Mouse intestinal microbiota reduction favors local intestinal immunity triggered by antigens displayed in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> biofilm

Background

We previously engineered Bacillus subtilis to express an antigen of interest fused to TasA in a biofilm. B. subtilis has several properties such as sporulation, biofilm formation and probiotic ability that were used for the oral application of recombinant spores harboring Echinococcus granulosus paramyosin and tropomyosin immunogenic peptides that resulted in the elicitation of a specific humoral immune response in a dog model.

Results

In order to advance our understanding of the research in oral immunization practices using recombinant B. subtilis spores, we describe here an affordable animal model. In this study, we show clear evidence indicating that a niche is required for B. subtilis recombinant spores to colonize the densely populated mice intestinal microbiota. The reduction of intestinal microbiota with an antibiotic treatment resulted in a positive elicitation of local humoral immune response in BALB/c mice after oral application of recombinant B. subtilis spores harboring TasA fused to E. granulosus (102-207) EgTrp immunogenic peptide. Our results were supported by a lasting prevalence of spores in mice feces up to 50 days after immunization and by the presence of specific secretory IgA, isolated from feces, against E. granulosus tropomyosin.

Conclusions

The reduction of mouse intestinal microbiota allowed the elicitation of a local humoral immune response in mice after oral application with spores of B. subtilis harboring immunogenic peptides against E. granulosus.

     

Mucosal delivery of anti-inflammatory IL-1Ra by sporulating recombinant bacteria

Background

Mucosal delivery of therapeutic protein drugs or vaccines is actively investigated, in order to improve bioavailability and avoid side effects associated with systemic administration. Orally administered bacteria, engineered to produce anti-inflammatory cytokines (IL-10, IL-1Ra), have shown localised ameliorating effects in inflammatory gastro-intestinal conditions. However, the possible systemic effects of mucosally delivered recombinant bacteria have not been investigated.

Results

B. subtilis was engineered to produce the mature human IL-1 receptor antagonist (IL-1Ra). When recombinant B. subtilis was instilled in the distal colon of rats or rabbits, human IL-1Ra was found both in the intestinal lavage and in the serum of treated animals. The IL-1Ra protein in serum was intact and biologically active. IL-1-induced fever, neutrophilia, hypoglycemia and hypoferremia were inhibited in a dose-dependent fashion by intra-colon administration of IL-1Ra-producing B. subtilis. In the mouse, intra-peritoneal treatment with recombinant B. subtilis could inhibit endotoxin-induced shock and death. Instillation in the rabbit colon of another recombinant B. subtilis strain, which releases bioactive human recombinant IL-1β upon autolysis, could induce fever and eventually death, similarly to parenteral administration of high doses of IL-1β.

Conclusions

A novel system of controlled release of pharmacologically active proteins is described, which exploits bacterial autolysis in a non-permissive environment. Mucosal administration of recombinant B. subtilis causes the release of cytoplasmic recombinant proteins, which can then be found in serum and exert their biological activity in vivo systemically.

     

Inhibition of Lipopolysaccharide-Induced Interleukin 8 in Human Adenocarcinoma Cell Line HT-29 by Spore Probiotics: <Emphasis Type="Italic">B. coagulans</Emphasis> and <Emphasis Type="Italic">B. subtilis</Emphasis> (natto)

