Principal Supervisor: Dr Chrystala Constantinidou - SLS
Co-supervisor: Dr Meera Unnikrishnan - WMS
PhD project title: A novel secretion system in Gram-negative bacteria
University of Registration: University of Warwick
Bacteria export proteins across membranes using a range of transport machineries. Gram-negative bacteria mediate protein translocation across their bilayer through six types of secretion systems, Types I to VI. In Gram-positive bacteria, it was thought for a long time that the Sec, Tat and flagellar systems accounted for all protein export: until the discovery of the Esx-1 system in Mycobacteria tuberculosis. It was soon shown that the secretion system was required for full virulence in M. tuberculosis. Further investigations revealed that Esx systems play significant roles not only in host-pathogen interactions, e.g. allowing Mycobacterium tuberculosis to escape the phagosome, but also in bacteria-bacteria interactions, e.g. in a special form of conjugation in Mycobacterium smegmatis.
Genome analyses suggested the existence of similar systems not only in Mycobacteria but also in other diverse groups of Gram-positive bacteria. Homologues (now known as Type VII secretion systems – T7SS) were found within the Actinobacteria (eg Corynebacterium diphtheriae and Streptomyces coelicolor) and Firmicutes (eg Staphylococcus aureus, Bacillus anthracis, Streptococcus agalactiae, Rhodococcus equi, Bacillus subtilis and Clostridium acetobutylicum). Although these appear to be less complex than their mycobacterial counterparts they have been shown to play important and diverse roles in host-pathogen interactions, DNA transfer, metal uptake, cell envelope integrity, sporulation and development, and inter-bacterial competition. Intriguingly, a more recent wide-ranging bioinformatics survey provides evidence for a link between T7SSs and toxin-antitoxin systems.
Unexpectedly, homologues have also been found in Gram-negative phyla, eg the Proteobacteria and the Cyanobacteria. Recent phylogenetic surveys in our lab, revealed the presence of WXG100 proteins in all five sub-divisions of the Proteobacteria, including several important pathogenic species such as Helicobacter pylori, Pseudomonas aeruginosa, Vibrio cholerae and Stenotrophomonas maltophilia. Excitedly, the putative T7SS cluster in H. pylori (containing homologues to the EsxA effector and an FtsK/SpoIIIE ATPase) is present in the majority of both complete and unfinished genomes of this species and our analysis indicates that it is associated with toxin-antitoxin domains.
This raises a number of intriguing questions: do these Gram-negative systems form functional T7SSs? If so, given that proteins secreted by Gram-negative bacteria face the additional barrier of the outer membrane, how is this achieved? Are the toxin-antitoxin domains observed, functional and dependent on the T7SS and how do they contribute to the virulence of these pathogenic bacteria? Under what conditions are they expressed and how are they regulated? Do they play a role in bacterial-host or bacterial-bacterial interactions? What is the phylogeny of T7SS amongst the numerous and novel Gram-negative genomes daily uploaded onto genome databases? How widespread is the association with toxin-antitoxin domains?
Initial studies will focus on Helicobacter pylori, an important pathogen that is strongly linked to the development of peptic ulcers and stomach cancer. In the stomach, H. pylori must survive the harsh, low pH environment, the host immune response and competition from other bacteria. Using a combination of approaches, including synthetic biology, proteomics, transcriptomics, cell biology, immunology and bioinformatics, we will assemble evidence that challenges the dogma that T7SSs belong solely in the realm of Gram-positive bacteria.
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
- Bioinformatics analysis
- Cloning and standard molecular biology techniques
- Mutagenesis; Proteomics
- DNA and RNA sequencing and data analysis
- Tissue culture and infection assays
- Setting up an in vivo tissue model
Contact: Dr Chrystala Constantinidou, University of Warwick