Periodic Reporting for period 1 - STICKY SUGARS (Sweet adhesins: Probing bacterial glycoproteins with novel tools to inspire future antibacterial strategies)
Berichtszeitraum: 2023-05-01 bis 2025-10-31
With bacteria increasingly resistant to current antibiotics, new strategies to fight infection are needed. Bacterial adhesin proteins, which play an important role in the establishment of infection, appear to be promising targets for novel therapies. Adhesins enable the first contact between bacteria and host cells. We know that some adhesins are modified with highly unique sugars that are important for adhesin stability and adherence. However, we do not know how widespread this modification is, and whether it constitutes a good target for antibacterial strategies. A lack of suitable methods for the detection of bacterial adhesin sugars has impeded the detailed study of these sugars, blocking further efforts to develop antibacterial strategies against bacterial adhesin sugars.
In this project, I aim to revolutionize our understanding of adhesin sugars, in terms of timing and prevalence of the modification, which is crucial information to start targeting adhesin sugars. To this end, I have designed an innovative approach that combines the strengths of carbohydrate chemistry, enzymology, microbiology and molecular dynamics to 1) establish straightforward methods to selectively visualize and quantify the adhesin sugars directly on the surface of bacterial cells; 2) detect the presence of adhesin sugars under changing growth conditions and in relevant clinical isolates; and 3) develop a computational method to predict the presence of sugars on adhesins that have not yet been identified. Recent findings from my team on the process of adhesin modification underscore the uniqueness of adhesin sugars, and this experience puts me in an ideal position to start this project.
The overarching goal of STICKY SUGARS is to lay the foundation for the exploitation of adhesin sugars as antibacterial targets. By visualizing adhesin sugars directly on bacteria, we can observe their response to antibacterial treatments and pave the way toward clinical applications in the future.
In this project, I aim to revolutionize our understanding of adhesin sugars, in terms of timing and prevalence of the modification, which is crucial information to start targeting adhesin sugars. To this end, I have designed an innovative approach that combines the strengths of carbohydrate chemistry, enzymology, microbiology and molecular dynamics to 1) establish straightforward methods to selectively visualize and quantify the adhesin sugars directly on the surface of bacterial cells; 2) detect the presence of adhesin sugars under changing growth conditions and in relevant clinical isolates; and 3) develop a computational method to predict the presence of sugars on adhesins that have not yet been identified. Recent findings from my team on the process of adhesin modification underscore the uniqueness of adhesin sugars, and this experience puts me in an ideal position to start this project.
The overarching goal of STICKY SUGARS is to lay the foundation for the exploitation of adhesin sugars as antibacterial targets. By visualizing adhesin sugars directly on bacteria, we can observe their response to antibacterial treatments and pave the way toward clinical applications in the future.
With the 2 PhD students appointed on this project, we have made significant strides to lay down the foundation for the project.
Towards objective 1, we have developed the chemistry to synthesize sugar-nucleotides that carry a chemical handle for later conjugation and visualization. These sugar-nucleotides will be used by the editing enzymes to specifically label adhesin sugars.
On the biochemical part of the project, we have identified the sugar specificity of a central glycosyltransferase enzyme in ETEC bacteria. In addition, we have started the expression of adhesin (glycoproteins) to serve as substrates for the development of our editing method. Gratifyingly, we have successfully performed the adhesin-sugar labeling in vitro on isolated proteins.
Towards objective 3, we have started to collect all relevant data on known glycosylation sites and developed computational methods to obtain insight into structural and chemical features of the sites to understand and predict site occupancy.
Towards objective 1, we have developed the chemistry to synthesize sugar-nucleotides that carry a chemical handle for later conjugation and visualization. These sugar-nucleotides will be used by the editing enzymes to specifically label adhesin sugars.
On the biochemical part of the project, we have identified the sugar specificity of a central glycosyltransferase enzyme in ETEC bacteria. In addition, we have started the expression of adhesin (glycoproteins) to serve as substrates for the development of our editing method. Gratifyingly, we have successfully performed the adhesin-sugar labeling in vitro on isolated proteins.
Towards objective 3, we have started to collect all relevant data on known glycosylation sites and developed computational methods to obtain insight into structural and chemical features of the sites to understand and predict site occupancy.
The identification and visualization of glycans on proteins is notoriously difficult to achieve on live cells. To tackle this issue, we are developing an editing method.
We have revealed that one of our predicted editing enzymes indeed accepts an unnatural sugar-nucleotide and transfers the sugar-derivative onto the adhesin glycoprotein to directly inform on the glycans on the protein surface.
Next steps include the application of our method on live cell surface glycoproteins, and developing other enzymes-sugarnucleotide pairs that also work.
We have revealed that one of our predicted editing enzymes indeed accepts an unnatural sugar-nucleotide and transfers the sugar-derivative onto the adhesin glycoprotein to directly inform on the glycans on the protein surface.
Next steps include the application of our method on live cell surface glycoproteins, and developing other enzymes-sugarnucleotide pairs that also work.