Gene expression in Lactococcus lactis

Abstract Lactic acid bacteria are of major economic importance, as they occupy a key position in the manufacture of fermented foods. A considerable body of research is currently being devoted to the development of lactic acid bacterial strains with improved characteristics, that may be used to make fermentations pass of more efficiently, or to make new applications possible. Therefore, and because the lactococci are designated 'GRAS' organisms ('generally recognized as safe') which may be used for safe production of foreign proteins, detailed knowledge of homologous and heterologous gene expression in these organisms is desired. An overview is given of our current knowledge concerning gene expression in Lactococcus lactis . A general picture of gene expression signals in L. lactis emerges that shows considerable similarity to those observed in Escherichia coli and Bacillus subtilis . This feature allowed the expression of a number of L. lactis -derived genes in the latter bacterial species. Several studies have indicated, however, that in spite of the similarities, the expression signals from E. coli, B. subtilis and L. lactis are not equally efficient in these three organisms.     

A组轮状病毒VP5*和VP8*的表达和免疫学性质研究

轮状病毒是引起婴幼儿腹泻的主要病原,VP4是RV重要的抗原蛋白,在早期病毒与细胞黏附过程中发挥重要作用,包括受体结合和细胞渗透。在细胞黏附过程中,VP4易被切割成VP5*和VP8*两个片段并以此增强病毒感染性。为了深入研究VP5*和VP8*的免疫学性质,进一步评价其应用前景,本研究从TB-Chen株RV基因组中编码VP4蛋白基因上克隆了VP5*和VP8*开放读码框核苷酸序列,构建了表达质粒,在原核大肠杆菌系统中重组表达了VP5*和VP8*蛋白,进一步分析了它们的免疫学性质。结果显示,VP5*和VP8*可在E.coli中高效表达,重组蛋白VP5* (rVP5*)和VP8* (rVP8*)可诱导免疫豚鼠产生特异性血清抗体,这些抗体可特异性识别自身蛋白（rVP5*或rVP8*）,可识别来自的TB-Chen株重组VP4蛋白,并可识别SA11和Wa感染的MA104细胞中合成的病毒VP4蛋白。这些结果表明,rVP5*和rVP8*蛋白具有较高的免疫原性,抗rVP5*和抗rVP8*的抗体具有高度特异性和交叉反应性。     

Secretion of biologically active murine interleukin-10 by Lactococcus lactis

We investigated the ability of Lactococcus lactis to secrete biologically active, murine interleukin-10 (mIL-10). mIL-10 was synthesized as a fusion protein, consisting of the mature part of the eukaryotic protein fused to the secretion signal of the lactococcal Usp45 protein. The secreted protein was analyzed by PAGE, ELISA and bioassay.We show that L. lactis can efficiently secrete biologically active, murine IL-10. Determination of the N-terminal amino acid sequence confirmed correct processing of the fusion polypeptide by the lactococcal signal peptidase. The amount of mIL-10, accumulating in the medium, could be increased by a factor of ten by growing the cells in an optimized medium, buffered at near-neutral pH. Under these conditions, up to 30 mg of mIL-10 was obtained from a 10-litre fermentation.     

乳酸乳球菌作为黏膜免疫活载体疫苗传递抗原的研究进展

乳酸菌是人和动物肠道内的常见细菌,被公认为安全级(generally recognized as safe,GRAS)微生物。近年来,对于乳酸菌作为宿主菌表达外源蛋白或抗原的研究取得了一定进展。乳酸乳球菌(Lactococcus lactis)是乳酸菌的代表菌种,以其生长迅速、易于操作等优点成为表达外源抗原,作为黏膜免疫活载体疫苗的理想菌株。随着对乳酸乳球菌基因工程的研究逐渐深入,已发展了一系列组成型和诱导型乳酸乳球菌表达系统以及蛋白定位系统。破伤风毒素片段C、布氏杆菌L7/L12蛋白等多种病原微生物抗原已成功在乳酸乳球菌中表达,并已证明部分重组乳酸乳球菌作为黏膜免疫疫苗可以同时刺激局部黏膜免疫应答和系统免疫应答。目前,如何使活载体乳酸乳球菌以最佳方式向黏膜免疫系统提呈抗原继而诱导有效免疫反应是该领域的研究热点,也是巨大挑战。实现外源抗原在乳酸乳球菌中的准确定位及与细胞因子的共表达是未来研究的重要方向之一。乳酸乳球菌作为黏膜免疫活载体疫苗传递外源抗原具有广阔的应用前景。     

Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli

Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure-function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, full-length VP8* cDNA, including the entire trypsin cleavage-encoding region in gene 4, was synthesized and amplified by RT-PCR from total RNA purified from bovine rotavirus strain C486 propagated in MA104 cell culture. The extended VP8* cDNA (VP8ext) was cloned into the pGEM-T Easy plasmid and subcloned into the Escherichia coli expression plasmid pET28a(+). The correspondent 30 kDa protein was overexpressed in E. coli BL21(DE3)pLysS cells under the control of the T7 promoter. The identity and the antigenicity of VP8ext were confirmed on Western blots using anti-His and anti-rotavirus antibodies. Immobilized Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 4 mg of VP8ext per liter of induced E. coli culture. Our results indicate that VP8ext maintained its native antigenicity and specificity, providing a good source of antigen for the production of P type-specific immune reagents. Detailed structural analysis of pure recombinant VP8 subunit should allow a better understanding of its role in cell attachment and rotavirus tropism. Application of similar procedure to distinct rotavirus P serotypes should provide valuable P serotype-specific immune reagents for rotavirus diagnostics and epidemiologic surveys.     

Genetic relatedness of VP1 genes of Australian and Taiwanese rotavirus isolates

Gene 1 (which encodes the viral RNA-dependent RNA polymerase, VP1) of an atypical human reassortant rotavirus strain, E210 (serotype G2P1B), is unrelated to genes 1 of standard human rotaviruses. To ascertain the origin of this gene, we determined a partial sequence and found that it exhibited greatest identity to gene 1 of a Taiwanese isolate, TE83, which is representative of G2 strains that caused an epidemic of gastroenteritis in 1993. Limited sequence identity to genes 1 of standard human and animal viruses was observed. This was confirmed by phylogenetic analysis. However, hybridization analysis using an E210 gene 1-specific probe indicated that a related gene was found among other Australian G2 isolates and in a Japanese strain isolated in the 1970s.     

VP8* antigen produced in tobacco transplastomic plants confers protection against bovine rotavirus infection in a suckling mouse model

Group A rotavirus is a major leading cause of diarrhea in mammalian species worldwide. In Argentina, bovine rotavirus (BRV) is the main cause of neonatal diarrhea in calves. VP4, one of the outermost capsid proteins, is involved in various virus functions. Rotavirus infectivity requires proteolytic cleavage of VP4, giving an N-terminal non-glycosilated sialic acid-recognizing domain (VP8*), and a C-terminal fragment (VP5*) that remains associated with the virion. VP8* subunit is the major determinant of the viral infectivity and one of the neutralizing antigens.In this work, the C486 BRV VP8* protein was produced in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern blot, northern blot and western blot. VP8* was highly stable in the transplastomic leaves, and formed insoluble aggregates that were partially solubilized by sonication. The recombinant protein yield was 600 μg/g of fresh tissue (FT). Both the soluble and insoluble fractions of the VP8* plant extracts were able to induce a strong immune response in female mice as measured by ELISA and virus neutralization test. Most important, suckling mice born to immunized dams were protected against oral challenge with virulent rotavirus. Results presented here contribute to demonstrate the feasibility of using antigens expressed in transplastomic plants for the development of subunit vaccines.     

STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core

The VP8* subunit of rotavirus spike protein VP4 contains a sialic acid (Sia)-binding domain important for host cell attachment and infection. In this study, the binding epitope of the N-acetylneuraminic acid (Neu5Ac) derivatives has been characterized by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. From this STD NMR data, it is proposed that the VP8* core recognizes an identical binding epitope in both methyl alpha-D-N-acetylneuraminide (Neu5Acalpha2Me) and the disaccharide methyl S-(alpha-D-N-acetylneuraminosyl)-(2-->6)-6-thio-beta-D-galactopyranoside (Neu5Ac-alpha(2,6)-S-Galbeta1Me). In the VP8*-disaccharide complex, the Neu5Ac moiety contributes to the majority of interaction with the protein, whereas the galactose moiety is solvent-exposed. Molecular dynamics calculations of the VP8*-disaccharide complex indicated that the galactose moiety is unable to adopt a conformation that is in close proximity to the protein surface. STD NMR experiments with methyl 9-O-acetyl-alpha-D-N-acetylneuraminide (Neu5,9Ac(2)alpha2Me) in complex with rhesus rotavirus (RRV) VP8* revealed that both the N-acetamide and 9-O-acetate moieties are in close proximity to the Sia-binding domain, with the N-acetamide's methyl group being saturated to a larger extent, indicating a closer association with the protein. RRV VP8* does not appear to significantly recognize the unsaturated Neu5Ac derivative [2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en)]. Molecular modeling of the protein-Neu5Ac2en complex indicates that key interactions between the protein and the unsaturated Neu5Ac derivative when compared with Neu5Acalpha2Me would not be sustained. Neu5Acalpha2Me, Neu5Ac-alpha(2,6)-S-Galbeta1Me, Neu5,9Ac(2)alpha2Me, and Neu5Ac2en inhibited rotavirus infection of MA104 cells by 61%, 35%, 30%, and 0%, respectively, at 10 mM concentration. NMR spectroscopic, molecular modeling, and infectivity inhibition results are in excellent agreement and provide valuable information for the design of inhibitors of rotavirus infection.     

