Graduation Year

2010

Document Type

Thesis

Degree

M.S.

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Lindsey N. Shaw, Ph.D.

Co-Major Professor

Stanley M. Stevens, Ph.D.

Committee Member

James T. Riordan, Ph.D.

Keywords

Staphylococcus aureus, proteomics, protein extraction, community-acquired MRSA, hospital-acquired MRSA

Abstract

Staphylococcus aureus

is a bacterial pathogen that is believed to be the most common agent of human infectious disease, causing conditions ranging from common skin lesions to life-threatening illnesses. S. aureus has also shown a remarkable ability to develop resistance to antimicrobial treatment, making infections difficult to treat. In the post-genomic era, proteomic studies analyzing the protein complement of a genome in a particular organism at any given time, have gained real significance. This result is largely due to dynamic changes in protein expression profiles which can lead wide alterations in physiology and behavior. For proteomics, it is necessary to maximize protein concentration and to devise a method that can be easily employed and provide reproducible results. Most proteomic studies of S. aureus involve 2D gel electrophoresis (2-DE); however, 2-DE has many drawbacks. Proteins that are too large, hydrophobic, acidic, or basic are poorly resolved. Multi-dimensional protein identification (MudPIT) allows complex protein samples to be analyzed in solution. As yet, there has not been a study involving solely 2D liquid chromatography followed by mass spectrometric analysis in S. aureus

; therefore we sought to catalogue the intracellular proteome and secretome of a commonly used and well-studied lab strain, SH1000. This was conducted during post-exponential and stationary phases of growth so as to understand its adaptation over time by utilizing differential protein synthesis. We found cytoplasmic proteins involved in glycolysis to be highly expressed in post-exponential phase while proteins involved in tricarboxylic acid cycle to be prevalent in stationary phase. We also found

production of

agr-regulated secreted toxins and proteases to be upregulated in stationary phase. In addition to this we employed proteomic approaches to quantitatively profile the secretomes of leading clinical isolates of S. aureus, as such a study is currently lacking. These included the two most common hospital-associated S. aureus strains (USA100 and USA200), and the two most common community-associated S. aureus strains (USA300 and USA400). We found agr-regulated proteins are generally upregulated in CA-MRSA strains USA300 and USA400 and surface-associated proteins to be upregulated in HA-MRSA strains USA100 and USA200. This finding concurs with literature regarding transcriptomic studies showing a hyperactive agr in CA-MRSA strains compared to HA-MRSA strains.

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