Evaluation of the Immunogenic Response of a Novel Enterobactin Conjugate Vaccine in Chickens for the Production of Enterobactin-Specific Egg Yolk Antibodies.

Passive immunization with specific egg yolk antibodies (immunoglobulin Y, IgY) is emerging as a promising alternative to antibiotics to control bacterial infections. Recently, we developed a novel conjugate vaccine that could trigger a strong immune response in rabbits directed against enterobactin (Ent), a highly conserved siderophore molecule utilized by different Gram-negative pathogens. However, induction of Ent-specific antibodies appeared to be affected by the choice of animal host and vaccination regimen. It is still unknown if the Ent conjugate vaccine can trigger a specific immune response in layers for the purpose of production of anti-Ent egg yolk IgY. In this study, three chicken vaccination trials with different regimens were performed to determine conditions for efficient production of anti-Ent egg yolk IgY. Purified Ent was conjugated to three carrier proteins, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and CmeC (a subunit vaccine candidate), respectively. Intramuscular immunization of Barred Rock layers with KLH-Ent conjugate four times induced strong immune response against whole conjugate vaccine but the titer of Ent-specific IgY did not change in yolk with only a 4 fold increase detected in serum. In the second trial, three different Ent conjugate vaccines were evaluated in Rhode Island Red pullets with four subcutaneous injections. The KLH-Ent or CmeC-Ent conjugate consistently induced high level of Ent-specific IgY in both serum (up to 2,048 fold) and yolk (up to 1,024 fold) in each individual chicken. However, the Ent-specific immune response was only temporarily and moderately induced using a BSA-Ent vaccination. In the third trial, ten White Leghorn layers were subcutaneously immunized three times with KLH-Ent, leading to consistent and strong immune response against both whole conjugate and the Ent molecule in each chicken; the mean titer of Ent-specific IgY increased approximately 32 and 256 fold in serum and yolk, respectively. Consistent with its potent binding to various Ent derivatives, the Ent-specific egg yolk IgY also inhibited in vitro growth of a representative Escherichia coli strain. Together, this study demonstrated that the novel Ent conjugate vaccine could induce strong, specific, and robust immune response in chickens. The Ent-specific hyperimmune egg yolk IgY has potential for passive immune intervention against Gram-negative infections.

Enterobactin-Specific Antibodies Induced by a Novel Enterobactin Conjugate Vaccine.

Enterobactin (Ent)-mediated high-affinity iron acquisition is critical for Gram-negative bacteria to survive in the host. Given the bacteriostatic effect of lipocalin resulting from its potent Ent-binding ability, immune intervention directly targeting Ent is promising for iron-dependent pathogen control. Recently, an Ent conjugate vaccine was reported, but it still has several significant weaknesses. In this study, we sought to develop an innovative Ent conjugate vaccine that can induce a high level of antibodies directed against Ent and to provide solid evidence demonstrating siderophore-binding capacity of Ent-specific antibodies. Using a simple method, we successfully conjugated purified Ent to different carriers, including keyhole limpet hemocyanin (KLH), bovine serum albumin, and CmeC, a vaccine candidate for Campylobacter control. Subcutaneous immunization of rabbits with the KLH-Ent conjugate triggered a strong systemic IgG immune response with an up to 16,384-fold increase in IgG titer directed against whole conjugate and an up to 4,096-fold increase in the level of specific anti-Ent IgG. To evaluate the ability of Ent-specific IgG to bind to the Ent derivatives present in vivo, various Ent derivatives were chemically synthesized and a unique enzyme-linked immunosorbent assay method was developed. The Ent-specific IgG also displayed exceptional reactivity to ferric Ent, a linear trimer of Ent, and different salmochelins. Growth assays further demonstrated that the Ent-specific antibodies significantly inhibited Ent-dependent growth of Campylobacter spp. and Escherichia coli Collectively, this study reports an efficient method to prepare a new type of Ent conjugate vaccines for inducing a high level of Ent-specific antibodies, which can bind to various Ent derivatives and display lipocalin-like bacteriostatic features.IMPORTANCE Ent-mediated high-affinity iron acquisition is a universal and critical contributor for Gram-negative pathogens to survive and infect hosts. Published information has supported an innovative immune intervention strategy that directly targets Ent to starve pathogens by limiting the availability of iron to be utilized. Compared to a recently published Ent conjugate, there are three advantages of the vaccine described in this study: ease of preparation, induction of high titer of anti-Ent IgG, and the ability of Ent-specific antibodies to bind various Ent derivatives, including the salmochelins that help enteric pathogens evade sequestration of siderophores by host lipocalins. In addition, the Ent-specific antibodies were demonstrated to function similarly to lipocalin to interfere with the Ent-dependent growth of Campylobacter and E. coli under iron-restricted conditions. This study has significant potential for broader applications to prevent and control various Gram-negative infections in humans and animals.

Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection.

Competition for iron influences host-pathogen interactions. Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron. In response, the host secretes siderophore-binding proteins, such as lipocalin 24p3, which limit siderophore-mediated iron import into bacteria. Mammals produce 2,5-dihydroxy benzoic acid, a compound that resembles a bacterial siderophore. Our data suggest that bacteria use both mammalian and bacterial siderophores. In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro. In addition, mice lacking the mammalian siderophore resist E. coli infection. Finally, we show that the host responds to infection by suppressing siderophore synthesis while up-regulating lipocalin 24p3 expression via TLR signaling. Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

Production and immunogenicity of recombinant ferric enterobactin protein (FepA).

OBJECTIVES: Ferric siderophore complexes are produced by most bacteria to acquire iron, a vital element. These complexes are transported across the outer membrane by receptor proteins commonly known as FepA (ferric enterobactin protein). In this study we attempted to evaluate the immunogenicity of the membrane protein FepA, aiming at inhibition of iron uptake to protect invasion of the host by the bacterium. METHODS: The genomic fepA gene was amplified from Escherichia coli O157:H7. The PCR product was ligated into pET28a and was then expressed in E. coli BL21(DE3). The recombinant protein purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography was injected into BALB/C mice to induce immunity. Antibody titer was determined by ELISA. Mouse groups were challenged with various doses of E. coli O157:H7, Shigella flexneri, Klebsiella pneumoniae, and Salmonella typhi to study immune response. RESULTS: An 85-kDa recombinant protein was expressed and purified. Immunogenicity of the recombinant protein was determined by injecting BALB/C mice. The antibody produced therein could efficiently recognize and bind ferric enterobactin binding protein. Immunized mice challenged with higher doses of selected bacteria survived. CONCLUSIONS: Significant recognition by the antibody of ferric enterobactin binding protein may lead to its application in the restriction of Enterobacteriaceae propagation.

Mucosal lipocalin 2 has pro-inflammatory and iron-sequestering effects in response to bacterial enterobactin.

Nasal colonization by both gram-positive and gram-negative pathogens induces expression of the innate immune protein lipocalin 2 (Lcn2). Lcn2 binds and sequesters the iron-scavenging siderophore enterobactin (Ent), preventing bacterial iron acquisition. In addition, Lcn2 bound to Ent induces release of IL-8 from cultured respiratory cells. As a countermeasure, pathogens of the Enterobacteriaceae family such as Klebsiella pneumoniae produce additional siderophores such as yersiniabactin (Ybt) and contain the iroA locus encoding an Ent glycosylase that prevents Lcn2 binding. Whereas the ability of Lcn2 to sequester iron is well described, the ability of Lcn2 to induce inflammation during infection is unknown. To study each potential effect of Lcn2 on colonization, we exploited K. pneumoniae mutants that are predicted to be susceptible to Lcn2-mediated iron sequestration (iroA ybtS mutant) or inflammation (iroA mutant), or to not interact with Lcn2 (entB mutant). During murine nasal colonization, the iroA ybtS double mutant was inhibited in an Lcn2-dependent manner, indicating that the iroA locus protects against Lcn2-mediated growth inhibition. Since the iroA single mutant was not inhibited, production of Ybt circumvents the iron sequestration effect of Lcn2 binding to Ent. However, colonization with the iroA mutant induced an increased influx of neutrophils compared to the entB mutant. This enhanced neutrophil response to Ent-producing K. pneumoniae was Lcn2-dependent. These findings suggest that Lcn2 has both pro-inflammatory and iron-sequestering effects along the respiratory mucosa in response to bacterial Ent. Therefore, Lcn2 may represent a novel mechanism of sensing microbial metabolism to modulate the host response appropriately.

