Randall K. Holmes

MEMBRANE TRAFFIC AND THE CELLULAR UPTAKE OF CHOLERA TOXIN

In nature, cholera toxin (CT) and the structurally related E. coli heat labile toxin type I (LTI) must breech the epithelial barrier of the intestine to cause the massive diarrhea seen in cholera. This requires endocytosis of toxin-receptor complexes into the apical endosome, retrograde transport into Golgi cisternae or endoplasmic reticulum (ER), and finally transport of toxin across the cell to its site of action on the basolateral membrane. Targeting into this pathway depends on toxin binding ganglioside GM1 and association with caveolae-like membrane domains. Thus to cause disease, both CT and LTI co-opt the molecular machinery used by the host cell to sort, move, and organize their cellular membranes and substituent components.

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.

Biology and Molecular Epidemiology of Diphtheria Toxin and the tox Gene

Diphtheria toxin (DT) is an extracellular protein of Corynebacterium diphtheriae that inhibits protein synthesis and kills susceptible cells. The gene that encodes DT (tox) is present in some corynephages, and DT is only produced by C. diphtheriae isolates that harbor tox+ phages. The diphtheria toxin repressor (DtxR) is a global regulatory protein that uses Fe2+ as co-repressor. Holo-DtxR represses production of DT, corynebacterial siderophore, heme oxygenase, and several other proteins. Diagnostic tests for toxinogenicity of C. diphtheriae are based either on immunoassays or on bioassays for DT. Molecular analysis of tox and dtxR genes in recent clinical isolates of C. diphtheriae revealed several tox alleles that encode identical DT proteins and multiple dtxR alleles that encode five variants of DtxR protein. Therefore, recent clinical isolates of C. diphtheriae produce a single antigenic type of DT, and diphtheria toxoid continues to be an effective vaccine for immunization against diphtheria.

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.

Three-dimensional structure of the diphtheria toxin repressor in complex with divalent cation co-repressors

BACKGROUND When Corynebacterium diphtheriae encounters an environment with a low concentration of iron ions, it initiates the synthesis of several virulence factors, including diphtheria toxin. The diphtheria toxin repressor (DtxR) plays a key role in this iron-dependent, global regulatory system and is the prototype for a new family of iron-dependent repressor proteins in Gram-positive bacteria. This study aimed to increase understanding of the general regulatory principles of cation binding to DtxR. RESULTS The crystal structure of dimeric DtxR holo-repressor in complex with different transition metals shows that each subunit comprises an amino-terminal DNA-binding domain, an interface domain (which contains two metal-binding sites) and a third, very flexible carboxy-terminal domain. Each DNA-binding domain contains a helix-turn-helix motif and has a topology which is very similar to catabolite gene activator protein (CAP). Molecular modeling suggests that bound DNA adopts a bent conformation with helices alpha 3 of DtxR interacting with the major grooves. The two metal-binding sites lie approximately 10 A apart. Binding site 2 is positioned at a potential hinge region between the DNA-binding and interface domains. Residues 98-108 appear to be crucial for the functioning of the repressor; these provide four of the ligands of the two metal-binding sites and three residues at the other side of the helix which are at the heart of the dimer interface. CONCLUSIONS The crystal structure of the DtxR holorepressor suggests that the divalent cation co-repressor controls motions of the DNA-binding domain. In this way the metal co-repressor governs the distance between operator recognition elements in the two subunits and, consequently, DNA recognition.

Cellular internalization of cytolethal distending toxin: a new end to a known pathway

The cytolethal distending toxins (CDTs) are unique in their ability to induce DNA damage, activate checkpoint responses and cause cell cycle arrest or apoptosis in intoxicated cells. However, little is known about their cellular internalization pathway. We demonstrate that binding of the Haemophilus ducreyi CDT (HdCDT) on the plasma membrane of sensitive cells was abolished by cholesterol extraction with methyl‐β‐cyclodextrin. The toxin was internalized via the Golgi complex, and retrogradely transported to the endoplasmic reticulum (ER), as assessed by N‐linked glycosylation. Further translocation from the ER did not require the ER‐associated degradation (ERAD) pathway, and was Derlin‐1 independent. The genotoxic activity of HdCDT was dependent on its internalization and its DNase activity, as induction of DNA double‐stranded breaks was prevented in Brefeldin A‐treated cells and in cells exposed to a catalytically inactive toxin. Our data contribute to a better understanding of the CDT mode of action and highlight two important aspects of the biology of this bacterial toxin family: (i) HdCDT translocation from the ER to the nucleus does not involve the classical pathways followed by other retrogradely transported toxins and (ii) toxin internalization is crucial for execution of its genotoxic activity.

Inhibition of endoplasmic reticulum-associated degradation in CHO cells resistant to cholera toxin, Pseudomonas aeruginosa exotoxin A, and ricin.

ABSTRACT Many plant and bacterial toxins act upon cytosolic targets and must therefore penetrate a membrane barrier to function. One such class of toxins enters the cytosol after delivery to the endoplasmic reticulum (ER). These proteins, which include cholera toxin (CT), Pseudomonas aeruginosa exotoxin A (ETA), and ricin, move from the plasma membrane to the endosomes, pass through the Golgi apparatus, and travel to the ER. Translocation from the ER to the cytosol is hypothesized to involve the ER-associated degradation (ERAD) pathway. We developed a genetic strategy to assess the role of mammalian ERAD in toxin translocation. Populations of CHO cells were mutagenized and grown in the presence of two lethal toxins, ETA and ricin. Since these toxins bind to different surface receptors and attack distinct cytoplasmic targets, simultaneous acquisition of resistance to both would likely result from the disruption of a shared trafficking or translocation mechanism. Ten ETA- and ricin-resistant cell lines that displayed unselected resistance to CT and continued sensitivity to diphtheria toxin, which enters the cytosol directly from acidified endosomes, were screened for abnormalities in the processing of a known ERAD substrate, the Z form of α1-antitrypsin (α1AT-Z). Compared to the parental CHO cells, the rate of α1AT-Z degradation was decreased in two independent mutant cell lines. Both of these cell lines also exhibited, in comparison to the parental cells, decreased translocation and degradation of a recombinant CTA1 polypeptide. These findings demonstrated that decreased ERAD function was associated with increased cellular resistance to ER-translocating protein toxins in two independently derived mutant CHO cell lines.

