Oral administration of Lactococcus lactis expressing Helicobacter pylori Cag7-ct383 protein induces systemic anti-Cag7 immune response in mice

To express the 3'-region (1152 bp) of the cag7 gene of Helicobacter pylori 51 strain, encoding the C-terminal 383 amino acid (ct383 aa) region of Cag7 protein that is known to cover the needle region of T4SS, in a live delivery vehicle Lactococcus lactis , the cag7-ct383 gene was amplified by PCR. DNA sequence analysis revealed that the amino acid sequence of Cag7-ct383 of H. pylori 51 shared 98.4% and 97.4% identity with H. pylori 26695 and J99, respectively. Intramuscular injection of the GST-Cag7-ct383 fusion protein into a rat could raise the anti-Cag7 antibody, indicating the immunogenicity of the Cag7-ct383 protein. When the cag7-ct383 gene was cloned in Escherichia coli–L. lactis shuttle vector (pMG36e) and transformed into L. lactis , the transformant could produce the Cag7-ct383 protein, as evidenced by Western blot analysis. The Cag7-ct383 protein level in the L. lactis transformant reached a maximum at the early stationary phase without extracellular secretion. The oral administration of the L. lactis transformant into mice generated anti-Cag7 antibody in serum in five of five mice. These results suggest that L. lactis transformant expressing Cag7-ct383 protein may be applicable as an oral vaccine to induce mucosal and systemic immunity to H. pylori .     

Optimized Expression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis><Emphasis Type="Italic">ureB</Emphasis> Gene in the <Emphasis Type="Italic">Lactococcus lactis</Emphasis> Nisin-Controlled Gene Expression (NICE) System and Experimental Study of Its Immunoreactivity

In the development of an oral vaccine against Helicobacter pylori, H. pylori urease subunit B (UreB) was expressed in a food-grade delivery vehicle, Lactococcus lactis NZ3900. The ureB gene (Genbank accession no. FJ436980) was amplified by polymerase chain reaction (PCR) from MEL-Hp27. The PCR-amplified ureB gene was cloned in the E. coli–L. lactis shuttle vector pNZ8110 and transformed into E. coli MC1061. After the transformant had been identified, the recombinant plasmid was purified and electrotransformed into L. lactis NZ3900. The conditions of UreB expression in the L. lactis transformant were optimized by orthogonal experiment. The maltose binding protein (MBP)-UreB fusion protein expressed by TB₁/pMAL-c2X-ureB was used to cultivate mice polyclonal anti-UreB serum after purification by the amylose prepacked column. The Western blot method was adopted to confirm whether the UreB expressed by L. lactis transformant had immunoreactivity. The optimized conditions for UreB expression were as follows. Nisin 40 ng/ml was added to the medium when the recombinant grew to OD₆₀₀≈0.30–0.40 and the induction time lasted 5 h. As a result, the maximum yield of UreB was 27.26 μg/mL of medium, and the maximum percentage of UreB in cell extracts of the L. lactis transformant reached its peak at 20.19%. Western blot analysis showed that the UreB protein expressed by L. lactis transformant had favorable immunoreactivity. All these results make an appealing case for construction of the food-grade vaccine for H. pylori.     

Food-Grade Expression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> UreB Subunit in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and its Immunoreactivity

Helicobacter pylori is the principal cause of chronic active gastritis, peptic ulcer, and gastric cancer. To develop an oral vaccine against H. pylori infection, we had expressed the H. pylori ureB gene (Genbank accession no. FJ436980) in nisin-controlled expression vectors using Lactococcus lactis NZ3900 as host. The ureB gene was amplified by PCR from a H.pylori strain MEL-Hp27. Then the ureB gene was fused translationally downstream of the nisin-inducible promoter nisA in a L. lactis plasmid pNZ8149. Lactose utilization based on the complementation of the lacF gene was used as a dominant selection marker for the food-grade expression system employing L. lactis NZ3900. The conditions of UreB expression in this system were optimized by orthogonal experiment. The optimized conditions have been determined as follows: induction of expression was carried out at the cells density of OD₆₀₀ ≈ 0.4 with 25 ng/ml nisin, and harvest after 5 h. The maximum percentage of recombinant UreB was estimated to be 7% of total soluble cellular proteins and the yield was 12.9 μg/ml. Western blot demonstrated that the UreB protein was expressed in the L. lactis transformant and had favorable immunoreactivity. These results indicated that the lactococci-derived vaccines could be promising candidates as alternative vaccine strategies for preventing H. pylori infection.     

