Brucellae are highly infectious intracellular pathogens found in a wide range of mammals including humans causing abortion, infertility and undulant fever. Unlike other intracellular pathogens, Brucella spp does not encode classical virulent factors. However, chronic intracellular persistence in host cells indicates Brucella effectors likey interact with unidentified eukaryotic targets to modify the cellular environment for bacterial survival. Minimal information is available on Brucella proteins that contribute to bacterial virulence indicating an immediate need to explore the mechanisms of Brucella pathogenesis. Brucella spp encodes a TIR domain containing protein (TcpB) to subvert host innate immune responses to create a less hostile environment for its survival and replication. TcpB inhibits NF-?B activation and pro-inflammatory cytokine secretion mediated by Toll-like receptor (TLR) 2 and 4. TcpB deficient B.melitensis presents an attenuated phenotype in mouse model indicating that TcpB is an important virulent factor of Brucella. TcpB targets a TLR adaptor protein TIRAP/MAL to inhibit TLR2 &4 signaling. TcpB induces ubiquitination and targeted degradation of TIRAP employing an unidentified ubiquitin ligase. Our preliminary studies indicated that TcpB interacts with the microtubule associated protein, CLIP170 and the over expression of CLIP170 induces ubiquitination of TIRAP. The overall objective of this RO3 grant proposal is to determine whether TcpB employs CLIP170 for TLR inhibition though the ubiquitination and targeted degradation of TIRAP.
Specific aims of the present proposal are:
Aim 1 : To examine enhanced ubiquitination of TIRAP by CLIP170. In vivo and in vitro ubiquitination assays will be employed to analyze the ubiquitination of TIRAP by CLIP170. Experimental data will confirm the ubiquitin ligase property of CLIP170 with specificity to TIRAP.
Aim 2 : To analyze degradation of TIRAP by CLIP170. We will determine the CLIP170 induced degradation of TIRAP by co-transfection and western analysis. Decreasing concentrations of TIRAP with increasing amounts of CLIP170 will indicate the degradation of TIRAP.
Aim 3 : To determine inhibition of TLR2 and 4 signaling by CLIP170. We will assess the TLR suppression property of CLIP by NF-?B reporter assays using HEK-TLR2 or 4 cells. A dose dependent inhibition of NF-?B expression will indicate TLR suppression property of CLIP170. Since CLIP170 interacts with TcpB and TIRAP, the experimental data will confirm that TcpB recruits CLIP170 for the inhibition of TLR signaling through the targeted degradation of TIRAP.
The Problem-Minimal information on virulent determinants of Brucella Brucellosis is one of the most common zoonotic diseases in the world and poses a major threat to human health and animal agriculture. Minimal information is available on Brucella proteins that contribute to bacterial virulence indicating an immediate need to explore the mechanisms of Brucella pathogenesis. The Solution-Brucella encoded protein, TcpB contribule to the virulence of Brucella This RO3 grant proposal aims to investigate the mechanism of TcpB mediated suppression of host innate immune responses. The experimental data would provide novel information on how an intracellular pathogen exploits host cell machinery to suppress the innate immune recognition.