Biochemical and structural characterization of novel antibacterial and antifungal bacterial toxins

Antimicrobial-resistant bacterial pathogens constitute a major risk to public health and caused 1.27 million deaths in 2019. As only a few antimicrobials have been successfully developed recently, new strategies to fend off pathogenic bacteria are critically needed. To this aim, our collaborators at the Levy lab have developed a computational approach and discovered novel toxin domains of Polymorphic toxins (PTs) and their cognate immunity genes (PIMs) that can neutralize the toxin activities. PTs are multi-domain proteins that contain C-terminal toxin domains and are employed by microbes to kill competing microbes and can be utilized in various clinical and biotechnological applications. The toxicity of nine short novel toxins (PT1-9) that cause bacterial or yeast cell death was validated. The toxins likely act as enzymes that cause severe damage to cell shape, membrane, and DNA.

To further investigate the novel PTs` enzymatic activity, we expressed three novel PTs as recombinant proteins. We demonstrated using biochemistry studies that the toxins have efficient metallo-DNAse activity. To investigate the structural basis for the PTs catalytic activity, we solved the crystal structure of two novel toxin-immunity protein complexes. The structures confirm the new protein topology of the PTs that do not resemble the topology and structure of any known protein. Also, the structures shed light on the enzymatic mechanism of the PTs and the neutralization activity of the PIMs. Hence, our work elucidates mechanistic details regarding activity of the novel toxins that can serve as the basis for developing new antibacterial and antifungal treatments.