Terry D. Connell

Phase variation of gonococcal protein II: Regulation of gene expression by slipped-strand mispairing of a repetitive DNA sequence

Expression of outer membrane protein II (P.II) of Neisseria gonorrhoeae is subject to reversible phase variation at a rate of 10(-3)-10(-4)/cell/generation. The signal peptide coding regions of P.II genes contain variable numbers of tandem repeats of the sequence CTCTT. Changes in the number of CTCTT units, leading to frameshifting within the gene, are responsible for changes in P.II expression. Phase variation mediated by the CTCTT repeat also occurred in E. coli, as assayed with a P.II-alkaline phosphatase (phoA) gene fusion. Phase variation in both the gonococcus and E. coli was recA-independent, occurred at similar rates, and involved insertions or deletions of one or more repeat units. The characteristics of the phase variation process were consistent with a model in which expression of P.II genes is regulated by slipped-strand mispairing of the DNA in the CTCTT repeat region.

Ganglioside-Linked Terminal Sialic Acid Moieties on Murine Macrophages Function as Attachment Receptors for Murine Noroviruses

ABSTRACT Noroviruses are the major cause of nonbacterial gastroenteritis in humans. However, little is known regarding the norovirus life cycle, including cell binding and entry. In contrast to human noroviruses, the recently discovered murine norovirus 1 (MNV-1) readily infects murine macrophages and dendritic cells in culture. Many viruses, including the related feline calicivirus, use terminal sialic acids (SA) as receptors for infection. Therefore, we tested whether SA moieties play a role during MNV-1 infection of murine macrophages. Competition with SA-binding lectins and neuraminidase treatment led to a reduction in MNV-1 binding and infection in cultured and primary murine macrophages, suggesting a role for SA during the initial steps of the MNV-1 life cycle. Because SA moieties can be attached to glycolipids (i.e., gangliosides), we next determined whether MNV-1 uses gangliosides during infection. The gangliosides GD1a, GM1, and asialo-GM1 (GA1) are natural components of murine macrophages. MNV-1 bound to ganglioside GD1a, which is characterized by an SA on the terminal galactose, but not to GM1 or asialo-GM1 in an enzyme-linked immunosorbent assay. The depletion of gangliosides using an inhibitor of glycosylceramide synthase (d-threo-P4) led to a reduction of MNV-1 binding and infection in cultured and primary murine macrophages. This defect was specifically rescued by the addition of GD1a. A similar phenotype was observed for MNV field strains WU11 (GV/WU11/2005/USA) and S99 (GV/Berlin/2006/DE). In conclusion, our data indicate that MNV can use terminal SA on gangliosides as attachment receptors during binding to murine macrophages.

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.

Enterotoxin-Based Mucosal Adjuvants Alter Antigen Trafficking and Induce Inflammatory Responses in the Nasal Tract

ABSTRACT The safety of nasal vaccines containing enterotoxin-based mucosal adjuvants has not been studied in detail. Previous studies have indicated that native cholera toxin (nCT) can alter antigen trafficking when applied nasally. In this study, we determined the enterotoxin-based variables that alter antigen trafficking. To measure the influence of enterotoxin-based mucosal adjuvants on antigen trafficking in the nasal tract, native and mutant enterotoxins were coadministered with radiolabeled tetanus toxoid (TT). The nCT and heat-labile enterotoxin type 1 (LTh-1) redirected TT into the olfactory neuroepithelium (ON/E). Antigen redirection occurred mainly across the nasal epithelium without subsequent transport along olfactory neurons into the olfactory bulbs (OB). Thus, no significant accumulation of the vaccine antigen TT was observed in the OB when coadministered with nCT. In contrast, neither mutant CT nor mutant LTh-1, which lack ADP-ribosyltransferase activity, redirected TT antigen into the ON/E. Thus, ADP-ribosyltransferase activity was essential for antigen trafficking across the olfactory epithelium. Accumulation of TT in the ON/E was also due to B-subunit binding to GM1 gangliosides, as was demonstrated (i) by redirection of TT by LTh-1 in a dose-dependent manner, (ii) by ganglioside inhibition of the antigen redirection by LTh-1 and nCT, and (iii) by the use of LT-IIb, a toxin that binds to gangliosides other than GM1. Redirection of TT into the ON/E coincided with elevated production of interleukin 6 (IL-6) but not IL-1β or tumor necrosis factor alpha in the nasal mucosa. Thus, redirection of TT is dependent on ADP-ribosyltransferase activity and GM1 binding and is associated with production of the inflammatory cytokine IL-6.

