Factors Affecting Inducible Expression of Outer Membrane Protein A (OmpA) of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> Type-1 in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> Using Nisin Inducible Controlled Expression (NICE)

Potential use of Lactococcus lactis (L. lactis) as a heterologous protein expression host as well as for delivery of multiple therapeutic proteins has been investigated extensively using Nisin Inducible Controlled Expression (NICE) system. Optimum inducible expression of heterologous protein by NICE system in L. lactis depends on multiple factors. To study the unexplored role of factors affecting heterologous protein expression in L. lactis using NICE, the present study outlines the optimization of various key parameters such as inducer concentration, host’s proteases and precipitating agent using Outer membrane protein A (OmpA). For efficient expression and secretion of OmpA, pSEC:OmpA vector was successfully constructed. To circumvent the troubles encountered during detection of expressed OmpA, the precipitating agent was switched from TCA to methanol. Nevertheless, detection was achieved accompanied by degraded protein products. Speculating the accountability of observed degradation at higher inducer concentration, different nisin concentrations were evaluated. Lower nisin concentrations were found desirable for optimum expression of OmpA. Consistently observed degradation was eliminated by incorporation of protease inhibitor cocktail which inhibits intracellular proteases and expression in VEL1153 (NZ9000 ΔhtrA) strain which inhibits extracellular protease leading to optimum expression of OmpA. Versatility and complexity of NICE system in L. lactis requires fine-tuning of target protein specific parameters for optimum expression.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0556-2) contains supplementary material, which is available to authorized users.     

PpiA, a surface PPIase of the cyclophilin family in Lactococcus lactis

Background

Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes.

Results

In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H₂O₂) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H₂O₂. Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities.

Conclusions

Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro.     

Construction of a recombinant allergen-producing probiotic bacterial strain: Introduction of a new line for a live oral vaccine against Chenopodium album pollen allergy

Background:

During the last two decades, significant advances have been made in the fields of lactococcal genetics and protein expression. Lactococcus lactis (L. lactis) is an effective vector for protein expression and can be used as an antigen delivery system. Hence, L. lactis is an ideal candidate for mucosal immunotherapy. Profilin (Che a 2), the major allergen in Chenopodium album, is one of the most important causes of allergic diseases in desert and semi-desert areas, especially in Iran, Saudi Arabia, and Kuwait that was cloned and expressed in L. lactis for the first time.

Methods:

To construct L. lactis that expressed Che a 2, a DNA sequence was cloned and used to transform bacteria. Expression of Che a 2 was analyzed via monitoring of related RNA and protein. Hydrophobicity, adherence to HT-29 cells, antibiotic resistance, resistance to gastrointestinal contents, pH, and bile salt in recombinant and native L. lactis were evaluated.

Results:

Immunoblot analyses demonstrated that recombinant Che a 2 is expressed as a 32 kDa dimeric protein immunological studies showed it can bind human IgE. Both native and recombinant bacteria were sensitive to low pH and simulated gastric conditions. Bacterial survival was reduced 80-100% after 2 h of exposure to pH 1.5-2. Both native and recombinant bacteria were able to grow in 0.3 and 2% bile salts. After incubation of recombinant L. lactis in simulated gastric and intestinal juices for one and two hours, respectively, cell survival was reduced by 100%. Adhesion capability in both strains was minimal and there were no significant differences in any of our tests between native and recombinant bacteria.

Conclusion:

Successfully recombinant L. lactis with capability of expression Che a 2 was produced and revealed it is sensitive to gastrointestinal contents. Key Words: Recombinant L. lactis, Probiotic bacteria, Chenopodium pollen allergen, Oral vaccines     

Amino Acid Accumulation Limits the Overexpression of Proteins in Lactococcus lactis

Background

Understanding the biogenesis pathways for the functional expression of recombinant proteins, in particular membrane proteins and complex multidomain assemblies, is a fundamental issue in cell biology and of high importance for future progress in structural genomics. In this study, we employed a proteomic approach to understand the difference in expression levels for various multidomain membrane proteins in L. lactis cells grown in complex and synthetic media.

Methodology/Principal Findings

The proteomic profiles of cells growing in media in which the proteins were expressed to high or low levels suggested a limitation in the availability of branched-chain amino acids, more specifically a too limited capacity to accumulate these nutrients. By supplying the cells with an alternative path for accumulation of Ile, Leu and/or Val, i.e., a medium supplement of the appropriate dipeptides, or by engineering the transport capacity for branched-chain amino acids, the expression levels could be increased several fold.

Conclusions

We show that the availability of branched chain amino acids is a critical factor for the (over)expression of proteins in L. lactis. The forward engineering of cells for functional protein production required fine-tuning of co-expression of the branched chain amino acid transporter.     

