Crystal structure of a new heat-labile enterotoxin, LT-IIb.

BACKGROUND: Cholera toxin from Vibrio cholerae and the type I heat-labile enterotoxins (LT-Is) from Escherichia coli are oligomeric proteins with AB5 structures. The type II heat-labile enterotoxins (LT-IIs) from E. coli are structurally similar to, but antigenically distinct from, the type I enterotoxins. The A subunits of type I and type II enterotoxins are homologous and activate adenylate cyclase by ADP-ribosylation of a G protein subunit, G8 alpha. However, the B subunits of type I and type II enterotoxins differ dramatically in amino acid sequence and ganglioside-binding specificity. The structure of LT-IIb was determined both as a prototype for other LT-IIs and to provide additional insights into structure/function relationships among members of the heat-labile enterotoxin family and the superfamily of ADP-ribosylating protein toxins. RESULTS: The 2.25 A crystal structure of the LT-IIb holotoxin has been determined. The structure reveals striking similarities with LT-I in both the catalytic A subunit and the ganglioside-binding B subunits. The latter form a pentamer which has a central pore with a diameter of 10-18 A. Despite their similarities, the relative orientation between the A polypeptide and the B pentamer differs by 24 degrees in LT-I and LT-IIb. A common hydrophobic ring was observed at the A-B5 interface which may be important in the cholera toxin family for assembly of the AB5 heterohexamer. A cluster of arginine residues at the surface of the A subunit of LT-I and cholera toxin, possibly involved in assembly, is also present in LT-IIb. The ganglioside receptor binding sites are localized, as suggested by mutagenesis, and are in a position roughly similar to the sites where LT-I binds its receptor. CONCLUSIONS: The structure of LT-IIb provides insight into the sequence diversity and structural similarity of the AB5 toxin family. New knowledge has been gained regarding the assembly of AB5 toxins and their active-site architecture.

Immunomodulation with enterotoxins for the generation of secretory immunity or tolerance: applications for oral infections.

The heat-labile enterotoxins, such as cholera toxin (CT), and the labile toxins types I and II (LT-I and LT-II) of Escherichia coli have been extensively studied for their immunomodulatory properties, which result in the enhancement of immune responses. Despite superficial similarity in structure, in which a toxic A subunit is coupled to a pentameric binding B subunit, different toxins have different immunological properties. Administration of appropriate antigens admixed with or coupled to these toxins by oral, intranasal, or other routes in experimental animals induces mucosal IgA and circulating IgG antibodies that have protective potential against a variety of enteric, respiratory, or genital infections. These include the generation of salivary antibodies that may protect against colonization with mutans streptococci and the development of dental caries. However, exploitation of these adjuvants for human use requires an understanding of their mode of action and the separation of their desirable immunomodulatory properties from their toxicity. Recent findings have revealed that adjuvant action is not critically dependent upon the enzymic activity of the A subunits, and that the isolated B subunits may exert different effects on cells of the immune system than do the intact toxins. Interaction of the toxins with immunocompetent cells is not exclusively dependent upon their conventional ganglioside receptors. Immunomodulatory effects have been observed on dendritic cells, macrophages, CD4(+) and CD8(+) T-cells, and B-cells. Numerous factors-including the precise form of the toxin adjuvant, properties of the antigen, whether and how they are coupled, route of administration, and species of animal model-affect the outcome, whether this is enhanced humoral and cellular immunity, or specific induced tolerance toward the antigen.

Regulation of myosin isozyme expression by vitamin D3 deficiency and 1,25-dihydroxyvitamin D3 in the rat heart.

In this report, we demonstrate a significant inverse correlation between contractility and serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels and no correlation between contractility and serum levels of calcium, phosphate, or PTH. We also examined myosin isozyme distribution in vitamin D3-deficient rats, because myosin isozyme distribution can alter contractility. There was a significant increase in the levels of the V1 myosin isozyme in animals raised on a vitamin D3-deficient diet that maintained normal serum calcium and phosphate levels. There was no difference in the relative myosin isozyme distribution in animals raised on a hypocalcemia-yielding vitamin D3-deficient diet vs. animals raised on a control diet. As increased contractility has been observed in both groups of vitamin D3-deficient animals, a shift in myosin isozyme distribution cannot solely explain the increase in contractility previously observed in the vitamin D3-deficient rat heart. To determine whether 1,25-(OH)2D3 directly regulates myosin isozyme levels, we analyzed myosin isozyme distribution in primary cultures of ventricular myocytes. We found that 1,25-(OH)2D3 reduces total myosin levels, but does not alter myosin isozyme distribution. Thus, we show that the influence of vitamin D3 status on myosin isozyme expression in the intact rat involves a complex regulatory system of direct and indirect effects.

