The Brucella TIR domain containing proteins BtpA and BtpB have a structural WxxxE motif important for protection against microtubule depolymerisation

Abstract

BackgroundThe TIR domain-containing proteins BtpA/Btp1/TcpB and BtpB are translocated into host cells by the facultative intracellular bacterial pathogen Brucella. Here, they interfere with Toll like receptor signalling to temper the host inflammatory response. BtpA has also been found to modulate microtubule dynamics. In both proteins we identified a WxxxE motif, previously shown to be an essential structural component in a family of bacterial type III secretion system effectors that modulate host actin dynamics by functioning as guanine nucleotide exchange factors of host GTPases. We analysed a role for the WxxxE motif in association of BtpA and BtpB with the cytoskeleton.ResultsUnlike BtpA, ectopically expressed BtpB did not show a tubular localisation, but was found ubiquitously in the cytoplasm and the nucleus, and often appeared in discrete punctae in HeLa cells. BtpB was able to protect microtubules from drug-induced destabilisation similar to BtpA. The WxxxE motif was important for the ability of BtpA and BtpB to protect microtubules against destabilising drugs. Surprisingly, ectopic expression of BtpA, although not BtpB, in HeLa cells induced the formation of filopodia. This process was invariably dependent of the WxxxE motif. Our recent resolution of the crystal structure of the BtpA TIR domain reveals that the motif positions a glycine residue that has previously been shown to be essential for interaction of BtpA with microtubules.ConclusionsOur results suggest a structural role for the WxxxE motif in the association of BtpA and BtpB with microtubules, as with the WxxxE GEF family proteins where the motif positions an adjacent catalytic loop important for interaction with specific Rho GTPases. In addition, the ability of ectopically expressed BtpA to induce filopodia in a WxxxE-dependent manner suggests a novel property for BtpA. A conserved WxxxE motif is found in most bacterial and several eukaryotic TIR domain proteins. Despite the similarity between ectopically expressed BtpA and WxxxE GEFs to modulate host actin dynamics, our results suggest that BtpA is not part of this WxxxE GEF family. The WxxxE motif may therefore be a more common structural motif than thus far described. BtpA may provide clues to cross-talk between the TLR and GTPase signalling pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0053-y) contains supplementary material, which is available to authorized users.

The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility

“…We found that ectopically expressed TirS accumulated in filament-like structures of irregular shapes within the host cytosol but that do not co-localize with typical cytoskeleton components (microtubules, actin or intermediate filaments). This observation is different from those previously described for other bacterial TIR proteins from Brucella [8,16] and BaTdp from B . anthracis [18].…”

“…In addition to its ability to interfere with TLR signaling, the bacterial TIR effector protein from Brucella BtpA targets and modulates microtubules [16] through a WxxxE motif [17] as well as the recently identified TIR protein from Bacillus anthracis referred to as BaTcp [18]. Since the WxxxE is also present in TirS, we investigated the intracellular localization of ectopically expressed TirS by confocal microscopy on HeLa cells transfected with myc-TirS and indirect immuno-fluorescence staining with anti-myc antibodies.…”

“…In addition to control of inflammatory responses, some TIR domain-containing proteins such as BtpA or BaTcp have been shown to target and modulate microtubules [18,24] through a WxxxE motif that is also present in TirS and involved in cross-talk between the TLR and GTPase signaling pathways [16]. We found that ectopically expressed TirS accumulated in filament-like structures of irregular shapes within the host cytosol but that do not co-localize with typical cytoskeleton components (microtubules, actin or intermediate filaments).…”

A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors

“…Indeed, recently the TIR domain of TcpC has been shown to directly interact with the NACHT leucin-rich repeat PYD protein 3 (NLRP3) inflammasome and caspase-1, besides MyD88, to perturb inflammasome activation (Waldhuber et al, 2016). There are also additional potential targets yet to be identified for BtpA/TcpB since it interferes with microtubule dynamics (Radhakrishnan et al, 2011;Felix et al, 2014) and induces unfolded protein response (Smith et al, 2013). The EMBO Journal UBAP1 targeting by Pseudomonas TIR domain protein Paul RC Imbert et al This notion that bacterial TIR domains provide a broad interaction platform is supported by our observations.…”

The TIR-domain containing effectors BtpA and BtpB fromBrucella abortusblock energy metabolism

“…However, preferential binding to MyD88 was also demonstrated [16]. It is likely that these Brucella TIR-containing proteins display additional targets or functions, as they modulate microtubule dynamics when ectopically expressed [17, 18] and BtpA was shown to induce the unfolded protein response [19].…”