A Novel Two-Component System Involved in Secretion Stress Response in Streptomyces lividans

Background

Misfolded proteins accumulating outside the bacterial cytoplasmic membrane can interfere with the secretory machinery, hence the existence of quality factors to eliminate these misfolded proteins is of capital importance in bacteria that are efficient producers of secretory proteins. These bacteria normally use a specific two-component system to respond to the stress produced by the accumulation of the misfolded proteins, by activating the expression of HtrA-like proteases to specifically eliminate the incorrectly folded proteins.

Methodology/Principal Findings

Overproduction of alpha-amylase in S. lividans causing secretion stress permitted the identification of a two-component system (SCO4156-SCO4155) that regulates three HtrA-like proteases which appear to be involved in secretion stress response. Mutants in each of the genes forming part of the two-genes operon that encodes the sensor and regulator protein components accumulated misfolded proteins outside the cell, strongly suggesting the involvement of this two-component system in the S. lividans secretion stress response.

Conclusions/Significance

To our knowledge this is the first time that a specific secretion stress response two-component system is found to control the expression of three HtrA-like protease genes in S. lividans, a bacterium that has been repeatedly used as a host for the synthesis of homologous and heterologous secretory proteins of industrial application.     

Heterologous expression of membrane proteins: choosing the appropriate host

Background

Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein.

Methodology/Principal Findings

The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts.

Conclusions/Significance

Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.     

Genetically Engineered Lactococcus lactis Protect against House Dust Mite Allergy in a BALB/c Mouse Model

Background

Mucosal vaccine based on lactic acid bacteria is an attractive concept for the prevention and treatment of allergic diseases, but their mechanisms of action in vivo are poorly understood. Therefore, we sought to investigate how recombinant major dust mite allergen Der p2-expressing Lactococcus lactis as a mucosal vaccine induced the immune tolerance against house dust mite allergy in a mouse model.

Methods

Three strains of recombinant L. lactis producing Der p2 in different cell components (extracellular, intracellular and cell wall) were firstly constructed. Their prophylactic potential was evaluated in a Der p2-sensitised mouse model, and immunomodulation properties at the cellular level were determined by measuring cytokine production in vitro.

Results

Der p2 expressed in the different recombinant L. lactis strains was recognized by a polyclonal anti-Der p2 antibody. Oral treatment with the recombinant L. lactis prior sensitization significantly prevented the development of airway inflammation in the Der p2-sensitized mice, as determined by the attenuation of inflammatory cells infiltration in the lung tissues and decrease of Th2 cytokines IL-4 and IL-5 levels in bronchoalveolar lavage. In addition, the serum allergen-specific IgE levels were significantly reduced, and the levels of IL-4 in the spleen and mesenteric lymph nodes cell cultures were also markedly decreased upon allergen stimulation in the mice fed with the recombinant L. lactis strains. These protective effects correlated with a significant up-regulation of regulatory T cells in the mesenteric lymph nodes.

Conclusion

Oral pretreatment with live recombinant L. lactis prevented the development of allergen-induced airway inflammation primarily by the induction of specific mucosal immune tolerance.     

Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA

Background

The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized.

Principal Findings

The binding of glycine betaine to purified OpuA and OpuAC (K_D = 4–6 µM) did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 Å) and closed-liganded (2.3 Å) conformation. The binding pocket is formed by three tryptophans (Trp-prism) coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher.

Conclusions

Ionic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site.     

Membrane Protein Biogenesis in Ffh- or FtsY-Depleted Escherichia coli

Background

The Escherichia coli version of the mammalian signal recognition particle (SRP) system is required for biogenesis of membrane proteins and contains two essential proteins: the SRP subunit Ffh and the SRP-receptor FtsY. Scattered in vivo studies have raised the possibility that expression of membrane proteins is inhibited in cells depleted of FtsY, whereas Ffh-depletion only affects their assembly. These differential results are surprising in light of the proposed model that FtsY and Ffh play a role in the same pathway of ribosome targeting to the membrane. Therefore, we decided to evaluate these unexpected results systematically.

Methodology/Principal Findings

We characterized the following aspects of membrane protein biogenesis under conditions of either FtsY- or Ffh-depletion: (i) Protein expression, stability and localization; (ii) mRNA levels; (iii) folding and activity. With FtsY, we show that it is specifically required for expression of membrane proteins. Since no changes in mRNA levels or membrane protein stability were detected in cells depleted of FtsY, we propose that its depletion may lead to specific inhibition of translation of membrane proteins. Surprisingly, although FtsY and Ffh function in the same pathway, depletion of Ffh did not affect membrane protein expression or localization.

