Porphyromonas gingivalis is a keystone bacterium found in chronic periodontal disease, which is characterised by severe inflammation and destruction of supportive tooth structures. The disease has also been linked to systemic pathologies including cardiovascular diseases and autoimmune disorders. P. gingivalis growth is dependent on the acquisition of environmental haem for its iron and porphyrin requirements. As such, the bacterium has an array of proteins for haem acquisition, including HusA (Heme uptake system protein A). In previous work1, we have shown that HusA directly binds haem and is essential for P. gingivalis to grow under haem-limiting conditions, such as those found inside plaques and in saliva. Therefore, HusA may represent a therapeutic target for treating chronic periodontitis and possibly other associated systemic diseases.
As first steps towards developing compounds that target HusA and P. gingivalis, we have characterised the haem-binding mode of HusA using a combined mutagenesis and biophysical approach. We show that HusA binds haem with micromolar affinity. Four residues significantly affect haem binding when mutated singly or in pairs, and these residues map to a shallow hydrophobic pocket on the surface of the solution structure of HusA. To probe HusA binding specificity, the binding of HusA to a range of haem analogues was tested. We report that HusA binds deuteroporphyrin-IX (DPIX) ~20 times tighter than for haem. Based on these findings, and as a proof of principle, we have begun to design DPIX-antibiotic conjugates that target HusA. Preliminary results from one DPIX-antibiotic conjugate show that it can bind HusA in vitro and diminish P. gingivalis population numbers inside human cells. In summary, our work has shown that targeting and manipulating HusA:porphyrin interactions is a promising avenue to developing new therapeutics against P. gingivalis.