Evaluation of the Immunogenicity and Protective Efficacy of an Enterotoxigenic Escherichia coli CFA/I Adhesin-Heat-Labile Toxin Chimera.

Recent efforts to develop an enterotoxigenic Escherichia coli (ETEC) vaccine have focused on the antigenically conserved tip adhesins of colonization factors. We showed previously that intranasal immunization with dsc19CfaE, a soluble variant of the in cis donor strand-complemented tip adhesin of a colonization factor of the class 5 family (CFA/I) fimbria, is highly immunogenic and protects against oral challenge with CFA/I-positive (CFA/I+) ETEC strain H10407 in the Aotus nancymaae nonhuman primate. We also reported a cholera toxin (CT)-like chimera (called dsc19CfaE-CTA2/CTB) in which the CTA1 domain of CT was replaced by dsc19CfaE that was strongly immunogenic when administered intranasally or orogastrically in mice. Here, we evaluate the immunogenicity and protective efficacy (PE) of a refined and more stable chimera comprised of a pentameric B subunit of ETEC heat-labile toxin (LTB) in lieu of the CTB pentamer and a donor strand truncation (dsc14) of CfaE. The refined chimera, dsc14CfaE-sCTA2/LTB, was highly immunogenic in mice when administered intranasally or intradermally, eliciting serum and fecal antibody responses against CfaE and LTB, as well as strong hemagglutination inhibition titers, a surrogate for neutralization of intestinal adhesion mediated by CfaE. Moreover, the chimera was safe and highly immunogenic when administered intradermally to guinea pigs. In A. nancymaae, intradermal (i.d.) immunization with chimera plus single-mutant heat-labile toxin [LT(R192G)] elicited strong serum anti-CfaE and anti-LTB antibody responses and conferred significant reduction of diarrhea compared to phosphate-buffered saline (PBS) controls (PE = 84.1%; P < 0.02). These data support the further evaluation of dsc14CfaE-sCTA2/LTB as an ETEC vaccine in humans.

Biochemical and immunological characterization of an ETEC CFA/I adhesin cholera toxin B subunit chimera.

Surface-expressed colonization factors and their subunits are promising candidates for inclusion into a multivalent vaccine targeting enterotoxigenic Escherichia coli (ETEC), a leading cause of acute bacterial diarrhea in developing regions. However, soluble antigens are often poorly immunogenic in the absence of an adjuvant. We show here that the serum immune response to CfaE, the adhesin of the ETEC colonization factor CFA/I, can be enhanced in BALB/c mice by immunization with a chimeric antigen containing CfaE and pentameric cholera toxin B subunit (CTB) of cholera toxin from Vibrio cholerae. We constructed this antigen by replacing the coding sequence for the A1 domain of the cholera toxin A subunit (CTA) with the sequence of donor strand complemented CfaE (dscCfaE) within the cholera toxin operon, resulting in a dscCfaE-CTA2 fusion. After expression, via non-covalent interactions between CTA2 and CTB, the fusion and CTB polypeptides assemble into a complex containing a single dscCfaE-CTA2 protein bound to pentameric CTB (dscCfaE-CTA2/CTB). This holotoxin-like chimera retained the GM1 ganglioside binding activity of CTB, as well as the ability of CfaE to mediate the agglutination of bovine red blood cells when adsorbed to polystyrene beads. When administered intranasally to mice, the presence of CTB in the chimera significantly increased the serum immune response to CfaE compared to dscCfaE alone, stimulating a response similar to that obtained with a matched admixture of dscCfaE and CTB. However, by the orogastric route, immunization with the chimera elicited a superior functional immune response compared to an equivalent admixture of dscCfaE and CTB, supporting further investigation of the chimera as an ETEC vaccine candidate.

Glycolipid Crosslinking Is Required for Cholera Toxin to Partition Into and Stabilize Ordered Domains.

Current models of lipid rafts propose that lipid domains exist as nanoscale compositional fluctuations and these fluctuations can potentially be stabilized into larger domains, consequently better compartmentalizing cellular functions. However, the mechanisms governing stabilized raft assembly and function remain unclear. Here, we test the role of glycolipid crosslinking as a raft targeting and ordering mechanism using the well-studied raft marker cholera toxin B pentamer (CTxB) that binds up to five GM1 glycosphingolipids to enter host cells. We show that when applied to cell-derived giant plasma membrane vesicles, a variant of CTxB containing only a single functional GM1 binding site exhibits significantly reduced partitioning to the ordered phase compared to wild-type CTxB with five binding sites. Moreover, monovalent CTxB does not stabilize membrane domains, unlike wild-type CTxB. These results support the long-held hypothesis that CTxB stabilizes raft domains via a lipid crosslinking mechanism and establish a role for crosslinking in the partitioning of CTxB to ordered domains.

Whole-Genome Sequencing Identifies In Vivo Acquisition of a blaCTX-M-27-Carrying IncFII Transmissible Plasmid as the Cause of Ceftriaxone Treatment Failure for an Invasive Salmonella enterica Serovar Typhimurium Infection.

