The work presented herein details post-translational modifications (PTMs) in Staphylococcus aureus that are involved in mediating the stress response and normal cellular processes. The first PTM that was investigated is regulated intramembrane proteolysis (RIP) for the activation of the ECF sigma factor σS. We achieved this by analyzing the role of the site-1 protease, which we termed “putative regulator of sigmaS” (PrsS), as it is predicted to be the first enzyme in the RIP cascade, leading to the activation of σS. It was determined that the putative site-1 protease, prsS, mimics transcriptional profiles of sigS; with expression low in all strains examined other than in the highly mutagenic strain RN4220. Moreover, up-regulation of the protease was observed in response to cell wall-targeting antibiotics, DNA-damaging agents, and during infection in human serum and RAW 264.7 cells, similar to that previously demonstrated for sigS. It was further determined that prsS mutants, like sigS mutants, are more sensitive to cell wall-targeting antibiotics and DNA-damaging agents, which is explained, in part, by alterations in altered abundance of proteins in the prsS mutant that mediate antibiotic resistance (Pbp2a, FemB, and HmrA) and the response to DNA damage (BmrA, Hpt, and Tag). Importantly, transcriptional analyses of proteins affected in the protease mutant, revealed that their expression is decreased in both prsS and sigS mutants, suggesting that this is a result of sigS-mediated regulation. Lastly, it was determined that PrsS, similar to σS, is required for infection in whole human blood and murine models of virulence. Next, since the abundance of a stress response protease, HtrA1, was altered in prsS mutants, we aimed to assess the roles of this enzyme, and its homolog HtrA2 in S. aureus. Interestingly, we first determined that unlike that previously described for the HtrA enzymes, these proteases do not have a role in Agr-mediated virulence regulation. We attribute this finding to unintended mutations likely introduced during strain construction, which is common for S. aureus strains. We next used transcription profiling of the htrA genes in order to understand their role in the cell, and found that they are moderately expressed under standard conditions, and are up-regulated in response to both in vitro and ex vivo stressors that lead to cell protein, DNA, and cell envelope damage. Further to this, the protease mutants are more sensitive to numerous conditions that affect macromolecular stability, including elevated temperature, alterations in pH, reactive oxygen species, DNA damage, and antimicrobial stress. In order to further explore these sensitivities and gain insight into putative substrates, we employed a yeast-2 hybrid screen, and identified numerous proteins that interact with HtrA1 and HtrA2, including those that mediate the response to stress and normal cellular homeostasis. Taken together, we provide evidence to suggest the HtrA proteases in S. aureus are required both during standard conditions and in stress-inducing environments to mediate protein folding and proteolysis of a broad range of substrates. Finally, we performed the first examination of prenylation in a bacterial organism. Prenylation is a well-studied post-translational modification (PTM) in eukaryotes, wherein a prenyl group is added to a metabolite or the C-terminal “CAAX” motif of a protein. Interestingly, the machinery exists for this PTM in a wide variety of prokaryotic species, thus we set out to investigate its impact in S. aureus. To achieve this, we disrupted prenyl group synthesis by inactivating ispA, the gene encoding a prenyl synthetase. The abrogation of prenylation ensued in striking alterations in the cell, including lack of pigmentation and smaller colony size, similar to small-colony variants (SCVs) of S. aureus. In addition to this, the ispA mutant displayed a growth defect, as a result of lower ATP levels. Moreover, the prenylation mutant displayed alterations in resistance to antibiotics, including increased resistance to aminoglycosides and antimicrobial peptides (AMPs), yet elevated sensitivity to cell wall-targeting antibiotics. These differences in susceptibility to cell envelope targeting antibiotics are a result of alterations in cell envelope architecture, including variations in fatty acid composition and increased membrane fluidity. Collectively, the pleotropic consequences of the disruption of prenylation indicate that this process is key to maintaining cellular homeostasis in S. aureus, and perhaps other bacterial species.

Streptococcus pneumoniae is a significant human pathogen and the leading cause of community-acquired pneumonia and acute otitis media. One of the primary defense mechanisms of the human immune system against pneumococcal infection involves granule-mediated killing of bacterial cells by neutrophils. While this mechanism has previously been shown to kill about half of pneumococci in vitro, we hypothesized that some pneumococcal strains have evolved to be more resistant to this granule-mediated killing. Clinical isolates demonstrated a varying range of sensitivity to neutrophil granules. Additionally, we established that the absence of the capsule may affect sensitivity as unencapsulated isolates showed a higher average survival than encapsulated isolates. Finally, pneumococcal surface protease HtrA was found to potentially serve as a protective factor as many knockouts were more sensitive than the wildtypes, recombinant HtrA protected wildtype TIGR4, and a resistant isolate showed higher htrA expression levels than sensitive isolates.