Inhibition of cardiac myocyte maturation by 1,25-dihydroxyvitamin D3.

The signals that regulate cardiac myocyte maturation in the neonatal heart are not completely understood. In our study we examined the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on primary cultures of ventricular myocytes isolated from neonatal rat hearts. Our data show that 1,25-(OH)2D3 inhibited an increase in both the muscle-specific form of creatine kinase and V1 myosin isoenzyme levels in myocytes induced to mature by serum withdrawal. Thus, in contrast to other cell types studied to date, in the heart, 1,25-(OH)2D3 blocks cell maturation. Treating cultures with phorbol 12-myristate 13-acetate induced a decrease in the muscle-specific isoenzyme of creatine kinase similar to the effects of 1,25-(OH)2D3. Interestingly, we found that staurosporine, a protein kinase-C inhibitor, blunts the effects of 1,25-(OH)2D3 and has the opposite effect of phorbol 12-myristate 13-acetate on cultured myocytes induced to mature by serum withdrawal. Thus, our data identify a novel role for 1,25-(OH)2D3 in the regulation of myocardial development and suggest that 1,25-(OH)2D3 may be acting through a protein kinase-dependent mechanism to maintain cardiac myocytes in an immature state.

1,25-Dihydroxyvitamin D3-regulated binding of nuclear proteins to a c-myc intron element.

The steroid hormone 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] induces maturation of many cells, including HL-60 promyelocytic leukemia cells that are differentiated to monocytes/macrophages. This process involves changes in transcription of genes such as c-myc. We investigated the effects of 1,25-(OH)2D3 on nuclear protein binding to the c-myc intron element (MIE), a region of DNA within the c-myc gene. A mutation in this MIE sequence has been shown to be associated with uncontrolled expression of the c-myc gene in various cell lines. In this report, we demonstrate for the first time that 1,25-(OH)2D3 induces increased binding of nuclear proteins to this MIE in HL-60 cells. The major MIE-binding proteins were approximately 32 kDa in size. Interestingly, phorbol 12-myristate 13-acetate induced a similar increase in binding of the 32-kDa doublet protein to the MIE. In addition, we showed that the level of 138-kDa MIE-binding protein was increased by 1,25-(OH)2D3 and phorbol 12-myristate 13-acetate. However, the extent of MIE binding by the 138-kDa protein is significantly less than that of the 32-kDa doublet binding species. MIE binding by the 32-kDa doublet protein was significantly increased within 12 h of 1,25-(OH)2D3 treatment. The time course of this increase was similar to that of 1,25-(OH)2D3-induced inhibition of c-myc gene transcription. We also demonstrated that dephosphorylation of the 32-kDa doublet protein inhibited its binding to the MIE. Thus, this study shows that the mechanism employed by 1,25-(OH)2D3 for regulation of c-myc expression may involve an increase in protein binding to the MIE, which has been shown to be the site for control of c-myc gene expression.

A new mobilizable cosmid vector for use in Vibrio cholerae and other gram-negative bacteria.

A new mobilizable cosmid vector, pCOS5, was engineered for use in Vibrio cholerae (Vc). Plasmid pCOS5 is small in size (7286 bp), contains the oriT from plasmid RK2, and has several unique restriction sites. The complete nucleotide sequence of pCOS5 was deduced from the DNA fragments used in its construction. Biparental matings using Escherichia coli (Ec) SM10 and triparental matings using Ec DH5 alpha[pRK2013] were used to measure the conjugation frequency of pCOS5 and pAJM1, a clone containing a 40-kb insert of chromosomal DNA from Vc ligated into pCOS5. Transfer of pCOS5 or pAJM1 to Vc occurred at a frequency of between 10(-2)-10(-3) transconjugants per recipient cell. The promiscuous nature of RP4/RK2 transfer functions makes pCOS5 a potentially useful vector for mobilizing large fragments of cloned DNA between different Gram- bacteria that support replication of ColE1 plasmids or as a mobilizable suicide vector in Gram- bacteria where replication of ColE1 plasmids is not supported.

