Cloning, overexpression and purification of PBSs enzymes of Brucella abortus.

CASTILLO F; CARVELLI L; NOLLY MB; DAMIANI MT; SÁNCHEZ DG

San Juan

Congreso; XLI Reunión Científica Anual de la Sociedad de Biología de Cuyo.; 2023

Bacteria of the Brucella genus are the causative agents of brucellosis, a widely distributed zoonotic disease that affects a variety of animal species. This disease can lead to abortions and infertility, causing significant economic losses due to reduced livestock productivity. Brucella spp. can be transmitted to humans through contact with infected animals or the consumption of products derived from them, resulting in a debilitating febrile illness. Without proper treatment, it can become chronic, leading to complications such as spondylitis, endocarditis, and encephalitis. These bacteria are facultative intracellular pathogens, and the main structural component of their cell wall is a peptidoglycan (PG) polymer consisting of oligosaccharide chains. These chains contain a periodic disaccharide motif of N-acetylglucosamine (NAG) and N-acetylmuramic (NAM) linked together through attached peptide chains. The complete assembly of PG is a complex process involving approximately 20 enzymatic reactions that occur in both the cytoplasm and the interior and exterior of the cytoplasmic membrane. Key reactions include the activity of a glycosyltransferase (GTase), which polymerizes the oligosaccharide chains, and a transpeptidase (TPase), which catalyzes peptide cross-linking. These activities are performed by Penicillin-Binding Proteins (PBPs). A collaboration was established for the structural characterization of the enzymes involved in the formation of Brucella spp.´s PG, including pbp-2, pbp-1B, muaA, murD, murE, and murF. While the Mur proteins were successfully purified, the PBP proteins formed inclusion bodies. In this project, we aim to clone the PBPs into expression vectors that incorporate a solubility tag or facilitate their export to the bacterial periplasm. This approach will allow us to obtain soluble fractions of these proteins, enabling their proper folding and purification.