Catching a killing machine in the act: visualizing the membrane attack complex (MAC) in bacterial minicells. 

Dr. Doryen Bubeck; d.bubeck@imperial.ac.uk 

Complement is one of the immune system’s first responders to infection. Pathogen detection by any of the three complement cascades triggers formation of the membrane attack complex (MAC), a lytic pore that directly destroys Gram-negative bacteria.  MAC assembles in a sequential and irreversible pathway to form an 11 nm hole in the lipid bilayer. As the pore dimensions are only compatible with traversing the outer membrane of Gram-negative bacteria, it remains unclear how MAC damages the inner membrane to cause cell death. This project will explore structural biology approaches to investigate how MAC kills bacteria. In doing, so we will provide insight into the mechanism of action of an immune pore which has the potential to inform why some bacteria are resistant to MAC.  

References:  

Serna M, Giles JL, Morgan BP, Bubeck D. Structural basis of complement membrane attack complex formation. Nature Communications. 2016 Feb 4;7:10587. doi:10.1038/ncomms10587.   

Menny A, Serna M, Boyd C, Gardner S, Joseph AP, Morgan BP, Topf M, Brooks NJ, Bubeck D. CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers. Nature Communications. 2018 Dec 14; 9(1):5316. doi:10.1038/s41467-018-07653-5.   

Understanding the eco-evolutionary dynamics of overlapping plant species’ ranges. (Dr. Will Pearse; will.pearse@imperial.ac.uk)  

Understanding how species’ ranges change and evolve is a long-standing problem in ecology and evolution. It is widely thought that lineages of species whose traits evolve more rapidly compete less with one-another and so are more likely to have overlapping ranges, but directly testing such hypotheses is challenging. This project will leverage two statistical frameworks recently developed in the lab (Pearse et al. 2019) to understand how lineages’ traits and ranges co-evolve. Making use of global plant distribution, trait, and phylogenetic data, this project will explore how the ecological rules that determine species’ ranges have evolved. These joint eco-evolutionary models will permit more accurate forecasts of species’ ranges by using evolutionary information to highlight commonalities across species. These insights will be used to inform ongoing conservation planning, which has highlighted uncertainty in the mode of evolution of species’ traits as a major source of error when spatially prioritising global conservation (Mazel et al. 2018). 

References: 

Pearse WD, Legendre P, Neto PP & Davies TJ. The interaction of phylogeny and community structure: linking clades' ecological structures and trait evolution (2019). Global Ecology and Biogeography 28: 1499-1511. 

Mazel F, Pennell M, Cadotte M, Diaz S, Dalla Riva G, Grenyer R, Leprieur F, Mooers A, Mouillot D, Tucker C & Pearse WD (2018). Prioritizing phylogenetic diversity captures functional diversity unreliably. Nature Communications 9(1): 2888.

Structural and functional investigation of the human Vitamin C transporter. (Professor Bernadette Byrne; b.byrne@imperial.ac.uk) 

The human Vitamin C transporters, hSVCT1 and hSVCT2, belong to the Nucleobase Ascorbate transporter (NAT) family of proteins.  hSVCT2 is key in the anticancer effects of ascorbic acid. hSVCT1 has been expressed in both Xenopus oocytes and COS cells and the protein used to study function and obtain a low resolution structure.  However very little is known about these proteins, in particular how these differ in terms of substrate specificity and regulation compared with the NAT family proteins from bacteria and other eukaryotes. This project will initiate studies on the hSVCTs, designing and generating expression constructs and expressing the proteins in mammalian cells in order to allow us to obtain the most physiologically relevant material for study. The protein will be isolated using established detergent based extraction and purification protocols and the sample submitted to basic biophysical analyses to check for purity and stability. Earlier work in our laboratory has successfully stabilised a fungal NAT, UapA, for crystallographic purposes by the introduction of a single point mutation in the NAT signature motif sequence. We will attempt mutagenesis of the SVCTs based on this and other possible stabilisation sites. The most stable protein will be submitted to structural analysis using cryo-EM and if time and protein allows we will work with our collaborators at Kings College to attempt to understand the role of lipids in the structure and function of hSVCTs.   

References: 

Yilmaz Alguel, Sotiris Amilis, James Leung, George Lambrinidis, Stefano Capaldi, Nicola J. Scull, Gregory Craven, So Iwata, Alan Armstrong, Emmanuel Mikros, George Diallinas, Alexander D. Cameron, Bernadette Byrne*. (2016) The structure of UapA, a eukaryotic purine/H+ symporter, reveals a role for homodimerization in transport activity. Nature Commun. 7: 11336. 

Albertina Velho and Simon Jarvis (2009). Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting. Exp. Cell Res. 315: 2312–2321.