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Drug mechanism and protein function in live mycobacteria

Final Activity Report Summary - MYCOPRO (Drug mechanism and protein function in live mycobacteria)

Mycobacterium tuberculosis, the causative agent of TB, is responsible for 1.7 million deaths per year. In order to develop new and improved treatments for this disease, it is important to understand the biology of the bacterium itself and the mode of action of anti-tubercular drugs.

The main achievement of this work was the development of a sensor to study protein-protein interactions inside living bacteria. Mycobacterium smegmatis was chosen for this sensor, since these bacteria are related to M. tuberculosis, but are non-pathogenic and fast-growing. By studying the interaction of one protein with another, it is often possible learn about the function of these proteins, their role in the life of the bacterium, and their suitability as a target for new drugs The sensor developed in this work is a split-protein sensor named "Split-Trp", since it is based on Trp1p, an enzyme from the tryptophan biosynthetic pathway.

In order to develop this sensor, it was necessary to create a strain of M. smegmatis that lacks its own Trp1p and is therefore unable to synthesis tryptophan. This was achieved by gene knockout to give a new strain that can only grow when it is fed with tryptophan. The Split-Trp assay is based on a version of Trp1p that has been split into two fragments. The genes for test proteins are each fused to one gene fragment of Trp1p (named Ntrp and Ctrp in the figure). These fused genes are introduced into the new strain of M. smegmatis, where the corresponding fusion proteins are made. When the test proteins interact, they bring together the two halves of Trp1p and the M. smegmatis regain their ability to synthesise tryptophan. Protein-interaction can therefore be detected since they allow these bacteria to grow on agar plates lacking tryptophan (see figure). By contrast, if the proteins do not interact then there is no growth of the bacteria.

Well-known proteins were chosen in the first place, to demonstrate that protein-protein interactions reliably lead to growth, whereas there no growth is observed with pairs of proteins that do not interact. Once these demonstrations had been done there was interest from several research groups in using this method to study the proteins of M. tuberculosis. A number of proteins were studied in this way, including three proteins involved in synthesis of the bacterial cell wall. One of these proteins is an enzyme, and the other two proteins were shown to bind to this enzyme and thereby control its activity and specificity.

Also of interest is Protein kinase G. This protein is part of an unknown signalling system, which allows mycobacteria to sense and respond to their environment. Previous studies have shown that PknG is essential during M. tuberculosis infection in mice: gene knockout of PknG led to a less virulent strain of M. tuberculosis and longer survival of infected mice. Inhibition of PknG could represent a new strategy to treat tuberculosis, and in order to develop this strategy it is important to understand the contribution of PknG to bacterial viability and virulence. Using the Split-Trp assay it was shown that PknG binds to a protein named GarA. Classical biochemical methods were employed to confirm the interaction of PknG and GarA and uncover the role of GarA. Unmodified GarA binds to several mycobacterial enzymes, and is likely to regulate their activity. PknG modifies GarA by transferring a phosphate group to a particular target site. In its modified form GarA is "switched off" and no longer binds to these enzymes. Thus PknG and GarA constitute a system for control of metabolism.

The new assay has already proved useful for studying signalling and cell wall synthesis in mycobacteria. The aim is to make this tool available to the research community to aid studies of the fundamental biology of M. tuberculosis and new drug targets to help combat this disease organism.