Brucella abortus T4SS: characterization of four proteins translocated into host cells trough VirB secretion system

MARCHESINI M.I; HERRMANN, C. K.; COMERCI D. J

Buenos Aires

Conferencia; 2011 Brucellosis International Research Conference; 2011

Asociación Argentina de Microbiología

Type IV secretion systems (T4SS) are specialized membrane protein complexes used by many bacterial pathogens for the delivery of effector molecules that target and subvert diverse host cellular processes. It has been demonstrated that Brucella T4SS (VirB) is essential to evade lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic or proteinprotein interaction domains, among others, are good candidates for modulation of host cell functions. To identify these candidates, we performed a combined in silico-in vivo screening and by this approach we identified four proteins (BPE043, BPE005, BPE275 and BPE123) that are translocated into the eukaryotic cell cytoplasm in a VirB–dependent manner. BPE043 is a conserved hypothetical protein with prediction of two transmembrane domains and four apolipoprotein domains; BPE005 is annotated as a putative cyclic nucleotide-binding protein; BPE275 is a member of the rhomboid family and BPE123 is a hypothetical protein with prediction of a signal peptide and a coiled coil motif. Detailed inspection of BEP123 coiled coil motif revealed similarity to the helix domain of the eukaryotic protein cortactin, an actin-binding protein and a central regulator of the actin cytoskeleton. Importantly, cortactin is also a common target exploited by many microbes during infection. BEP123 ectopically expressed in mammalian cells promoted lamellipodia formation and co-localized with cortical F-actin and cortactin, suggesting a role for BPE123 in targeting and regulating actin cytoskeleton dynamics during B. abortus infection. In this regard, we demonstrate that inhibition of cortactin expression by interfering RNA strongly inhibited B. abortus replication. With regards to the other VirB-translocated proteins, immunofluorescence microscopy was performed on cell cultured macrophages infected with Brucella strains carrying 3XFLAG tagged proteins. Results obtained from these experiments indicate that fusion proteins localized to the Brucella containing vacuole, suggesting a possible role in regulation of host vesicular traffic that would allow the establishment of the replicative organelle.pathogens for the delivery of effector molecules that target and subvert diverse host cellular processes. It has been demonstrated that Brucella T4SS (VirB) is essential to evade lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic or proteinprotein interaction domains, among others, are good candidates for modulation of host cell functions. To identify these candidates, we performed a combined in silico-in vivo screening and by this approach we identified four proteins (BPE043, BPE005, BPE275 and BPE123) that are translocated into the eukaryotic cell cytoplasm in a VirB–dependent manner. BPE043 is a conserved hypothetical protein with prediction of two transmembrane domains and four apolipoprotein domains; BPE005 is annotated as a putative cyclic nucleotide-binding protein; BPE275 is a member of the rhomboid family and BPE123 is a hypothetical protein with prediction of a signal peptide and a coiled coil motif. Detailed inspection of BEP123 coiled coil motif revealed similarity to the helix domain of the eukaryotic protein cortactin, an actin-binding protein and a central regulator of the actin cytoskeleton. Importantly, cortactin is also a common target exploited by many microbes during infection. BEP123 ectopically expressed in mammalian cells promoted lamellipodia formation and co-localized with cortical F-actin and cortactin, suggesting a role for BPE123 in targeting and regulating actin cytoskeleton dynamics during B. abortus infection. In this regard, we demonstrate that inhibition of cortactin expression by interfering RNA strongly inhibited B. abortus replication. With regards to the other VirB-translocated proteins, immunofluorescence microscopy was performed on cell cultured macrophages infected with Brucella strains carrying 3XFLAG tagged proteins. Results obtained from these experiments indicate that fusion proteins localized to the Brucella containing vacuole, suggesting a possible role in regulation of host vesicular traffic that would allow the establishment of the replicative organelle.