Michael G. Jobling

Characterization of hapR, a positive regulator of the Vibrio cholerae HA/protease gene hap, and its identification as a functional homologue of the Vibrio harveyi luxR gene

The Vibrio cholerae HA/protease gene (hap) promoter is inactive in Escherichia coli. We cloned and sequenced the 0.7 kb hap promoter fragment from strain 3083‐2 and showed that hap is located immediately 3′ of ompW, encoding a minor outer membrane protein. A clone from a genomic library of strain 3083‐2 was isolated, which was required for activation of the hap promoter in E. coli. Expression from the hap promoter only occurred late in the growth phase. A single complete open reading frame (ORF) designated HapR was identified on a 1.7 kb DNA fragment that was required for activation. Allelic replacements showed that hapR was also essential for hap expression in V. cholerae. In El Tor, but not in classical biotypes of V. cholerae, hapR mutations also produced a rugose colonial phenotype. HapR was shown to encode a 203‐amino‐acid polypeptide with 71% identity to LuxR of V. harveyi, an essential positive regulator of the lux operon that has no previously identified homologues. The amino‐terminal domain (residues 21–68) showed significant homology to the TetR family of helix–turn–helix DNA‐binding domains and was 95% identical to the same domain of LuxR. HapR and LuxR activated both the hap and the lux promoters at near wild‐type levels, despite only limited homology in the promoter sequences (46% identity with 12 gaps over 420 bp). DNA sequences and ORFs 5′ (but not 3′) of the hapR and luxR loci were homologous, suggesting a common origin for these loci, and hapR‐hybridizing sequences were found in other vibrios. We conclude that HapR is absolutely required for hap expression and that HapR and LuxR form a new family of transcriptional activator proteins.

Analysis of structure and function of the B subunit of cholera toxin by the use of site-directed mutagenesis.

Oligonucleotide‐directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT‐B) altered at residues Cys‐9, Gly‐33, Lys‐34, Arg‐35, Cys‐86 and Trp‐88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT‐B poly‐peptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT‐B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly‐33 and negatively or positively charged residues for Trp‐88. Substitution of lysine or arginine for Gly‐33 did not affect immunoreactivity or GM1 ‐binding activity of CT‐B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg‐35 interfered with formation of holotoxin, but none of the observed substitutions for Lys‐34 or Arg‐35 affected binding of CT‐B to GM1. The Cys‐9, Cys‐86 and Trp‐88 residues were important for establishing or maintaining the native conformation of CT‐B or protecting the CT‐B polypeptide from rapid degradation in vivo.

LitR, a new transcriptional activator in Vibrio fischeri, regulates luminescence and symbiotic light organ colonization

Vibrio fischeri is the bacterial symbiont within the light‐emitting organ of the sepiolid squid Euprymna scolopes . Upon colonizing juvenile squids, bacterial symbionts grow on host‐supplied nutrients, while providing a bioluminescence that the host uses during its nocturnal activities. Mutant bacterial strains that are unable to emit light have been shown to be defective in normal colonization. A 606 bp open reading frame was cloned from V. fischeri that encoded a protein, which we named LitR, that had about 60% identity to four related regulator proteins: Vibrio cholerae HapR, Vibrio harveyi LuxR, Vibrio parahaemolyticus OpaR and Vibrio vulnificus SmcR. When grown in culture, cells of V. fischeri strain PMF8, in which litR was insertionally inactivated, were delayed in the onset of luminescence induction and emitted only about 20% as much light per cell as its parent. Protein‐binding studies suggested that LitR enhances quorum sensing by regulating the transcription of the luxR gene. Interestingly, when competed against its parent in mixed inocula, PMF8 became the predominant symbiont present in 83% of light organs. Thus, the litR mutation appears to represent a novel class of mutations in which the loss of a regulatory gene function enhances the bacteriums competence in initiating a benign infection.