Probiotics are used as a treatment for different intestinal disorders. They confer health benefits by different ways. This study was aimed to investigate immunomodulatory effect of Bacillus probiotic spores on the production of lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) in HT-29 intestinal epithelial cells. Differentiated intestinal epithelial cell line was used as a model for the study of colonization of purified spores (Bacillus subtilis (natto) and B. coagulans) and their anti-inflammatory effects. MTT assay and trypan blue staining were used for the detection of optimal concentration of the purified spores and LPS. Pre-treatment assay was done by treatment of the cells with the purified spores for 2 h, followed by challenges with LPS for 3 and 18 h. Post-treatment assay was done by initial treatment of the cells with LPS for 18 h, followed by the spores for 3 and 6 h. Levels of IL-8 secretion and its mRNA expression were measured by ELISA and relative Q real-time PCR. Our results showed similar rates of adherence to intestinal epithelial cells by the spore probiotics, while displaying no cytotoxic effect. In the pre-treatment assay, a significant decrease in IL-8, at both protein and mRNA levels, was measured for B. coagulans spores after the addition of LPS, which was higher than those observed for Bacillus subtilis (natto) spores. In the post-treatment assay, while Bacillus subtilis (but not B. coagulans) diminished the LPS-stimulated IL-8 levels after 3 h of incubation, the inhibitory effect was not constant. In conclusion, ability of Bacillus spore probiotics for adherence to intestinal epithelial cell and their anti-inflammatory effects, through interference with LPS/IL-8 signaling, was shown in this study. Further studies are needed to characterize responsible bacterial compounds associated with these effects.     

Mucosal Adjuvant Activity of IL-2 Presenting Spores of Bacillus subtilis in a Murine Model of Helicobacter pylori Vaccination

The endospores of Bacillus subtilis are now widely used as a platform for presentation of heterologous proteins and due to their safety record and high resistance to harsh environmental conditions can be considered as potential vehicles for oral vaccination. In this research we show that recombinant B. subtilis spores presenting a fragment of the Helicobacter acinonychis UreB protein and expressing the ureB gene under vegetative promoter elicit a strong cellular immune response in orally immunized mice when co-administered with spores presenting IL-2. We show for the first time the successful application of two types of recombinant spores, one carrying an antigen and the other an adjuvant, in a single oral immunization.     

Construction of malate-sensing <Emphasis Type="Italic">Escherichia coli</Emphasis> by introduction of a novel chimeric two-component system

In an attempt to develop a high-throughput screening system for screening microorganisms which produce high amounts of malate, a MalKZ chimeric HK-based biosensor was constructed. Considering the sequence similarity among Escherichia coli (E. coli) MalK with Bacillus subtilis MalK and E. coli DcuS, the putative sensor domain of MalK was fused with the catalytic domain of EnvZ. The chimeric MalK/EnvZ TCS induced the ompC promoter through the cognate response regulator, OmpR, in response to extracellular malate. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as malate concentration increased. By using this strategy, various chimeric TCS-based bacteria biosensors can be constructed, which may be used for the development of biochemical-producing recombinant microorganisms.     

Recombinant flagellin-PAL fusion protein of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> induced cell-mediated and protective immunity against bacteremia in BALB/c mice

We report a new recombinant fusion protein composed of full-length Legionella pneumophila flagellin A and peptidoglycan-associated lipoprotein (PAL), rFLA-PAL, capable of inducing protective immunity against L. pneumophila. The recombinant protein was over expressed in Escherichia coli strain BL21 (DE3) using pET-28a (+) expression vector (pET28a-flaA-pal) and purified by Ni²⁺ exchange chromatography. Immunological properties of rFLA-PAL were assessed in a mouse model. Female BALB/c mice, immunized with rFLA-PAL, exhibited a rapid increase in serum antibody concentration against each of its protein portions. Furthermore, a strong activation of both innate and adaptive cell-mediated immunity was observed as indicated by antigen-specific splenocyte proliferation, IFN-γ and IL-12 production, and early production of TNF-α in the serum and in splenocyte cultures which were separately assessed against PAL and FLA. BALB/c mice were challenged with a lethal dose of L. pneumophila intravenously. In a 10-days follow-up after intravenous lethal challenge with L. pneumophila, a 100% survival rate was observed for mice immunized with rFLA-PAL, same as for those immunized with a sublethal dose of L. pneumophila. Based on the potent immune responses observed in mice immunized with rFLA-PAL, this recombinant fusion protein could be a potential vaccine candidate against the intracellular pathogen L. pneumophila.     

