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Codon optimisation to improve expression of a Mycobacterium avium ssp. paratuberculosis- specific membrane-associated antigen by Lactobacillus salivarius
Abstract
Subunit and DNA-based vaccines against Mycobacterium avium ssp. paratuberculosis (MAP) attempt to overcome inherent issues associated with whole-cell formulations. However, these vaccines can be hampered by poor expression of recombinant antigens from a number of disparate hosts. The high G+C content of MAP invariably leads to a codon bias throughout gene expression. To investigate if the codon bias affects recombinant MAP antigen expression, the open reading frame of a MAP-specific antigen MptD (MAP3733c) was codon optimised for expression against a Lactobacillus salivarius host. Of the total 209 codons which constitute MAP3733c, 172 were modified resulting in a reduced G+C content from 61% for the native gene to 32.7% for the modified form. Both genes were placed under the transcriptional control of the PnisA promoter; allowing controlled heterologous expression in L. salivarius. Expression was monitored using fluorescence microscopy and microplate fluorometry via GFP tags translationally fused to the C-termini of the two MptD genes. A > 37-fold increase in expression was observed for the codon-optimised MAP3733synth variant over the native gene. Due to the low cost and improved expression achieved, codon optimisation significantly improves the potential of L. salivarius as an oral vaccine stratagem against Johne's disease.
Supplementary resource (1)
Nucleotide Sequence
January 2013
... MAP is particularly well-suited to Lactobacillus vector delivery because it is a pathogen transmitted by ingestion of contaminated feces or milk and passes through the GI tract where it infects the intestinal mucosa (Bannantine and Bermudez, 2013). However, despite the obvious advantages of Lactobacillus based mucosal immunization; encompassing the inherent ability of specific strains to survive gastric transit, adhere to the intestinal epithelium (Messaoudi et al., 2012), and stimulate both mucosal and systemic immune responses (Mohamadzadeh et al., 2009), there are known difficulties in expression of G+C rich coding sequences in the A+T rich Lactobacillus host (Johnston et al., 2013). Indeed, we recently determined that the significantly divergent genomic G+C content of MAP and Lactobacillus salivarius (69 and 33% respectively) leads to a disparity in codon usage, which significantly impedes recombinant MAP protein synthesis. ...
... To alleviate this translation inefficiency likely due to ribosomal pausing at rare codons (Buchan and Stansfield, 2007), codon optimization of a MAP gene (MAP3733c) was performed by introducing a series of synonymous mutations; modifying the coding region to better suit the codon bias of L. salivarius. It was shown that synthesis of a MAP specific membrane protein within L. salivarius could be markedly enhanced (>37fold) owing to codon optimization, resulting in the abundance of MAP-GFP protein fusion fluorescence in recombinant cells (Johnston et al., 2013). However, that protein was never confirmed to truly represent the native protein as no monoclonal antibodies or other specific detection reagents were developed against it. ...
... Plasmids pNZ9530 and pNZ8048 were originally obtained from the University College Cork culture collection, while pUC57 vectors containing codon-optimized synthetic GFP and MptDsynth genes were obtained from the Cork Institute of Technology culture collection (Johnston et al., 2013). Cultures of E. coli harboring individual vectors were supplemented with Erythromycin (Ery, 200 μg/ml), Chloramphenicol (Cm, 12.5 μg/ml), or Ampicillin (Amp, 200 μg/ml) for pNZ9530, pNZ8048, and pUC57 containing cells respectively. ...
It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.
... In L. lactis, the expression of heterologous genes depends on the distribution of rare codons and the codon usage [11,18], which makes codon usage the single most important factor in heterologous gene expression. Consequently, codon optimization is a simple and effective step to enhance protein production in L. lactis [19,20]. Signal peptide (SP) is an important factor that have a great impact on the secretion of a protein [7]. ...
... Codon usage variation among the genes is determined by compositional constraints, translational selection, and hydrophobicity of these genes [42]. The accumulation of rare codons in the microorganisms can lower the translation efficiency [19,20], and codons with high G+C makes mRNA fold into a stable secondary structure, thus inhibiting the translation initiation and reducing protein expression [41,43]. There is usually a strong association between codon usage bias and gene expression levels in many microorganisms [14]. ...
