Screening and identification of Brucella effector proteins secreted to the host cell

MARCHESINI, MARÍA INÉS; COMERCI, DIEGO; UGALDE, RODOLFO

Rosario, Argentina

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2006

SAIB

Brucella spp. are Gram-negative bacteria that cause a worldwide zoonosis called brucellosis. They are facultative intracellular pathogens that replicate inside professional and non-professional phagocytes. A type IV secretion system (T4SS, called VirB in Brucella) is essential to avoid lysosome fusion and to create a replicative niche inside cells. A possible role of T4SS is to deliver effector proteins that modulate intracellular trafficking to allow intracellular multiplication. To identify these effector proteins we took advantage of the completed genome of B. abortus S2308, and looked for proteins with eukaryotic domains assuming they are good candidates for modulation of host cell functions. All the open reading frames were scanned using web-based programs. Proteins with eukaryotic domains were selected for fusion with Bordetella pertussis Adenylate Cyclase catalytic subunit (CyaA), a reporter for protein translocation from bacteria to eukaryotic cells. When translocated to the host cell cytoplasm, the hybrid enzyme is activated by calmodulin (CaM) and synthesises cAMP. We infected macrophagic cells with bacteria expressing the fusion proteins and analyzed cAMP levels at different times post-infection. We identified two proteins that are delivered to the eukaryotic cytoplasm. One of them requires the presence of a functional T4SS and is the first Brucella effector protein identified so far.