Structural and Functional Studies on the Interaction of GspC and GspD in the Type II Secretion System

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspCHR) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspCHR adopts an all-β topology. N-terminal β-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC–GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspCHR–GspDN0 interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells SIGNAL TRANSDUCTION BY A PROTEASE-RESISTANT TOXIN VARIANT

Cholera and Escherichia coliheat-labile toxins (CT and LT) require proteolysis of a peptide loop connecting two major domains of their enzymatic A subunits for maximal activity (termed “nicking”). To test whether host intestinal epithelial cells may supply the necessary protease, recombinant rCT and rLT and a protease-resistant mutant CTR192H were prepared. Toxin action was assessed as a Cl− secretory response (Isc) elicited from monolayers of polarized human epithelial T84 cells. When applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 μA/cm2). Isc was reduced 2-fold, however, when toxins were applied to basolateral membranes. Pretreatment of wild type toxins with trypsin in vitro restored the “basolateral” secretory responses to “apical” levels. Toxin entry into T84 cells via apical but not basolateral membranes led to nicking of the A subunit by a serine-type protease. T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when applied to apical membranes. Thus, T84 cells express a serine-type protease(s) fully sufficient for activating the A subunits of CT and LT. The protease, however, is only accessible for activation when the toxin enters the cell via the apical membrane.

Attenuated Endocytosis and Toxicity of a Mutant Cholera Toxin with Decreased Ability To Cluster Ganglioside GM1 Molecules

ABSTRACT Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM1. The homopentomeric B-subunit of the toxin can bind up to five GM1 molecules at once. Here, we examined the role of polyvalent binding of GM1 in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM1. The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM1 when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM1 molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM1 molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.

Transfer of the Cholera Toxin A1 Polypeptide from the Endoplasmic Reticulum to the Cytosol Is a Rapid Process Facilitated by the Endoplasmic Reticulum-Associated Degradation Pathway

ABSTRACT The active pool of internalized cholera toxin (CT) moves from the endosomes to the Golgi apparatus en route to the endoplasmic reticulum (ER). The catalytic CTA1 polypeptide is then translocated from the ER to the cytosol, possibly through the action of the ER-associated degradation (ERAD) pathway. Translocation was previously measured indirectly through the downstream effects of CT action. We have developed a direct biochemical assay for CTA1 translocation that is independent of toxin activity. Our assay is based upon the farnesylation of a CVIM motif-tagged CTA1 polypeptide (CTA1-CVIM) after it enters the cytosol. When expressed from a eukaryotic vector in transfected CHO cells, CTA1-CVIM was targeted to the ER, but was not secreted. Instead, it was translocated into the cytosol and degraded in a proteosome-dependent manner. Translocation occurred rapidly and was monitored by the appearance of farnesylated CTA1-CVIM in the detergent phase of cell extracts generated with Triton X-114. Detergent-phase partitioning of CTA1-CVIM resulted from the cytoplasmic addition of a 15-carbon fatty acid farnesyl moiety to the cysteine residue of the CVIM motif. Our use of the CTA1-CVIM translocation assay provided supporting evidence for the ERAD model of toxin translocation and generated new information on the timing of CTA1 translocation.

A Class of Mutant CHO Cells Resistant to Cholera Toxin Rapidly Degrades the Catalytic Polypeptide of Cholera Toxin and Exhibits Increased Endoplasmic Reticulum‐Associated Degradation