Immunostimulatory activity of LT-IIa, a type II heat-labile enterotoxin of Escherichia coli.

Certain bacterial molecules potentiate immune responses to parenterally administered antigens. One such molecule that has been intensely investigated is cholera toxin, a type I heat-labile enterotoxin produced by the Gram-negative bacterium Vibrio cholerae. Immunization with a mixture of a foreign antigen and cholera toxin enhances the immune response to the antigen. Similar adjuvant activity is associated with LT-I, a closely related type I heat-labile enterotoxin produced by Escherichia coli. The adjuvant activities of LT-IIa, a member of the type II heat-labile enterotoxins produced by E. coli, have not been described. LT-IIa and CT differ significantly in amino acid sequence of the B polypeptides and in receptor binding affinity. In this study, rats were subcutaneously immunized with fimbrillin, a protein isolated from the bacterium Porphyromonas gingivalis, and with fimbrillin in combination with LT-IIa, the prototypical type II enterotoxin. Previous studies documented that fimbrillin administered alone is a poor immunogen. Animals immunized with the mixture of fimbrillin and LT-IIa produced high titers of specific IgG antibody directed against fimbrillin. Anti-fimbrillin antibody titers in sera from animals receiving the combination of LT-IIa + fimbrillin were comparable to those obtained from sera of animals immunized with cholera toxin + fimbrillin. The results of these experiments demonstrate that LT-IIa exhibits an adjuvant activity that is equal to that of cholera toxin. Recombinant methods have been established for producing large amounts of LT-IIa, an advantage that will likely provide an economic impetus to consider incorporating the enterotoxin as an immunostimulatory agent in future vaccines.

Promotion of HL-60 cell differentiation by 1,25-dihydroxyvitamin D3 regulation of protein kinase C levels and activity.

The hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] promotes differentiation of a number of cell types including HL-60 promyelocytic leukemia cells. It is now established that protein kinase Cbeta (PKCbeta) plays a critical role in HL-60 cell maturation to a monocyte/macrophage phenotype. In the present study, we investigated the importance of PKCbeta levels and activation in 1,25-(OH)2D3-mediated differentiation of HL-60 cells. Cell differentiation promoted by 1,25-(OH)2D3 at 48 hr was 39 +/- 3% (mean +/- SEM) nitroblue tetrazolium (NBT) positive and at 72 hr it was 35 +/- 2% NBT positive and 70% CD14 positive. Thus, promotion of cell differentiation by 20 nM 1,25-(OH)2D3 treatment was maximal at 48-72 hr. When PKCbeta levels and cell differentiation were assayed at 72 hr, treatment with 20 nM 1,25-(OH)2D3 for the initial 6 hr increased PKCbeta levels by 175% but had little effect on cell differentiation (7 +/- 2% NBT positive; 11% CD14 positive). The effect of ionomycin, a calcium ionophore, on PKCbeta levels and cell differentiation also was examined. Alone, 5 microM ionomycin promoted few cells (3% CD14 positive) to differentiate. In contrast, cells treated with 5 microM ionomycin for 66 hr after a 6-hr pretreatment with 20 nM 1,25-(OH)2D3 resulted in 34 +/- 5% NBT positive cells and 73% CD14 positive cells. Quantitatively, this induction of differentiation was identical to that observed in cultures continuously treated with 1,25-(OH)2D3 (35 +/- 2% NBT positive; 70% CD14 positive). Therefore, ionomycin seemed to replace the requirement for the continuous presence of 1,25-(OH)2D3. Chelerythrine chloride (3 microM), a specific PKC inhibitor, blocked differentiation promoted by 1,25-(OH)2D3 alone (82 +/- 2% inhibition) or in sequence with ionomycin (86 +/- 3% inhibition). Taken together, our data show that the capacity of 1,25-(OH)2D3 to both increase PKCbeta levels and activate PKC is utilized to promote HL-60 cell differentiation. These data further suggest that 1,25-(OH)2D3 has a genomic action to increase PKCbeta levels and also a nongenomic action requiring its continuous presence to promote HL-60 cell differentiation.

Interleukin-17-dependent CXCL13 mediates mucosal vaccine-induced immunity against tuberculosis.