Cloning and expression of <Emphasis Type="Italic">nlpA</Emphasis> gene as DNA vaccine candidate against <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis>

Acinetobacter baumannii is one of the highly antibiotic-resistant bacteria that cause infections with high rate of death. This bacterium is one the common causes of infection worldwide leading to endemic and epidemic nosocomial infections. Despite many efforts, there is no effective vaccine against A. baumannii. As NlpA is one of the important antigenic factors in biogenesis of outer membrane vesicles, and OMV-based reported vaccines in A. baumannii stimulated the immune responses, this study was aimed to clone and express nlpA gene in eukaryotic HDF cells and evaluate the induced immunization following the administration of resulting construct as DNA vaccine in BALB/c mice. The nlpA gene of A. baumannii was amplified using PCR. The PCR product was then cloned and subcloned into the pTZ57R/T and pEGFP-C2 vectors respectively. The cloning was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The pEGFP-C2-nlpA recombinant plasmid was transferred into the HDF cells using electroporation and the expression of target gene was validated by RT-PCR. The recombinant construct was injected to BALB/c mice through three IM injections and the levels of IgG, IgM, INF-γ, IL-2, IL-4, and IL-12 were determined using ELISA assay. The A. baumannii nlpA gene was amplified during PCR as 867 bp band which was successfully cloned in pEGFP-C2-nlpA vector. Obtained data from RT-PCR and presence of the 867 bp fragment in transformed HDF cells confirmed the nlpA gene expression. Following the injection of pEGFP-C2-nlpA showed the increased level of IgG, IgM, INF-γ, IL-2, IL-4, and IL-12 in serum of immunized mice. Overall, through this study recombinant pEGFP-C2-nlpA was generated and successfully expressed the A. baumannii nlpA gene in eukaryotic cells. Additionally, our in vivo study confirmed that the recombinant construct capable to induce the immune response in immunized mice. These findings suggest the pEGFP-C2-nlpA may be considered as DNA vaccine candidate against A. baumannii.     

Biosynthesis of secreted ribonucleases of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> and <Emphasis Type="Italic">Bacillus circulans</Emphasis> under nitrogen starvation

The level of biosynthesis of secreted guanyl-specific ribonucleases (RNases) of Bacillus intermedius (binases) and Bacillus circulans (RNases Bci) by recombinant B. subtilis strains increases under nitrogen starvation. The promoter of the binase gene carries the sequences homologous to the recognition sites of the regulatory protein TnrA, which regulates gene expression under growth limitation by nitrogen. Using the B. subtilis strain defective in protein TnrA, it has been shown that the regulatory protein TnrA is involved in the regulation of expression of the binase gene and the gene of RNase Bci. The TnrA regulation of expression of the RNase Bci gene is indirect, probably by means of the regulatory protein PucR. Thus, it has been established that at least two regulatory mechanisms activate the expression of the genes encoding the secreted RNases of spore-forming bacteria: a system of proteins homologous to the B. subtilis PhoP-PhoR, and regulation by a protein similar to the B. subtilis TnrA regulatory protein.     

Immunostimulatory characteristics of a novel adjuvant on the basis of cucumarioside A<Subscript>2</Subscript>-2 and monogalactosyldiacylgycerol

A novel antigen carrier has been formulated on the basis of a cucumarioside-A₂-2 triterpene glycoside (CD) complex with cholesterol and monogalactosyldiacylglycerol from Ahnfeltia tobuchiensis (MGDGAt) and Ulva fenestrate (MGDGUf). Morphological and immunostimulative characteristics of the carrier were studied. Electron microscopy experiments demonstrated the formation of homogeneous tubular structures in a mixture of CD, cholesterol, and MGDG in molar ratio of 1: 2: 3. In animals immunized by the carrier bearing pore forming protein monomer of pseudotuberculosis agent CD and MGDG synergically affected synthesis of specific antibodies, interleukin-2, and γ-interferon and delayed hypersensitivity reaction when compared to Freund’s complete adjuvant or to immunostimulatory complexes between Quillaja saponaria saponins and phosphatidylcholine from egg yolk. The immunostimulatory effect depends upon the composition of polyunsaturated fatty acids of MGDG. The new tubular adjuvant carrier is a competitive adjuvant, as it includes CD obtained from far-eastern sea cucumber commercial species Cucumaria japonica, and MGDG from seaweed.     

