Dr Edward J. Taylor
01904 328252
Email: edward.taylor@york.ac.uk
The Structural and Functional Analysis of a Medically Important Bacteriophage
My main interests lie in the interplay between certain pathogenic bacteria and their compatible bacteriophage. In some cases a complex chain of molecular events may result in the expression of bacteriophage encoded toxin genes or virulence factors. These have a direct effect, causing the symptoms of diseases such as diphtheria, cholera, dysentery, botulism, necrotizing pneumonia, toxic shock, food poisoning and scarlet fever. We have focused on the pathogenic bacterium Streptococcus pyogenes SF370 M1 (GAS) and one of its associated Siphovirida prophages SF370.1.
Background
Bacteriophages are viruses that infect bacteria. They do this by “hijacking” the bacterial metabolism in order to reproduce; the virus injects its own DNA in the bacteria. This DNA contains all the necessary information to produce new phage. Here the DNA is decoded by the bacteria to produce new virus particles. Finally the bacteria are burst open by enzymes called endolysins (Figure 1.) and the new virus particles are released. S. pyogenes M1 (GAS) is a gram-positive human pathogen responsible for a wide range of disease variants from mild infection such as cellulites, strep sore throat (pharyngitis) to the more invasive life threatening forms such as toxic shock and necrotizing fasciitis (flesh eating disease).
Figure 1. A lysed area (plaque) in a lawn of S.pyogenes, due to the activity of a phage endolysin expressed a part of the phage life cycle.
A fascinating three way interplay exists between the lysogenized “dormant” SF370.1 prophage, the bacterium and the human host. Phage induction results in phage proliferation and the expression of the two key phage encoded virulence factors the SpeC toxin and the Spd1 DNase (Figure 2.).
Figure 2.The phage mediated transfer of SpeC toxin and the Spd1 Dnase1 genes (red) in the Lysogenic (genome integrated) and lytic (none integrated) variants of the phage reproductive cycle. The blue arrow indicated the point in the cycle where the activity of Hylp1 helps in the transfection process. The green arrow indicated the point in the cycle where the SpeC toxin, Dnase1 and endolysin genes are expressed.
These proteins go on to interact with the human immune system. The SpeC toxin has a well documented action, which is to crosslink the major histocompatibility complex class ii with antigen presenting cells, causing massive cytokine production and many of the symptoms associated with scarlet fever and toxic shock syndrome. The virulence role of the streptococcal DNase, is less well defined, but they have been shown to protect GAS against neutrophil killing by degrading the DNA in neutrophil extracellular traps. This promotes S. pyogenes to adopt a much invasive and dangerous necrotizing mode of growth as well as probably helping the phage progeny to escape the immune system.
Research Interests
Our aim is to obtain the X-ray structures of key proteins encoded by the SF 370.1 phage genome (Figure 3.) and using molecular biology, biochemistry and microbiological techniques look at their interactions.
Figure 3. The SF370.1 phage genome showing the location of key genes of interest.
A typical study involves the cloning and expression of the target gene. The protein is then purified, screened and crystallized. Once protein crystals have been grown, X-ray diffraction is used to determine the protein structure. Elements within the structure may be seen to account for the enzyme’s function such as catalysis (Figure 4,a to d). Several themes have emerged from this work, which are the study of hyaluronate lyase (2) which help the phage transcend the bacterial hyaluronic acid capsule (6,10), the mechanisms of bacterial endolysins (3,5) and the Spd1 DNase1 virulence factor(1) (Figure 4.c and d).
Figure 4. The sequence of milestones leading to the structural solution of the Spd1 DNase virulence factor. Single protein crystals (a) causes X-rays to be diffracted (b) a series of these diffraction patterns are collected as a data set. This structure (c) was solved in combination with a derivative data set, and is shown here with modelled surface and the position of an active site residue in red(d)
Our general interests include the structure and mechanisms of carbohydrate active enzymes, particularly protein domains which bind to “non classical signature sugars”. These are typically associated with the cell walls of some problematic bacteria such as S. pyogenes. Specific interactions between these sugars may be used as the basis for a rapid identification technique, but little is known about how it works on a molecular level. Other past work includes the carbon fixing enzymes of the ribulose monophosphate pathway(11,13), enzymes which break down plant polysaccharides(8,9,12) and enzymes involved in the deglycosylation of proteins(4,7).
Selected Publications
- The structural characterisation of a prophage encoded extracellular DNase from Streptococcus pyogenes.
J E Korczynska, J P Turkenburg and E J Taylor, Nucleic Acids Research, 2012, 40, 928-938. - The X-ray crystal structure of an Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase reveals a (beta/alpha)(8) catalytic domain, two ancillary domains and active site residues key for transglycosylation activity.
Z Ling, M D Suits, R J Bingham, N C Bruce, G J Davies, A J Fairbanks, J W Moir and E J Taylor, J Mol Biol, 2009, 389, 1-9. - Structure and kinetic investigation of Streptococcus pyogenes family GH38 alpha-mannosidase.
M D Suits, Y Zhu, E J Taylor, J Walton, D L Zechel, H J Gilbert and G J Davies, PLoS One, 2010, 5, e9006. - The X-ray crystal structure of an Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase reveals a (beta/alpha)(8) catalytic domain, two ancillary domains and active site residues key for transglycosylation.
Z Ling, M D Suits, R J Bingham, N C Bruce, G J Davies, A J Fairbanks, J W Moir and E J Taylor, J Mol Biol, 2009, 389, 1-9.
