Localization studies of the FemXAB protein family in Staphylococcus aureus

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

The public health burden of methicillin-resistant Staphylococcus aureus (MRSA) strains and the emergence of vancomycin resistance in S. aureus call for alternative strategies in chemotherapy. The FemXAB family of proteins is responsible for the assembly of the characteristic pentaglycine cross-bridge in the peptidoglycan of S. aureus, which is essential both for cell viability and for the expression of high level methicillin resistance. Fem proteins are, therefore, good candidate targets for the development of pathogen specific antimicrobial compounds, acting alone or synergistically with -lactams. In this work, we characterized the localization of the FemX, FemA and FemB proteins (which add the 1st; 2nd and 3rd; 4th and 5th glycines of the crossbridge, respectively) during the bacterial cell cycle, using fluorescence microscopy. These proteins localized preferably at the membrane, including the septum, and de-localized in the absence of the peptidoglycan precursor. FemA and FemB proteins colocalized in all cells observed during the different stages of the cell cycle, whilst FemX and FemA proteins co-localized in 64% of the cells. To determine if the FemB protein was dependent on FemA for proper localization, different approaches were used to attempt inactivation of the femA gene, but all were unsuccessful, suggesting that femA may be a lethaltarget.