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Chemical Crosstalk: targets of interkingdom signals at the host-pathogen interface

Final Report Summary - CHEMICALCROSSTALK (Chemical Crosstalk: targets of interkingdom signals at the host-pathogen interface)

The emergence of multidrug resistant bacteria is a global concern, threatening a return to the pre-antibiotic era when infectious disease was a routine cause of death. There is an urgent need to discover novel antibiotics and explore new approaches to combat infection. Blocking microbial virulence or cell-cell communication have been proposed as potential therapeutic approaches; however, our understanding of the hugely complex interactions operating at the host-pathogen interface is still very limited.

Cells often communicate, and pathogen and host manipulate each other, using small molecule signals. This is fundamentally a chemical problem, yet there is a lack of molecular level detail in these fields: How are signals sensed? How do the signal transduction pathways operate? Do different species ‘eavesdrop’ on each other’s communication? In the project ChemicalCrosstalk we have explored the mode of action of an interkingdom signal using an interdisciplinary blend of chemistry and biology.

Dynorphin (DYN) is an important stress-related peptide hormone that binds opioid receptors in humans, and previous studies have shown that it induces virulence in the opportunistic bacterial pathogen, Pseudomonas aeruginosa (PAO). Our aim was to understand this interaction at the molecular level and identify potential receptors for DYN in PAO that could mediate this phenotype. In the first stage of the project, we designed and synthesised novel DYN-mimetic probes for chemical proteomics studies. Probes were equipped with both a photoreactive group and a tag for detection (Figure 1). The photoreactive group is inert under standard culture conditions but upon irradiation with UV light forms a highly reactive intermediate that crosslinks the probe to proteins or other biomolecules in close proximity – allowing us to ‘capture’ the binding partners of DYN for analysis. The tag we used was a terminal alkyne – a small, discrete functional group that can be reacted in a biorthogonal ligation with azide-functionalised labels (a ‘click’ reaction) for further analysis. Over the course of the project, a small library of ~30 compounds was prepared to explore structure-activity relationships and the nature of the photoreactive group.

The following workflow was optimised to analyse the binding of DYN-probes to PAO: bacteria were grown in a chemically defined medium to specific growth phases, and then live cells were incubated with probe at the different concentrations and for different times; bacteria were then irradiated with UV light to trigger the activation of the photoreactive group, harvested, lysed and lysates subject to click chemistry to attach a fluorophore for visualisation or an affinity handle for isolation/enrichment of probe-captured targets. Enriched proteins were then digested into peptides and analysed by high-resolution tandem LC-MS/MS proteomics; comparison with controls allowed us to identify those proteins that were bound to or close to the DYN-probe. We found that the probes bound to the lipopolysaccharide (LPS) molecules that are highly abundant in the membranes of PAO, as well as several membrane proteins –including receptors that PAO could be using to detect DYN. Interestingly, we observed that the exact choice of photoreactive group had a strong impact on the labelling. These observations are in-line with other reports and of general interest, since the use of photoprobes as tools to understand biology is currently a very active topic of research in the field of chemical biology. The increasing power of modern proteomics has the potential to greatly enhance the value of such tools.

In parallel research efforts, we explored the phenotype of DYN-treatment, such as the production of virulence factors (toxins etc that damage the host). Several large-scale analyses of the PAO proteome were also carried out to define the impact of DYN treatment on a global scale, revealing changes in specific secondary metabolic pathways and proteins involved in LPS modification. Combined with the chemical proteomic studies described above, our data suggest that DYN may trigger a response in PAO similar to that of antimicrobial peptides (human endogenous defence molecules). To explore this hypothesis, we initiated the study of PAO mutant strains lacking the putative receptor for DYN. Although further validation is required, this project has been successful in its key goal of using an innovative and unbiased chemical proteomics approach to reveal a candidate receptor for an interkingdom signal.

In summary, we have synthesised novel chemical probes and applied them to discover the potential protein binding partners of DYN in live bacterial cells. Combining these tools with global proteomic and phenotypic analyses of bacteria treated with DYN sheds new light on the mode of action of this human signalling molecule in bacterial infection. This study has relevance in a world where understanding host-pathogen interactions has never been more important.

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