Expression of green fluorescent protein in Lactococcus lactis

The gfp gene from Aequorea victoria, encoding the green fluorescent protein (GFP) has been expressed in Lactococcus lactis subsp. lactis biovar cremoris MG1363, upon construction and introduction of plasmid pLS1GFP into this host. GFP was monitored in living cells during growth to evaluate its use in molecular and physiological studies. Quantification of the levels of GFP expressed by cultures was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed us to distinguish, in mixed cultures, lactococcal cells expressing GFP. Our results indicate that GFP can be used as a reporter in L. lactis.     

食品级分泌表达载体的构建及报告蛋白在乳酸乳球菌中的表达

为构建乳酸乳球菌食品级分泌表达载体,通过PCR扩增质粒pMG36e的p32启动子片段及乳酸乳球菌MG1363未知分泌蛋白(Usp45)基因的核糖体结合位点、分泌信号肽和成熟肽前11个氨基酸的编码序列(SPusp45),克隆到食品级载体pSH91中,构建食品级分泌性表达载体pSQ;克隆报告基因金黄色葡萄球菌核酸酶(NucA)成熟肽的编码序列nucA到pSQ中分泌信号后,转化乳酸乳球菌MBP71,构建了乳酸乳球菌食品级分泌性表达系统L lactis/pSQ-nucA;通过TB-D法和酶谱法检测L lactis/pSQ-nucA的表达形式、表达量并与以前构建的L lactis/pSQZ-nucA系统表达能力进行比较,结果发现L lactis/pSQ-nucA能够分泌性表达NucA,分泌性表达的NucA量大约是胞内NucA的10倍;L lactis/pSQ-nucA的表达量高于lactis/pSQZ-nucA.为进一步目的蛋白的的分泌性表达及食品级疫苗的研制奠定了基础.     

Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice

The VP8* fragment from the rotavirus spike protein was expressed as a fusion protein with two different cell wall proteins of Saccharomyces cerevisiae, Icwp (Ssr1p) and Pir4, to achieve cell wall targeting or secretion to the growth medium of the fusion proteins. Two different host strains were used for the expression of the fusion proteins, a standard S. cerevisiae strain and a mnn9 glycosylation deficient strain, the later to reduce hyper-glycosylation. The Icwp-VP8* fusion could only be detected in the growth medium, indicating that the presence of the VP8* moiety interferes with the anchorage of Icwp to the cell wall. In the case of the Pir4-VP8* fusion proteins, we achieved cell wall targeting or secretion depending on how the gene fusion had been performed. In all cases, the fusion proteins expressed in the mnn9 strain showed a reduced level of glycosylation. Mice were inoculated intraperitoneally either with Pir4-VP8* or Icwp-VP8* fusion proteins purified from the growth medium of mnn9 strains expressing them or with whole cells of an mnn9 strain expressing a Pir4-VP8 fusion protein on its cell walls. Hundred percent of mice inoculated with the Pir4-VP8* fusion protein and 25% of those inoculated with the Icwp-VP8* fusion protein showed high titers of anti-VP8* antibodies. No specific immune response was detected in those mice inoculated with whole cells. Finally, susceptibility to rotavirus infection of the offspring of immunized dams was determined and protection was found in a percentage of approximately 60% with respect to the control group.     