We report a case of ceftriaxone treatment failure for bacteremia caused by Salmonella enterica subsp. enterica serovar Typhimurium, due to the in vivo acquisition of a blaCTX-M-27-encoding IncFII group transmissible plasmid. The original ß-lactamase-susceptible isolate ST882S was replaced by the resistant isolate ST931R during ceftriaxone treatment. After relapse, treatment was changed to ciprofloxacin, and the patient recovered. Isolate ST931R could transfer resistance to Escherichia coli at 37°C. We used whole-genome sequencing of ST882S and ST931R, the E. coli transconjugant, and isolated plasmid DNA to unequivocally show that ST882S and ST931R had identical chromosomes, both having 206 identical single-nucleotide polymorphisms (SNPs) versus S Typhimurium 14028s. We assembled a complete circular genome for ST931R, to which ST882S reads mapped with no SNPs. ST882S and ST931R were isogenic except for the presence of three additional plasmids in ST931R. ST931R and the E. coli transconjugant were ceftriaxone resistant due to the presence of a 60.5-kb IS26-flanked, blaCTX-M-27-encoding IncFII plasmid. Compared to 14082s, ST931R has almost identical Gifsy-1, Gifsy-2, and ST64B prophages, lacks Gifsy-3, and instead carries a unique Fels-2 prophage related to that found in LT2. ST882S and ST931R both had a 94-kb virulence plasmid showing >99% identity with pSLT14028s and a cryptic 3,904-bp replicon; ST931R also has cryptic 93-kb IncI1 and 62-kb IncI2 group plasmids. To the best of our knowledge, in vivo acquisition of extended-spectrum ß-lactamase resistance by S Typhimurium and blaCTX-M-27 genes in U.S. isolates of Salmonella have not previously been reported.

Complete Genome Sequence of Escherichia coli ER1821R, a Laboratory K-12 Derivative Engineered To Be Deficient in All Methylcytosine and Methyladenine Restriction Systems.

We present here the complete genomic sequence of a rifampin-resistant derivative of the Escherichia coli K-12 laboratory strain ER1821, engineered to be deficient in all known restriction systems, making it suitable for generating unbiased libraries from organisms with non-K-12 methylation patterns. The ER1821R genome is most closely related to that of DH1, another popular cloning strain (both derived from MM294), but is deleted for the e14 prophage (McrA(-)) and the immigration control (McrBC(-) EcoKI R(-) M(-) Mrr(-)) loci.

The chromosomal nature of LT-II enterotoxins solved: a lambdoid prophage encodes both LT-II and one of two novel pertussis-toxin-like toxin family members in type II enterotoxigenic Escherichia coli.

Heat-labile enterotoxins (LT) of enterotoxigenic Escherichia coli (ETEC) are structurally and functionally related to cholera toxin (CT). LT-I toxins are plasmid-encoded and flanked by IS elements, while LT-II toxins of type II ETEC are chromosomally encoded with flanking genes that appear phage related. Here, I determined the complete genomic sequence of the locus for the LT-IIa type strain SA53, and show that the LT-IIa genes are encoded by a 51 239 bp lambdoid prophage integrated at the rac locus, the site of a defective prophage in E. coli K12 strains. Of 50 LT-IIa and LT-IIc, 46 prophages also encode one member of two novel two-gene ADP-ribosyltransferase toxin families that are both related to pertussis toxin, which I named eplBA or ealAB, respectively. The eplBA and ealAB genes are syntenic with the Shiga toxin loci in their lambdoid prophages of the enteric pathogen enterohemorrhagic E. coli. These novel AB(5) toxins show pertussis-toxin-like activity on tissue culture cells, and like pertussis toxin bind to sialic acid containing glycoprotein ligands. Type II ETEC are the first mucosal pathogens known to simultaneously produce two ADP-ribosylating toxins predicted to act on and modulate activity of both stimulatory and inhibitory alpha subunits of host cell heterotrimeric G-proteins.

A mutational analysis of residues in cholera toxin A1 necessary for interaction with its substrate, the stimulatory G protein Gsα.

Pathogenesis of cholera diarrhea requires cholera toxin (CT)-mediated adenosine diphosphate (ADP)-ribosylation of stimulatory G protein (Gsα) in enterocytes. CT is an AB5 toxin with an inactive CTA1 domain linked via CTA2 to a pentameric receptor-binding B subunit. Allosterically activated CTA1 fragment in complex with NAD+ and GTP-bound ADP-ribosylation factor 6 (ARF6-GTP) differs conformationally from the CTA1 domain in holotoxin. A surface-exposed knob and a short α-helix (formed, respectively, by rearranging "active-site" and "activation" loops in inactive CTA1) and an ADP ribosylating turn-turn (ARTT) motif, all located near the CTA1 catalytic site, were evaluated for possible roles in recognizing Gsα. CT variants with one, two or three alanine substitutions at surface-exposed residues within these CTA1 motifs were tested for assembly into holotoxin and ADP-ribosylating activity against Gsα and diethylamino-(benzylidineamino)-guanidine (DEABAG), a small substrate predicted to fit into the CTA1 active site). Variants with single alanine substitutions at H55, R67, L71, S78, or D109 had nearly wild-type activity with DEABAG but significantly decreased activity with Gsα, suggesting that the corresponding residues in native CTA1 participate in recognizing Gsα. As several variants with multiple substitutions at these positions retained partial activity against Gsα, other residues in CTA1 likely also participate in recognizing Gsα.