To achieve the food-grade expression of bile salt hydrolase (BSH, EC 3.5.1.24) from Lactobacillus plantarum BBE7, the nisin controlled gene expression system (NICE), food-grade selection maker and signal peptide of Lactococcus lactis were used in this study. The open reading frame of BSH was optimized based on the codon bias of L. lactis, resulting in 12-fold and 9.5% increases in the intracellular and extracellular BSH activities, respectively. Three synthetic propeptides, LEISSTCDA (acidic), LGISSTCNA (neutral) and LKISSTCHA (basic) were also fused with signal peptide SPusp45 of vector pNZ8112 and introduced into the food-grade expression vector pNZ8149, respectively. Among these propeptides, acidic propeptide was effective in increasing the secretion efficiency and yield of BSH in recombinant bacteria , while neutral propeptide had no significant effect on the secretion of BSH. In contrast, basic propeptide strongly reduced the extracellular expression of BSH. By using codon optimization and the acidic propeptide together, the extracellu-lar BSH activity was increased by 11.3%, reaching its maximum of 3.56 U/mg. To the best of our knowledge, this is the first report on the intracellular and extracellular expression of BSH using food-grade expression system, which would lay a solid foundation for large-scale production of BSH and other heterologous proteins in L. lactis.
... In L. lactis, the expression of heterologous genes depends on the distribution of rare codons and the codon usage [11,18], which makes codon usage the single most important factor in heterologous gene expression. Consequently, codon optimization is a simple and effective step to enhance protein production in L. lactis [19,20]. Signal peptide (SP) is an important factor that have a great impact on the secretion of a protein [7]. ...
... Codon usage variation among the genes is determined by compositional constraints, translational selection, and hydrophobicity of these genes [42]. The accumulation of rare codons in the microorganisms can lower the translation efficiency [19,20], and codons with high G+C makes mRNA fold into a stable secondary structure, thus inhibiting the translation initiation and reducing protein expression [41,43]. There is usually a strong association between codon usage bias and gene expression levels in many microorganisms [14]. ...
To achieve the food-grade expression of bile salt hydrolase (BSH, EC 3.5.1.24) from Lactobacillus plantarum BBE7, the nisin controlled gene expression system (NICE), food-grade selection maker and signal peptide of Lactococcus lactis were used in this study. The open reading frame of BSH was optimized based on the codon bias of L. lactis, resulting in 12-fold and 9.5% increases in the intracellular and extracellular BSH activities, respectively. Three synthetic propeptides, LEISSTCDA (acidic), LGISSTCNA (neutral) and LKISSTCHA (basic) were also fused with signal peptide SPusp45 of vector pNZ8112 and introduced into the food-grade expression vector pNZ8149, respectively. Among these propeptides, acidic propeptide was effective in increasing the secretion efficiency and yield of BSH in recombinant bacteria, while neutral propeptide had no significant effect on the secretion of BSH. In contrast, basic propeptide strongly reduced the extracellular expression of BSH. By using codon optimization and the acidic propeptide together, the extracellular BSH activity was increased by 11.3%, reaching its maximum of 3.56 U/mg. To the best of our knowledge, this is the first report on the intracellular and extracellular expression of BSH using food-grade expression system, which would lay a solid foundation for large-scale production of BSH and other heterologous proteins in L. lactis.
... On the other hand, other studies have observed significantly improved protein expression following codon optimization, including expression of Mycobacterium avium ssp. paratuberculosis antigen in Lactobacillus salivarius (Johnston et al. 2013) and expression of fluorescent proteins in gram-positive bacteria (Sastalla et al. 2009). In our study, codon optimization improved the expression of 3D8 scFv L. paracasei in both the secreted (Fig. 1c, d) and cell wall-anchored forms (Fig. 1e). ...
