Professor Colin Kleanthous joins the department

The department’s most recent recruit, Colin Kleanthous, has joined as the new Iveagh Professor of Microbial Biochemistry.

Professor Kleanthous is a protein chemist whose main interest is protein-protein interactions - how proteins form complexes with each other and how this communicates information. He works largely with bacterial systems, covering a range of approaches including structural studies and in vivo work.

Colin Kleanthous

Professor Colin Kleanthous

‘I am trying to link the structural biochemistry of the protein complexes we are interested in with the cellular information they convey,’ says Professor Kleanthous, who also exploits the systems he studies to ask more general questions about protein complex specificity. ‘We are particularly interested in understanding the molecular mechanisms by which proteins associate with each other since this will ultimately help us understand the complexity of protein networks within cells.’

Moving from the University of York where he had been for the past 9 years, he is looking forward to the new opportunities at Oxford. ‘One of the main attractions of coming to Oxford was the opportunity of working with pioneering scientists, in Biochemistry and the many departments on South Parks Road,’ he comments.

Two bacterial systems have formed the core of Professor Kleanthous’ studies over the years. One is a family of protein antibiotics known as colicins that are part of a large family of anti-bacterial proteins that target Escherichia coli. Bacteriocins (as colicins are more generically known) are produced by many Gram-negative bacteria and are used to target neighbouring organisms competing for the same resources.

‘It’s a form of aggressive defence,’ explains Professor Kleanthous. ‘They are antibiotics but unlike small molecule antibiotics like penicillin, these are plasmid-encoded proteins that are released during times of cellular stress.’

‘I’m interested in how colicins get into cells because this is contingent on lots of protein-protein interactions. Colicins bind to specific receptor proteins on the cell surface and then assemble a complex nanomachine that links the outside of the cell to the inside. Once these connections are made, the colicin is able to transit into the cell although how this occurs is not understood.’

‘I want to answer the basic question of how you get a folded protein to cross the membrane barriers of a bacterial cell. If we can answer that then we might be able to understand similar processes in, for example, protein import in mitochondria.’

Professor Kleanthous’ interests also extend to the systems that colicins parasitize. While serving important functions, for example in the maintenance of the cell’s defences, they are often poorly understood in terms of their endogenous cellular functions, so the group uses colicins to probe these functions.

Another angle to his studies on colicin transport is the role of unstructured (unfolded) parts of proteins. These regions attain fold when they bind to another protein and in this way acquire a function. Unfolded regions give functional versatility to a protein - cell cycle regulators are a classic example of this.

Professor Kleanthous’ group has found that unfolded regions can also help a protein get into cells – as seen for colicins.

‘We found that unfolded regions of colicins are used to pass a ‘message’ across the bacterial membrane. An unfolded region can thread through protein pores naturally resident in the cell membrane. These small pores are there to allow the normal diffusion of nutrients and metabolites into and out of the cell and to exclude large toxic molecules such as folded proteins.’

‘Antibacterial proteins have evolved to exploit these holes in order to pass a signal directly into the cell. The signal seems to act as a trigger for toxin entry.’

Figure showing an antibacterial colicin (brown) binding to its receptor BtuB and then the pore of an OmpF trimer in the outer membrane (left hand side). This initial binding allows the unstructured regions of the colicin to pass through to the periplasm (right hand side), the capture of TolB and induced contact with TolA  all of which together trigger translocation of the colicin across the outer membrane

Figure showing an antibacterial colicin (brown) binding to its receptor BtuB and then the pore of an OmpF trimer in the outer membrane (left hand side). This initial binding allows the unstructured regions of the colicin to pass through to the periplasm (right hand side), the capture of TolB and induced contact with TolA – all of which together trigger translocation of the colicin across the outer membrane (Click to enlarge)

The other system which Professor Kleanthous’ group has been studying is a transcriptional regulator from the Gram-positive bacterium Streptomyces coelicolor known as sigma R, a member of the ExtraCytoplasmic Function sigma factor family. ECF sigma factors respond to environmental stresses to activate transcription from selected promoters and so respond to or counteract the stress.

These sigma factors are held in check by small inhibitor proteins, or anti-sigma factors, as Professor Kleanthous explains: ‘Sigma R controls a regulon that touches all aspects of the cell biology of the organism. It is normally sequestered in a complex with a zinc-containing anti-sigma factor called RsrA.’

‘We have been studying how RsrA inhibits the activity of sigma R and how RsrA responds to oxidative stress. When RsrA becomes oxidised, it dissociates and releases sigma R to activate transcription. We are trying to uncover the mechanism by which RsrA senses oxidative stress and how this impacts on its protein-protein interaction with sigma R.’

‘There is a large family of anti-sigma proteins that appear to respond to different types of stress. We would like to link particular cellular stresses to the specific sigma factor/anti-sigma factor pairings to see how complexes, which appear to be related, are responding to different stresses.’

The core of the group’s work on these bacterial protein complexes is classic biochemistry - studying isolated proteins in vitro, their structures, binding affinities, kinetic mechanisms and stoichiometries. Professor Kleanthous comments that the department combines strengths in cellular studies with excellent structural biology. He brings complementary strengths in protein chemistry and biophysics – areas within the department he hopes to develop and expand in the coming years.

As part of his efforts to link the biochemical and biophysical studies of the complexes with what is going on in vivo, his group wants to exploit the strength in microscopy at Oxford. ‘Ultimately, we want to study the import of a single molecule into a living bacterium, to disentangle the mechanics of that process,’ he says.

‘For example, one of the proteins that is parasitised during colicin import is TolB. We discovered that when colicins contact TolB they modify its allosteric behaviour. This led us into single molecule studies using fluorescently-labelled TolB to investigate this allosteric transition in vitro. I’d like to extend this work by investigating such processes in vivo, which we can do by using the sophisticated imaging technologies available here in Oxford.’

Microscopy and other approaches will boost the little that is known about what TolB and other proteins that are parasitized by colicins do. ‘I’m hoping that being in this new setting will allow us to probe in vivo aspects of the biology we are interested in and bring it more into a biomedical arena,’ says Professor Kleanthous.‘The proteins that we study are antibacterial but have not been used as antibiotics in mammals, and this is something we’d like to explore in the future.’

‘Gut microbes are known to make colicin-like molecules, though their involvement in pathogenicity has always been controversial. But the systems they parasitize are known virulence factors – for example, in E.coli 157. So maybe by understanding the interactions of colicins we might be able to target the systems they parasitize with small molecule antibiotics. Oxford provides a perfect environment to test these ideas.’

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