Understanding the Role of Peptidoglycan Metabolism in Bacterial Predation

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

We are studying the natural, friendly bacterium Bdellovibrio bacteriovorus - roughly translated as "leech-like bacteria-eater" - which is able to kill other less-friendly bacteria. The control of bacterial populations is important in many areas of human existence - namely tackling undesirable pathogenic species in healthcare (e.g. superbugs), crop pestilence, food safety, biofouling and water quality management. The potential exists to use i)knowledge gained from Bdellovibrio study, ii)protein products (enzybiotics) or iii)whole cells/cultures in a therapeutic manner. Unlike other predatory bacteria, Bdellovibrio kills its targets from within - entering them, breaking them down, reproducing inside and then bursting them to release daughter cells and begin the cycle anew.

Bdellovibrio arose from a non-predatory ancestor bacterium, and thus developed specialized tools that allow it to enter and kill other bacteria. These tools are protein enzymes - we aim to investigate the form and function of these so that we understand the killing process better, and perhaps even enhance the potential of Bdellovibrio as an antibacterial agent. Such proteins can be very useful to let us target the invasion of pathogen cells and break them in a controlled way.

Prior investigation by our laboratories revealed that one such enzyme (Bd3459) was targeted to the prey, and acted to change prey shape. We showed that Bd3459 achieved this by cutting particular regions of the prey wall (known as peptidoglycan), causing the wall to partially collapse and so alter the shape of the bacterium that it previously supported - much like sawing away at the support walls of a house! The shape change serves to provide the optimal space for Bdellovibrio invasion of prey, which also signals to fellow invaders that this particular "home" is occupied and that further entry would be wasteful. There are more "special" Bdellovibrio enzymes that "chip away" at the cell walls of bacterial prey- we would like to work on these to develop a fuller picture of what goes on when Bdellovibrio starts to kill its prey and to allow people to use these for biotechnology.

We will look at the enzymes themselves in atomic detail (known as x-ray crystallography), using fluorescent versions of the enzymes to track where they exert their effects (do they "chew up" the host or prevent unwanted destruction of self?), monitoring the precise nature of the function (known as enzyme assays), and also testing the enzyme:location:function relationship by constructing mutant strains of bacteria (lacking the enzymes) to confirm/dispel the hypotheses arising.

The investigation of peptidoglycan-targeting enzymes has a very practical application - an intact wall is essential to most medically-relevant bacteria, and forms the basis of action of several very successful antibiotics (e.g. penicillin, vancomycin). Results from our study may inform on this process, and also have implications for microbial physiology (form and function) in general.

Technical Summary

Bdellovibrio bacteriovorus is the model organism for investigating predatory bacteria; it breaches the outer membrane of Gram-negative prey (examples include many pathogens) and reproduces within the periplasm, modifying but not destroying the prey cell wall until it is lysed at the end of Bdellovibrio reproduction. Bdellovibrio has many specialized proteins for this purpose, one such example of which is the specialized peptidoglycan endopeptidase, Bd3459. We showed recently that this enzyme is the elusive peptidase that converts the rod-shaped prey into the spherical niche upon invasion. Our studies revealed that Bd3459 is a member of the DacB/PBP4 DD-transpeptidase family, but had several modifications presumably related to its promiscuous use in hydrolysing prey walls. This work represented the first molecular-level investigation into the evolution of predation, and we aim to further explore the processes of Bdellovibrio prey-wall-modification/self-protection.
We will:-
1)Determine which features of Bd3459 (deviations/modifications from "housekeeping" DacBs especially) are most important for substrate recognition and cell-rounding function
2)Investigate whether the co-expressed Bd3460 ankyrin protein is responsible for self-protection or further prey manipulation (bioinformatics strongly suggests a protein:protein interaction role for Bd3460)
3)Test the predatory roles of specifically predatorily upregulated peptidoglycan LD-transpeptidases (localization, structural features, specific activity)
4)Determine how peptidoglycan deacetylases act on prey peptidoglycan and whether this regulates its degradation (pertaining to a phenotype described ~30 years ago to which no gene product has been ascribed) -including proteins Bd0468/Bd3279. We have an interesting double knockout of these that suggests they are indeed the sought-after deacetylases. These enzymes act to modify host wall hydrolytic sensitivity and putatively aid in differentiating host versus self.

Planned Impact

Bdellovibrio-based projects have significant relevance to almost all of the strategic priorities of the BBSRC - the natural antibiotic action of this species pertinent to animal health, food security and ageing-related disease (long term bacterial infections such as diabetic ulcers). Bdellovibrio prey of particular interest include Pseudomonas, Acinetobacter, Burkholderia, Proteus, Salmonella and Klebsiella. The diverse, bacterially-degredative enzymes of Bdellovibrio may have uses and impacts outside of whole cell applications, and the advent of synthetic biology means that engineered therapeutic strains are now possible (e.g. the recent use of pyocin-producing E. coli).

Industry: Antibacterial usage of Bdellovibrio has potential benefits in healthcare, farming (crops and livestock) and bioremediation. The lifestyle of Bdellovibrio means that it is also a rich source of unique enzymes, with potential for technology development (the bacterium itself representing a natural nanoscience solution to bacterial control and manipulation).