The entD gene of the Escherichia coli K12 enterobactin gene cluster.

The Escherichia coli entD gene encodes a product necessary for the synthesis of the iron-chelating and transport molecule enterobactin (Ent); cells harbouring entD mutations fail to grow in iron-deficient environments. For unknown reasons, it has not been possible to identify the entD product. The nucleotide sequence of the entD region has now been determined. An open reading frame extending in the same direction as the adjacent fepA gene and capable of encoding an approximately 24 kDa polypeptide was found; it contained a high percentage of rare codons and two possible translational start sites. Complementation data suggested that EntD proteins truncated at the carboxy terminus retain some activity. Two REP sequences were present upstream of entD and an IS186 sequence was observed downstream. RNA dot-blot hybridizations demonstrated that entD is transcribed from the strand predicted by the sequencing results. An entD-lacZ recombinant plasmid was constructed and shown to express low amounts of a fusion protein of the anticipated size (approximately 125 kDa). The evidence suggests a number of possible explanations for difficulties in detecting the entD product. Sequence data indicate that if entD has its own promoter, it is weak; the REP sequences suggest that entD mRNA may be destabilized; and translation may be slow because of the frequency of rare codons and a possible unusual start codon (UUG). The data are also consistent with previous evidence that the entD product is unstable.

Specific inhibition of Escherichia coli ferrienterochelin uptake by a normal human serum immunoglobulin.

Normal human serum contains an enterochelin-specific antibody which presumably acts with transferrin to hinder iron assimilation by enterochelin-producing pathogens. This antibody can be isolated from serum by sodium sulfate fractionation or affinity chromatography by employing an enterochelin-derived ligand (2,3-dihydroxy-N-benzoyl-L-serine) attached to aminohexyl Sepharose 4B. In assays of iron uptake by whole cells, the antibody inhibited enterochelin-directed uptake but not that mediated by citrate or ferrichrome. Also, the growth stimulatory effect of enterochelin on an Ent- strain of Escherichia coli was blocked by the immunoglobulin. This antibody has a high affinity for enterochelin; various elution procedures employing high salt concentrations and low pH failed to remove it from affinity columns. Elution with 3 M sodium thiocyanate or 13 mM 2,3-dihydroxybenzoic acid proved successful. Two pieces of evidence indicate the enterochelin-specific antibody is primarily of the immunoglobulin A (IgA) isotype. It could be removed from serum with goat antihuman IgA and was present only in sodium sulfate fractions of serum known to contain IgA.

Bacteriostatic enterochelin-specific immunoglobulin from normal human serum.

Heat-inactivated normal human serum produces iron-reversible bacteriostasis of a number of microorganisms. This inhibitory effect was abolished by adsorption of serum with ultraviolet-killed cells of species that produce the siderophore enterochelin. Bacteriostasis also was alleviated by adsorption of serum with 2,3-dihydroxy-N-benzoyl-L-serine, a degradation product of enterochelin, bound to the insoluble matrix AH-Sepharose 4B. The adsorption process did not add iron or enterochelin to serum, nor did it remove transferrin. The immunoglobulin fraction from normal human serum was isolated; when added to a defined medium (M199) prepared so as to mimic normal human serum, the immunoglobulin rendered the medium inhibitory to an enterochelin-defective strain of Salmonella typhimurium. Adsorption of this medium with AH-Sepharose 4B-2,3-dihydroxy-N-benzoyl-L-serine removed the inhibition. Our results indicate that enterochelin-specific immunoglobulins exist in normal human serum. These immunoglobulins may act synergistically with transferrin to effect bacteriostasis of enterochelin-producing pathogens.