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.     

Surface expression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> urease subunit B gene E fragment on <Emphasis Type="Italic">Lactococcus lactis</Emphasis> by means of the cell wall anchor of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> protein A

Fragment E of ureB (ureBE) was cloned from a clinical isolate of Helicobacter pylori. A prokaryotic expression vector, pAMJ399, with the ureB fragment E and the Staphylococcus aureus protein A anchor fragment (spaX), was constructed. The fusion protein was expressed under the control of the P170 promoter in Lactococcus lactis. Western blot assay of lactococcal cell wall extracts with a polyclonal chicken antiserum confirmed the immunity of the expressed recombinant protein which was located on the cell surface. These results provide the first report of a surface display system in lactic acid bacteria for the delivery of oral vaccines against Helicobacter pylori.     

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.

     

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.     

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.

     

Cloning and Characterization of a Novel <Emphasis Type="Italic">tuf</Emphasis> Promoter from <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> IL1403

Genetic engineering of lactic acid bacteria (LAB) requires a reliable gene expression system. Especially, a stable promoter is an important genetic element to induce gene expression in such a system. We report on a novel tuf promoter (Ptuf) of Lactococcus lactis subsp. lactis IL1403 that was screened and selected through analysis of previously published microarray data. Ptuf activity was examined and compared with three other known lactococcal promoters (PdnaJ, PpfkA, and Pusp45) using different bacteria as expression hosts. Each promoter was, respectively, fused to the promoterless and modified bmpB gene as a reporter, and we estimated promoter activity through BmpB expression. All promoters were active in IL1403, and Ptuf activity was strongest among them. The activity of each promoter differed by host bacteria (Lactobacillus plantarum Lb25, Lactobacillus reuteri ATCC23272, and Escherichia coli Top10F’). Ptuf had the highest activity in IL1403 when growth reached late log phase. The activity of each promoter correlated with the expression of each cognate gene in the microarray data (R ² = 0.7186, P = 0.06968). This study revealed that novel food-grade promoters such as IL1403 Ptuf can be selected from microarray data for food-grade microorganisms and Ptuf can be used to develop a reliable gene expression system in L. lactis.     

Nisin-controlled extracellular production of apidaecin in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Apidaecins are heat-stable, nonhelical antibacterial peptides isolated from lymph fluid of the honeybee (Apis mellifera). These peptides are active against a wide range of gram-negative bacteria and they are the most prominent components of the honeybee humoral defense against microbial invasion. In the present study, one isoform of apidaecin, apidaecin Ho, was expressed extracellularly in the food-grade bacterium Lactococcus lactis. Results showed that expression driven by the lactococcal nisA promoter and Usp45 signal peptide resulted in efficient secretion of apidaecin in L. lactis subsp. cremoris NZ9000. Recombinant apidaecin was purified by gel filtration and semipreparative RP-HPLC, and about 10 mg active recombinant apidaecin was obtained from 1,000 ml culture. This is the first report on the nisin-controlled extracellular production of active apidaecin in L. lacits. The expression and delivery of apidaecin in the food-grade L. lactis may provide a clue to facilitate the widespread application of apidaecin in the control and prevention of gram-negative bacteria infections of human and animals.     

Codon optimization of the calf prochymosin gene and its expression in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Chymosin as an important industrial enzyme widely used in cheese manufacture. The yeast Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, low yields (80 U/ml in shake flask cultures) were obtained when the K. lactis strain GG799 was used to express chymosin. We hypothesized that the codon-usage bias of the host may have resulted in inefficient translation and chymosin production. To improve expression efficiency of recombinant calf chymosin in K. lactis strain GG799, we designed and synthesized a DNA sequence encoding calf prochymosin using optimized codons, while keeping the G + C content relatively low. We altered 333 nucleotides to optimize codons encoding 315 amino acids. In shaking flask culture, chymosin activity was 575 U/ml in the strain expressing the optimized gene, a sevenfold higher expression level compared with the non-optimized control. SDS–PAGE analysis revealed that the purified recombinant calf chymosin had a molecular mass of 35.6 kDa, the same as the molecular weight of native calf chymosin. Alpha-casein, beta-casein, and kappa-casein were incubated with the recombinant calf chymosin from K. lactis strain GG799 or chymosin from calf stomach and the breakdown products were analyzed by SDS–PAGE. Both the recombinant calf chymosin and the native calf chymosin specifically hydrolyzed kappa-casein. Our results show that codon optimization of the calf chymosin gene improves expression in K. lactis strain GG799. Genetic manipulation to optimize codon usage has important applications for industrial chymosin production.     