The protein 3D8 single-chain variable fragment (3D8 scFv) has potential anti-viral activity due to its ability to penetrate into cells and hydrolyze nucleic acids. Probiotic
Lactobacillus paracasei engineered to secrete 3D8 scFv for oral administration was used to test the anti-viral effects of 3D8 scFv against gastrointestinal virus infections. We found that injection of 3D8 scFv into the intestinal lumen resulted in
the penetration of 3D8 scFv into the intestinal villi and lamina propria. 3D8 scFv secreted from engineered L. paracasei retained its cell-penetrating and nucleic acid-hydrolyzing activities, which were previously shown with 3D8 scFv expressed in Escherichia coli. Pretreatment of RAW264.7 cells with 3D8 scFv purified from L. paracasei prevented apoptosis induction by murine norovirus infection and decreased messenger RNA (mRNA) expression of the viral capsid protein VP1. In a mouse model, oral administration of the engineered L. paracasei prior to murine norovirus infection reduced the expression level of mRNA encoding viral polymerase. Taken together, these results suggest that L. paracasei secreting 3D8 scFv provides a basis for the development of ingestible anti-viral probiotics active against gastrointestinal viral infection.
... The overall production yields of proteins are generally correlated with translation efficiency that is determined by translation initiation and elongation rates. Although we successfully demonstrated that our synthetic parts (eSD2) in the BCD system are highly effective on the translation initiation of target genes, those synthetic parts cannot control the translation elongation rate which is mainly dependent on the sequences of target gene (codon usage); the presence of rare codons or codon bias in the target gene, often cause the slow translation elongation 44,45 . By the modification of target gene sequence (i.e codon optimization), the translation elongation rate can be increased, and the overall production yield can be further increased by the combination with our synthetic parts. ...
The lactic acid bacteria (LAB) Leuconostoc citreum are non-sporulating hetero-fermentative bacteria that play an important role in the fermented food industry. In this study, for the enhanced and reliable production of recombinant proteins in L. citreum, we developed a bicistronic design (BCD) expression system which includes a short leader peptide (1st cistron) followed by target genes (2nd cistron) under the control of a single promoter. Using superfolder green fluorescent protein (sfGFP) as a reporter, the functionality of BCD in L. citreum was verified. Further, to improve the expression in BCD, we tried to engineer a Shine-Dalgarno sequence (SD2) for the 2nd cistron and a promoter by FACS screening of random libraries, and both strong SD2 (eSD2) and promoter (P710V4) were successfully isolated. The usefulness of the engineered BCD with P710V4 and eSD2 was further validated using three model proteins-glutathione-s-transferase, human growth hormone, and α-amylase. All examined proteins were successfully produced with levels highly increased compared with those in the original BCD as well as the monocistronic design (MCD) expression system.
... In the context of vectored subunit vaccines, success has been achieved in the expression of MAP antigens in Lactobacillus salivarius using a codon optimization strategy (151,152). Multi-antigen virally vectored vaccines were also engineered and tested in mice and cattle. In this context, the vaccine designated HAV is a fusion of two secreted and two cell surface MAP proteins whose DNA coding sequence was inserted into the human adenovirus 5 and Modified Vaccinia Ankara delivery vectors. ...
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants causing chronic diarrhea, malnutrition, and muscular wasting. Neonates and young animals are infected primarily by the fecal–oral route. MAP attaches to, translocates via the intestinal mucosa, and is phagocytosed by macrophages. The ensuing host cellular immune response leads to granulomatous enteritis characterized by a thick and corrugated intestinal wall. We review various tissue culture systems, ileal loops, and mice, goats, and cattle used to study MAP pathogenesis. MAP can be detected in clinical samples by microscopy, culturing, PCR, and an enzyme-linked immunosorbent assay. There are commercial vaccines that reduce clinical disease and shedding, unfortunately, their efficacies are limited and may not engender long-term protective immunity. Moreover, the potential linkage with Crohn’s disease and other human diseases makes MAP a concern as a zoonotic pathogen. Potential therapies with anti-mycobacterial agents are also discussed. The completion of the MAP K-10 genome sequence has greatly improved our understanding of MAP pathogenesis. The analysis of this sequence has identified a wide range of gene functions involved in virulence, lipid metabolism, transcriptional regulation, and main metabolic pathways. We also review the transposons utilized to generate random transposon mutant libraries and the recent advances in the post-genomic era. This includes the generation and characterization of allelic exchange mutants, transcriptomic analysis, transposon mutant banks analysis, new efforts to generate comprehensive mutant libraries, and the application of transposon site hybridization mutagenesis and transposon sequencing for global analysis of the MAP genome. Further analysis of candidate vaccine strains development is also provided with critical discussions on their benefits and shortcomings, and strategies to develop a highly efficacious live-attenuated vaccine capable of differentiating infected from vaccinated animals.
