Taken together, these findings suggest an unappreciated role of the Staphopains in biofilm maturation.

Studies in Chapter III examine the role of the transcriptional regulator rot in biofilm formation. Using both coated and uncoated biofilm assays, Rot was found to be essential for S. aureus biofilm formation. The expression of RNAIII, the major effecter molecule of the agr quorum-sensing system, inversely correlated with intracellular Rot protein concentration. Rot protein levels were found to be high under biofilm conditions and modulation of rot expression demonstrated that intracellular levels of Rot modulate biofilm formation. Examination of protease expression, production, and activity revealed that production of secreted proteases are inhibited by Rot and electrophoretic mobility shift assays demonstrated that inhibition was due to the direct binding of Rot to protease promoters. Chemical and biochemical protease inhibitors were able to restore the capacity to form a biofilm to rot and follow-up mutation of protease genes confirmed these findings. Finally, Δ rot mutant is attenuated in a murine catheter model of infection. These findings suggest an essential role for Rot as an inhibitor of proteases to modulate biofilm formation, and demonstrate the importance of coordinated regulation during infection.

Keywords

biofilm, protease, regulation, Rot, Staphopain, Staphylococcus aureus

Pages

x, 125 pages

Bibliography

Includes bibliographical references (pages 110-125).

Copyright

Copyright 2013 Joe Michael Mootz

Included in

Microbiology Commons

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