Improved acid-stress tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000 and <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 by overexpression of the anti-acid component <Emphasis Type="Italic">recT</Emphasis>

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60 min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.     

Enhanced expression of heterologous inulinase in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> by disruption of <Emphasis Type="Italic">hap1</Emphasis> gene

Inulinase gene (Kcinu) derived from Kluyveromyces cicerisporus was expressed extracellularly in Kluyveromyces lactis using an episomal vector directed by Kcinu promoter. The influence of hap1 gene disruption on the expression of inulinase was studied. Inulinase activity in the supernatant of the recombinant Klhap1Δ strain was 391 U ml⁻¹ after cultured 120 h, which was 2.2-fold that of the wild type host. The relative inulinase mRNA level of the Klhap1Δ strain was 11.3-fold that of the wild type strain, and the expression plasmid was more stable in the mutant host. Based on these results, the disruption of hap1 facilitated the high and stable expression of inulinase controlled by Kcinu promoter in K. lactis.     

Use of the <Emphasis Type="Italic">usp45</Emphasis> lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Replacement of the signal peptide (SP) of the bacteriocins enterocin P (EntP) and hiracin JM79 (HirJM79), produced by Enterococcus faecium P13 and Enterococcus hirae DCH5, respectively, by the signal peptide of Usp45 (SP _usp45 ), the major Sec-dependent protein secreted by Lactococcus lactis, permits the production, secretion, and functional expression of EntP and HirJM79 by L. lactis. Chimeric genes encoding the SP _usp45 fused to either mature EntP (entP), with or without the immunity gene (entiP) or to mature HirJM79 (hirJM79), with or without the immunity gene (hiriJM79), were cloned into the expression vector pMG36c, carrying the P₃₂ constitutive promoter, and into pNZ8048 under control of the inducible PnisA promoter. The production of EntP and HirJM79 by most of the L. lactis recombinant strains was 1.5- to 3.7-fold higher and up to 3.6-fold higher than by the E. faecium P13 and E. hirae DCH5 control strains, respectively. However, the specific antimicrobial activity of the recombinant EntP was 1.1- to 6.2-fold higher than that produced by E. faecium P13, while that of the HirJM79 was a 40% to an 89% of that produced by E. hirae DCH5. Chimeras of SP _usp45 fused to mature EntP or HirJM79 drive the production and secretion of these bacteriocins in L. lactis in the absence of specific immunity and secretion proteins. The supernatants of the recombinant L. lactis NZ9000 strains, producers of EntP, showed a much higher antimicrobial activity against Listeria spp. than that of the recombinant L. lactis NZ9000 derivatives, producers of HirJM79.     

Transgenic Peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) Expressing the Urease Subunit B Gene of <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

Helicobacter pylori (H. pylori) has been identified as the main pathogenic factors of chronic gastritis and peptic ulcer, and the Class I carcinogen of gastric cancer by WHO. Vaccine has become the most effective measure to prevent and cure H. pylori infection. The UreB is the most effective and common immunogen of all strains of H. pylori and may stimulate the immunoresponse protecting the human body against the challenge of H. pylori. UreB antigen gene was cloned into the binary vector pBI121 which contains a seed-specific promoter Oleosin of peanut and a kanamycin resistance gene, and then UreB gene was transformed into peanut embryo leaflets by Agrobacter-mediated method. The putative transgenic plants were examined for the presence of UreB in the nuclear genome of peanut plants by PCR analysis. Expression of UreB gene in plants was identified by RT-PCR and Western blot analysis. These results suggest that the UreB transgenic peanut can be potentially used as an edible vaccine for controlling H. pylori.     

High-level expression and characterization of a highly thermostable chitosanase from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The sequence of an endo-chitosanase gene (CSN) from Aspergillus fumigatus was optimized based on the preferred codons of Pichia pastoris and synthesized in vitro through overlapping PCR (CSN-P). The gene was cloned into a yeast expression vector, pHBM905A, and secretorily expressed in Pichia pastoris GS115. The yield of CSN-P reached ~3 mg/ml with a high-density fermentation in a 14 l fermenter and the enzyme activity was ~25,000 U/ml. The enzyme had half-lives of 2.5 h at 80°C, 1 h at 90°C and 32 min at 100°C. It retained 70% activity after incubation with 10 M urea at room temperature for 30 min. This enzyme was used for a large-scale preparation of oligosaccharides: 3 g enzyme converted 200 kg chitosan into oligosaccharides in 24 h at 60°C.     