Exploitation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> as a robust workhorse for production of heterologous proteins and beyond

Bacillus subtilis, belonging to the type species of Bacillus, is a type of soil-derived, low %G+C, endospore-forming Gram-positive bacterium. After the discovery of B. subtilis 168 that displayed natural competence, this bacterium has been intensively considered to be an ideal model organism and a robust host to study several basic mechanisms, such as metabolism, gene regulation, bacterial differentiation, and application for industrial purposes, such as heterologous protein expression and the overproduction of an array of bioactive molecules. Since the first report of heterologous overproduction of recombinant proteins in this strain, the bulk production of a multitude of valuable enzymes, especially industrial enzymes, has been performed on a relatively large scale. Since B. subtilis can non-specifically secrete recombinant proteins using various signal peptides, it has tremendous advantages over Gram-negative bacterial hosts. Along with the report of the complete genome sequence of B. subtilis, a number of genetic tools, including diverse types of plasmids, bacterial promoters, regulatory elements, and signal peptides, have been developed and characterized. These novel genetic elements tremendously accelerated genetic engineering in B. subtilis recombinant systems. In addition, with the development of several complex gene expression systems, B. subtilis has performed a number of more complex functions. This ability enables it to be a substantial chassis in synthetic biology rather than just a workhorse for the overproduction of recombinant proteins. In this review, we review the progress in the development of B. subtilis as a universal platform to overproduce heterologous diverse high-value enzymes. This progress has occurred from the development of biological parts, including the characterization and utilization of native promoters, the fabrication of synthetic promoters and regulatory elements, and the assembly and optimization of genetic systems. Some important industrial enzymes that have been produced in large quantities in this host are also summarized in this review. Furthermore, the ability of B. subtilis to serve as a cellular tool was also briefly recapitulated and reviewed.     

Surface display of lipolytic enzyme,Lipase A and Lipase B of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> spore

For the enhancement of lipase stability in organic solvent containing reaction, live immobilization method, using Bacillus subtilis spore as a display vehicle was attempted. Bacillus subtilis coat protein cotE was used as an anchoring motif for the display of lipA and lipB of Bacillus subtilis. Using this motif, lipolytic enzyme Lipase A and Lipase B were functionally displayed on the surface of Bacillus subtilis spore. Purified spore displaying CotE-LipB fusion protein showed higher lipolytic activity compared to that of CotE-LipA fusion protein. The surface localization of Lipase B was verified with flow cytometry and protease accessibility experiment. Spore displayed lipase retained its activity against acetone and benzene which completely deactivated free soluble lipase in the same reaction condition.     

Engineering chimeric two-component system into <Emphasis Type="Italic">Escherichia coli</Emphasis> from <Emphasis Type="Italic">Paracoccus denitrificans</Emphasis> to sense methanol

Escherichia coli does not have the methanol sensing apparatus, was engineered to sense methanol by employing chimeric two-component system (TCS) strategy. A chimeric FlhS/EnvZ (FlhSZ) chimeric histidine kinase (HK) was constructed by fusing the sensing domain of Paracoccus denitrificans FlhS with the catalytic domain of E. coli EnvZ. The constructed chimeric TCS FlhSZ/OmpR could sense methanol by the expression of ompC and gfp gene regulated by ompC promoter. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed the dynamic response of the chimeric TCS to methanol. The expression of ompC and the gfp fluorescence was maximum at 0.01 and 0.5% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric HK FlhSZ. This strategy can be employed for the construction of several chimeric TCS based bacterial biosensors for the development of biochemical producing recombinant microorganisms.     

Conversion of xylan by recyclable spores of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> displaying thermophilic enzymes

Background

The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-d-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-d-xylans to remove successive d-xylose residues from the non-reducing termini.

Results

We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation.

Conclusion

Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions.

