Final Report Summary - TRIMBAT (Trimeric bacterial autotransporters)
Bacterial adhesins play a crucial role in pathogen-host interactions, initiating infections by adhesion to the extracellular matrix and to cell surface proteins prior to invasion. A new family of non-fimbrial adhesins, the trimeric autotransporter adhesins (TAAs), has recently been described in gram-negative pathogens. The common biological role of all these TAAs is to mediate the interaction of pathogenic bacteria with their hosts. They represent viable drug targets, as their interaction with the host often determines the outcome of infection with pathogenic bacteria. They are candidates for novel drugs targeting specifically the gam-negative pathogens and are potential candidates for generating new vaccines.
Project TRIMBAT aimed to increase knowledge of the TAAs family through structural and biochemical studies. In order to address project objectives series of expression constructs were prepared. The base constructs contain a full YadA beta-barrel domain and different length of the stalk domain connecting the YadA head domain. Four different length of the stalk domain were selected. In the second step the base constructs were modified to contain an artificial head domain as a replacement of the native passenger domain head. The following 'heads' were selected: a Cherry tag and a GFP, GST, MBP and Halo-tag for purification and membrane localisation studies.
The initial small-scale expression tests showed that of the selected artificial heads, at least the Cherry tag, is exported to the extracellular space. At this point the initial set of the research techniques was expanded and in cooperation with our partners at the Max-Planck Institute for Developmental Biology in Tuebingen, we used electron microscopy for confirmation of initial observations.
In order to achieve stable expression and maximise yields initial constructs were moved into the CcdA/CcdB toxin-antitoxin expression system. The expression levels improved and the recombinant protein was detectable on SDS gels. However, the purification of the expressed protein proved to be extremely difficult. The established protocols proved to be inadequate and significant effort was put into improving them to be applicable to the task at hand.
Alternative approaches to investigating project objectives were investigated. We have focused our attention on the construct containing MPB as the artificial head domain. We have modified the MBP so that it folding rate was decreased and demonstrated that the mutated MBP is in contrast to its native form exported into extracellular space.
In parallel I have also further characterised TAA family. We have managed to crystallise and solve the structure of E. coli immunoglobulin binding protein D (EibD, manuscript in preparation). The structure represents a complete passenger domain of the TAAs. It is the first structure to present transition of the passenger stalk from right to left supercoiling.
The obtained results fit well with the project objectives. We were able to design and successfully express hybrid trimeric autotransoporters usable for the in-vivo mechanistic studies and once the purification protocols are fully established the construct will provide solid base for the structural investigations. We were able to demonstrate that selected hybrid constructs are functional and the passenger domain is exported to the extracellular space. Furthermore, by engineering MBP in order to slow down its folding rate we were able to demonstrate that in such a circumstances the YadA is capable of exporting significantly larger then its natural head domain into the extracellular space.
Project TRIMBAT aimed to increase knowledge of the TAAs family through structural and biochemical studies. In order to address project objectives series of expression constructs were prepared. The base constructs contain a full YadA beta-barrel domain and different length of the stalk domain connecting the YadA head domain. Four different length of the stalk domain were selected. In the second step the base constructs were modified to contain an artificial head domain as a replacement of the native passenger domain head. The following 'heads' were selected: a Cherry tag and a GFP, GST, MBP and Halo-tag for purification and membrane localisation studies.
The initial small-scale expression tests showed that of the selected artificial heads, at least the Cherry tag, is exported to the extracellular space. At this point the initial set of the research techniques was expanded and in cooperation with our partners at the Max-Planck Institute for Developmental Biology in Tuebingen, we used electron microscopy for confirmation of initial observations.
In order to achieve stable expression and maximise yields initial constructs were moved into the CcdA/CcdB toxin-antitoxin expression system. The expression levels improved and the recombinant protein was detectable on SDS gels. However, the purification of the expressed protein proved to be extremely difficult. The established protocols proved to be inadequate and significant effort was put into improving them to be applicable to the task at hand.
Alternative approaches to investigating project objectives were investigated. We have focused our attention on the construct containing MPB as the artificial head domain. We have modified the MBP so that it folding rate was decreased and demonstrated that the mutated MBP is in contrast to its native form exported into extracellular space.
In parallel I have also further characterised TAA family. We have managed to crystallise and solve the structure of E. coli immunoglobulin binding protein D (EibD, manuscript in preparation). The structure represents a complete passenger domain of the TAAs. It is the first structure to present transition of the passenger stalk from right to left supercoiling.
The obtained results fit well with the project objectives. We were able to design and successfully express hybrid trimeric autotransoporters usable for the in-vivo mechanistic studies and once the purification protocols are fully established the construct will provide solid base for the structural investigations. We were able to demonstrate that selected hybrid constructs are functional and the passenger domain is exported to the extracellular space. Furthermore, by engineering MBP in order to slow down its folding rate we were able to demonstrate that in such a circumstances the YadA is capable of exporting significantly larger then its natural head domain into the extracellular space.