Biochemical insights into unique Bacteroidetes pore-forming toxins

Abstract

The human gut microbiota is one of the densest and most complex microbial ecosystems on earth, harbouring hundreds of different species. This high microbial density and species diversity leads to fierce competition for nutrients and space, hence promoting bacterial antagonism. The Gram-negative Bacteroidetes are prominent long-term colonisers of the intestinal flora and represent on average 50% of all bacterial isolates within the human gut. They possess anti-inflammatory, immunomodulatory and metabolic properties and hence play a pivotal role in human health. To outmatch competitors, Bacteroidetes have evolved strategies to directly antagonize opponents through the production of antimicrobial molecules. Among these, the recently discovered Bacteroidales-secreted antimicrobial proteins (BSAPs) are of special interest as they form a novel class of bacterial pore-forming toxins (PFTs). PFTs are produced by a wide range of bacteia and possess the remarkable ability to transition from inert monomeric water-soluble proteins into integral membrane oligomers to form lytic pores. Uniquely, BSAPs possess a Membrane Attack Complex/Perforin (MACPF) domain usually found in eukaryotic pore-forming innate immunity proteins. Incubation of BSAP-sensitive cells with purified BSAPs causes uptake of the membrane-impermeable DNA dye propidium iodide, and co-culture experiments demonstrate that BSAP-producing strains kill sensitive cells in a BSAP-dependent manner. BSAPs are hence the first bacterial MACPF proteins with bactericidal activity. However, the underlying molecular mechanisms of lytic pore formation and receptor recognition remain cryptic. Here, we present first biochemical insights into BSAP receptor recognition and oligomerization, as well as preliminary structural data of a BSAP/receptor complex. Our findings indicate that highly variable BSAP C-terminal domains, located downstream of the MACPF domain, are the sole factor responsible for receptor recognition and binding. Structural characterization of a BSAP/receptor complex further dissects this interaction and how receptor binding affects positioning of the MACPF domain in respect to the membrane plane. Taken together, our data provide first biochemical insights into these novel and poorly characterized PFTs