Surface Display of the Receptor-Binding Region of the Lactobacillusbrevis S-Layer Protein in Lactococcuslactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.     

Gene cloning, functional expression and secretion of the S-layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a in Lactococcus lactis

The ~93-kDa surface layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a forms a regular crystalline array providing a nanopatterned matrix for the future display of biologically relevant molecules. Lactococcus lactis NZ9000 was established as a safe expression host for the controlled targeted production of SgsE based on the broad host-range plasmid pNZ124Sph, into which the nisA promoter was introduced. SgsE devoid of its signal peptide-encoding sequence was cloned into the new vector and purified from the cytoplasm at a yield of 220 mg l- of expression culture. Secretion constructs were based on the signal peptide of the Lactobacillus brevis SlpA protein or the L. lactis Usp45 protein, allowing isolation of 95 mg of secreted rSgsE l-1. N-terminal sequencing confirmed correct processing of SgsE in L. lactis NZ9000. The ability of rSgsE to self-assemble in suspension and to recrystallize on solid supports was demonstrated by electron and atomic force microscopy.     

Identification by flagellum display of an epithelial cell- and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis

Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of Lactobacillus brevis ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin. For functional analysis of the SlpA surface protein, different regions of the slpA gene were expressed as internal in-frame fusions in the variable region of the fliC(H7) gene of Escherichia coli. The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size. The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence. Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella. The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein. The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin. The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells. In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells. The results identify the S-layer of L. brevis ATCC 8287 as an adhesin with affinity for human epithelial cells and fibronectin and locate the receptor-binding region within a fragment of 81 amino acids in the N-terminal part of the molecule, which in native S-layer seems inaccessible to antibodies.     

Receptor binding domain of Escherichia coli F18 fimbrial adhesin FedF can be both efficiently secreted and surface displayed in a functional form in Lactococcus lactis

Adherence of F18 fimbrial Escherichia coli to porcine intestinal epithelial cells is mediated by the adhesin (FedF) of F18 fimbriae. In a previous study, we demonstrated the specificity of the amino acid residues between 60 and 109 as the receptor binding domain of FedF. In this study, different expression, secretion, and anchoring systems for the receptor binding domain of the FedF adhesin in Lactococcus lactis were evaluated. Two partially overlapping receptor binding domains (42 and 62 amino acid residues) were expressed as fusions with L. lactis subsp. cremoris protein PrtP for evaluation of secretion efficiency. To evaluate the cell surface display of these FedF-PrtP fusions, they were further combined with different lengths of PrtP spacers fused with either the L. lactis AcmA anchor or the PrtP cell wall binding domain. An HtrA-defective L. lactis NZ9000 mutant was constructed to determine its effect on the level of secreted or anchored fusion proteins. Recombinant L. lactis clones secreting the receptor binding domain of F18 fimbriae as a fusion with the H domains of L. lactis protein PrtP were first constructed by using two different signal peptides. FedF-PrtP fusions, directed by the signal sequence of L. brevis SlpA, were throughout found to be secreted at significantly higher quantities than corresponding fusions with the signal peptide of L. lactis Usp45. In the surface display systems tested, the L. lactis AcmA anchor performed significantly better, particularly in the L. lactis NZ9000DeltahtrA strain, compared to the L. lactis PrtP anchor region. Of the cell surface display constructs with the AcmA anchor, only those with the longest PrtP spacer regions resulted in efficient binding of recombinant L. lactis cells to porcine intestinal epithelial cells. These results confirmed that it is possible to efficiently produce the receptor binding domain of the F18 adhesin in a functionally active form in L. lactis.     