The variable efficacy of tuberculosis (TB) vaccines and the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) emphasize the urgency for not only generating new and more effective vaccines against TB but also understanding the underlying mechanisms that mediate vaccine-induced protection. We demonstrate that mucosal adjuvants, such as type II heat labile enterotoxin (LT-IIb), delivered through the mucosal route induce pulmonary Mtb-specific T helper type 17 (Th17) responses and provide vaccine-induced protection against Mtb infection. Importantly, protection is interferon-γ (IFNγ)-independent but interleukin-17 (IL-17)-dependent. Our data show that IL-17 mediates C-X-C motif chemokine ligand 13 (CXCL13) induction in the lung for strategic localization of proinflammatory cytokine-producing CXCR5+ (C-X-C motif chemokine receptor 5-positive) T cells within lymphoid structures, thereby promoting early and efficient macrophage activation and the control of Mtb. Our studies highlight the potential value of targeting the IL-17-CXCL13 pathway rather than the IFNγ pathway as a new strategy to improve mucosal vaccines against TB.

Novel iron-regulated and Fur-regulated small regulatory RNAs in Aggregatibacter actinomycetemcomitans.

Iron can regulate biofilm formation via non-coding small RNA (sRNA). To determine if iron-regulated sRNAs are involved in biofilm formation by the periodontopathogen Aggregatibacter actinomycetemcomitans, total RNA was isolated from bacteria cultured with iron supplementation or chelation. Transcriptional analysis demonstrated that the expression of four sRNA molecules (JA01-JA04) identified by bioinformatics was significantly upregulated in iron-limited medium compared with iron-rich medium. A DNA fragment encoding each sRNA promoter was able to titrate Escherichia coli ferric uptake regulator (Fur) from a Fur-repressible reporter fusion in an iron uptake regulator titration assay. Cell lysates containing recombinant AaFur shifted the mobility of sRNA-specific DNAs in a gel shift assay. Potential targets of these sRNAs, determined in silico, included genes involved in biofilm formation. The A. actinomycetemcomitans overexpressing JA03 sRNA maintained a rough phenotype on agar, but no longer adhered to uncoated polystyrene or glass, although biofilm determinant gene expression was only modestly decreased. In summary, these sRNAs have the ability to modulate biofilm formation, but their functional target genes remain to be confirmed.

Mutational analysis of the ganglioside-binding activity of the type II Escherichia coli heat-labile enterotoxin LT-IIb.

The Escherichia coli type II heat-labile enterotoxin LT-IIb IIb consists of a single A polypeptide and five B polypeptides. The A polypeptide is responsible for the toxic activity, and the B polypeptides function to bind the toxin to gangliosides on the surface of the plasma membrane. Previous studies on the related type II enterotoxin LT-IIa demonstrated the importance of threonine (Thr) residues at positions 13, 14, and 34 in the mature B polypeptide for ganglioside GD1bp-binding activity. In this study, we used sitespecific mutagenesis to investigate ganglioside GD1a-binding activity of the B polypeptide of LT-IIb. We determined that Thr-13 and Thr-14 were involved in binding of ganglioside GD1a by the B polypeptides of LT-IIb but that Thr-34 was not essential. Substitution of serine, but not other amino acids, at position 13 or 14 in the B polypeptide of LT-IIb resulted in retention of ganglioside-binding activity equivalent to that of the wild-type enterotoxin, providing strong evidence that the hydroxyl groups of threonine or serine at positions 13 and 14 are important for the ganglioside-binding activity of LT-IIb. Chimeric genes that expressed hybrids of the B polypeptides of LT-IIb and LT-IIa were also constructed, and analysis of the hybrids showed that the specificity of their ganglioside-binding activity was determined by the N-terminal half of the molecule.

Characterization of hybrid toxins produced in Escherichia coli by assembly of A and B polypeptides from type I and type II heat-labile enterotoxins.

