Activation mechanism of Pertussis-like toxins

Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. While the role of pertussis toxin in whooping cough is well established, pertussislike toxins produced by other bacteria are less studied and their mechanisms of action are unclear. Here, we report that some extraintestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro. We found that this protein, EcPlt is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADPribosyltransfersase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, while pertussis toxin targets a cysteine residue within the α-subunit of http://www.jbc.org/cgi/doi/10.1074/jbc.M117.796094 The latest version is at JBC Papers in Press. Published on June 29, 2017 as Manuscript M117.796094 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 2, 2017 hp://w w w .jb.org/ D ow nladed from Activation mechanism of Pertussis-like toxins 2 inhibitory trimeric G proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity. Pathogenic Escherichia coli strains preferentially inhabit different sites within their hosts, with enteric or extra-intestinal niches presenting distinctly different challenges. Infection by enteric or diarrheagenic E. coli can result in gastroenteritis but seldom spreads beyond the intestinal tract except in immunocompromised individuals(1,2). In contrast, extra-intestinal E. coli (ExPEC) strains such as uropathogenic E. coli (UPEC) or neonatal meningitis E. coli (NMEC) can reside passively within the gut until conditions permit their expansion into the urinary tract, blood or nervous system where they may potentially cause lifethreatening disease(3). To do so, ExPEC strains express a range of virulence factors, often encoded on mobile genetic elements, including AB5 toxins. Such phage-encoded toxins are secreted by several major bacterial pathogens including: enterotoxigenic E. coli (ETEC), and enterohemorrhagic E. coli (EHEC)(4), Vibrio cholerae(5), Shigella(6), Salmonella(7) and Bordetella pertussis(8,9). Modern sequencing techniques frequently identify novel AB5 toxins related to those previously studied, but their conservation at the level of function remains to be determined. AB5 virulence factors bind to glycans present on the surface of eukaryotic host cell membranes resulting in their internalization. Once inside the host cell the enzymatic A subunits are released allowing them to disrupt host biochemistry and physiology. Host cell recognition and internalization is mediated by the toxin B subunits that share a unique pentameric ring-like assembly. This juxtaposes with the A subunit cargos that are class-specific and structurally divergent, their activation inside mammalian cells occurs through distinct intracellular detection and release mechanisms(10). Five AB5 toxin families currently exist: the enzymatic components of the Subtilase cytotoxin(11) and EcxAB toxin(12) are proteases while those of the Shiga group(6) are ribosome inhibitors; cholera and heat labile enterotoxins(4,5) carry related ADPribosyltransferases, as do the related Pertussis(8,9) and typhoid toxin (7) proteins. Toxin ADP-ribosyltransferases (ARTs) hydrolyze the nicotinamide group from NAD and transfer the ADP-ribose moiety onto specific host proteins. Pertussis toxin specifically targets inhibitory trimeric G-proteins by modifying a conserved cysteine located four residues from the C-termini of the Gα subunit(13). This modification renders the Gαi/o subunits unable to associate with their cognate G-protein coupled receptors (GPCRs) thus modulating the host’s immune response. A series of related pertussislike (Plt) toxins exist within the genomes of pathogenic bacteria including strains of E. coli, Salmonella and Yersinia. The bacterial strains harboring such virulence factors are diverse and their evolutionary relationship complicated due to the spread of these genes on mobile genetic elements. Proteins within the Plt family include by gest on N ovem er 2, 2017 hp://w w w .jb.org/ D ow nladed from Activation mechanism of Pertussis-like toxins 3 the atypical archetype member whose glycan binding B-subunits have expanded and diverged within the B. pertussis genome to form four separate genes yielding a pseudopentameric glycan-binding platform. In contrast, other pertussis-like toxins display a homopentameric glycan-binding stoichiometry. All Plt proteins nonetheless carry a conserved catalytic A subunit. In the secreted state the enzymatic ART domain of pertussis-like toxins lies atop the 5 glycan-binding subunits allowing its C-terminus to thread through a U-shaped NAD-binding cleft before plunging into the pore of the B subunit pentamer(7,9). When these C-terminal residues are truncated from Pertussis toxin its ART domain is constitutively active in vitro but is unable to associate with its pseudopentamer or enter cells(14). Enzymatic activation in vivo requires proteolytic separation of the inhibitory C-terminus and reduction of a connecting disulfide. While the molecular mechanisms underlying an alternate activation mechanism of cholera toxin are understood(15) the changes that occur following activation of a Pertussis-like protein have not previously been characterized. While the association of Pertussis toxin with whooping cough is well established, orthologous pertussis-like toxins present within other pathogenic bacterial infections are less studied and their mechanism(s) of action are unclear(7). Here we identify a Pertussis-like AB5 protein (EcPltAB) from clinical E. coli isolates that is related to the Typhoid and ArtAB toxins observed in typhoidal and nontyphoidal Salmonella serovars. We provide structures that confirm that Pertussis-like toxins are secreted as inactive forms in which an intramolecular disulfide holds an occluding C-terminal tail within the NAD binding site. This conserved disulfide also serves as a redox-switch that senses host cell entry, with reduction of the bond allowing displacement of the occluding C-terminal residues, facilitating NAD binding and maturation of catalytic activity. At a global level, the existence of EcPltAB type proteins expands the family of known bacterial Pertussis-like toxins capable of modulating the human immune system. Furthermore, we show that while AB5 proteins are segregated into evolutionarily related enzyme families these may not always modify the same residues within host proteins.