Basic Scientists: A substantial direct impact would be on predatory bacteria researchers - Bdellovibrio is the model organism in this field and our wider aim is to annotate the features revealed by its genome sequence and transcriptome analysis.
Of equal significance are the insights we will obtain into its prey, as E. coli is undoubtedly "the" model bacterium and we will uncover details of its cell wall physiology and regulation. The relationship between predator and prey is also relevant as a "simplified" model of intracellular growth adaptations, and as such will impact related pathogenesis/symbiosis fields.

Evolutionary microbiologists will be very interested in the series of peptidoglycan modofying enzymes that are required to firstly enter diverse bacterial cells and keep their walls expanded yet stable and also to "prepare" them for later synchronous lysis. Evolution for predation has required a large complement of such peptidoglycan active enzymes.

Students: Elements of the proposal (and associated spin-off findings) will make excellent small-scale lab projects for students - both the Lovering and Sockett groups find that Bdellovibrio elicits an enthusiastic and often awed response from students and we look upon this work as an ideal vehicle for fostering wider student appreciation of microbiology and structural biology.

Publications

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Cadby IT (2014) Life in the "old bag" yet: structure of peptidoglycan L,D-carboxypeptidases. in Structure (London, England : 1993)

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Tyson J (2017) Predatory Bacteria: Moving from Curiosity Towards Curative. in Trends in microbiology

 
Description Annual Report for Understanding the Role of Peptidoglycan Metabolism in Bacterial Predation - BB/J015229/1

Work arising from section 2.2.1
We have made excellent progress with the central aim of the proposal - namely, the research question "How do Bdellovibrio protect themselves when forming the prey cell niche?" We have obtained three crucial structures that explain this phenomenon, detailing inhibition of the niche forming enzymes. The Sockett laboratory has confirmed this from analysis of mutants that (i) remove protection, markedly affecting Bdellovibrio and (ii) knock in (E coli) phenocopy invasion of prey by Bdellovibrio with mutations in the niche forming enzymes. We are collating this work, with the aim of submission to Nature in 2014. We are also continuing in our efforts to understand peptidoglycan hydrolase substrate specificity, and currently have these proteins in complex with beta-lactams and are investigating whether we can complex active site knockouts with cell wall fragments.

Work arising from section 2.2.2
Likewise, we have made significant progress on a second question "Investigating the staged degradation of prey cell walls"; we have a high resolution structure for the cell wall modifying enzyme, confirmed activity for both modification homologues, and are just finishing the manuscript that describes how knock out of these agents leaves behind residual prey material - this is now to be submitted in the last period of 2014 to target journal PNAS, article titled "Bdellovibrio GlcNAc Deacetylases prime invaded prey-bacterial cell walls for final destruction and can be leveraged to produce ghosts with intact membrane and wall".

Work arising from section 2.2.3
Work on the alternate peptidoglycan hydrolases that have undergone significant gene duplication/expansion in Bdellovibrio is ongoing, and we have cloned, expressed and purified a representative of each family, with a view to completing structural studies and characterization of these in the second half of the grant timeline. Multiple gene knockouts for these enzymes are being produced and fluorescent fusion tags, which show for the first three different family members that they are expressed during predatory growth.
We have taken a member from each grouping and scanned for hydrolytic activity using peptidoglycan zymogram gels; initial results indicate that at least two family members are able to degrade cell wall material and as such we will prioritise these for further characterisation.
We have studied microscopically the mcherry-fusion-tagged enzymes from each grouping (as mentioned in the construction earlier, (correlating with those used for zymograms) . for each we have now made a detailed study of their location and timing of expression during the 4 hour predatory cycle. We have determined their expression pattern during deconstruction of bacterial prey cells. This has been very illuminating (and in some cases surprising) as to the timing of their action upon prey. We have been encouraged by these results to construct more mcherry fusions to other endopeptidases as we are revealing a pattern of systematic cell -wall modification processes. This endopeptidase work will make a paper later in 2015.

Outreach & Related Activities
Invited talks have been given by both Professor Sockett and Dr Lovering in 2013/2014, at prestigious conferences and locations (e.g. Gordon Research Conferences, EMBL, Institute Pasteur, Cambridge University) and we have acknowledged the BBSRC for the support of this project.
Prof Sockett hosted a lower 6th school student for a week in the lab June 2014 to do experiments on bacterial 2 hybrid interactions of Bdellovibrio proteins.
Prof Sockett and Dr Lovering hosted an undergraduate student Richard Acton on a 6 week Nuffield Summer Bursary position (3 weeks at each lab) studying the genetics and biochemistry of a Bdellovibrio cell wall adhesion gene-product.
Prof Sockett and Dr Lovering have been invited to co-author a review on Bdellovibrio for Nature Reviews Microbiology