Analysis of diphtheria toxin repressor‐operator interactions and characterization of a mutant repressor with decreased binding activity for divalent metals

The diphtheria toxin repressor (DtxR) is an Fe2+‐activated protein with sequence‐specific DNA‐binding activity for the diphtheria toxin (tox) operator. Under high‐iron conditions in Corynebacterium diphtheriae, DtxR represses toxin and siderophore biosynthesis as well as iron uptake. DtxR and a mutant repressor with His–47 substituted for Arg–47, designated DtxR‐R47H, were purified and compared. Six different divalent cations (Cd2+, Co2+, Fe2+, Mn2+, Ni2+, and Zn2+) activated the sequence‐specific DNA‐binding activity of DtxR and enabled it to protect the fox operator from DNase I digestion, but Cu2+ failed to activate DtxR. Hydroxyl radical footprinting experiments indicated that DtxR binds symmetrically about the dyad axis of the tox operator. Methylation protection experiments demonstrated that DtxR binding alters the susceptibility to methylation of three G residues within the AT‐rich tox operator. These findings suggest that two or more monomers of DtxR are involved in binding to the tox operator, with symmetrical DNA‐protein interactions occurring at each end of the palindromic operator. In this regard, DtxR resembles several other well‐characterized prokaryotic repressor proteins but differs dramatically from the Fe2+‐activated ferric uptake repressor protein (Fur) of Escherichia coli. The concentration of Co2+ required to activate DtxR‐R47H was at least 10‐foid greater than that needed to activate DtxR, but the sequence‐specific DNA binding of activated DtxR‐R47H was indistinguishable from that of wild‐type DtxR. The markedly deficient repressor activity of DtxR‐R47H is consistent with a significant decrease in its binding activity for divalent cations.

Identification of a DtxR-regulated operon that is essential for siderophore-dependent iron uptake in Corynebacterium diphtheriae.

The diphtheria toxin repressor (DtxR) uses Fe(2+) as a corepressor and inhibits transcription from iron-regulated promoters (IRPs) in Corynebacterium diphtheriae. A new IRP, designated IRP6, was cloned from C. diphtheriae by a SELEX-like procedure. DtxR bound to IRP6 in vitro only in the presence of appropriate divalent metal ions, and repression of IRP6 by DtxR in an Escherichia coli system was iron dependent. The open reading frames (ORFs) downstream from IRP6 and previously described promoter IRP1 were found to encode proteins homologous to components of ATP-binding cassette (ABC) transport systems involved in high-affinity iron uptake in other bacteria. IRP1 and IRP6 were repressed under high-iron conditions in wild-type C. diphtheriae C7(beta), but they were expressed constitutively in C7(beta) mutant strains HC1, HC3, HC4, and HC5, which were shown previously to be defective in corynebactin-dependent iron uptake. A clone of the wild-type irp6 operon (pCM6ABC) complemented the constitutive corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a clone of the wild-type irp1 operon failed to complement any of these strains. Complementation by subclones of pCM6ABC demonstrated that mutant alleles of irp6A, irp6C, and irp6B were responsible for the phenotypes of HC1, HC4, and HC5, respectively. The irp6A allele in HC1 and the irp6B allele in HC5 encoded single amino acid substitutions in their predicted protein products, and the irp6C allele in HC4 caused premature chain termination of its predicted protein product. Strain HC3 was found to have a chain-terminating mutation in dtxR in addition to a missense mutation in its irp6B allele. These findings demonstrated that the irp6 operon in C. diphtheriae encodes a putative ABC transporter, that specific mutant alleles of irp6A, irp6B, and irp6C are associated with defects in corynebactin-dependent iron uptake, and that complementation of these mutant alleles restores repression of corynebactin production under high-iron growth conditions, most likely as a consequence of restoring siderophore-dependent iron uptake mediated by the irp6 operon.

Coordinate regulation of siderophore and diphtheria toxin production by iron in Corynebacterium diphtheriae.

Iron is an environmental signal which regulates the coordinate expression of genes associated with virulence in many pathogenic bacteria. In response to iron-deprivation, lysogenic Corynebacterium diphtheriae C7 (beta) synthesizes and secretes diphtheria toxin and siderophore and induces a high-affinity iron uptake system. Diphtheria toxin is encoded by beta phage, but genes for siderophore production are encoded on the bacterial chromosome. Diphtheria toxin and siderophore production were shown to be coordinately induced during late logarithmic phase growth of wild-type C7(beta) in iron-limited medium. C. diphtheriae mutant C7hm723 produced siderophore and toxin constitutively under low-iron and high-iron conditions, but in mutants HC1, HC3, HC4, and HC5 their synthesis was partially repressed under high-iron conditions. The phenotypes of HC1, HC3, HC4, and HC5 are consistent with their severe defects in iron uptake, but the phenotype of C7hm723 is more likely to be explained by inactivation of the repressor for the iron regulon of C. diphtheriae.