High-level heterologous expression of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> PG37 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI, was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut⁺) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His⁺, Mut⁺). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that (10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at a broad pH range of 7.0–10.5 and at a temperature of 30°C or below.     

Nisin inducible production of listeriolysin O in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000

Background  

Listeria monocytogenesis a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactisNZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter.     

High efficiency electrotransformation of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>cells pretreated with lithium acetate and dithiothreitol

Background  

A goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria.     

A food-grade delivery system for<Emphasis Type="Italic"> Lactococcus lactis</Emphasis> and evaluation of inducible gene expression

The genetic improvement of Lactococcus lactis is a matter of biotechnological interest in the food industry and in the pharmaceutical and medical fields. However, to construct a food-grade delivery system, both the presence of antibiotic markers or plasmid sequences should be avoided and the maintenance and expression of the cloned gene should be guaranteed. The objective of this work was to produce crossover mutants of L. lactis with a reporter gene under the control of an inducible promoter in order to evaluate the level of gene expression. We utilized a nuclease gene of Staphylococcus aureus as a reporter gene, P _nisA as the nisin-inducible promoter, a non-essential gene involved in histidine biosynthesis of L. lactis as the site for homologous recombination, and pRV300 as a suicide vector for the genomic integration in L. lactis NZ9000. Single- and double-crossover mutants were identified by genotype and phenotype. Relative to episomal transformants of L. lactis, the level of expression of the heterologous protein after nisin induction was similar in the crossover mutants, suggesting that a single copy of the heterologous gene can be used to produce the protein of interest.     

An Epidemiologic Study on the Correlation Between Oral <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and Gastric <Emphasis Type="Italic">H. pylori</Emphasis>

The current study was conducted to determine the prevalence of Helicobacter pylori in the oral cavity of a Chinese population. Nested polymerase chain reaction (PCR) was used to investigate whether individuals with oral H. pylori show more possibility of gastric infection and to examine the relationship between gastric H. pylori infection and the presence of the bacteria in the oral cavity. A total of 443 dyspeptic patients participated in the study. Gastric H. pylori infection was detected by the rapid urease test and histology with Giemsa staining, PCR, or smear examination, whereas the presence of the bacteria in the oral cavity was observed by nested PCR from dental plaque. Relevant periodontal and dental parameters were recorded in the process of oral examination. Of the 443 study patients, oral H. pylori was found in the dental plaque of 263 (59.4%) and the stomach of 273 (61.6%). Additionally, in all four age groups, the prevalence of gastric infection was significantly higher among the patients with positive tests for H. pylori in their dental plaque than in the patients with no H. pylori in their dental plaque (P < 0.05). The oral cavity may be a potential reservoir for H. pylori, and the prevalence of oral H. pylori approximated that of gastric H. pylori in the studied population. Furthermore, a close relationship may exist between H. pylori in the oral cavity and bacteria in the stomach or gastric infection, and dyspeptic patients with gastric infection are more likely to harbor H. pylori in their oral cavity. Y. Liu and H. Yue contributed equally to the accomplishment of this study.     

Overexpression of <Emphasis Type="Italic">spoT</Emphasis> gene in coccoid forms of clinical <Emphasis Type="Italic">Helicobacter pylori</Emphasis> isolates

Helicobacter pylori (H. pylori) can convert to coccoid form in unfavorable conditions or as a result of antibiotic treatment. In order to adapt to harsh environments, H. pylori requires a stringent response which, encoded by the spoT gene, has a bifunctional enzyme possessing both (p)ppGpp synthetic and degrading activity. Our goal in this study was to compare spoT gene expression in spiral and induced coccoid forms of H. pylori with use of amoxicillin. First, clinical isolate coccoid forms were induced with amoxicillin; then, the viability test was analyzed by flow cytometer. After RNA extraction, cDNA synthesis and designing a specific primer for spoT gene, evaluation of the desired gene expression in both forms were studied. Bacterial isolates exposed to amoxicillin at MIC and 1/2 MIC induced morphological conversion better and faster than other MIC concentration. The expression of spoT gene was significantly downregulated in spiral forms of H. pylori, while the gene expression was upregulated and + 30.3-fold changes was seen in coccoid forms of bacterium. To summarize, spoT gene is one of the key factors for antibiotic resistance and its enhanced expression in coccoid form can be a valuable diagnostic marker for recognition of H. pylori during morphological conversion.