Daniel J. Metzger

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.

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.

Mutations in the Extracellular Protein Secretion Pathway Genes (eps) Interfere with Rugose Polysaccharide Production in and Motility of Vibrio cholerae

ABSTRACT Vibrio cholerae is the causal organism of the diarrheal disease cholera. The rugose variant of V. cholerae is associated with the secretion of an exopolysaccharide. The rugose polysaccharide has been shown to confer increased resistance to a variety of agents, such as chlorine, bioacids, and oxidative and osmotic stresses. It also promotes biofilm formation, thereby increasing the survival of the bacteria in the aquatic environments. Here we show that the extracellular protein secretion system (gene designated eps) is involved directly or indirectly in the production of rugose polysaccharide. A TnphoA insertion inepsD gene of the eps operon abolished the production of rugose polysaccharide, reduced the secretion of cholera toxin and hemolysin, and resulted in a nonmotile phenotype. We have constructed defined mutations of the epsD andepsE genes that affected these phenotypes and complemented these defects by plasmid clones of the respective wild-type genes. These results suggest a major role for the eps system in pathogenesis and environmental survival of V. cholerae.

BhuR, a Virulence-Associated Outer Membrane Protein of Bordetella avium, Is Required for the Acquisition of Iron from Heme and Hemoproteins

ABSTRACT Iron (Fe) is an essential element for most organisms which must be obtained from the local environment. In the case of pathogenic bacteria, this fundamental element must be acquired from the fluids and tissues of the infected host. A variety of systems have evolved in bacteria for efficient acquisition of host-bound Fe. The gram-negative bacterium Bordetella avium, upon colonization of the avian upper respiratory tract, produces a disease in birds that has striking similarity to whooping cough, a disease caused by the obligate human pathogen Bordetella pertussis. We describe a B. avium Fe utilization locus comprised of bhuR and six accessory genes (rhuIR and bhuSTUV). Genetic manipulations of B. avium confirmed that bhuR, which encodes a putative outer membrane heme receptor, mediates efficient acquisition of Fe from hemin and hemoproteins (hemoglobin, myoglobin, and catalase). BhuR contains motifs which are common to bacterial heme receptors, including a consensus FRAP domain, an NPNL domain, and two TonB boxes. An N-terminal 32-amino-acid segment, putatively required for rhuIR-dependent regulated expression of bhuR, is present in BhuR but not in other bacterial heme receptors. Two forms of BhuR were observed in the outer membrane of B. avium: a 91-kDa polypeptide consistent in size with the predicted mature protein and a smaller 82-kDa polypeptide which lacks the 104 amino acids found at the N terminus of the 91-kDa form. A mutation in hemA was engineered in B. avium to demonstrate that the bacterium transports heme into the cytoplasm in a BhuR-dependent manner. The role of BhuR in virulence was established in turkey poults by use of a competitive-infection model.

Recombinant Antigen-Enterotoxin A2/B Chimeric Mucosal Immunogens Differentially Enhance Antibody Responses and B7-Dependent Costimulation of CD4+ T Cells

ABSTRACT The ADP-ribosylating enterotoxins, cholera toxin (CT) and theEscherichia coli heat-labile toxin (LT-IIa), have been shown to enhance mucosal and systemic antibody (Ab) responses to coadministered antigens. The purpose of the present study was to compare the ability of the nontoxic A2/B subunits of these toxins, which have distinct targeting properties, to augment the immunogenicity of a genetically coupled protein antigen. Structurally similar chimeric proteins were generated by genetically replacing the toxic A1 subunit of CT or LT-IIa with the saliva-binding region (SBR) from the streptococcal adhesin AgI/II. Intranasal immunization of BALB/c mice with either chimeric protein induced significantly higher plasma and mucosal anti-SBR immunoglobulin A (IgA) and IgG Ab responses than SBR alone. Moreover, compared to SBR–LT-IIaA2/B, SBR-CTA2/B elicited significantly higher levels of plasma IgG1 and salivary IgA anti-SBR Ab responses. Ex vivo and in vitro experiments revealed that SBR-CTA2/B selectively up-regulated B7-2 expression on murine B cells isolated from both the nasal associated lymphoid tissue, cervical lymph nodes, and spleen. In contrast, SBR–LT-IIaA2/B had little effect on B7-1 or B7-2 expression on B220+, CD11b+, or CD11c+ cells. Analysis of the functional costimulatory activity of SBR-CTA2/B-treated B cells revealed a significant enhancement in anti-CD3-stimulated CD4+ T-cell proliferative responses, and this proliferation was significantly reduced by treatment with anti-B7-2 but not with anti-B7-1 or isotype control Abs. Thus, SBR-CTA2/B and SBR–LT-IIaA2/B exhibit distinct patterns of antibody responses associated with differential effects on B7-2 expression and subsequent costimulatory effects on CD4+ T cells.

Heme Utilization in Bordetella avium Is Regulated by RhuI, a Heme-Responsive Extracytoplasmic Function Sigma Factor

ABSTRACT Efficient utilization of heme as an iron (Fe) source byBordetella 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 thatrhuI 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 rhuIwas correlated with a dramatically increased expression of BhuR. A putative rhuI-dependent bhuR promoter was identified in the 199-bp region located proximal tobhuR. When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced intoEscherichia coli, promoter activity was evident, but only when rhuI was coexpressed in the cell. Sigma competition experiments in E. colidemonstrated 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 therhuI-dependent regulatory system. Fur titration assays indicated that expression of rhuI was probably Fur dependent.

Entry of Yersinia pestis into the Viable but Nonculturable State in a Low-Temperature Tap Water Microcosm

Yersinia pestis, the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism.

Distinct Cytokine Regulation by Cholera Toxin and Type II Heat-Labile Toxins Involves Differential Regulation of CD40 Ligand on CD4+ T Cells

ABSTRACT 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-α), 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-α 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.

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.

Identification of Hylemonella gracilis as an Antagonist of Yersinia pestis Persistence

Yersinia pestis , the etiological agent of plague, has garnered great interest in the Biological Defense community as its intentional release or use as a terror weapon could cause considerable morbidity while incurring incalculable financial costs for restoration and remediation efforts. The plague bacterium is thought to only persist within a host such as a flea or small mammal reservoir. Following an event such as an intentional release however, the plague bacterium would be spread throughout a number of atypical environments such as soil or water ecosystems. Recently, a small number of studies have been published describing the plague bacterium’s persistence in some of these atypical environments. Here we show that Y. pestis can colonize sterilized water microcosms for over 3 years yet when introduced to filtered fresh water microcosms the bacterium Hylemonella gracilis became the dominant bacterium in the microcosm, apparently preventing long-term Y. pestis persistence. The conditioning and outgrowth of H. gracilis on rich media is directly attributable and proportional to the introduction and concentration of Y. pestis to the microcosm.