We are recruiting BBSRC DTP students in other peptidoglycan-related projects, different from, but related to the BBSRC project and thus expertise is being cascaded in Knowledge Transfer.
Exploitation Route We anticipate that our findings will stimulate others working in the cell wall field on related (but non-predatory bacteria) projects
Sectors Agriculture, Food and Drink,Environment,Pharmaceuticals and Medical Biotechnology

 
Description They have been publicized by: -BBSRC website -BBSRC Business Magazine -A approx. 10 minute feature on Radio 4 "Inside Science" xmas eve special 2015
First Year Of Impact 2015
Sector Education,Culture, Heritage, Museums and Collections
Impact Types Cultural,Societal

 
Description Feature in BBSRC Business Magazine 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Article (and cover feature) on our research from this grant in Winter 2016 issue of BBSRC Business Magazine.
Year(s) Of Engagement Activity 2016
URL http://www.bbsrc.ac.uk/documents/bbsrc-business-winter-2016-pdf/
 
Description Feature on research included in segment on National radio 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Part of BBC Radio 4 Inside Science broadcast; reporting on use of predators in therapeutic context
Year(s) Of Engagement Activity 2016
 
Description Interview feature on National Radio 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview feature on our published work from this grant, conducted on Radio 4's "Inside Science" Christmas Eve special 2015.
Year(s) Of Engagement Activity 2015
 
Description Invited Presentations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Outreach & Related Activities
Invited talks have been given by both Professor Sockett and Dr Lovering in 2013-2015, at prestigious conferences and locations (e.g. Gordon Research Conferences, EMBL, Institute Pasteur, Cambridge University) and we have acknowledged the BBSRC for the support of this project.

Furthermore, both PIs (Sockett & Lovering) presented material at the 2015 FEMS International Congress , acknowledging BBSRC.

Dr Lovering is scheduled to present data arising from this award at the Prague Spring Protein Meeting (May 2016) and Canadian Society of Microbiology Annual Conference (Toronto, June 2016).


High interest from journals regarding invited reviews (documented elsewhere on outcomes from this proposal)
Year(s) Of Engagement Activity 2014,2015
 
Description Invited Speaker at International Meetings 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Outreach & Information Dissemination, raising awareness of predatory bacteria at the following:

2016, June: Gordon Conference on Bacterial Cell Surfaces, Mt Snow VT USA.
2017 April upcoming Falk Symposium 58 to Physicians, Edinburgh
2017 March upcoming Microbiology Society Meeting Edinburgh Bacterial Cell Surfaces
2016, June: HFSP Science Meeting, the Royal Society London.
2016, March: DFG SPP on "Phenotypic Heterogeneity in Bacteria" Dusseldorf Germany
2016, May: Prague Protein Meeting
2016, June: Canadian Society of Microbiology Annual Conference, Toronto
2017, June: Upcoming ASM Annual Meeting, New Orleans
2017 Sept: Upcoming Great Wall Symposium, Portugal
Year(s) Of Engagement Activity 2016,2017
 
Description Invited talks at Universities 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact The Institutions involved in 2016/7 were:

2017 January: University of Oslo Department of Biosciences
2016 December: University of Wurzburg Centre for Infectious Diseases
2016 November: University of Freiburg Graduate School of Biology and Medicine
2016 March: West Riding Microbiology Lecture University of Sheffield
2016 October: University of Bristol, Bristolbridge synthetic biology seminar series
2017 February: University of Warwick, Department of Biosciences
Year(s) Of Engagement Activity 2016,2017
 
Description Lead feature on BBSRC website 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Feature from the BBSRC on their website, describing our published research from this grant.
Year(s) Of Engagement Activity 2015
URL http://www.bbsrc.ac.uk/news/health/2015/151202-pr-how-bacterial-predators-kill-other-bacteria/
 
Description Professor Liz Sockett: Invited panellist June 2016 and contributor to Pew Foundation meeting on the Antimicrobial Pipeline Report, Boston USA 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact To assist Pew working group on aspects of therapeutic use of predators - in particular with reference to goring threat of antimicrobial resistance
Year(s) Of Engagement Activity 2016
 
Description School Visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk & discussion, both science-specific and also participation of women in science (talk at all female school)

Students have undertaken summer placements in my lab
Year(s) Of Engagement Activity 2014
 
Description Summer Student (Nuffield) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Prof Sockett and Dr Lovering hosted an undergraduate student Richard Acton on a 6 week Nuffield Summer Bursary position (3 weeks at each lab) studying the genetics and biochemistry of a Bdellovibrio cell wall adhesion gene-product.

Work undertaken by Richard will form part of a peer-reviewed publication; Richard's training will aid & influence his own research career
Year(s) Of Engagement Activity 2014
 
Description Summer student (schools) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Participants in your research and patient groups
Results and Impact Prof Sockett hosted a lower 6th school student for a week in the lab June 2014 to do experiments on bacterial 2 hybrid interactions of Bdellovibrio proteins

Fostering appreciation of science in younger generation
Year(s) Of Engagement Activity 2014
 
Description Talk to school students on research science as a career option 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Pupils attended a presentation about microbial diversity and then spoke in small groups over 2 and a half hours about careers in research science, specifically outside medicine
Year(s) Of Engagement Activity 2017