Identification of candidate carrier proteins for surface display on Lactococcus lactis by theoretical and experimental analyses of the surface proteome

Lactococcus lactis is a lactic acid bacterium of proven safety for use in human oral applications. For this purpose, surface display of recombinant proteins is important, and new approaches for it are being sought. Analysis of the bacterial surface proteome is essential in identifying new candidate carrier proteins for surface display. We have made two different predictions of surface-associated proteins of L. lactis MG1363 by using Augur and LocateP software, which yielded 666 and 648 proteins, respectively. Surface proteins of L. lactis NZ9000, a derivative of MG1363, were identified by using a proteomics approach. The surface proteins were cleaved from intact bacteria, and the resulting peptides were identified by mass spectrometry. The latter approach yielded 80 proteins, 34 of which were not predicted by either software. Of the 80 proteins, 7 were selected for further study. These were cloned in frame with a C-terminal hexahistidine tag and overexpressed in L. lactis NZ9000 using nisin-controlled expression. Proteins of correct molecular weight carrying a hexahistidine tag were detected. Their surface localization was confirmed with flow cytometry. Basic membrane protein A (BmpA) was exposed at the highest level. To test BmpA as a candidate carrier protein, the hexahistidine tag was replaced by the B domain of staphylococcal protein A in the genetic construct. The B domain was displayed on the surface with BmpA as a carrier. The advantage of covalent BmpA binding was demonstrated. BmpA was thus shown to be a suitable candidate for a carrier protein in lactococcal surface display.     

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。     

Influence of lipoteichoic acid D-alanylation on protein secretion in Lactococcus lactis as revealed by random mutagenesis

Lactococcus lactis, a food-grade nonpathogenic lactic acid bacterium, is a good candidate for the production of heterologous proteins of therapeutic interest. We examined host factors that affect secretion of heterologous proteins in L. lactis. Random insertional mutagenesis was performed with L. lactis strain MG1363 carrying a staphylococcal nuclease (Nuc) reporter cassette in its chromosome. This cassette encodes a fusion protein between the signal peptide of the Usp45 lactococcal protein and the mature moiety of a truncated form of Nuc (NucT). The Nuc secretion efficiency (secreted NucT versus total NucT) from this construct is low in L. lactis (approximately 40%). Twenty mutants affected in NucT production and/or in secretion capacity were selected and identified. In these mutants, several independent insertions mapped in the dltA gene (involved in D-alanine transfer in lipoteichoic acids) and resulted in a NucT secretion defect. Characterization of the dltA mutant phenotype with respect to NucT secretion revealed that it is involved in a late secretion stage by causing mature NucT entrapment at the cell surface.     

表达猪传染性胃肠炎病毒S基因的重组乳酸乳球菌的构建及免疫原性分析

根据猪传染性胃肠炎病毒纤突(S)蛋白的全基因序列及表达载体质粒的基因融合特点,设计一对引物,进行PCR扩增,获得含有TGEVS基因4个主要抗原位点的约2000bp的目的片段,将其与分泌表达的载体质粒pNZ8112进行连接,通过电击转化进入宿主菌乳酸乳球菌NZ9000细胞内,在乳链菌肽(Nisin)的诱导下进行表达,通过SDS-PAGE和Western blot分析,表明TGEVS蛋白在乳酸乳球菌中获得表达,所表达的TGEVS蛋白具有与TGE病毒一样的抗原特异性。间接免疫荧光试验表明重组菌表达蛋白定位于菌体表面。将表达TGEVS蛋白的重组乳酸乳球菌及空质粒菌株分别口服免疫BALB/c小鼠,收集粪便样品进行抗体检测,结果表明分泌型的重组菌pNZ8112-Sa/NZ9000免疫小鼠能够产生明显的抗TGEVsIgA抗体。     

Binding characteristics of the Lactobacillus brevis ATCC 8287 surface layer to extracellular matrix proteins

Self-assembling proteins that form crystalline surface layers on many microorganisms can be involved in bacterial-host adhesion via specific interactions with components of the extracellular matrix. Here, we describe the interaction of the Lactobacillus brevis ATCC 8287 surface-layer protein SlpA with fibronectin, laminin, fibrinogen and collagen using surface plasmon resonance. SlpA was found to interact with high affinity to fibronectin and laminin, with a respective binding constant of 89.8 and 26.7 nM. The interaction of SlpA with collagen and fibrinogen was found to be of much lower affinity, with respective binding constants of 31.8 and 26.1 microM. The serine protease inhibitor benzamidine greatly reduced the affinity of SlpA for fibronectin, whereas the affinity for laminin remained unaffected. No protease activity of the purified SlpA protein could be detected. These data suggest that L. brevis may interact with host cells directly through high affinity interactions with laminin and fibronectin predominantly, involving distinct regions of the SlpA protein.     

