Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of fatal bacterial infections in hospitals and has become a global health threat. Multi-drug resistance is the main reason for the failure of therapy. Although the antibiotic resistance mechanisms in bacteria have been studied for decades, there are few attempts at systems-wide investigations into how the bacteria respond to antibiotic stress. In this thesis, spectral counting-based label-free quantitative proteomics has been applied to study global responses in S. aureus treated with antibiotics. We developed a simple and easily repeated sample preparation procedure that is effective for extracting surface-associated proteins for the following spectral counting-based label-free quantitative proteomics experiments....[ Read more ]

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of fatal bacterial infections in hospitals and has become a global health threat. Multi-drug resistance is the main reason for the failure of therapy. Although the antibiotic resistance mechanisms in bacteria have been studied for decades, there are few attempts at systems-wide investigations into how the bacteria respond to antibiotic stress. In this thesis, spectral counting-based label-free quantitative proteomics has been applied to study global responses in S. aureus treated with antibiotics. We developed a simple and easily repeated sample preparation procedure that is effective for extracting surface-associated proteins for the following spectral counting-based label-free quantitative proteomics experiments.

In our first experiment, sub-inhibitory dose of oxacillin, which is a first-line β-lactam antibiotic, was first applied to the MRSA and methicillin-susceptible S. aureus (MSSA). On average, 1025 and 1013 proteins were identified at a false discovery rate threshold of 0.01, in the untreated groups of MRSA and MSSA. Differentially expressed proteins were obtained by comparing the spectral counts of identified proteins between the treated group and the untreated control group. Among the differentially expressed proteins, β-lactamase and penicillin-binding protein 2a (PBP2a) were observed up-regulated uniquely in oxacillin-treated MRSA, which is consistent with the known β-lactam resistant mechanisms of S. aureus. More interestingly, the peptidoglycan biosynthesis pathway and the pantothenate and CoA biosynthesis pathway were found up-regulated in both oxacillin-treated groups, indicating the common tolerance mechanism for both MRSA and MSSA

In our second experiment, proteomics was employed to study the synergistic mechanism of a drug combination -- a new erythromycin derivative SIPI-8294 and oxacillin -- against MRSA. A systematic experimental workflow has been designed, in which cultured MRSA was exposed to sub-inhibitory doses of oxacillin, SIPI-8294, erythromycin, and combinations of SIPI-8294/oxacillin (SIPI-8294/Oxa) and erythromycin/oxacillin (Ery/Oxa, which do not show synergistic effect). Among the differentially expressed proteins, the expression levels of PBP2a and β-lactamase were four times lower in the SIPI-8294/Oxa than in the Ery/Oxa treatment group. The results suggest that the synergistic mechanism may relate to interference with the known oxacillin resistance mechanism. Moreover, hierarchical clustering analysis for the differentially expressed proteins shows that SIPI-8294/Oxa elicits very different responses in the cell than those by the individual drugs or the Ery/Oxa combination. The data shows that the differentially expressed proteins potentially related to synergistic effect may impact oxidation-reduction homeostasis and cell wall biosynthesis.

In our third experiment, in order to mimic the clinical situation for treating infection, a lethal dose of SIPI-8294/Oxa was applied to MRSA cells, and the proteome responses were measured at 0.5 hour and 1 hour after treatment. A lethal dose of oxacillin alone, and a non-synergistic combination of Ery/Oxa at the same dose were used as the controls. Among the interesting findings, PBP2a and β-lactamase were not detected nor found differentially expressed in the cells treated with lethal doses of SIPI-8294/Oxa or oxacillin, in contrast to the situation when sub-inhibitory doses were used. In the SIPI-8294/Oxa group, peptidoglycan hydrolase proteins, which are involved in cell wall degradation and remodeling, were up-regulated significantly. Interestingly, the proteome responses appear to be completely different between MRSA treated with a lethal dose and a sub-inhibitory dose of SIPI-8294/Oxa.

This work is among the first attempts to study antibiotic responses in S. aureus by state-of-the-art quantitative proteomics. The data obtained offered a more complete view of the cellular responses to antibiotics at different doses, and may be useful in guiding the development of new antibiotics or treatment strategies.

Keywords:

Antibiotic resistance; Staphylococcus aureus; Label-free quantitative proteomics; Oxacillin, Synergistic effect; Mass spectrometry; sub-inhibitory dose response; lethal dose antibiotic response