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Bacterial Adhesion Control through Surface TArgeted Regulation

Periodic Reporting for period 1 - BAC-STAR (Bacterial Adhesion Control through Surface TArgeted Regulation)

Reporting period: 2017-09-01 to 2019-08-31

Medical implants have been optimised with micro- and nano-rough surfaces for enhanced bone intergration; however, such surfaces also promote the unwanted adhesion of pathogens, resulting in an inflammatory response, causing the loss of supporting bone, and ultimately leading to implant failure. Once a biofilm has formed, it can be very difficult to clinically remove it, which may result in recurring infection and progressive damage to both implant and supporting bone.

The model system for this grant, Actinobacillus/Aggregatibacter actinomycetemcomitans (A.ac.) is a Gram-negative bacterium associated with aggressive periodontitis (an inflammatory condition that causes the degradation of the tissues surrounding the teeth) and with failing implants. Complications of A.ac. infections include endocarditis (inflammation of the inner tissues of the heart, most commonly affecting the aortic valve), brain and subcutaneous abscesses, and osteomyelitis (bone infection). The mortality rate from A.ac. endocarditis is approximately 18%. It is therefore desirable to prevent an infection with A.ac. before it can gain the resistance advantages from biofilm formation and maximised pathogenicity.

Understanding the adhesion mechanisms of A.ac. and of its major adhesins (the molecules that mediate adhesion to surfaces) can help to understand the adhesion and biofilm formation strategies of a whole library of pathogens. The similarity between these adhesins implicates the potential to prevent the adhesion of further pathogens by minor modification to the disruption mechanism for A.ac. adhesion.

We intended to drive the development of novel antibacterial surfaces through creating more detailed knowledge about bacterial adhesion mechanisms. Bactericidal approaches promote the evolution of resistance mechanisms, and generally anti-adhesive surfaces are of limited use in the medical field, since such surfaces also prohibit host-tissue integration. Anti-adhesive surface modifications therefore needed to become more specific to achieve selective adhesion.

We therefore wanted to establish a bottom-up approach, starting with the assay development for molecular biology studies of bacterial adhesion factors, and of their interaction with surfaces. The aim was to create a feedback-loop that translates assay results into increasingly specific surface modifications, using iterative test runs that combine identified anti-adhesive conditions for a particular adhesion protein. Novel approaches to assay development and rapid translation of fundamental research into applied surface modifications were expected from the collaboration between a materials science group (BIOMAT: Department of Biomaterials) and a molecular biology group (IBV: Department of Biosciences). The collaboration between these groups is a unique opportunity to obtain both an understanding of adhesin function and the direct implementation of this knowledge to novel surface modifications. This project therefore promoted interdisciplinary expertise and has the strong potential to generate further multidisciplinary collaborations with opportunities for interdisciplinary training.
Substantial research infrastructure and expertise for investigations on anaerobic pathogens has been established within the scope of this project. The bacterial cell surface group at the University of Oslo and its collaboration partner, BIOMAT, have strengthened their synergetic research approach and expanded their network for more efficient investigations on societal problems, such as infection control on implant surfaces.

The adhesin-gene of interest for this project, extracellular matrix protein adhesin A (emaA), has been disrupted in multiple A.ac. serotypes. Adhesion from this library of knockout mutants is compared to wild type adhesion in order to elucidate the fundamental interactions of emaA with abiotic surfaces. Training in cloning and protein overexpression was performed on a related adhesin from the same protein family (type Vc trimeric autotransporters), and yielded valuable insights into the autotransport process of this adhesin. These results were published in Molecular Microbiology.

Static adhesion assays were developed for this purpose, and are continuously improved with focus on reproducibility and increasing selectivity for adhesion-disruptive interactions. The acquired experience on quantitative methods for cell-adhesion and survival has been applied to ion-uptake studies, which revealed promising regulation strategies for ion-uptake and reduction within cells.

The work package on surface modification required competence building on surface characterization techniques, which enabled more in-depth investigations on pathogen adhesion to abiotic surfaces in general. The resulting collaboration improved our understanding of a related adhesin family (type Ve autotransporters), which contribute to the virulence of fish pathogens and therefore has a substantial impact on the fish farming industry. The corresponding publication has been submitted to Environmental Microbiology.

The work has already resulted in two publications:
1) “Insights into the autotransport process of a trimeric autotransporter, Yersinia Adhesin A (YadA)” in Molecular Microbioly, 2019. 111: 844-862.
2) “The inverse autotransporters of Yersinia ruckeri YrInv and YrIlm contribute to biofilm formation and virulence” submitted to Environmental Microbiology and Environmental Microbiology Reports.

Additionally, two more publications are planned:
1) “Crucial substrate characteristics for emaA-mediated adhesion”
2) “Divalent cations influence cellular palladium uptake and regulate the characteristics of reduced palladium nanoparticles in cells”
Our interdisciplinary collaboration enabled scientific advances in the fields of assay and method development for research on infection resistant materials. Genetic manipulation techniques for difficult targets have been developed, and will benefit further investigations on A.ac. and on related pathogens in the future. The methods for detection and quantification of pathogens on prototype materials have also been optimized and standardized, allowing for accelerated progress in material research for medical applications. Implants with faster integration rates and lower risks of failure can be achieved by tuning their surface features towards greater selectivity for host cell integration and pathogen repulsion. We have therefore laid the groundwork for smart surfaces that repress virulence factors without killing the pathogens, thereby avoiding the occurrence of resistance mechanisms. The generated expertise from this project may even be used to investigate biologically mediated corrosion, and could therefore serve as development platform for corrosion-resistant materials. Such research is especially valuable for applications in aquatic environments, for example the shipping and oil industry, fish farming, off-shore wind power, bio-fuel cells, and hydroponics.

The research in this project has focused on the interactions of a specific bacterial adhesin, and fundamental insights about its function are expected from the experiments that can be performed in the remaining funding time. Additionally, the assays, local infrastructure, and international collaboration that have been developed as a result of this project will continue to foster interdisciplinary research on cell-surface interactions.