Effects of carbohydrates and lectins on cryptosporidial sporozoite penetration of cultured cell monolayers.

Cryptosporidium parvum first interacts with enterocytes when sporozoites penetrate the host plasma membrane. We have developed a shell vial assay using human embryonic Intestine 407 cells and purified C. parvum sporozoites to study this process. Sporozoites were incubated in culture medium with various carbohydrates and lectins, and the suspensions were then added to the cell monolayers. Following incubation, the monolayers were fixed and stained and the number of schizonts were counted. No decreases in sporozoite motility or Intestine 407 cell viability were observed with carbohydrate or lectin treatment. N-Acetyl-D-glucosamine, chitobiose and chitotriose inhibited C. parvum infection, compared to 5 other tested carbohydrates. Wheat germ agglutinin reduced penetration and concanavalin A enhanced schizont formation, when compared to 8 other lectins. Next, we pretreated sporozoites or Intestine 407 cells with wheat germ agglutinin and concanaval in A prior to sporozoite inoculation. Wheat germ agglutinin treatment of sporozoites or cells equally caused a reduction in C. parvum infection, while enhancement was only observed when Intestine 407 cell were pretreated with concanavalin A. These data suggest that glycoproteins with terminal N-acetyl-D-glucosamine residues may play a role in C. parvum adhesion or penetration of enterocytes. Also, host glycoproteins with concanavalin A-like activity may play a role in these processes.     

天冬氨酸提高乳酸乳球菌Lactococcus lactis NZ9000酸胁迫抗性的作用机制

【背景】乳酸菌作为重要的发酵微生物在应用过程中面临广泛存在的酸胁迫。【目的】确认天冬氨酸可有效提高乳酸乳球菌的酸胁迫抗性,通过解析天冬氨酸的作用机制,为进一步提高乳酸菌酸胁迫抗性提供可借鉴的思路。【方法】通过荧光定量PCR比较胁迫条件下天冬氨酸对L.lactisNZ9000产能和氨基酸代谢途径中关键基因转录水平的影响,并通过过量表达天冬酰胺酶增加胞内天冬氨酸的含量。【结果】天冬氨酸主要是在转氨酶的作用下生成草酰乙酸和谷氨酸。草酰乙酸参与三羧酸循环,为细胞提供更多的能量;谷氨酸经谷氨酸脱羧酶途径提高细胞的酸胁迫抗性。经pH4.0胁迫处理后,天冬氨酸使糖酵解和三羧酸循环产能途径中关键基因转录上调,胞内ATP含量为对照组的42倍;胞内谷氨酸含量为对照的1.99倍。通过过量表达天冬酰胺酶获得的重组菌株,在pH3.6条件下胁迫0.5h后,存活率约为对照组的11.11倍。【结论】在L. lactis NZ9000中探究了天冬氨酸提高酸胁迫抗性的作用机理,进一步完善了氨基酸代谢提高乳酸菌酸胁迫抗性的理论基础。     

Introducing glutathione biosynthetic capability into Lactococcus lactis subsp. cremoris NZ9000 improves the oxidative-stress resistance of the host

