Rhox, A Rhomboid Protease Of Brucella abortus Involved In Pathogenesis

FUNGUEIRO MF; HERRMANN, C. K.; PANUZZI L.; MARCHESINI, M. I.; COMERCI D. J

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias. LIII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB).; 2017

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The intramembrane serine proteases from the rhomboid family are widespread among the different organisms and have many biological functions. Despite the lack of homology between their sequences, they all seem to cleave their substrates within the lipid bilayer by a conserved mechanisms. However, little is known about the role of most of these enzymes during intracellular lifestyle of many pathogens. Our group recently identified substrates of Brucella abortus VirB type IV secretion system (SST4) involved in pathogenesis. One of them, RhoX (BPE275), is the only rhomboid protease present in B. abortus whose function is still unknown. Deletion of RhoX affects Brucella internalization. Homology sequence analysis by ClustalW revealed that despite differences in their sequences, RhoX have conserved specific regions relevant for protein structure and a catalytic dyad (Ser153-His227) responsible for the enzymatic activity. Furthermore, activity assays using B. abortus membrane preparations demonstrate that RhoX is an active rhomboid protease able to cleave substrates of other members in the rhomboid family such as Gurken and Spitz of D. melanogaster, LacY of E. coli and TatA of P. stuartii. In an attempt to identify putative substrates for Rhox among Brucella abortus proteome, we developed a bioinformatic screening. To validate this screening experimentally, transmembrane domains (TMD) of the putative candidates were cloned into pMal2E vectors as fusion proteins containing, as well, the sequence to encode the maltose binding protein (MBP) and both Flag and His tags. These constructions were expressed in E.coli MG1655, a mutant deficient for rhomboid protease, to perform activity assays. In further studies, we aim to study the function and location of BPE275 and its substrates during Brucella abortus pathogenesis.