The genes encoding the individual A and B polypeptides of the type I enterotoxin LTp-I and type II enterotoxins LT-IIa and LT-IIb were cloned and tested for complementation in Escherichia coli. Each gene encoding an A polypeptide was cloned into pACYC184, and each gene encoding a B polypeptide was cloned into the compatible plasmid Bluescript KS+. In addition, operon fusions representing all combinations of A and B genes were constructed in Bluescript KS+. Extracts from strains of E. coli expressing each combination of A and B genes, either from compatible plasmids or from operon fusions, were tested for immunoreactive holotoxin by radioimmunoassays and for toxicity by Y1 adrenal cell assays. Biologically active holotoxin was detected in each case, but the toxicity of extracts containing the hybrid toxins was usually less than that of extracts containing the wild-type holotoxins. The ganglioside-binding activity of each holotoxin was tested, and in each case, the B polypeptide determined the ganglioside-binding specificity. The A and B polypeptides of the type II heat-labile enterotoxins were also shown to form holotoxin in vitro without exposure to denaturing conditions, in contrast to the polypeptides of the type I enterotoxins that failed to form holotoxin in vitro under comparable conditions. These findings suggest that type I and type II enterotoxins have conserved structural features that permit their A and B polypeptides to form hybrid holotoxins, although the B polypeptides of the type I and type II enterotoxins have very little amino acid sequence homology.

Molecular genetic analysis of ganglioside GD1b-binding activity of Escherichia coli type IIa heat-labile enterotoxin by use of random and site-directed mutagenesis.

Mutagenesis of the B-subunit gene of Escherichia coli heat-labile enterotoxin LT-IIa was performed in vitro with sodium bisulfite. Mutants were screened initially by radial passive immune hemolysis assays for loss of binding to erythrocytes. Mutant B polypeptides were characterized for immunoreactivity; for binding to gangliosides GD1b, GD1a, and GM1; for formation of holotoxin; and for biological activity. Mutant alleles that determined altered binding specificities were sequenced. Three such mutant alleles encoded Thr-to-Ile substitutions at residues 13, 14, and 34 in the mature B polypeptide of LT-IIa. Each mutant protein failed to bind to ganglioside GD1b, although the Ile-14 mutant retained the ability to bind to ganglioside GM1. Site-specific mutagenesis was used to construct mutants with various amino acid substitutions at residue 13, 14, or 34. Only those mutant proteins with Ser substituted for Thr at position 13, 14, or 34 retained the ability to bind to ganglioside GD1b, thereby suggesting a role for the hydroxyl group of Thr or Ser in ganglioside GD1b binding.

Characterization of the repertoire of hypervariable regions in the Protein II (opa) gene family of Neisseria gonorrhoeae.

P.II outer membrane proteins of Neisseria gonorrhoeae are encoded by a family of closely related genes. Although the genes are highly conserved, major differences in sequence among them occur in two short regions, designated hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of the hypervariable regions in the P.II genes of strains FA1090. The FA1090 chromosome contained at least eleven P.II loci, having six different versions each of HV1 and HV2 among them. Southern blotting with HV-specific oligonucleotides showed that each version was present in one to three copies, and that there were nine unique combinations of HV1 and HV2 in the P.II genes. Although each of the versions of HV1 or HV2 had a unique DNA sequence, there were some similarities among them, particularly when certain ones were compared. Restriction fragments containing only the HV regions were cloned into an expression vector to demonstrate that the epitopes recognized by a set of monoclonal antibodies specific for different FA1090 P.II proteins were completely encoded by either HV1 or HV2.

Initial studies of the structural signal for extracellular transport of cholera toxin and other proteins recognized by Vibrio cholerae.

The specificity of the pathway used by Vibrio cholerae for extracellular transport of cholera toxin (CT) and other proteins was examined in several different ways. First, V. cholerae was tested for the ability to secrete the B polypeptides of the type II heat-labile enterotoxins of Escherichia coli. Genes encoding the B polypeptide of LT-IIb in pBluescriptKS- phagemids were introduced into V. cholerae by electroporation. Culture supernatants and periplasmic extracts were collected from cultures of the V. cholerae transformants, and the enterotoxin B subunits were measured by an enzyme-linked immunosorbent assay. Results confirmed that the B polypeptides of both LT-IIa and LT-IIb were secreted by V. cholerae with efficiencies comparable to that measured for secretion of CT. Second, the plasmid clones were introduced into strain M14, an epsE mutant of V. cholerae. M14 failed to transport the B polypeptides of LT-IIa and LT-IIb to the extracellular medium, demonstrating that secretion of type II enterotoxins by V. cholerae proceeds by the same pathway used for extracellular transport of CT. These data suggest that an extracellular transport signal recognized by the secretory machinery of V. cholerae is present in LT-IIa and LT-IIb. Furthermore, since the B polypeptide of CT has little, if any, primary amino acid sequence homology with the B polypeptide of LT-IIa or LT-IIb, the transport signal is likely to be a conformation-dependent motif. Third, a mutant of the B subunit of CT (CT-B) with lysine substituted for glutamate at amino acid position 11 was shown to be secreted poorly by V. cholerae, although it exhibited immunoreactivity and ganglioside GM1-binding activity comparable to that of wild-type CT-B. These findings suggest that Glu-11 may be within or near the extracellular transport motif of CT-B. Finally, the genetic lesion in the epsE allele of V. cholerae M14 was determined by nucleotide sequence analysis.