Conclusions

Our results suggest that indeed, while FtsY-depletion affects earlier steps in the pathway (possibly translation), Ffh-depletion disrupts membrane protein biogenesis later during the targeting pathway by preventing their functional assembly in the membrane.     

Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli

Background

N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogeneous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose.

Methodology/Principal Findings

Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium.

Conclusions/Significance

The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.     

Genes but not genomes reveal bacterial domestication of Lactococcus lactis

Background

The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST) scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE).

Methodology/Principal Findings

The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content) did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST) differing by up to 230 kb in genome size.

Conclusion/Significance

The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between “environmental” strains, the main contributors to the genetic diversity within the subspecies, and “domesticated” strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the “domesticated” strains essentially arose through substantial genomic flux within the dispensable genome.     

Genome-Wide Identification and Analysis of the TIFY Gene Family in Grape

Background

The TIFY gene family constitutes a plant-specific group of genes with a broad range of functions. This family encodes four subfamilies of proteins, including ZML, TIFY, PPD and JASMONATE ZIM-Domain (JAZ) proteins. JAZ proteins are targets of the SCF^COI1 complex, and function as negative regulators in the JA signaling pathway. Recently, it has been reported in both Arabidopsis and rice that TIFY genes, and especially JAZ genes, may be involved in plant defense against insect feeding, wounding, pathogens and abiotic stresses. Nonetheless, knowledge concerning the specific expression patterns and evolutionary history of plant TIFY family members is limited, especially in a woody species such as grape.

Methodology/Principal Findings

A total of two TIFY, four ZML, two PPD and 11 JAZ genes were identified in the Vitis vinifera genome. Phylogenetic analysis of TIFY protein sequences from grape, Arabidopsis and rice indicated that the grape TIFY proteins are more closely related to those of Arabidopsis than those of rice. Both segmental and tandem duplication events have been major contributors to the expansion of the grape TIFY family. In addition, synteny analysis between grape and Arabidopsis demonstrated that homologues of several grape TIFY genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of lineages that led to grape and Arabidopsis. Analyses of microarray and quantitative real-time RT-PCR expression data revealed that grape TIFY genes are not a major player in the defense against biotrophic pathogens or viruses. However, many of these genes were responsive to JA and ABA, but not SA or ET.

Conclusion

The genome-wide identification, evolutionary and expression analyses of grape TIFY genes should facilitate further research of this gene family and provide new insights regarding their evolutionary history and regulatory control.     

Design Parameters to Control Synthetic Gene Expression in Escherichia coli

Background

Production of proteins as therapeutic agents, research reagents and molecular tools frequently depends on expression in heterologous hosts. Synthetic genes are increasingly used for protein production because sequence information is easier to obtain than the corresponding physical DNA. Protein-coding sequences are commonly re-designed to enhance expression, but there are no experimentally supported design principles.

Principal Findings

To identify sequence features that affect protein expression we synthesized and expressed in E. coli two sets of 40 genes encoding two commercially valuable proteins, a DNA polymerase and a single chain antibody. Genes differing only in synonymous codon usage expressed protein at levels ranging from undetectable to 30% of cellular protein. Using partial least squares regression we tested the correlation of protein production levels with parameters that have been reported to affect expression. We found that the amount of protein produced in E. coli was strongly dependent on the codons used to encode a subset of amino acids. Favorable codons were predominantly those read by tRNAs that are most highly charged during amino acid starvation, not codons that are most abundant in highly expressed E. coli proteins. Finally we confirmed the validity of our models by designing, synthesizing and testing new genes using codon biases predicted to perform well.

Conclusion

The systematic analysis of gene design parameters shown in this study has allowed us to identify codon usage within a gene as a critical determinant of achievable protein expression levels in E. coli. We propose a biochemical basis for this, as well as design algorithms to ensure high protein production from synthetic genes. Replication of this methodology should allow similar design algorithms to be empirically derived for any expression system.     

Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling

Background

Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines.

Methodology/Principal findings

We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T_H1 CD4⁺ and CD8⁺ T cells and a systemic LACK-specific T_H1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T_H1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T_H1 response.

Conclusions/Significance

This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.     

Drosophila photoreceptor cells exploited for the production of eukaryotic membrane proteins: receptors, transporters and channels

Background

Membrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies.

Principal Findings

We describe the homologous and heterologous expression of 10 different targets from the three major MP classes - G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2–0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter - both involved in synaptic transmission - showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies.

Significance

We demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of eukaryotic MPs that have so far not been accessible to biochemical and biophysical studies.