Delivery of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> HpaA to gastrointestinal mucosal immune sites using <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and its immune efficacy in mice

Objective

To develop a safe and effective oral vaccine against Helicobacter pylori using its HpaA protein expressed in Lactococcus lactis.

Results

The gene encoding HpaA was obtained by PCR and ligated to pNZ8110-lysM following digestion with NaeI + SphI. The recombinant plasmid was transferred into E. coli for multiplication, and then into L. lactis. The recombinant L. lactis was induced to express HpaA, resulting in two products of 29 and 25 kDa, both of which yielded positive immunoreaction with mouse antisera against H. pylori, as confirmed by immunoblot assays. The 29 kDa product constituted 12% of the cell lysates. Oral inoculation with the engineered L. lactis evoked significantly elevated serum IgG level in mice (P < 0.05).

Conclusions

A novel engineered L. lactis strain was developed that efficiently produces whole HpaA protein with desired antigenicity and potent immunogenicity. It provides a basis for approaches to L. lactis-delivered anti-H. pylori vaccination.

     

Characterization of peptidoglycan hydrolase in Cag pathogenicity island of Helicobacter pylori

The Cag Type IV secretion apparatus proteins in Helicobacter pylori can mediate the injection of effector CagA protein into eukaryotic target cells. Although this apparatus forms an important pathway for bacterium–host interaction, its assembly process in vivo is poorly understood, and the proteins which contribute to break the bacterial cell walls in Cag-PAI have not yet been identified. The cagγ gene in Cag-PAI is a unique member that contains a conserved SLT catalysis domain, which makes it an attracting question whether cagy gene has the capacity to digest the bacterial cell wall. In the current study, therefore, the cagγ gene was cloned from the H. pylori NCTC 11637 and expressed in Escherichia coli, and its lytic effect on cell walls in vitro was observed. Results indicated that Cagγ protein has a lytic activity against bacterial cell walls. An allelic-exchange mutant (Δcagγ) was further constructed to investigate the relationship between Cagγ and effector CagA translocation. These results suggested that Cagγ contributed to the assembly of Cag Type IV secretion apparatus by digesting the peptidoglycan meshwork of bacterial cell walls.     

Investigation of the immunomodulatory effects of Lactobacillus casei and Bifidobacterium lactis on Helicobacter pylori infection

Background: Lactobacillus and Bifidobacterium species have shown beneficial effects in the treatment of Helicobacter pylori infection; however, the mechanisms behind such effects are not fully understood. In this study, we have investigated the immunomodulatory effects of probiotics in a mouse model of H. pylori infection. Materials and methods: H. pylori‐infected C57BL/6 mice were treated with L. casei L26, B. lactis B94, or no probiotics for 5 weeks, respectively. Mice not infected with H. pylori were included as normal controls. Gastric histology, protein levels of interleukin (IL)‐1β, IL‐10, IL‐12/23p40, and H. pylori colonization density in the gastric tissues, as well as H. pylori‐specific antibodies were examined. Results: In mice receiving L. casei L26 and B. lactis B94, gastric neutrophil infiltration and IL‐1β were significantly decreased and IL‐10 was significantly increased as compared with mice receiving no probiotics. In mice receiving B. lactis B94, IL‐12/23p40 was significantly increased and H. pylori IgG was significantly reduced as compared with mice receiving no probiotics. No significant difference of H. pylori colonization was observed among the three groups of mice. Conclusion: The reduced level of IL‐1β and neutrophil infiltration observed in mice infected with H. pylori following treatment with L. casei L26 and B. lactis B94 resulted from a modulation of immune response rather than a decrease of H. pylori colonization. Furthermore, B. lactis B94 has the intrinsic ability to promote a Th1 immune response through an increase in IL‐12/IL‐23.     