... Although vaccine development to date has focused on the use of attenuated pathogens, the attendant risk of such constructs reverting to a virulent phenotype remains a very 16.4 Sola-Oladokun · Culligan · Sleator Changes may still occur before final publication online and in print real possibility. Employing live vectors, particularly lactic acid bacteria (LAB), as vaccine delivery vehicles has the advantage of oral administration and mucosal immune stimulation ( Johnston et al. 2013( Johnston et al. , 2014. The gut-associated lymphoid tissue (GALT) is exploited to induce humoral and cellular immune responses ( Johnston et al. 2010). ...
Bioengineered probiotics represent the next generation of whole cell-mediated biotherapeutics. Advances in synthetic biology, genome engineering, and DNA sequencing and synthesis have enabled scientists to design and develop probiotics with increased stress tolerance and the ability to target specific pathogens and their associated toxins, as well as to mediate targeted delivery of vaccines, drugs, and immunomodulators directly to host cells. Herein, we review the most significant advances in the development of this field. We discuss the critical issue of biological containment and consider the role of synthetic biology in the design and construction of the probiotics of the future. Expected final online publication date for the Annual Review of Food Science and Technology Volume 8 is February 28, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... On the other hand, other studies have observed significantly improved protein expression following codon optimization, including expression of Mycobacterium avium ssp. paratuberculosis antigen in Lactobacillus salivarius (Johnston et al. 2013) and expression of fluorescent proteins in gram-positive bacteria (Sastalla et al. 2009). In our study, codon optimization improved the expression of 3D8 scFv L. paracasei in both the secreted (Fig. 1c, d) and cell wall-anchored forms (Fig. 1e). ...
The protein 3D8 single-chain variable fragment (3D8 scFv) has potential anti-viral activity due to its ability to penetrate into cells and hydrolyze nucleic acids. Probiotic Lactobacillus paracasei engineered to secrete 3D8 scFv for oral administration was used to test the anti-viral effects of 3D8 scFv against gastrointestinal virus infections. We found that injection of 3D8 scFv into the intestinal lumen resulted in the penetration of 3D8 scFv into the intestinal villi and lamina propria. 3D8 scFv secreted from engineered L. paracasei retained its cell-penetrating and nucleic acid-hydrolyzing activities, which were previously shown with 3D8 scFv expressed in Escherichia coli. Pretreatment of RAW264.7 cells with 3D8 scFv purified from L. paracasei prevented apoptosis induction by murine norovirus infection and decreased messenger RNA (mRNA) expression of the viral capsid protein VP1. In a mouse model, oral administration of the engineered L. paracasei prior to murine norovirus infection reduced the expression level of mRNA encoding viral polymerase. Taken together, these results suggest that L. paracasei secreting 3D8 scFv provides a basis for the development of ingestible anti-viral probiotics active against gastrointestinal viral infection.
... It has been demonstrated that the expression level of genes containing codons matching those of a given bacterial host is much higher than that of genes containing a different set of codons (21). Additionally, Johnston and co-workers (22) demonstrated that by changing codon sequence of the gene coding for the MAP-specific antigen of Mycobacterium avium ssp. paratuberculosis (MptD), the production yield of MptD peptide in Lactobacillus salivarius was enhanced over 37 times, as compared to that obtained with its native codon sequence. ...
Lactic acid bacteria (LAB), a large group of food grade microorganisms, have recently been developed to express reactive antigens, which are intended to be used for vaccination. Immunological effects of these vaccines depend on the amount of the expressed antigenic protein loaded on the strains utilized for the delivery. Several factors including gene choice, cloning vector, and expression system could account for the different yield of antigens. Moreover, appropriate cellular location of the expressed gene product, either in the cytoplasm, the cell wall, or secreted into the medium can also affect the immune response. This review addresses the current knowledge of vaccine development by using lactic acid bacteria as an antigen-delivery system. The principal aim of this manuscript is to inform the reader on the processes of constructing recombinant LAB (rLAB) expressing about particular heterologous, immunologically reactive proteins, which can then be used as advantageous live vaccines.