Genetic characterization of wild-type and mutant fur genes of Bordetella avium.

For most, if not all, organisms, iron (Fe) is an essential element. In response to the nutritional requirement for Fe, bacteria evolved complex systems to acquire the element from the environment. The genes encoding these systems are often coordinately regulated in response to the Fe concentration. Recent investigations revealed that Bordetella avium, a respiratory pathogen of birds, expressed a number of Fe-regulated genes (T. D. Connell, A. Dickenson, A. J. Martone, K. T. Militello, M. J. Filiatraut, M. L. Hayman, and J. Pitula, Infect. Immun. 66:3597-3605, 1998). By using manganese selection on an engineered strain of B. avium that carried an Fe-regulated alkaline phosphatase reporter gene, a mutant was obtained that was affected in expression of Fe-regulated genes. To determine if Fe-dependent regulation in B. avium was mediated by a fur-like gene, a fragment of the B. avium chromosome, corresponding to the fur locus of B. pertussis, was cloned by PCR. Sequencing revealed that the fragment from B. avium encoded a polypeptide with 92% identity to the Fur protein of B. pertussis. In vivo experiments showed that the cloned gene complemented H1780, a fur mutant of Escherichia coli. Southern hybridizations and PCRs demonstrated that the manganese mutant had a deletion of 2 to 3 kbp of nucleotide sequence in the region located immediately 5' of the fur open reading frame. A spontaneous PCR-derived mutant of the B. avium fur gene was isolated that encoded a Fur protein in which a histidine was substituted for an arginine at amino acid position 18 (R18H). Genetic analysis showed that the R18H mutant gene when cloned into a low-copy-number vector did not complement the fur mutation in H1780. However, the R18H mutant gene was able to complement the fur mutation when cloned into a high-copy-number vector. The cloned wild-type fur gene will be useful as a genetic tool to identify Fur-regulated genes in the B. avium chromosome.

Comparative analysis of the mucosal adjuvanticity of the type II heat-labile enterotoxins LT-IIa and LT-IIb.

Cholera toxin (CT) and the heat-labile enterotoxin of Escherichia coli (LT-I) are members of the serogroup I heat-labile enterotoxins (HLT) and can serve as systemic and mucosal adjuvants. However, information is lacking with respect to the structurally related but antigenically distinct serogroup II HLT, LT-IIa and LT-IIb, which have different binding specificities for ganglioside receptors. The purpose of this study was to assess the effectiveness of LT-IIa and LT-IIb as mucosal adjuvants in comparison to the prototypical type I HLT, CT. BALB/c mice were immunized by the intranasal (i.n.) route with the surface protein adhesin AgI/II of Streptococcus mutans alone or supplemented with an adjuvant amount of CT, LT-IIa, or LT-IIb. Antigen-specific antibody responses in saliva, vaginal wash, and plasma were assayed by enzyme-linked immunosorbent assay. Mice given AgI/II with LT-IIa or LT-IIb by the i.n. route had significantly higher mucosal and systemic antibody responses than mice immunized with AgI/II alone. Anti-AgI/II immunoglobulin A (IgA) antibody activity in saliva and vaginal secretions of mice given AgI/II with LT-IIa or LT-IIb was statistically similar in magnitude to that seen in mice given AgI/II and CT. LT-IIb significantly enhanced the number of AgI/II-specific antibody-secreting cells in the draining superficial cervical lymph nodes compared to LT-IIa and CT. LT-IIb and CT induced significantly higher plasma anti-AgI/II IgG titers compared to LT-IIa. When LT-IIb was used as adjuvant, the proportion of plasma IgG2a relative to IgG1 anti-AgI/II antibody was elevated in contrast to the predominance of IgG1 antibodies promoted by AgI/II alone or when CT or LT-IIa was used. In vitro stimulation of AgI/II-specific cells from the superficial lymph nodes and spleen revealed that LT-IIa and LT-IIb induced secretion of interleukin-4 and significantly higher levels of gamma interferon compared to CT. These results demonstrate that the type II HLT LT-IIa and LT-IIb exhibit potent and distinct adjuvant properties for stimulating immune responses to a noncoupled protein immunogen after mucosal immunization.

