No fitness cost entailed by type VI secretion system synthesis, assembly, contraction, or disassembly in enteroaggregative Escherichia coli.

IMPORTANCE: Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Due to its size and mechanism of action, the T6SS was intuitively thought to be energetically costly. Here, using a combination of mutants and growth measurements in liquid medium, on plates, and in competition experiments, we show that the T6SS does not entail a growth cost to enteroaggregative Escherichia coli.

Intracellular Delivery of Human Purine Nucleoside Phosphorylase by Engineered Diphtheria Toxin Rescues Function in Target Cells.

Despite a wealth of potential applications inside target cells, protein-based therapeutics are largely limited to extracellular targets due to the inability of proteins to readily cross biological membranes and enter the cytosol. Bacterial toxins, which deliver a cytotoxic enzyme into cells as part of their intoxication mechanism, hold great potential as platforms for delivering therapeutic protein cargo into cells. Diphtheria toxin (DT) has been shown to be capable of delivering an array of model proteins of varying sizes, structures, and stabilities into mammalian cells as amino-terminal fusions. Here, seeking to expand the utility of DT as a delivery vector, we asked whether an active human enzyme, purine nucleoside phosphorylase (PNP), could be delivered by DT into cells to rescue PNP deficiency. Using a series of biochemical and cellular readouts, we demonstrate that PNP is efficiently delivered into target cells in a receptor- and translocation-dependent manner. In patient-derived PNP-deficient lymphocytes and pluripotent stem cell-differentiated neurons, we show that human PNP is efficiently translocated into target cells by DT, where it is able to restore intracellular hypoxanthine levels. Further, through replacement of the native receptor-binding moiety of DT with single-chain variable fragments that were selected to bind mouse HBEGF, we show that PNP can be retargeted into mouse splenocytes from PNP-deficient mice, resulting in restoration of the proliferative capacity of T-cells. These findings highlight the versatility of the DT delivery platform and provide an attractive approach for the delivery of PNP as well as other cytosolic enzymes implicated in disease.

Corrigendum: Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

[This corrects the article on p. 2 in vol. 7, PMID: 28144586.].

Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

The only recognized virulence factor of enterotoxigenic Bacteroides fragilis (ETBF) that accompanies bloodstream infections is the zinc-dependent non-lethal metalloprotease B. fragilis toxin (BFT). The isolated toxin stimulates intestinal secretion, resulting in epithelial damage and necrosis. Numerous publications have focused on the interrelation of BFT with intestinal inflammation and colorectal neoplasia, but nothing is known about the mechanism of its secretion and delivery to host cells. However, recent studies of gram-negative bacteria have shown that outer membrane vesicles (OMVs) could be an essential mechanism for the spread of a large number of virulence factors. Here, we show for the first time that BFT is not a freely secreted protease but is associated with OMVs. Our findings indicate that only outer surface-exposed BFT causes epithelial cell contact disruption. According to our in silico models confirmed by Trp quenching assay and NMR, BFT has special interactions with outer membrane components such as phospholipids and is secreted during vesicle formation. Moreover, the strong cooperation of BFT with polysaccharides is similar to the behavior of lectins. Understanding the molecular mechanisms of BFT secretion provides new perspectives for investigating intestinal inflammation pathogenesis and its prevention.

Host-cell specificity and transcytosis of nontoxic nonhemagglutinin protein of botulinum neurotoxin serotype D.

Serotype D botulinum toxin (BoNT) complex (TC), a causative agent of foodborne botulism in animals, traverses the gastrointestinal tract and circulation, eventually becoming localized in neuromuscular junctions, where the serotype D BoNT cleaves SNARE substrate synaptobrevin II involved in neurotransmitter release. During this process, BoNT must pass through cells, thus from the intestinal lumen to the cells of the intestinal tract and blood vessels. The botulinum TC is formed by association of the BoNT with at least one nontoxic protein, which may be a nontoxic nonhemagglutinin (NTNHA). In this work, we examined the binding and transcytosis of serotype D NTNHA protein in epithelial and endothelial cells to clarify the role played by the protein in toxin delivery. Our studies showed that NTNHA bound to and transcytosed across rat intestinal epithelial (IEC-6) and bovine aortic endothelial (BAEC) cells. While NTNHA also bound to canine renal (MDCK) or human colon carcinoma (Caco-2) cells, but it did not traverse across MDCK or Caco-2 cells. Such specificity of NTNHA protein transcytosis may explain why only some animals are sensitive to botulinum toxin. The sensitivity depends on the toxin serotype in play, and the route of toxin delivery.