Cloning and Characterization of a 22 kDa Outer-Membrane Protein (Omp22) from Helicobacter pylori

Helicobacter pylori is a causative agent of gastritis and peptic ulceration in humans. As the first step towards development of a vaccine against H. pylori infection, we have attempted to identify protective antigens. A potential target of vaccine development would be a H. pylori specific protein, which is surface-exposed and highly antigenic. We identified a 22 kDa outer-membrane protein (Omp22) from H. pylori, which was highly immunoreactive. By screening a H. pylori genomic DNA library with rabbit anti-H. pylori outer-membrane protein antibodies, the omp22 gene was cloned and 1.4 kb of the nucleotide sequence was determined. One open reading frame, encoding a 179-residue polypeptide, was identified and the amino acid sequence deduced showed homology with peptidoglycan-associated lipoproteins. The sequence was conserved among other H. pylori strains. Omp22 protein is expressed as a precursor polypeptide of 179 residues and undergoes lipid modification and cleavage of an 18 amino acid signal peptide to yield a mature protein. Omp22 protein in H. pylori as well as recombinant Omp22 protein expressed in E. coli was localized into the outer membrane and exposed on the cell surface. Omp22 may have the potential as a target antigen for the development of a H. pylori vaccine.     

Reduction of lactate production in Lactococcus lactis, a combined strategy: metabolic engineering by introducing foreign alanine dehydrogenase gene and hemin addition

An approach to decreasing the lactate production in Lactococcus lactis by metabolic engineering is presented. The inhibitory effects of a low pH due to the accumulation of lactate on cell growth and nisin production in L. lactis are well known. To avoid such inhibitory effects, a new strategy by rerouting carbon flow was considered. In an effort to suppress lactate production, a new gene was introduced into L. lactis to create a novel pathway for alanine synthesis to reroute the metabolic flow of lactate. Alanine dehydrogenase (E.C.1.4.1.1) encoded by alaD from Bacillus sphaericus was expressed in L. lactis. The enzyme was expressed to a specific activity of nearly 0.39 U/mg protein in the transformant. Hemin addition was also considered to decrease the lactate production in L. lactis. The effect of hemin on the alanine production in the transformant was investigated. This study showed that using the combined strategy, stronger effects on lactate and alanine productions were observed in the transformant.     

Immunological features and the ability of inhibitory effects on enzymatic activity of an epitope vaccine composed of cholera toxin B subunit and B cell epitope from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> urease A subunit

Epitope vaccine based on urease of Helicobacter pylori is a promising option for prophylactic and therapeutic vaccination against H. pylori infection. In this study, we constructed an epitope vaccine with mucosal adjuvant cholera toxin B subunit (CTB) and an epitope (UreA_183-203) of H. pylori urease A subunit named CTB-UA. The CTB-UA fusion protein was expressed in Escherichia coli, and the purified protein was used for intraperitoneal immunization experiments in BALB/c mice. The experimental results indicated that anti-CTB-UA antibody could recognize both H. pylori urease A subunit (UreA) and urease B subunit (UreB). Besides, the CTB-UA epitope vaccine had good immunogenicity and immunoreactivity and could induce specific neutralizing antibodies which showed effectively inhibitory effect on the enzymatic activity of H. pylori urease. CTB-UA is a promising molecule to be investigated as H. pylori vaccine antigen candidate.     

Expression of UreB and HspA of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in silkworm pupae and identification of its immunogenicity

For mass production of urease B subunit (UreB) and heat shock protein A subunit (HspA) of Helicobacter pylori with Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) and to determine whether they could be used as an oral vaccine against H. pylori, besides, to determine the time course of expressed recombinant protein and the optimum acquisition time directly through green fluorescence, HspA and enhanced green fluorescence protein (EGFP) genes were cloned into vector pFastBacDual to form donor vector pFastBacDual-(EGFP) (HspA), UreB gene was cloned into vector pFastBacDual to form donor vector pFastBacDual-UreB,then they were transformed into E. coli BmDH10Bac to obtain the recombinant Bacmid-(EGFP) (HspA) and Bacmid-UreB respectively. They were used to transfect BmN cells and generated the recombinant baculovirus BmNPV-(EGFP) (HspA) and BmNPV-UreB. Using these recombinant baculovirus BmNPV-(EGFP) (HspA) and BmNPV-UreB inoculated the silkworm pupae, a recombinant HspA and UreB protein were expressed in silkworm pupae, which were around 13 and 62 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. After oral immunization of mice, serum specific IgG antibodies against HspA and UreB in vaccine group were much higher than that in mock and native silkworm powder control groups. The results indicated that the expressed recombinant HspA and UreB in silkworm pupae would possess good immunogenicity. In addition, when EGFP and HspA proteins were expressed, a direct correlation between the increase in intensity of fluorescence and HspA concentration.     