Recombination among Protein II genes of Neisseria gonorrhoeae generates new coding sequences and increases structural variability in the Protein II family

Expression of Neisseria gonorrhoeae Protein II (P.II) is subject to phase variation and antigenic variation. The P.II proteins made by one strain possess both unique and conserved antigenic determinants. To study the mechanism of antigenic variation, we cloned several P.II genes, using as probes a panel of monoclonal antibodies (MAbs) specific for unique determinants. The DNA sequences of three P.II genes showed that they shared a conserved framework, with two short hypervariable (HV) regions being responsible for most of the differences among them. We demonstrated that unique epitopes recognized by the MAbs were at least partially encoded by one of the HV regions. Moreover, we found that reassortment of the two HV regions among P.II genes occurs, generating increased structural and antigenic variability in the P.II protein family.

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.

Toll-Like Receptor 2 Mediates Cellular Activation by the B Subunits of Type II Heat-Labile Enterotoxins

ABSTRACT The type II heat-labile enterotoxins (LT-IIa and LT-IIb) of Escherichia coli have an AB5 subunit structure similar to that of cholera toxin (CT) and other type I enterotoxins, despite significant differences in the amino acid sequences of their B subunits and different ganglioside receptor specificities. LT-II holotoxins and their nontoxic B subunits display unique properties as immunological adjuvants distinct from those of CT and its B subunits. In contrast to type II holotoxins, the corresponding pentameric B subunits, LT-IIaB and LT-IIbB, stimulated cytokine release in both human and mouse cells dependent upon Toll-like receptor 2 (TLR2). Induction of interleukin-1β (IL-1β), IL-6, IL-8, or tumor necrosis factor alpha in human THP-1 cells by LT-IIaB or LT-IIbB was inhibited by anti-TLR2 but not by anti-TLR4 antibody. Furthermore, transient expression of TLR1 and TLR2 in human embryonic kidney 293 cells resulted in activation of a nuclear factor-κB-dependent luciferase gene in response to LT-IIaB or LT-IIbB. Moreover, peritoneal macrophages from TLR2-deficient mice failed to respond to LT-IIaB or LT-IIbB, in contrast to wild-type or TLR4-deficient cells. These results demonstrate that besides their established binding to gangliosides, the B subunits of type II enterotoxins also interact with TLR2. Although a ganglioside-nonbinding mutant (T34I) of LT-IIaB effectively induced cytokine release, a phenotypically similar point mutation (T13I) in LT-IIbB abrogated cytokine induction, suggesting a variable requirement for gangliosides as coreceptors in TLR2 agonist activity. TLR2-dependent activation of mononuclear cells by type II enterotoxin B subunits appears to be a novel mechanism whereby these molecules may exert their immunomodulatory and adjuvant activities.

Comparison of the genome sequence of the poultry pathogen Bordetella avium with those of B. bronchiseptica, B. pertussis, and B. parapertussis reveals extensive diversity in surface structures associated with host interaction.

Bordetella avium is a pathogen of poultry and is phylogenetically distinct from Bordetella bronchiseptica, Bordetella pertussis, and Bordetella parapertussis, which are other species in the Bordetella genus that infect mammals. In order to understand the evolutionary relatedness of Bordetella species and further the understanding of pathogenesis, we obtained the complete genome sequence of B. avium strain 197N, a pathogenic strain that has been extensively studied. With 3,732,255 base pairs of DNA and 3,417 predicted coding sequences, it has the smallest genome and gene complement of the sequenced bordetellae. In this study, the presence or absence of previously reported virulence factors from B. avium was confirmed, and the genetic bases for growth characteristics were elucidated. Over 1,100 genes present in B. avium but not in B. bronchiseptica were identified, and most were predicted to encode surface or secreted proteins that are likely to define an organism adapted to the avian rather than the mammalian respiratory tracts. These include genes coding for the synthesis of a polysaccharide capsule, hemagglutinins, a type I secretion system adjacent to two very large genes for secreted proteins, and unique genes for both lipopolysaccharide and fimbrial biogenesis. Three apparently complete prophages are also present. The BvgAS virulence regulatory system appears to have polymorphisms at a poly(C) tract that is involved in phase variation in other bordetellae. A number of putative iron-regulated outer membrane proteins were predicted from the sequence, and this regulation was confirmed experimentally for five of these.

Comparative Analysis of the Mucosal Adjuvanticity of the Type II Heat-Labile Enterotoxins LT-IIa and LT-IIb

ABSTRACT Cholera toxin (CT) and the heat-labile enterotoxin ofEscherichia 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 ofStreptococcus 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.

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.