Identification of new proteins in Staphylococci involved in bacterial metabolism and adaptation

Abstract

Bacteria of the genus Staphylococcus are of particular importance because of their widespread presence in the environment and their ability to cause skin and soft tissue infections in humans and animals. These infections are often difficult to treat because staphylococcal strains are or can become resistant to antibiotics. In this work, we studied different aspects of bacterial behavior with a focus on Staphylococcus aureus and Staphylococcus pseudintermedius. Single-domain antibodies (VH/VHH) have desirable molecular features, such as small size, high tissue penetration, and stability, which make them suitable as potent inhibitors of enzymes and toxins. In chapter 1, we studied the potential of penicillin binding protein 2 (PBP2)-specific VH/VHH to sensitize S. aureus in combination with b- lactam antibiotics. Our results indicate that the selected antibodies have little inhibitory effect on S. aureus under the tested conditions and show no synergistic effect in combination with b-lactams. Hence, the tested VH/VHH cannot serve as promising drugs against S. aureus. In chapter 2, we observed an effect of the two nuclease genes encoded in S. aureus genome on the expression of some virulence factors. In an attempt to study the underlying mechanisms, we found out that the observed effects result from secondary mutations in the genome. In chapter 3, we identified a serotonin N-acetyltransferase (SNAT) in S. pseudintermedius based on sequence similarities to known SNAT proteins. In mammals, SNAT catalyze the acetylation of neurochemicals such as serotonin and tryptamine, a reaction that modulates and regulates different functions in the body. The activity of the newly identified SNAT in S. pseudintermedius was confirmed in vitro using recombinant putative SNAT proteins and in vivo using a knockout deletion mutant of the wildtype strain. We also found that SNAT homologues are widely distributed among staphylococci. SNAT products are important antioxidants in bacteria but are also known to induce different responses in the human body. This indicates that SNAT expression in staphylococci could potentially lead to bidirectional signaling with the host. In the final chapter, we aimed to identify the function of the S. aureus gene cluster that bears homology to the cold-sensitive DesKR operon in Bacillus subtilis. In S. aureus the cluster encodes the two-component system TCS-7 and two effector proteins. Based on our results, this four-gene cluster plays a role in cold temperature adaptation in S. aureus as a cluster deletion mutant does not survive a shift to 20°C. However, the predicted effector proteins of TCS-7 share no similarity with DesKR which is a membrane-associated fatty acids desaturase. Accordingly, the effects of TCS-7 activation and their underlying mechanism remain to be identified. The major goal of this work was to gain insight into the physiology of staphylococci and explore new therapeutic avenues against these opportunistic pathogens. The main findings discussed here are the identification of SNAT, the enzyme that enable staphylococci to produce neuromodulating molecules with the potential to interfere with signaling pathways in the colonized host and, revealing the involvement of the proteins encoded in the gene cluster of TCS-7 in S. aureus cold adaptation.