Endochitinase is transported to the extracellular milieu by the eps-encoded general secretory pathway of Vibrio cholerae.

The chiA gene of Vibrio cholerae encodes a polypeptide which degrades chitin, a homopolymer of N-acetylglucosamine (GlcNAc) found in cell walls of fungi and in the integuments of insects and crustaceans. chiA has a coding capacity corresponding to a polypeptide of 846 amino acids having a predicted molecular mass of 88.7 kDa. A 52-bp region with promoter activity was found immediately upstream of the chiA open reading frame. Insertional inactivation of the chromosomal copy of the gene confirmed that expression of chitinase activity by V. cholerae required chiA. Fluorescent analogues were used to demonstrate that the enzymatic activity of ChiA was specific for beta,1-4 glycosidic bonds located between GlcNAc monomers in chitin. Antibodies against ChiA were obtained by immunization of a rabbit with a MalE-ChiA hybrid protein. Polypeptides with antigenic similarity to ChiA were expressed by classical and El Tor biotypes of V. cholerae and by the closely related bacterium Aeromonas hydrophila. Immunoblotting experiments using the wild-type strain 569B and the secretion mutant M14 confirmed that ChiA is an extracellular protein which is secreted by the eps system. The eps system is also responsible for secreting cholera toxin, an oligomeric protein with no amino acid homology to ChiA. These results indicate that ChiA and cholera toxin have functionally similar extracellular transport signals that are essential for eps-dependent secretion.

Distinct cytokine regulation by cholera toxin and type II heat-labile toxins involves differential regulation of CD40 ligand on CD4(+) T cells.

Cholera toxin (CT) and the type II heat-labile enterotoxins (HLT) LT-IIa and LT-IIb act as potent systemic and mucosal adjuvants and induce distinct T-helper (Th)-cell cytokine profiles. In the present study, CT and the type II HLT were found to differentially affect cytokine production by anti-CD3-stimulated human peripheral blood mononuclear cells (PBMC), and the cellular mechanisms responsible were investigated. CT suppressed interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-alpha), and IL-12 production by PBMC cultures more than either LT-IIa or LT-IIb. CT but not LT-IIa or LT-IIb reduced the expression of CD4(+) T-cell surface activation markers (CD25 and CD69) and subsequent proliferative responses of anti-CD3-stimulated T cells. CT but not LT-IIa or LT-IIb significantly reduced the expression of CD40 ligand (CD40L) on CD4(+) T cells. In a coculture system, CT-treated CD4(+) T cells induced significantly less TNF-alpha and IL-12 p70 production by both autologous monocytes and monocyte-derived dendritic cells than either LT-IIa- or LT-IIb-treated CD4(+) T cells. These findings demonstrate that CT, LT-IIa, and LT-IIb differentially affect CD40-CD40L interactions between antigen-presenting cells and T cells and help explain the distinct cytokine profiles observed with type I and type II HLT when used as mucosal adjuvants.

Heme utilization in Bordetella avium is regulated by RhuI, a heme-responsive extracytoplasmic function sigma factor.

Efficient utilization of heme as an iron (Fe) source by Bordetella avium requires bhuR, an Fe-regulated gene which encodes an outer membrane heme receptor. Upstream of bhuR is a 507-bp open reading frame, hereby designated rhuI (for regulator of heme uptake), which codes for a 19-kDa polypeptide. Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized that rhuI encoded a potential in-trans regulator of the heme receptor gene in trans. Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of bhuR. Experimental evidence for the regulatory activities of rhuI was first revealed by recombinant experiments in which overproduction of rhuI was correlated with a dramatically increased expression of BhuR. A putative rhuI-dependent bhuR promoter was identified in the 199-bp region located proximal to bhuR. When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced into Escherichia coli, promoter activity was evident, but only when rhuI was coexpressed in the cell. Sigma competition experiments in E. coli demonstrated that rhuI conferred biological properties on the cell that were consistent with RhuI having sigma factor activity. Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of the rhuI-dependent regulatory system. Fur titration assays indicated that expression of rhuI was probably Fur dependent.