     

Sporulation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in binary cultures with ultramicrobacteria

The effect of ultramicrobacterial epibionts of the genera Kaistia (strain NF1), Chryseobacterium (strain NF4), and Stenotrophomonas (strain FM3) on the process of sporulation of Bacillus subtilis ATCC 6633 was studied. The investigated strains of ultramicrobacteria (UMB) were found to inhibit the sporulation process of B. subtilis ATCC 6633 in binary mixed cultures, exhibiting a 3-day delay of the onset of sporulation compared to the control one, an extended period of the prospore maturation, formation of the fraction of immature spores, and development of ultrastructural defects in many endospores. Thus, investigation of binary mixed cultures of B. subtilis and UMB revealed that, apart from suppression of reproduction and lysis of host vegetative cells, inhibition of spore formation and destruction of endospores was yet another feature of intermicrobial parasitism. The UMB parasites of the studied genera are assumed to participate in the regulation of development and reproduction of B. subtilis in natural habitats of this spore-forming bacterium.     

Heterologous expression of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> gene of glutamyl endopeptidase in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains defective in regulatory proteins

Expression of the gene of glutamyl endopeptidase from Bacillus intermedius (gseBi) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5–5-fold decrease in glutamyl endopeptidase activity. It has been concluded that expression of the B. intermedius glutamyl endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.     

Gamma-irradiation of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> for the use as an immunogenic whole cell vaccine

Streptococcus pneumoniae is a major respiratory pathogen that causes millions of deaths worldwide. Although subunit vaccines formulated with the capsular polysaccharides or their protein conjugates are currently-available, low-cost vaccines with wide serotype coverage still remain to be developed, especially for developing countries. Recently, gamma- irradiation has been considered as an effective inactivation method to prepare S. pneumoniae vaccine candidate. In this study, we investigated the immunogenicity and protective immunity of gamma-irradiated S. pneumoniae (r-SP), by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP), both of which were made by traditional inactivation methods. Intranasal immunization of C57BL/6 mice with r-SP in combination with cholera toxin as an adjuvant enhanced S. pneumoniaespecific antibodies on the airway mucosal surface and in sera more potently than that with h-SP or f-SP under the same conditions. In addition, sera from mice immunized with r-SP potently induced opsonophagocytic killing activity more effectively than those of h-SP or f-SP, implying that r-SP could induce protective antibodies. Above all, immunization with r-SP effectively protected mice against S. pneumoniae infection. Collectively, these results suggest that gamma- irradiation is an effective method for the development of a killed whole cell pneumococcal vaccine that elicits robust mucosal and systemic immune responses.     

Molecular characterization of <Emphasis Type="Italic">bmyC</Emphasis> gene of the mosquito pupicidal bacteria, <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> (VCRC B483) and in silico analysis of bacillomycin D synthetase C protein

A strain of Bacillus amyloliquefaciens (VCRC B483) exhibiting mosquito pupicidal, keratinase and antimicrobial activities was isolated from mangrove forest ecosystem of Andaman and Nicobar Islands. Molecular characterization of the strain showed the presence of lipopeptide encoding bmyC gene. Phylogenetic tree based on protein sequence of this gene exhibited homology with mycosubtilin synthetase of Bacilus atropheus and Iturin synthetase of Bacillus subtilis and B. amyloliquefaciens. This is the first report on the evolutionary conservation of amino acids concerned with the function and structure of bmyC protein of B. amyloliquefaciens. The presence of valine at the 1197th position in our strain was found to be unique and different from the existing strains of B. subtilis and B. amyloliquefaciens. Molecular modelling studies revealed significant changes in the structure of epimerization domain of the bmyC protein with A1197V variation. Crude metabolite of this strain exhibited antifungal activity against Fusarium sp. and Carvularia sp.     

Significantly enhancing recombinant alkaline amylase production in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization

Background

Alkaline amylase has significant potential for applications in the textile, paper and detergent industries, however, low yield of which cannot meet the requirement of industrial application. In this work, a novel ARTP mutagenesis-screening method and fermentation optimization strategies were used to significantly improve the expression level of recombinant alkaline amylase in B. subtilis 168.