Construction of a recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strain expressing a fusion protein of Omp22 and HpaA from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> for oral vaccine development

Objectives

To develop orally administrated anti-Helicobacter pylori vaccination, a Lactococcus lactis strain was genetically constructed for fusion expression of H. pylori protective antigens HpaA and Omp22.

Results

The fusion gene of omp22 and hpaA with an adapter encoding three glycines was cloned from a plasmid pMAL-c2x-omp22-hpaA into Escherichia coli MC1061 and L. lactis NZ3900 successively using a shutter vector pNZ8110. Expression of the fusion gene in L. lactis was induced with nisin resulting in production of proteins with molecular weights of 50 and 28 kDa. Both of them were immunoreactive with mouse anti-H. pylori sera as determined via western blotting. Oral vaccination of BALB/c mice using the L. lactis strain carrying pNZ8110-omp22-hpaA elicited significant systematic humoral immune response (P < 0.05).

Conclusions

This is the first report showing that a fusion protein of two H. pylori antigens was efficiently expressed in L. lactis with immunogenicity. This is a considerable step towards H. pylori vaccines.

     

Secretory expression of K88 (F4) fimbrial adhesin FaeG by recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> for oral vaccination and its protective immune response in mice

K88 (F4) fimbrial adhesin, FaeG, was expressed extracellularly in Lactococcus lactis using a nisin-controlled gene expression system. The antibody response and protective efficacy of the recombinant bacteria (L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C₈₃₅₄₉ challenge were evaluated in ICR mice. Mice vaccinated with L. lactis [spNZ8048-faeG] had a significantly increased antigen-specific IgG level in the serum and decreased mortality rate (P < 0.05) compared with the control. This indicates that oral immunization of L. lactis [spNZ8048-faeG] can induce an immune-response protection upon challenge with live ETEC in ICR mice. An erratum to this article can be found at     

Cloning and Production of a Novel Bacteriocin, Lactococcin K, from Lactococcus lactis Subsp. lactis MY23

A gene encoding the antimicrobial peptide, lactococcin K, was isolated from Lactococcus lactis subsp. lactis MY23 then cloned and expressed in Escherichia coli. Because the expressed lactococcin K was formed as an inclusion body in recombinant E. coli, a fusion protein containing lactococcin K and maltose-binding protein (MBP) was produced in a soluble form. For high-level production of lactococcin K, we performed a pH-stat fed-batch culture to produce 43,000 AU lactococcin K ml⁻¹ in 12 h. Revisions requested 3 November 2005; Revisions received 7 December 2005     

<Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> as a live vehicle for oral immunization against chicken anemia virus

The AcmA binding domains of Lactococcus lactis were used to display the VP1 protein of chicken anemia virus (CAV) on Lactobacillus acidophilus. One and two repeats of the cell wall binding domain of acmA gene were amplified from L. lactis MG1363 genome and then inserted into co-expression vector, pBudCE4.1. The VP1 gene of CAV was then fused to the acmA sequences and the VP2 gene was cloned into the second MCS of the same vector before transformation into Escherichia coli. The expressed recombinant proteins were purified using a His-tag affinity column and mixed with a culture of L. acidophilus. Whole cell ELISA and immunofluorescence assay showed the binding of the recombinant VP1 protein on the surface of the bacterial cells. The lactobacilli cells carrying the CAV VP1 protein were used to immunize specific pathogen-free chickens through the oral route. A moderate level of neutralizing antibody to CAV was detected in the serum of the immunized chickens. A VP1-specific proliferative response was observed in splenocytes of the chickens after oral immunization. The vaccinated groups also showed increased levels of Th1 cytokines interleukin (IL)-2, IL-12, and IFN-γ. These observations suggest that L. acidophilus can be used in the delivery of vaccines to chickens.     