After binding to the eukaryotic cell surface, cholera toxin undergoes retrograde transport to the endoplasmic reticulum. The catalytic A1 polypeptide of cholera toxin (CTA1) then crosses the endoplasmic reticulum membrane and enters the cytosol in a process that may involve the quality control mechanism known as endoplasmic reticulum‐associated degradation. Other toxins such as Pseudomonas exotoxin A and ricin are also thought to exploit endoplasmic reticulum‐associated degradation for entry into the cytosol. To test this model, we mutagenized Chinese hamster ovary cells and selected clones that survived a prolonged coincubation with Pseudomonas exotoxin A and ricin. These lethal endoplasmic reticulum‐translocating toxins bind different surface receptors and target different cytosolic substrates, so resistance to both would likely result from disruption of a shared trafficking or translocation event. Here we characterize two Pseudomonas exotoxin A/ricin‐resistant clones that exhibited increased endoplasmic reticulum‐associated degradation. Both clones acquired the following unselected traits: (i) resistance to cholera toxin; (ii) increased degradation of an endoplasmic reticulum‐localized CTA1 construct; (iii) increased degradation of an established endoplasmic reticulum‐associated degradation substrate, the Z variant of α1‐antitrypsin (α1AT‐Z); and (iv) reduced secretion of both α1AT‐Z and the transport‐competent protein α1AT‐M. Proteosome inhibition partially rescued the α1AT‐M secretion deficiencies. However, the mutant clones did not exhibit increased proteosomal activity against cytosolic proteins, including a second CTA1 construct that was expressed in the cytosol rather than in the endoplasmic reticulum. These results suggested that accelerated endoplasmic reticulum‐associated degradation in the mutant clones produced a cholera toxin/Pseudomonas exotoxin A/ricin‐resistant phenotype by increasing the coupling efficiency between toxin translocation and toxin degradation.

Use of Translational Fusion of the MrpH Fimbrial Adhesin-Binding Domain with the Cholera Toxin A2 Domain, Coexpressed with the Cholera Toxin B Subunit, as an Intranasal Vaccine To Prevent Experimental Urinary Tract Infection by Proteus mirabilis

ABSTRACT This is a follow-up to our previous study using an intranasal vaccine composed of MrpH, the tip adhesin of the MR/P fimbria, and cholera toxin to prevent urinary tract infection by Proteus mirabilis (X. Li, C. V. Lockatell, D. E. Johnson, M. C. Lane, J. W. Warren, and H. L. Mobley, Infect. Immun. 72:66-75, 2004). Here, we have expressed a cholera toxin-like chimera in which the MrpH adhesin-binding domain (residues 23 to 157) replaces the cholera toxin A1 ADP-ribosyltransferase domain. This chimera, when administered intranasally without additional adjuvant, is sufficient to induce protective immunity in mice.

The Cholera Toxin A13 Subdomain Is Essential for Interaction with ADP-Ribosylation Factor 6 and Full Toxic Activity but Is Not Required for Translocation from the Endoplasmic Reticulum to the Cytosol

ABSTRACT Cholera toxin (CT) moves from the plasma membrane to the endoplasmic reticulum (ER) by retrograde vesicular traffic. In the ER, the catalytic CTA1 polypeptide dissociates from the rest of the toxin and enters the cytosol by a process that involves the quality control mechanism of ER-associated degradation (ERAD). The cytosolic CTA1 then ADP ribosylates Gsα, resulting in adenylate cyclase activation and intoxication of the target cell. It is hypothesized that the C-terminal A13 subdomain of CTA1 plays two crucial roles in the intoxication process: (i) it contains a hydrophobic domain that triggers the ERAD mechanism and (ii) it facilitates interaction with the cytosolic ADP-ribosylation factors (ARFs) that serve as allosteric activators of CTA1. In this study, we examined the role(s) of the CTA13 subdomain in CT intoxication. Full-length CTA1 constructs and truncated CTA1 constructs lacking the A13 subdomain were generated and used to conduct two-hybrid studies of interactions with ARF6, in vitro enzyme assays, in vivo toxicity assays, and in vivo processing/degradation assays. Direct, plasmid-mediated expression of CTA1 constructs in the ER or cytosol of transfected CHO cells was used to perform the in vivo assays. With these methods, we found that the A13 subdomain of CTA1 is important both for interaction with ARF6 and for full expression of enzyme activity in vivo. Surprisingly, however, the A13 subdomain was not required for ERAD-mediated passage of CTA1 from the ER to the cytosol. A possible alternative trigger for CTA1 to activate the ERAD mechanism is discussed.