Functional characterization of Vip3Aa from Bacillus thuringiensis reveals the contributions of specific domains to its insecticidal activity.

Microbially derived, protein-based biopesticides offer a more sustainable pest management alternative to synthetic pesticides. Vegetative insecticidal proteins (Vip3), multidomain proteins secreted by Bacillus thuringiensis, represent a second-generation insecticidal toxin that has been preliminarily used in transgenic crops. However, the molecular mechanism underlying Vip3's toxicity is poorly understood. Here, we determine the distinct functions and contributions of the domains of the Vip3Aa protein to its toxicity against Spodoptera frugiperda larvae. We demonstrate that Vip3Aa domains II and III (DII-DIII) bind the midgut epithelium, while DI is essential for Vip3Aa's stability and toxicity inside the protease-enriched host insect midgut. DI-DIII can be activated by midgut proteases and exhibits cytotoxicity similar to full-length Vip3Aa. In addition, we determine that DV can bind the peritrophic matrix via its glycan-binding activity, which contributes to Vip3Aa insecticidal activity. In summary, this study provides multiple insights into Vip3Aa's mode-of-action which should significantly facilitate the clarification of its insecticidal mechanism and its further rational development.

Unique features in the intracellular transport of typhoid toxin revealed by a genome-wide screen.

Typhoid toxin is a virulence factor for Salmonella Typhi and Paratyphi, the cause of typhoid fever in humans. This toxin has a unique architecture in that its pentameric B subunit, made of PltB, is linked to two enzymatic A subunits, the ADP ribosyl transferase PltA and the deoxyribonuclease CdtB. Typhoid toxin is uniquely adapted to humans, recognizing surface glycoprotein sialoglycans terminated in acetyl neuraminic acid, which are preferentially expressed by human cells. The transport pathway to its cellular targets followed by typhoid toxin after receptor binding is currently unknown. Through a genome-wide CRISPR/Cas9-mediated screen we have characterized the mechanisms by which typhoid toxin is transported within human cells. We found that typhoid toxin hijacks specific elements of the retrograde transport and endoplasmic reticulum-associated degradation machineries to reach its subcellular destination within target cells. Our study reveals unique and common features in the transport mechanisms of bacterial toxins that could serve as the bases for the development of novel anti-toxin therapeutic strategies.

Generation and Characterization of Typhoid Toxin-Neutralizing Human Monoclonal Antibodies.

Typhoid toxin is a virulence factor of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, and is thought to be responsible for the symptoms of severe disease. This toxin has a unique A2B5 architecture with two active subunits, the ADP ribosyl transferase PltA and the DNase CdtB, linked to a pentameric B subunit, which is alternatively made of PltB or PltC. Here, we describe the generation and characterization of typhoid toxin-neutralizing human monoclonal antibodies by immunizing genetically engineered mice that have a full set of human immunoglobulin variable region genes. We identified several monoclonal antibodies with strong in vitro and in vivo toxin-neutralizing activity and different mechanisms of toxin neutralization. These antibodies could serve as the basis for the development of novel therapeutic strategies against typhoid fever.

Mechanism of substrate recognition and transport by an amino acid antiporter.

In extremely acidic environments, enteric bacteria such as Escherichia coli rely on the amino acid antiporter AdiC to expel protons by exchanging intracellular agmatine (Agm(2+)) for extracellular arginine (Arg(+)). AdiC is a representative member of the amino acid-polyamine-organocation (APC) superfamily of membrane transporters. The structure of substrate-free AdiC revealed a homodimeric assembly, with each protomer containing 12 transmembrane segments and existing in an outward-open conformation. The overall folding of AdiC is similar to that of the Na(+)-coupled symporters. Despite these advances, it remains unclear how the substrate (arginine or agmatine) is recognized and transported by AdiC. Here we report the crystal structure of an E. coli AdiC variant bound to Arg at 3.0 A resolution. The positively charged Arg is enclosed in an acidic binding chamber, with the head groups of Arg hydrogen-bonded to main chain atoms of AdiC and the aliphatic portion of Arg stacked by hydrophobic side chains of highly conserved residues. Arg binding induces pronounced structural rearrangement in transmembrane helix 6 (TM6) and, to a lesser extent, TM2 and TM10, resulting in an occluded conformation. Structural analysis identified three potential gates, involving four aromatic residues and Glu 208, which may work in concert to differentially regulate the upload and release of Arg and Agm.

Bacteroides fragilis ubiquitin homologue drives intraspecies bacterial competition in the gut microbiome.

Interbacterial antagonism and associated defensive strategies are both essential during bacterial competition. The human gut symbiont Bacteroides fragilis secretes a ubiquitin homologue (BfUbb) that is toxic to a subset of B. fragilis strains in vitro. In the present study, we demonstrate that BfUbb lyses certain B. fragilis strains by non-covalently binding and inactivating an essential peptidyl-prolyl isomerase (PPIase). BfUbb-sensitivity profiling of B. fragilis strains revealed a key tyrosine residue (Tyr119) in the PPIase and strains that encode a glutamic acid residue at Tyr119 are resistant to BfUbb. Crystal structural analysis and functional studies of BfUbb and the BfUbb-PPIase complex uncover a unique disulfide bond at the carboxy terminus of BfUbb to mediate the interaction with Tyr119 of the PPIase. In vitro coculture assays and mouse studies show that BfUbb confers a competitive advantage for encoding strains and this is further supported by human gut metagenome analyses. Our findings reveal a previously undescribed mechanism of bacterial intraspecies competition.

Structural insight into Bacillus thuringiensis Sip1Ab reveals its similarity to ETX_MTX2 family beta-pore-forming toxin.