This study describes how a metabolic engineering approach can be used to improve bacterial stress resistance. Some Lactococcus lactis strains are capable of taking up glutathione, and the imported glutathione protects this organism against H(2)O(2)-induced oxidative stress. L. lactis subsp. cremoris NZ9000, a model organism of this species that is widely used in the study of metabolic engineering, can neither synthesize nor take up glutathione. The study described here aimed to improve the oxidative-stress resistance of strain NZ9000 by introducing a glutathione biosynthetic capability. We show that the glutathione produced by strain NZ9000 conferred stronger resistance on the host following exposure to H(2)O(2) (150 mM) and a superoxide generator, menadione (30 microM). To explore whether glutathione can complement the existing oxidative-stress defense systems, we constructed a superoxide dismutase deficient mutant of strain NZ9000, designated as NZ4504, which is more sensitive to oxidative stress, and introduced the glutathione biosynthetic capability into this strain. Glutathione produced by strain NZ4504(pNZ3203) significantly shortens the lag phase of the host when grown aerobically, especially in the presence of menadione. In addition, cells of NZ4504(pNZ3203) capable of producing glutathione restored the resistance of the host to H(2)O(2)-induced oxidative stress, back to the wild-type level. We conclude that the resistance of L. lactis subsp. cremoris NZ9000 to oxidative stress can be increased in engineered cells with glutathione producing capability.     

Production of biological active murine IFN-gamma by recombinant Lactococcus lactis.

IFN-gamma is a cytokine produced primarily by both T lymphocytes and natural killer cells and it is considered to be an attractive therapeutic molecule. In the present study, a DNA sequence encoding the mature murine IFN-gamma (muIFN-gamma) protein was cloned and expressed in the food-grade lactic acid bacterium Lactococcus lactis. The activity of recombinant muIFN-gamma produced by genetically engineered L. lactis was confirmed in an antiviral assay using MoV cells infected with Vesicular Stomatitis Virus. The data provide the first demonstration that a Gram-positive bacterium, L. lactis, is able to produce functional muIFN-gamma. This recombinant strain could lead to the development of a new, well-tolerated vector to deliver active muIFN-gamma at the mucosal level.     

Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA

Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactis cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters.     

Lactococcus lactis, an efficient cell factory for recombinant protein production and secretion

The use of Gram-positive bacteria for heterologous protein production proves to be a useful choice due to easy protein secretion and purification. The lactic acid bacterium Lactococcus lactis emerges as an attractive alternative to the Gram-positive model Bacillus subtilis. Here, we review recent work on the expression and secretion systems available for heterologous protein secretion in L. lactis, including promoters, signal peptides and mutant host strains known to overcome some bottlenecks of the process. Among the tools developed in our laboratory, inactivation of HtrA, the unique housekeeping protease at the cell surface, or complementation of the Sec machinery with B. subtilis SecDF accessory protein each result in the increase in heterologous protein yield. Furthermore, our lactococcal expression/secretion system, using both P(Zn)zitR, an expression cassette tightly controlled by environmental zinc, and a consensus signal peptide, SP(Exp4), allows efficient production and secretion of the staphylococcal nuclease, as evidenced by protein yields (protein amount/biomass) comparable to those obtained using NICE or P170 expression systems under similar laboratory conditions. Finally, the toolbox we are developing should contribute to enlarge the use of L. lactis as a protein cell factory.     

Expression of the Staphylococcus aureus surface proteins HtrA1 and HtrA2 in Lactococcus lactis

Staphylococcus aureus encodes two HtrA-like serine surface proteases, called HtrA1 and HtrA2. The roles of these HtrA homologs were distinguished by expression studies in a heterologous host, Lactococcus lactis, whose single extracellular protease, HtrA(Ll), was absent. HtrA(Ll) is involved in stress resistance, and processing and/or degradation of extracellular proteins. Controlled expression of staphylococcal htrA1 and htrA2 was achieved in L. lactis strain NZ9000 DeltahtrA, as confirmed with anti-HtrA1 and anti-HtrA2 specific antibodies. HtrA1 fully restored thermo-resistance to the htrA-defective L. lactis strain, despite a poor capacity to degrade abnormal or truncated proteins. We therefore propose that activities of HtrA1 other than proteolysis may be sufficient for high-temperature growth complementation. HtrA2 is 36% identical to HtrA(Ll), and was highly expressed in L. lactis; nevertheless, it displayed nearly no detectable activities. The poor proteolytic activities of staphylococcal HtrA proteins in L. lactis may reflect a requirement for S. aureus-specific co-factors.