Microtubule motors power plasma membrane tubulation in clathrin-independent endocytosis.

How the plasma membrane is bent to accommodate clathrin-independent endocytosis remains uncertain. Recent studies suggest Shiga and cholera toxin induce membrane curvature required for their uptake into clathrin-independent carriers by binding and cross-linking multiple copies of their glycosphingolipid receptors on the plasma membrane. But it remains unclear if toxin-induced sphingolipid crosslinking provides sufficient mechanical force for deforming the plasma membrane, or if host cell factors also contribute to this process. To test this, we imaged the uptake of cholera toxin B-subunit into surface-derived tubular invaginations. We found that cholera toxin mutants that bind to only one glycosphingolipid receptor accumulated in tubules, and that toxin binding was entirely dispensable for membrane tubulations to form. Unexpectedly, the driving force for tubule extension was supplied by the combination of microtubules, dynein and dynactin, thus defining a novel mechanism for generating membrane curvature during clathrin-independent endocytosis.

ADP-ribosylation factor 6 acts as an allosteric activator for the folded but not disordered cholera toxin A1 polypeptide.

The catalytic A1 subunit of cholera toxin (CTA1) has a disordered structure at 37°C. An interaction with host factors must therefore place CTA1 in a folded conformation for the modification of its Gsα target which resides in a lipid raft environment. Host ADP-ribosylation factors (ARFs) act as in vitro allosteric activators of CTA1, but the molecular events of this process are not fully characterized. Isotope-edited Fourier transform infrared spectroscopy monitored ARF6-induced structural changes to CTA1, which were correlated to changes in CTA1 activity. We found ARF6 prevents the thermal disordering of structured CTA1 and stimulates the activity of stabilized CTA1 over a range of temperatures. Yet ARF6 alone did not promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted disordered CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6. Thus, ARF alone is unable to activate disordered CTA1 at physiological temperature: additional host factors such as lipid rafts place CTA1 in the folded conformation required for its ARF-mediated activation. Interaction with ARF is required for in vivo toxin activity, as enzymatically active CTA1 mutants that cannot be further stimulated by ARF6 fail to intoxicate cultured cells.

A single native ganglioside GM1-binding site is sufficient for cholera toxin to bind to cells and complete the intoxication pathway.

Cholera toxin (CT) from Vibrio cholerae is responsible for the majority of the symptoms of the diarrheal disease cholera. CT is a heterohexameric protein complex with a 240-residue A subunit and a pentameric B subunit of identical 103-residue B polypeptides. The A subunit is proteolytically cleaved within a disulfide-linked loop to generate the A1 and A2 fragments. The B subunit of wild-type (wt) CT binds 5 cell surface ganglioside GM(1) (GM(1)) molecules, and the toxin-GM(1) complex traffics from the plasma membrane (PM) retrograde through endosomes and the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the enzymatic A1 fragment retrotranslocates to the cytosol to cause disease. Clustering of GM(1) by multivalent toxin binding can structurally remodel cell membranes in ways that may assist toxin uptake and retrograde trafficking. We have recently found, however, that CT may traffic from the PM to the ER by exploiting an endogenous glycosphingolipid pathway (A. A. Wolf et al., Infect. Immun. 76:1476-1484, 2008, and D. J. F. Chinnapen et al., Dev. Cell 23:573-586, 2012), suggesting that multivalent binding to GM(1) is dispensable. Here we formally tested this idea by creating homogenous chimeric holotoxins with defined numbers of native GM(1) binding sites from zero (nonbinding) to five (wild type). We found that a single GM(1) binding site is sufficient for activity of the holotoxin. Therefore, remodeling of cell membranes by mechanisms that involve multivalent binding of toxin to GM(1) receptors is not essential for toxicity of CT. Through multivalent binding to its lipid receptor, cholera toxin (CT) can remodel cell membranes in ways that may assist host cell invasion. We recently found that CT variants which bind no more than 2 receptor molecules do exhibit toxicity, suggesting that CT may be able to enter cells by coopting an endogenous lipid sorting pathway without clustering receptors. We tested this idea directly by using purified variants of CT with zero to five functional receptor-binding sites (BS). One BS enabled CT to intoxicate cells, supporting the conclusion that CT can enter cells by coopting an endogenous lipid-sorting pathway. Although multivalent receptor binding is not essential, it does increase CT toxicity. These findings suggest that achieving higher receptor binding avidity or affecting membrane dynamics by lipid clustering and membrane remodeling may be driving forces for evolution of AB(5) subunit toxins that can bind multivalently to cell membrane lipid receptors.