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theses

Biofilms are communities of microorganisms attached to a surface or each other. Biofilm associated cells are the etiologic agents of recurrent Staphylococcus aureus infections. Oxygen is utilized by S. aureus as a terminal electron acceptor (TEA). Infected human tissues are hypoxic or anoxic. S. aureus increases biofilm formation in response to hypoxia, but how this occurs is unknown. This thesis reports that oxygen influences biofilm formation in its capacity as a TEA for cellular respiration. Genetic, physiological, or chemical inhibition of respiratory processes elicited increased biofilm formation. Impaired respiration led to increased cell lysis via divergent regulation of two processes: increased expression of the AtlA murein hydrolase and decreased expression of the AtlA-inhibitory glycopolymers, WTA. The AltA-dependent release of cytosolic DNA contributed to increased biofilm formation. The fibronectin binding protein A, which is known to interact with AtlA, was also found to be involved in fermentative biofilm formation. Further, cell lysis and biofilm formation were governed by the SrrAB and the SaeRS two-component regulatory systems (TCRS). Genetic evidence suggests that SrrAB-dependent biofilm formation occurs in response to the accumulation of reduced menaquinone. SaeRS-dependent biofilm formation also occurred in response to changes in the respiratory status of the cell, via an as yet undefined signal molecule(s). Further, a high cellular titer of phosphorylated SaeR is required for biofilm formation. Epistasis analyses found that SaeRS and SrrAB influence biofilm formation independent of one another, in vitro. SrrAB and SaeRS governed host colonization in vivo, in the context of a mouse model of orthopedic implant-associated biofilm formation. Of these two TCRS, SrrAB is the dominant system driving biofilm formation in vivo. Biofilms impart protection from innate immunity as well as therapeutic agents. Data presented suggest that pre-formed biofilms, established by fermenting S. aureus, can be prompted to detach and disperse upon exposure to a TEA (oxygen or nitrate). Exposure to oxygen (reaeration) results in increased growth but decreased transcription of atlA and decreased release of DNA. Reaeration is also accompanied by increased transcription of sspA which encodes for a protease capable of cleaving AtlA. Biofilm dispersal was blocked in a strain that is incapable of respiration, suggesting changes in cellular respiratory status are being sensed to trigger dispersal. The transcription of atlA and sspA upon reaeration was modulated in a divergent manner by SrrAB. Data presented suggest that SrrAB achieves divergent regulation of atlA, in two separate growth conditions, via the small RNA, rsaE, as an intermediary. In summation, the results presented define the bases for how oxygen dictates the lifestyle choices of S. aureus. The studies also establish the mechanistic and regulatory bases underlying the formation of anaerobic and fermentative biofilms by S. aureus.

ETD_7894

Ph.D.

Includes bibliographical references

by Ameya Ashutosh Mashruwala

doi:10.7282/T3RX9FZK

ETD doctoral

The author owns the copyright to this work.