Results

The activity of alkaline amylase in mutant B. subtilis 168 mut-16# strain was 1.34-fold greater than that in the wild-type, and the highest specific production rate was improved from 1.31 U/(mg·h) in the wild-type strain to 1.57 U/(mg·h) in the mutant strain. Meanwhile, the growth of B. subtilis was significantly enhanced by ARTP mutagenesis. When the agitation speed was 550 rpm, the highest activity of recombinant alkaline amylase was 1.16- and 1.25-fold of the activities at 450 and 650 rpm, respectively. When the concentration of soluble starch and soy peptone in the initial fermentation medium was doubled, alkaline amylase activity was increased 1.29-fold. Feeding hydrolyzed starch and soy peptone mixture or glucose significantly improved cell growth, but inhibited the alkaline amylase production in B. subtilis 168 mut-16#. The highest alkaline amylase activity by feeding hydrolyzed starch reached 591.4 U/mL, which was 1.51-fold the activity by feeding hydrolyzed starch and soy peptone mixture. Single pulse feeding-based batch feeding at 10 h favored the production of alkaline amylase in B. subtilis 168 mut-16#.

Conclusion

The results indicated that this novel ARTP mutagenesis-screening method could significantly improve the yield of recombinant proteins in B. subtilis. Meanwhile, fermentation optimization strategies efficiently promoted expression of recombinant alkaline amylase in B. subtilis 168 mut-16#. These findings have great potential for facilitating the industrial-scale production of alkaline amylase and other enzymes, using B. subtilis cultures as microbial cell factories.

     

<Emphasis Type="Italic">Brucella abortus</Emphasis> phosphoglyceromutase and dihydrodipicolinate reductase induce Th1 and Th2-related immune responses

Brucellae are intracellular bacterial pathogens that cause Brucellosis, bringing great economic burdens to developing countries. The pathogenic mechanisms of Brucella are still poorly understood. Earlier immune response plays an important role in the Brucella infection. Phosphoglyceromutase (PGM) and dihydrodipicolinate reductase (DapB) were cloned, expressed, purified, and their immunocompetence was analyzed. Cytokines were detected by murine macrophages (RAW 264.7) and splenocytes that stimulated with the two recombinant proteins. The immune responses were analyzed by ELISA from mice with the two recombinant proteins immunized. TNF-α, IL-6 and IL-8 were produced in stimulated RAW 264.7 cells and splenocytes. Th1-type cytokines, IFN-γ and IL-2, induced in RAW 264.7 cells and splenocytes were higher then Th2-type cytokines, IL-4 and IL-5. Th2-related immune response was induced in splenocytes obtained 35 days after mice immunized with the two proteins. The production of IgG1 was higher than IgG2a in immunized mice. Taken together, our results demonstrated that the two proteins could induce Th1 and Th2-type immune responses in vivo and in vitro.     

Defense Responses and Changes in Symbiotic Gut Microflora in the Colorado Potato Beetle <Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis> under the Effect of Endophytic Bacteria from the Genus Bacillus

Phytophagous insects and host plants have a complex of microsymbionts and make up a united co-evolving system with them. Microsymbiotic complexes are actively involved in stress responses of macrosymbionts. We established that a treatment of potato plants with endophytic bacterial strains Bacillus thuringiensis var. thuringiensis-5689, B. th. var. kurstaki-5351, and Bacillus subtilis 26D decreased the survival rate of the plant feeder, Colorado potato beetle Leptinotarsa decemlineata Say. The B. th. strains suppressed phenoloxidase and acetylcholinesterase activities in the beetle hemolymph. An antagonistic relationship was found between endophytic bacteria B. subtilis 26D and beetle symbiotic bacteria from the genera Acinetobacter and Enterobacter, with the former being able to suppress the growth of endophytic colonies. The recombinant B. subtilis strain 26D Cry, containing the B. th. var. kurstaki δ-endotoxin cry1Ia gene, combined the ability of the original B. subtilis 26D strain to suppress the development of beetle symbionts and immune responses with a production of the Cry toxin, thus leading to a high mortality of the phytophage.