Simvastatin, a commonly prescribed statin, has exhibited several unexpected non-cholesterol lowering benefits. For example, patients taking statins have a decreased mortality rate due to bactereamia, a systemic bacterial infection commonly caused by Staphylococcus aureus (S. aureus). To investigate statin protection during bactereamia, human umbilical vein endothelial cells (HUVEC) were pre-treated with simvastatin followed by infection with S. aureus, and infection was significantly decreased. Simvastatin inhibits the cholesterol biosynthesis pathway. Therefore, the protective effect of simvastatin may be due to isoprenoid inhibition, specifically, farnesyl pyrophosphate (Fpp) and geranylgeranyl pyrophosphate (GGpp). Fpp and GGpp prenylate small G-proteins that function in cytoskeletal rearrangement and endocytosis. When Fpp and GGpp were replenished, infection was not significantly reduced. Furthermore, when farnesyl transferase and geranylgeranyl transferase, enzymes essential to transfer isoprenoid group during prenylation, were inhibited a significant decrease in infection was observed. The data indicates that Fpp and GGpp are essential for S. aureus infection. / Department of Biology

It is important to understand the mechanism of endocytic invasion into the host cell by Staphylococcus aureus. Activation of phosphoinositide-3-kinase (PI3K) is essential to S. aureus invasion. In a normal cell, the p85 subunit of PI3K is bound at the Rho GTPase activating protein (RhoGAP) domain to small guanosine triphosphate binding proteins (GTPases), which are attached to the cell membrane by a prenyl group. This association anchors PI3K near the cellular membrane. PI3K must be anchored near the membrane in order to phosphorylate its substrate. The hypothesis for this project is that deletion of the binding domain between PI3K and small GTPases will block endocytic bacterial invasion by sequestering PI3K in the cytosol. To investigate this hypothesis, the RhoGAP binding domain of PI3K p85 was mutated using site-directed mutagenesis and S. aureus invasion was reduced by up to 86% (p<0.05), which shows that this domain is important to bacterial invasion. / Department of Biology

Staphylococcus aureus, the most prevalant etiologic agent causing sepsis (a damaging inflammatory response), is traditionally cleared with antibiotics. Increased numbers of antibiotic-resistant strains mandate additional treatments to clear infections and prevent sepsis. There is evidence that suggests the lipid-lowering drug simvastatin may be beneficial for treating S. aureus infections due to its anti-inflammatory and immunomodulatory effects. In this study we pretreated 8-13 week old, male and female Balb/c and C57BL/6 mice with 1000 ng/g [BW] simvastatin in ethanol at 18 and 3 hours prior to S. aureus infection. We subsequently administered 10 mg/kg [BW] gentamicin in saline at 3, 6, 12, 24, and 48 hour timepoints. Another group of mice did not receive simvastatin treatment, and the final group received control treatments and was not infected with S. aureus. Our studies demonstrate that simvastatin may down-regulate sepsis-inducing inflammatory responses in S. aureus-infected C57BL/6 mice. / Department of Biology

Access to abstract restricted until May 2015 / Access to thesis restricted until May 2015 / Department of Biology

Sepsis is a life threatening condition caused by infectious agents, including the Gram-positive bacterium Staphylococcus aureus. Symptoms of sepsis often include intravascular coagulation and organ failure. Tissue factor (TF), the initiator of coagulation, may contribute to fibrin deposition in the lungs of patients with sepsis. We have found that lung epithelial cells constitutively express TF on the cell surface and in intracellular pools. Levels of TF diminished in response to S. aureus invasion possibly indicating a release in the form of shedding vesicles. TF levels diminish in response to viable bacteria, but not in response to heat killed (HK) bacteria. Our studies indicate that bacterial attachment at the host cell surface is insufficient to diminish levels of constitutive TF. Finally, we established that levels of constitutive intracellular TF diminish in response to the bacterial toxin, α-hemolysin, alone. This approach may provide a basis for understanding the role of TF in coagulation seen in sepsis. / Department of Biology

Staphylococcus aureus infections have become a widespread problem. Simvastatin decreases S. aureus invasion. Simvastatin use reduces prenylation of target proteins, including CDC42. Prenylated CDC42 is active at the cell membrane. Our hypothesis is that CDC42 activity at the cell membrane is needed for endocytic S. aureus invasion. The prenylation site on CDC42 was deleted and mutant CDC42 (CDC42C5O7V/V5) was transfected into mammalian cells, which were exposed to S. aureus. Decreased bacterial infection of up to 90% was seen in cells stably expressing CDC42C507V/V5. Mammalian cells were treated with secramine A, an inhibitor of CDC42 activity, and exposed to S. aureus. Decreased bacterial invasion of 70% in these cells was seen. These findings suggest that CDC42 activity at the cell membrane is needed for S. aureus cell invasion. These findings increase understanding of the mechanism of S. aureus cell invasion and could be used to develop new treatment or prevention methods. / Department of Biology

Staphylococcus aureus is a Gram positive potentially pathogenic bacterium which has a propensity to gather resistance determinants. Mupirocin is a novel topical antibiotic active against many Gram positive species, including staphylococci and effective in the treatment and prevention of staphylococcal infections. Mupirocin acts by competitively inhibiting the charging of isoleucyl tRNA-synthetase (IRS) with isoleucine. Resistance has been observed in Staphylococcus aureus and coagulase negative staphylococci. Intermediate-level resistance (MIC >8μg ml^-1 >512μg ml^-1) is thought to be due to spontaneous mutations in the native IRS. High-level resistance (>512μg ml^-1) is conferred by a second IRS protein, encoded by mupA which has a much lower affinity for mupirocin than isoleucine. The mupirocin resistance gene (mupA) is usually found on a 4.05kb EcoRI fragment of plasmids of otherwise varied EcoRI restriction pattern which are easily transferred between strains by filter mating. Prior to the onset of these studies, mupirocin resistance had not been found linked to another resistance determinant. Initial investigations intended to identify mechanisms of gene flux resident on mupA plasmids revealed a family of related mupA plasmids, the p3356 family which includes three plasmid types: p3356, p3356D and p3358. p3356 contains a single copy mupA flanked by direct repeats of the staphylococcal insertion sequence IS257. p3356D is identical to p3356 except for the duplication of a "mupA-IS257" cassette in tandem repeat. p3358 is related to p3356D by the insertion of a pT181-like plasmid (tetracycline resistant) accompanied by the duplication of an IS257 in direct repeat to flank the inserted pT181; thus p3358 is the first documented example of linked resistance between mupirocin and another resistance determinant, namely tetracycline. IS257 has been implicated as the recombinogenic site in the gene duplication event involved in the evolution of p3356D from p3356. IS257 co-integrative transposition has been demonstrated to allow the co-integration of pOX7 with p3356 to generate a p3358-type plasmid in which pT181 is replaced by pOX7. Therefore, it is concluded that IS257 transposition and recombination is a mechanism by which staphylococcal replicons can evolve to form multiply resistant replicons.

Thesis (DClinPsychol)--British Psychological Society. BLDSC no. DX233166.

Thesis (Ph. D., Animal Physiology)--University of Idaho, January 2007. / Major professor: Mark A. McGuire. Includes bibliographical references. Also available online (PDF file) by subscription or by purchasing the individual file.

Please read the abstract (Summary) in the section, 00front of this document / Thesis (DPhil)--University of Pretoria, 2008. / Pharmacology / DPhil / Unrestricted