| Sumario: |
Members of the genus Brucella are intracellular -Proteobacteria
responsible for brucellosis, a chronic disease
of humans and animals. Little is known about Brucella
virulence mechanisms, but the abilities of these bacteria
to invade and to survive within cells are decisive factors
for causing disease. Transmission electron and fluorescence
microscopy of infected nonprofessional phagocytic
HeLa cells revealed minor membrane changes accompanied
by discrete recruitment of F-actin at the site of Brucella
abortus entry. Cell 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. Inactivation of Rho small GTPases
using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522,
and TcdA significantly reduced the uptake of B. abortus
by HeLa cells. In contrast, cytotoxic necrotizing factor
from Escherichia coli, known to activate Rho, Rac, and
Cdc42 small GTPases, increased the internalization of
both virulent and non-virulent B. abortus. Expression of
dominant-positive Rho, Rac, and Cdc42 forms in HeLa
cells promoted the uptake of B. abortus, whereas expression
of dominant-negative forms of these GTPases in
HeLa 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
approach used to discern the molecular events leading to
Brucella internalization provides new alternatives for exploring
the complexity of the signals required by intracellular
pathogens for cell invasion.
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