Heterologous protein secretion in Lactococcus lactis is enhanced by the Bacillus subtilis chaperone-like protein PrsA

The Bacillus subtilis lipoprotein PrsA enhances the yield of several homologous and heterologous exported proteins in B. subtilis by being involved in the posttranslocational stage of the secretion process. In this work, we have studied the effect of B. subtilis PrsA on the secretion of Bacillus amyloliquefaciens α-amylase (AmyQ), a target protein for PrsA, and Bacillus licheniformis penicillinase (PenP) a nontarget protein for PrsA, in Lactococcus lactis. Two compatible plasmids were constructed and introduced into L. lactis strain NZ9000: one high copy plasmid, expressing the AmyQ gene (amyQ) or the PenP gene (penP), and one low copy plasmid, expressing the PrsA encoding gene (prsA). When amyQ and prsA were simultaneously expressed under the nisin-inducible promoter P _nisA , Western blotting experiments revealed a 15- to 20-fold increase in the total yield of AmyQ and a sixfold increase in secreted AmyQ activity, compared to a control strain lacking prsA. When expressed under the same induction conditions, PrsA had no effect on the secretion or total yield of PenP. These results show that the secretion yield of some heterologous proteins can be significantly increased in L. lactis when coproduced with the B. subtilis PrsA protein.     

Efficient secretion of a modified E7 protein from human papilloma virus type‐16 by Lactococcus lactis

Aims: To create and provide a strain of the food‐grade bacterium Lactococcus lactis able to efficiently secrete a modified form of the E7 protein from the human papilloma virus (HPV) type‐16. Methods and Results: We cloned the coding sequence of a modified E7 (E7m) from the HPV‐16 in a plasmid regulated by the strong expression promoter p59. Secretion of the E7m was made by the signal peptide of the usp45 gene. The E7m was detected by Western blot in the cell‐free‐medium fraction, showing no degradation or aberrant forms. Conclusions: We constructed a strain of L. lactis able to secrete efficiently a HPV‐16 E7 modified protein with diminished transforming activity. Significance and Impact of the Study: Human papilloma virus infection is associated with more than 99% of cervical cancers. Immunotherapy targeting E7 to treat HPV‐associated cervical malignancies has been demonstrated to be highly efficient. However, native E7 maintains transforming activity. We present this new strain of a food‐grade bacterium able to efficiently secrete a HPV‐16 E7‐modified protein with diminished transforming activity. This new strain could be used as a live vaccine to deliver E7 at a mucosal level and generate antitumour immune responses against HPV‐associated tumours.     

Genome plasticity in Lactococcus lactis

Comparative genome analyses contribute significantly to our understanding of bacterial evolution and indicate that bacterial genomes are constantly evolving structures. The gene content and organisation of chromosomes of lactic acid bacteria probably result from a strong evolutionary pressure toward optimal growth of these microorganisms in milk. The genome plasticity of Lactococcus lactis was evaluated at inter- and intrasubspecies levels by different experimental approaches. Comparative genomics showed that the lactococcal genomes are not highly plastic although large rearrangements (a.o. deletions, inversions) can occur. Experimental genome shuffling using a new genetic strategy based on the Cre-loxP recombination system revealed that two domains are under strong constraints acting to maintain the original chromosome organisation: a large region around the replication origin, and a smaller one around the putative terminus of replication. Future knowledge of the rules leading to an optimal genome organisation could facilitate the definition of new strategies for industrial strain improvement.     

Calcium influences the stability and conformation of rotavirus SAH glycoprotein VP7 expressed in Dictyostelium discoideum

We have previously reported expression of the rotavirus outer capsid glycoprotein, VP7, in the relatively new expression host, Dictyostelium discoideum. To optimise yields of recombinant VP7, we examined the role of Ca²⁺ since stability of both VP7 and mature rotavirus during a rotavirus infection are calcium-dependent. Low micromolar levels of free extracellular Ca²⁺ were required to maximise yields of VP7 in D. discoideum whilst levels of VP7 were reduced following depletion of intracellular Ca²⁺ reserves using A23187 and EGTA. Immunoblot analysis suggested that VP7 was being degraded in an intracellular compartment. Immunoprecipitation with a conformation-dependent neutralising antibody confirmed that EGTA-induced Ca²⁺ chelation alters the conformation of VP7. These results suggest that stability of VP7 is dependent on maintaining adequate levels of intracellular Ca²⁺ and that conformational changes in VP7 which occur following depletion of Ca²⁺ reserves induce rapid proteolysis of the protein. Since these results establish conditions for expressing optimal levels of VP7 in the correct conformation they have important implications for the development of a subunit vaccine based on recombinant VP7.     