Production and Targeting of the Brucella abortus Antigen L7/L12 in Lactococcus lactis: a First Step towards Food-Grade Live Vaccines against Brucellosis

Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.     

Expression of the Lactobacillus casei lactate dehydrogenase gene in Bacillus subtilis

Summary A gene for allosteric lactate dehydrogenase (LDH) of Lactobacillus casei ATCC393 was transferred into Bacillus subtilis. The LDH was produced in a growth-associated type, and comprised up to 40 % of the total cellular protein. The maximum specific activity in the transformant was 208 U/mg protein which was approximately 16 times higher than in L. casei or in the previously constructed Escherichia coli transformant.     

Survival of Helicobacter pylori in well water

The mortality of a clinical Helicobacter pylori strain was assessed by inoculating it in untreated well water, filtered well water, and autoclaved well water. Two different temperatures (5 and 25 °C) were used during the experimental period. Because Escherichia coli is commonly used as indicator of faecal pollution of water, we compared the survival of H. pylori using E. coli as indicator of its persistence. H. pylori was not culturable 48 h after inoculation, whereas the population of E. coli, monitored at the same temperature, decreased slowly, especially in filtered water. In untreated water, both H. pylori and E. coli survived less well than in filtered and autoclaved water. In general the survival of H. pylori and E. coli was better in filtered water than in autoclaved water and the ability of H. pylori to survive several days in water at 5 °C is reported, supporting the observation that H. pylori survives better at 5 °C than at higher temperature. This suggests a possible faecal–oral transmission of H. pylori in the presence of a contaminated water.     

Reduced activation of inflammatory responses in host cells by mouse-adapted Helicobacter pylory isolates

Helicobacter pylori strains that harbour the Cag pathogenicity island (Cag PAI) induce interleukin (IL)-8 secretion in gastric epithelial cells, via the activation of NF- kappa B, and are associated with severe inflammation in humans. To investigate the influence of Cag PAI-mediated inflammatory responses on H. pylori adaptation to mice, a selection of H. pylori clinical isolates (n = 12) was cag PAI genotyped and tested in co-culture assays with AGS gastric epithelial cells, and in mouse colonization studies. Six isolates were shown to harbour a complete cag PAI and to induce NF- kappa B activation and IL-8 secretion in AGS cells. Of the eight isolates that spontaneously colonized mice, six had a cag PAI(-) genotype and did not induce pro-inflammatory responses in these cells. Mouse-to-mouse passage of the two cag PAI(+) -colonizing strains yielded host-adapted variants that infected mice with bacterial loads 100-fold higher than those of the respective parental strains (P= 0.001). These mouse-adapted variants were affected in their capacity to induce pro-inflammatory responses in host cells, yet no changes in cag PAI gene content were detected between the strains by DNA microarray analysis. This work provides evidence for in vivo selection of H. pylori bacteria with a reduced capacity to induce inflammatory responses and suggests that such bacteria are better adapted to colonize mice.     

Control of gene expression in Helicobacter pylori using the Tet repressor

The lack of a versatile system to control gene expression in Helicobacter pylori has hampered efforts to study H. pylori physiology and pathogenesis. To overcome these limitations, we evaluated the utility of an inducible system based on the well-characterized Tet repressor (TetR) and Tet operator (tetO). As validation of this system, we introduced three copies of tetO into the promoter region upstream of the cagUT operon (encoding two virulence factors required for function of the H. pylori Cag type IV secretion system) and expressed tetR by introducing a codon-optimized gene into the chromosomal ureA locus. Introduction of the tetO copies upstream of cagUT did not disrupt promoter activity, as determined by immunoblotting for CagT. The subsequent introduction of tetR, however, did repress CagT synthesis. Production of CagT was restored when strains were cultured in the presence of the inducer, anhydrotetracycline. To demonstrate one potential application of this new tool, we analyzed the function of the Cag type IV secretion system. When the modified H. pylori strains were co-cultured with AGS cells, activity of the Cag type IV secretion system was dependent on the presence of anhydrotetracycline as evidenced by inducer-dependent induction of IL-8 secretion, CagA translocation, and appearance of type IV secretion system pili at the bacteria–host interface. These studies demonstrate the effectiveness of the tetR–tetO system to control gene expression in H. pylori and provide an improved system for studying H. pylori physiology and pathogenesis.