Final Report Summary - S. AUREUS CLS (Staphylococcus aureus cardiolipin production and role in host-pathogen interactions)
Staphylococcus aureus infections are associated with significant morbidity and mortality in healthcare and community settings. Inside the host, invading S. aureus is challenged by host innate immune defenses that include professional phagocytes and an arsenal of secreted antimicrobials. Long term persistence of S. aureus may be due to adoption of quiescent physical and metabolic states, such as biofilms, endocardial vegetations, and small colony variants. The metabolic changes needed for adaptation and long term survival inside the host are largely unknown. Our preliminary studies have revealed a pronounced metabolic switch that resulted in a change of the membrane composition from the major phospholipid phosphatidylglycerol (PG) to cardiolipin (CL). This striking lipid change occurs when S. aureus is phagocytosed by neutrophils and can also be mimicked in vitro in the stationary phase of growth, or through ATP depletion. Our central hypothesis is that adaptive lipid changes are essential for optimal survival of S. aureus under adverse conditions. In bacteria, CL is mainly synthesised by membrane-bound cardiolipin synthase enzymes (Cls). S. aureus genome contains two open reading frames putatively encoding Cls, each with ~30% identity to the E. coli CLS enzymes. The function of the two S. aureus Cls have not been explored and will be the focus of this grant proposal. Towards this end, we will investigate the:
(i) function of CLS enzyme 1 and enzyme 2 in lipid metabolism,
(ii) role of CL in lipoteichoic acid generation,
(iii) requirement for CL during persistence and survival in planktonic cultures, in
(iv) endocardial vegetations, and
(v) interactions with host defenses. Understanding of the molecular mechanisms and regulation of CL synthesis in S. aureus may uncover novel targets for the development of anti-Staphylococcal agents.
The genome of all sequenced strains of S. aureus contains two open reading frames (orfs) predicting proteins with ~30% identity to the principal CL synthase (cls) of Escherichia coli. To test whether these orfs ('cls1', 'cls2') encode CL synthases and contribute to CL accumulation in S. aureus, we expressed these proteins in a cls strain of E. coli and created isogenic single and double mutants in S. aureus. Expression of either Cls1 or Cls2 in E. coli resulted in increased CL accumulation in stationary phase. S. aureus with deletion of both cls1/cls2 showed no detectable CL accumulation in stationary phase or following phagocytosis by neutrophils. CL accumulation in stationary phase was due almost solely to Cls2 whereas both Cls1 and Cls2 contributed to CL accumulation following phagocytosis by neutrophils. Differences in the relative contribution of Cls1 and Cls2 to CL accumulation under different triggering conditions suggest differences in the role and regulation of these two enzymes.
The final goal of the project is to identify the biological role of S. aureus CL. Our preliminary results suggest that CL may play an important role in defence against cationic antimicrobial protein group IIA phospholipase A2, in survival in a more complex environment such as endocardial vegetations, and for triggering host immune reponses. We would like to expand biochemical studies on S. aureus cardiolipin synthases. Purification should be greatly facilitated by tagging of Cls1 and Cls2 with His or HA tags. Our experiments as well as others (Tsai et al. 2011) suggested that CL accumulation may be beneficial for bacteria under very specific conditions.
(i) function of CLS enzyme 1 and enzyme 2 in lipid metabolism,
(ii) role of CL in lipoteichoic acid generation,
(iii) requirement for CL during persistence and survival in planktonic cultures, in
(iv) endocardial vegetations, and
(v) interactions with host defenses. Understanding of the molecular mechanisms and regulation of CL synthesis in S. aureus may uncover novel targets for the development of anti-Staphylococcal agents.
The genome of all sequenced strains of S. aureus contains two open reading frames (orfs) predicting proteins with ~30% identity to the principal CL synthase (cls) of Escherichia coli. To test whether these orfs ('cls1', 'cls2') encode CL synthases and contribute to CL accumulation in S. aureus, we expressed these proteins in a cls strain of E. coli and created isogenic single and double mutants in S. aureus. Expression of either Cls1 or Cls2 in E. coli resulted in increased CL accumulation in stationary phase. S. aureus with deletion of both cls1/cls2 showed no detectable CL accumulation in stationary phase or following phagocytosis by neutrophils. CL accumulation in stationary phase was due almost solely to Cls2 whereas both Cls1 and Cls2 contributed to CL accumulation following phagocytosis by neutrophils. Differences in the relative contribution of Cls1 and Cls2 to CL accumulation under different triggering conditions suggest differences in the role and regulation of these two enzymes.
The final goal of the project is to identify the biological role of S. aureus CL. Our preliminary results suggest that CL may play an important role in defence against cationic antimicrobial protein group IIA phospholipase A2, in survival in a more complex environment such as endocardial vegetations, and for triggering host immune reponses. We would like to expand biochemical studies on S. aureus cardiolipin synthases. Purification should be greatly facilitated by tagging of Cls1 and Cls2 with His or HA tags. Our experiments as well as others (Tsai et al. 2011) suggested that CL accumulation may be beneficial for bacteria under very specific conditions.