谷胱甘肽在乳酸乳球菌抵抗氧胁迫中的保护作用

为研究谷胱甘肽(GSH)在乳酸乳球菌NZ9000抗氧胁迫中的生理作用,以能够生物合成GSH的重组菌NZ9000(pNZ3203)为实验菌株进行了研究。结果表明,在较高H2O2胁迫剂量(150mmol/L H2O2,15min)下,前培养3h、5h和7h(即乳酸链球菌素诱导1h、3h和5h)时的重组菌细胞的存活率分别是处于相应生长时期对照菌NZ9000(pNZ8148)的1.8±0.1倍、2.6±0.1倍和2.9±0.3倍。表明GSH可以提高宿主菌NZ9000对H2O2所引发氧胁迫的抗性。GSH还可以提高宿主菌NZ9000对其它化学物质(如超氧阴离子自由基生成剂———甲萘醌)所引发氧胁迫的抗性。这表现在经20mmol/L甲萘醌处理60min后,前培养5h(即乳酸链球菌素诱导3h)时重组菌细胞的存活率是对照菌的6.2±0.1倍。由此表明,通过代谢工程手段在菌株NZ9000中引入GSH合成能力,可以提高宿主菌对氧胁迫的抗性。     

轮状病毒vp8基因在原核系统中的初步表达

通过RT—PCR反应获得轮状病毒Wa株vp8基因的cDNA片段,将其克隆入pGEX—5X—1表达载体中,构建重组质粒pGEX—VP8,转化大肠杆菌JM109,筛选阳性克隆子并对插入片段vp8进行序列测定,诱导后通过SDS—PAGE检测重组蛋白,并观察表达量随时间变化的特征。结果显示,测序结果与vp8序列一致,VP8蛋白的表达量在诱导后6—8h达到高峰。     

一株能够利用血红素进行有氧呼吸的乳酸乳球菌

[目的]乳酸乳球菌在呼吸状态下,生长速度快,生物产量大,是改善发酵剂生产效率的潜在途径之一,本研究旨在观察由传统乳制品中分离得到的乳酸乳球菌在血红素存在状态下有氧呼吸情况以及代谢的变化.[方法]对本实验室保藏的12株乳酸乳球菌进行有氧培养实验,比较其生物量和代谢产物的差异.观测在4℃下储藏30 d后的活菌数差异.[结果]筛选出一株在血红素存在条件下进行有氧呼吸的菌株KLDS 4.0316,与没有添加血红素的原菌株相比生物量增长了50%,在4℃储藏30 d后,添加血红素并振荡的活菌数依然维持在100 CFU/mL,而未添加血红素未振荡检测不到活菌.在血红素存在下,KLDS 4.0316代谢产物发生了变化,与没有添加血红素的原菌株相比乳酸产量减少了48%.[结论]KLDS 4.0316在血红素存在条件下能够进行有氧呼吸,乳酸产生减少,生物量增加.     

Conversion of methionine to methional by Lactococcus lactis

Lactic acid bacteria were screened for methional production from 4-methylthio-2-ketobutanoate. Only Lactococcus lactis IFPL730 produced high amounts of methional. It was demonstrated that production of this compound was an exclusively enzymatic reaction. The present work describes for the first time that L. lactis can convert enzymatically methionine to methional in a process mediated by aminotransferase and alpha-ketoacid decarboxylase activities. The activity seems to be strain dependent.     

Acidic proteome of growing and resting Lactococcus lactis metabolizing maltose

The acidic proteome of Lactococcus lactis grown anaerobically was compared for three different growth conditions: cells growing on maltose, resting cells metabolizing maltose, and cells growing on glucose. In maltose metabolizing cells several proteins were up-regulated compared with glucose metabolizing cells, however only some of the up-regulated proteins had apparent relation to maltose metabolism. Cells growing on maltose produced formate, acetate and ethanol in addition to lactate, whereas resting cells metabolizing maltose and cells growing on glucose produced only lactate. Increased levels of alcohol-acetaldehyde dehydrogenase (ADH) and phosphate acetyltransferase (PTA) in maltose-growing cells compared with glucose-growing cells coincided with formation of mixed acids in maltose-growing cells. The resting cells did not grow due to lack of an amino acid source and fermented maltose with lactate as the sole product, although ADH and PTA were present at high levels. The maltose consumption rate was approximately three times lower in resting cells than in exponentially growing cells. However, the enzyme levels in resting and growing cells metabolizing maltose were similar, which indicates that the difference in product formation in this case is due to regulation at the enzyme level. The levels of 30S ribosomal proteins S1 and S2 increased with increasing growth rate for resting cells metabolizing maltose, maltose-growing cells and glucose-growing cells. A modified form of HPr was synthesized under amino acid starvation. This is suggested to be due to alanine misincorporation for valine, which L. lactis is auxotrophic for. L. lactis conserves the protein profile to a high extent, even after prolonged amino acid starvation, so that the protein expression profile of the bacterium remains almost invariant.