The family of Deg/HtrA proteases is present in a wide range of organisms from bacteria, archaea to eukaryota. These ATP-independent serine endopeptidases play key roles in the cellular protein quality control. The cyanobacterium Synechocystis sp. PCC 6803, a model organism for studies on photosynthesis, metabolism and renewable energy, contains three Deg proteases known as HhoA, HhoB and HtrA. The three proteases are important for survival in stress conditions, such as high light or temperature. In my work the biochemical characteristics of each protease were revealed in vitro and in vivo. In vitro studies performed using recombinant Synechocystis Deg proteases allowed conclusions about their oligomerization states, proteolytic activities and tertiary structure. The in vivo studies addressed their sub-cellular localization, expression and physiological importance by comparing wild-type Synechocystis cells with the three single mutants lacking one of the Deg proteases. HhoA seems to be involved in the cytoplasmic protein quality control. This protease is regulated post-transcriptional and post-translational: oligomerization, pH and/or cation-binding are some of the important factors to stimulate its proteolytic activity. Instead HhoB acts on periplasmic proteins and seems to be important for the transportation/secretion of proteins. While it has low proteolytic capacity, it may act as a chaperone. The stress-induced HtrA functions in the cellular tolerance against photosynthetic stress; additionally it might act as a protease partner of HhoB, generating a protease/chaperone complex. The results presented in this thesis lay the foundation for a better understanding of the dynamic protein quality control in cyanobacteria, which is undoubtedly important for various cellular metabolic pathways.

This thesis will be organized into three chapters discussing the mechanism underlying the onset and progression of osteoarthritis (OA) in the temporomandibular joint (TMJ). Understanding the mechanism of OA development in the TMJ helps in understanding how OA progresses and how to treat this disease. The goal of this investigation is to examine the process of cartilage degeneration and OA biomarker expression in the TMJ to understand their role in TMJ OA onset and development.Chapter one covers mechanisms that are altered in TMJ OA during disease progression. Using animal models with different stressors such as mechanical disturbances, direct injury, and changes in the extracellular matrix composition revealed the role of the different mechanisms that are up-regulated and down regulated during cartilage destruction. Chapter two will cover a paper I wrote that introduces a novel non-invasive technique applied to mice, which induces an early onset of OA in the TMJ. I developed this technique with the aim to provide a new mouse model where the onset and progression of OA more closely mimic the natural TMJ OA progression in humans. The histopathological analysis of the cartilage demonstrates that onset of OA starts at 2 weeks after treatment induction and is aggravated by week eight. This data demonstrated the effectiveness of our technique in inducing OA in the TMJ. Chapter three will cover a second paper I wrote on the association of RAGE with the progression of OA in the TMJ of mice by using mice with and without RAGE expression. RAGE has been show to contribute to the progression of OA by releasing several pro-inflammatory and catalytic cytokines. Additionally, RAGE has been shown to modulate the expression of specific OA biomarkers, including HtrA-1, Mmp-13, and Tgf-β1 in knee cartilage. The objective of this study was to study the effect of knocking out RAGE on the expression of Mmp-1 3, HtrA-1, and Tgf-β1 in the TMJ. After histophatological and quantitative analysis of biomarkers expression, the results demonstrated for the first time that absence of RAGE expression in the TMJ provides a protective effect against development of TMJ OA in mice.

Adaptation to environmental changes is important for the survival of living organisms. Under extreme abiotic conditions, organic molecules (such as lipids, proteins and nucleic acids) are prone to damage. Under these conditions stress response mechanisms are activated, either to prevent the source of damage or to promote the rapid turnover of damaged molecules. Like all photoautotrophic organisms, cyanobacteria are sensitive to high light intensity and oxidative stress, which induces damage to the photosynthetic apparatus. My thesis is divided in two subjects related to particular stress responses in the cyanobacterium Synechocystis sp. PCC 6803: 1) the role of Deg/HtrA proteases and 2) investigations on the small CAB-like proteins. Deg/HtrA proteases are ATP-independent serine endopeptidases with a characteristic C-terminal PDZ domain. These proteases are largely dispersed among living organisms, with many different functions, mostly involved in protein quality control. The genome of Synechocystis sp. PCC 6803 contains three genes coding for Deg/HtrA proteases: HtrA, HhoA and HhoB. These proteases are essential for survival under high light and heat stress, and may overlap in their functions. During my Ph.D. studies I focused on the identification of the precise localization of the Deg/HtrA proteases in the cyanobacterial cell, analyzed the biochemical properties of recombinant Synechocystis Deg/HtrA proteases in vitro and adopted proteomic and metabolomic approaches to study the physiological importance of these proteases. My data show that Deg/HtrA proteases are not only important in stress response mechanisms under adverse conditions, but are also involved in the stabilization of important physiological processes, such as polysaccharides biosynthesis and peptidoglycan turnover. The small CAB-like proteins (SCPs) belong to the light harvesting-like family of stress induced proteins and are thought to be involved in the photoprotection of the photosynthetic apparatus. Five small CAB-like proteins where identified in Synechocystis sp. PCC 6803 (ScpA-E). In my studies I identified another relative to the SCPs, LilA, which I found to be co-transcribed with ScpD. I also focused on the subcellular localization and identification of potential interaction partners of the SCPs.

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