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VIral and BacteRial Adhesin Network Training

Periodic Reporting for period 1 - ViBrANT (VIral and BacteRial Adhesin Network Training)

Reporting period: 2018-01-01 to 2019-12-31

Infectious diseases, particularly drug resistant pathogens, were one of the key issues on the agenda of the G7- Summit in Germany in 2015. Every major health organisation from WHO onwards has identified infectious diseases and the decreasing efficacy of antibiotics in general as a major concern: classic antimicrobial therapy is failing; and deadly viruses are (re)emerging – the most recent example of which is of course Covid-19. Experts agree that new, rapid diagnostics and new antimicrobials are urgently required to prevent infections and therefore lower the demand for therapeutic treatments, reducing use of antimicrobials and so slowing the rise of drug resistance. Understanding the molecular basis of pathogen adhesion will lead to the development of novel anti-adhesion strategies and new diagnostic tools. It is therefore essential that Europe trains researchers in this important emerging area. Adhesion is at the heart of virulence: it plays the first and decisive role in the infection process of pathogens. Bacteria and viruses adhere to organic or inorganic surfaces, to each other, to host molecules or to host cells. Pathogens use adhesins to colonize tissues and cause infections, to bind host molecules for immune evasion, and in the case of bacteria, to bind to each other to create e.g. antibiotic-resistant biofilms on implanted devices. In ViBrANT we will adopt a highly cross-disciplinary, translational approach to improve our understanding of adhesion biology, to detect infections at the earliest possible stage, and thus to meet the threat posed by new and (re)emerging pathogens. Our aims are:
1) Optimal training through research for future leaders in a collaborative, interdisciplinary environment drawing on engineering, physical and biomedical sciences from academia and industry.
2) Promoting structured ESR training on an EU-wide basis through individual career and training plans, international placement of PhD students and frequent international exchanges in their working environment.
3) Cross-fertilisation of projects by sharing knowledge and experience as well as resources and samples.
4) European intersectoral collaborations on pathogen adhesion
Recent advances in the understanding of the importance of adhesion, in instrumentation for structure elucidation, and in nanobiosensors allow us to move towards meaningful systematic study of the molecular basis of pathogen adhesion and towards new diagnostic tools. ViBrANT will provide integrated Europe-wide world-class training through bottom-up research in adhesion, from clinical microbiological research, through functional studies of adhesins, to the elucidation of structures and the design of new diagnostic tests and devices.
During the first period of the Project, a key aim has been to ensure that the ViBrANT consortium maximises the collaborative environment fostered between the universities, commercial partners and the ESRs. We have arranged five different training workshops both in Germany and in the UK for the ESRs and they also took part in an international science conference FEMS2019 in Glasgow showing their posters there.
Local and international outreach and engagement has been actively encouraged with the ESRs: they contribute regular blog posts to the ViBrANT website, also in two other languages, visit schools and public events to tell people about ViBrANT and its impact.
During the first two years all the ESRs have made good progress with their projects. They have all published their first articles in Medical Microbiology and Immunology. Most of the ESRs have had their first secondments to other participants in ViBrANT, both industrial and academic to widen their knowledge and experience and produce concrete results for the projects.

Key research performed during this period includes:
In Work Package 1, the University of Leeds and Oslo University together (including industrial secondments) have devised the first concrete biosensor measurements of the trimeric autotransporter adhesin (TAA) Yersinia entercolitica YadA binding to collagen and vitronectin, extracellular matrix (ECM) molecules in humans. These impedance-based biosensors allowed us to show that the YadA-vitronectin interaction is not as expected. Frankfurt University resequenced the TAA BadA from Bartonella henselae showing that it was actually more than 3,900, not 3,082, residues long – and allowing the first definition of how this protein binds to fibronectin, another ECM protein.
In Work Package 2, the University of Leeds has made meaningful progress on key structural work that will underpin the rest of their projects. At the University of Leeds, individual sections of the TAA BpaC from Burkholderia pseudomallei were purified. These include novel repeats of unknown structure and function from the stalk region. The structure of these at 1.4 Å resolution (Fig. 1) showed that they adopted the canonical “YadA” head structure, and this has allowed us to build a model of the entire BpaC head and stalk.
Work Package 3 has focussed on use of these adhesins in microfluidic setups and the development of antiadhesive properties. One of the industrial beneficiaries, CeNTI, has made good progress on devising coatings that prevent bacteria adhering to plastic surfaces – either by making the surface super-hydrophilic or by making it superhydrophobic. The coated surfaces survive washing. The University of Minho and the University of Hull have developed techniques for detecting bacteria in microfluidic setups: we can now using magnetic nanoparticles to separate bacteria expressing YadA from those that do not; and the University of Hull has developed a microfluidic version of the “FISH” assay to label and visualise bacteria in very small scale. Such miniaturisation is essential for the development of point-of-care diagnostics.
The chief result of Work Package 4 has been the development of robust epidemiological markers for Staphylococcus aureus (i.e. the same bacterium as MRSA), which is an opportunistic pathogen in hospital settings (see below for more details). This work has already been published.
In terms of progress beyond state of the art, we have three significant results:
1) We have demonstrated that biosensors can be used to detect the presence of these trimeric autotransporter adhesins (TAAs) on the outside of bacteria, thus opening a mechanism for point-of-care pathogen diagnostics.
2) By following the genomic evolution of Staphylococcus aureus in the nose for up to 36 months, we have determined how many single nucleotide polymorphisms (SNPs) need to be used to identify specific S. aureus strains (e.g. to distinguish between pathogenic (MRSA) and non-pathogenic strains). By whole-genome and other sequencing approaches, we showed that 20 SNPs is enough to identify the strain of S. aureus during outbreaks of infection (Goyal et al. Front Microbiol 2019 10:1525).
It is worth remarking that the work described above is both beyond state-of-the-art and is likely to be translated within the time-frame of ViBrANT or shortly thereafter into diagnostics with societal impact and thus socio-economic benefits.
Fig. 1 Structure of residues 741-1054 of the BpaC C-terminal region