GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes - Direct activation of Cdc42

Members of the genus Brucella are intracellular a-Proteobacteria responsibl e for brucellosis, a chronic disease of humans and animals. Little is known about Brucella virulence mechanisms, but the abilities of these bacteria t o invade and to survive within cells are decisive factors for causing disea se. Transmission electron and fluorescence microscopy of infected nonprofes sional phagocytic HeLa cells revealed minor membrane changes accompanied by discrete recruitment of F-actin at the site of Brucella abortus entry. Cel l uptake of B. abortus was negatively affected to various degrees by actin, actin-myosin, and microtubule chemical inhibitors. Modulators of MAPKs and protein-tyrosine kinases hampered Brucella cell internalization. Inactivat ion of Rho small GTPases using clostridial toxins TcdB-10463, TcdB-1470, Tc sL-1522, and TcdA significantly reduced the uptake of B. abortus by HeLa ce lls. In contrast, cytotoxic necrotizing factor from Escherichia coli, known to activate Rho, Rac, and Cdc42 small GTPases, increased the internalizati on of both virulent and non-virulent B. abortus. Expression of dominant-pos itive Rho, Rac, and Cdc42 forms in HeLa cells promoted the uptake of B. abo rtus, whereas expression of dominant-negative forms of these GTPases in HeL a cells hampered Brucella uptake. Cdc42 was activated upon cell contact by virulent B. abortus, but not by a noninvasive isogenic strain, as proven by affinity precipitation of active Rho, Rac, and Cdc42. The polyphasic appro ach used to discern the molecular events leading to Brucella internalizatio n provides new alternatives for exploring the complexity of the signals req uired by intracellular pathogens for cell invasion.