BACKGROUND: Microbially derived, protein-based biopesticides are an important approach for sustainable pest management. The secreted insecticidal proteins (Sips) produced by the bacterium Bacillus thuringiensis exhibit potent insecticidal activity against coleopteran pests and are, therefore, attractive as candidate biopesticides. However, the modes-of-action of Sips are unclear as comprehensive structural information for these proteins is lacking. RESULTS: Using X-ray crystallography, we elucidated the structure of monomeric Sip1Ab at 2.28 Å resolution. Structural analyses revealed that Sip1Ab has the three domains and conserved fold characteristic of other aerolysin-like beta-pore-forming toxins (ß-PFTs). Based on the sequence and structural similarities between Sip1Ab and other ETX_MTX2 subfamily toxins, we suggested the mechanism of these proteins and proposed that it is common to them all. CONCLUSION: The atomic-level structural data for Sip1Ab generated by the present study could facilitate future structural and mechanistic research on Sips as well as their application in sustainable insect pest management. © 2023 Society of Chemical Industry.

Molecular Insights into the Assembly and Functional Diversification of Typhoid Toxin.

Typhoid toxin is an A2B5 protein toxin and an important virulence factor for the human-adapted bacterial pathogen Salmonella enterica serovar Typhi, the causative agent of typhoid fever. Typhoid toxin contains two enzymatic subunits, PltA and CdtB, which dock onto a pentameric delivery platform composed of the protein PltB. It was recently reported that the same enzymatic subunits can assemble with a different delivery platform composed of the protein PltC, forming a distinct version of typhoid toxin. However, the differences in structure and receptor specificity between the PltC and PltB typhoid toxins remain unknown. Here, we determined atomic-level structures of the pentameric PltC subunit, the fully assembled PltC typhoid toxin, and the PltC pentamers in complex with glycan receptors. Biochemical and structural analyses indicate that PltB and PltC are unable to form heteromeric delivery complexes due to electrostatic repulsion at the subunit interface and thus form separate toxins only. We further observed that, despite low sequence similarity between PltB and PltC, they interact with PltA in a similar manner but that PltC exhibits stronger electrostatic interactions with PltA, enabling it to outcompete PltB in toxin assembly. The ligand-bound atomic structures of PltC show an additional glycan binding site not found in PltB and glycan array analysis indicates that PltB and PltC exhibit significant differences in glycan binding specificity. Collectively, this study offers atomic-level insights into how S. Typhi produces two distinct versions of typhoid toxin, thereby generating functional diversity in this key virulence factor. IMPORTANCE Typhoid fever is a devastating disease that kills more than 115,000 people every year and is caused by Salmonella Typhi. Typhoid toxin, exclusively produced by S. Typhi, was demonstrated to be responsible for the pathogenesis of typhoid fever. Typhoid toxin consists of a pentameric delivery B subunit to transport the catalytic A subunits into the host cell through binding of the glycan receptors. Recent study shows that S. Typhi encodes two homologous delivery B subunits that are able to associate with the same active subunits to produce alternative toxins with distinct functional characteristics. Here, we show that the two delivery subunits can form only homopentameric delivery platforms that compete to associate with typhoid toxin's active subunits and that the two resulting toxins have distinct glycan-binding properties that confer distinct functional traits. These findings highlight the unique assembly and functional diversification of typhoid toxins.

Alternate subunit assembly diversifies the function of a bacterial toxin.

Bacterial toxins with an AB5 architecture consist of an active (A) subunit inserted into a ring-like platform comprised of five delivery (B) subunits. Salmonella Typhi, the cause of typhoid fever, produces an unusual A2B5 toxin known as typhoid toxin. Here, we report that upon infection of human cells, S. Typhi produces two forms of typhoid toxin that have distinct delivery components but share common active subunits. The two typhoid toxins exhibit different trafficking properties, elicit different effects when administered to laboratory animals, and are expressed using different regulatory mechanisms and in response to distinct metabolic cues. Collectively, these results indicate that the evolution of two typhoid toxin variants has conferred functional versatility to this virulence factor. More broadly, this study reveals a new paradigm in toxin biology and suggests that the evolutionary expansion of AB5 toxins was likely fueled by the plasticity inherent to their structural design coupled to the functional versatility afforded by the combination of homologous toxin components.

Investigation of the role of typhoid toxin in acute typhoid fever in a human challenge model.

Salmonella Typhi is a human host-restricted pathogen that is responsible for typhoid fever in approximately 10.9 million people annually1. The typhoid toxin is postulated to have a central role in disease pathogenesis, the establishment of chronic infection and human host restriction2-6. However, its precise role in typhoid disease in humans is not fully defined. We studied the role of typhoid toxin in acute infection using a randomized, double-blind S. Typhi human challenge model7. Forty healthy volunteers were randomized (1:1) to oral challenge with 104 colony-forming units of wild-type or an isogenic typhoid toxin deletion mutant (TN) of S. Typhi. We observed no significant difference in the rate of typhoid infection (fever ≥38 °C for ≥12 h and/or S. Typhi bacteremia) between participants challenged with wild-type or TN S. Typhi (15 out of 21 (71%) versus 15 out of 19 (79%); P = 0.58). The duration of bacteremia was significantly longer in participants challenged with the TN strain compared with wild-type (47.6 hours (28.9-97.0) versus 30.3(3.6-49.4); P ≤ 0.001). The clinical syndrome was otherwise indistinguishable between wild-type and TN groups. These data suggest that the typhoid toxin is not required for infection and the development of early typhoid fever symptoms within the context of a human challenge model. Further clinical data are required to assess the role of typhoid toxin in severe disease or the establishment of bacterial carriage.