Mycobacterium tuberculosis (Mtb) is the pathogen that causes tuberculosis and develops resistance to many of the existing drugs. The sole licensed TB vaccine, BCG, is unable to provide a comprehensive defense. So, it is crucial to maintain the immunological response to eliminate tuberculosis. Our previous in silico study reported five uncharacterized proteins as potential vaccine antigens. In this article, we considered the uncharacterized Mtb H37Rv regions of difference (RD-2) Rv1987 protein as a promising vaccine candidate. The vaccine quality of the protein was analyzed using reverse vaccinology and immunoinformatics-based quality-checking parameters followed by an ex vivo preliminary investigation. In silico analysis of Rv1987 protein predicted it as surface localized, secretory, single helix, antigenic, non-allergenic, and non-homologous to the host protein. Immunoinformatics analysis of Rv1987 by CD4 + and CD8 + T-cells via MHC-I and MHC-II binding affinity and presence of B-cell epitope predicted its immunogenicity. The docked complex analysis of the 3D model structure of the protein with immune cell receptor TLR-4 revealed the protein’s capability for potential interaction. Furthermore, the target protein-encoded gene Rv1987 was cloned, over-expressed, purified, and analyzed by mass spectrometry (MS) to report the target peptides. The qRT-PCR gene expression analysis shows that it is capable of activating macrophages and significantly increasing the production of a number of key cytokines (TNF-α, IL-1β, and IL-10). Our in-silico analysis and ex vivo preliminary investigations revealed the immunogenic potential of the target protein. These findings suggest that the Rv1987 be undertaken as a potent subunit vaccine antigen and that further animal model immuno-modulation studies would boost the novel TB vaccine discovery and/or BCG vaccine supplement pipeline.
Photosynthetic organisms are one of the best sources for a sustainable life. Microalgae is one of these sources, which found wide application in the production processes for food, feed, pharmaceuticals, nutraceuticals, biofuel, bioethanol, bioplastics, etc. They can convert stress-related effects such as excessive heat and light or nutrient limitation to high-value molecules including fatty acids, antioxidant pigments, and proteins. Studies on using microalgae as a recombinant production platform are gaining momentum. The platform provides a safe, economic, and efficient environment to produce proteins in the correct conformation and bioactive form. To date, microalgal cell has been a host to produce many proteins with therapeutic effects, such as vaccine subunits, human growth factors, antimicrobial peptides, enzymes, and antibodies. Recent advancements in the genetic engineering of microalgal genomes are getting closer to making microalgae more useful for industrial scale production. In this chapter, advances and limitations of the microalgae platform were discussed from the perspective of recombinant therapeutics.KeywordsMicroalgae Recombinant protein production Therapeutics Health Green technology
Current developments in microbial research and synthetic biology have motivated the investigators to develop a diverse range of engineered microbes expressing valuable biotherapeutics targeting a range of human clinical applications. Probiotics, developed through a recombinant approach, represent the new era of whole cell-mediated biotherapeutics. These engineered probiotics are target specific and have high endurance capability, which makes them a perfect vaccine or drug carrying vehicle in the host system. This chapter outlines the diverse capabilities of biotherapeutics expressed through engineered microbes as a potent engineered probiotic, for the treatment of different chronic human pathologies including metabolic disorder, malignancy, chronic inflammation bowel disease concerned with the gastrointestinal tract and autoimmune disorders, respectively. This chapter also entails the significant advancement in the area of reprogrammed microbes used as an effective bio-scavenger, for the remediation of toxic heavy metals from the environment. The sustainable production of target specific biotherapeutics from a recombinant microbiome will enable us to overcome the burden of expensive drugs with severe side effects. This modern concept of engineered biotherapeutics will narrow down many technical and economic barriers associated with drug development globally. The modern era on microbial biotherapeutics will ensure healthy lives and promote the well-being of the global population.
Lactic acid bacteria (LAB) have been utilized since the 1990s for therapeutic heterologous gene expression. The ability of LAB to elicit an immune response against expressed foreign antigens has led to their exploration as potential mucosal vaccine candidates. LAB vaccine vectors offer many attractive advantages: simple, noninvasive administration (usually oral or intranasal), the acceptance and stability of genetic modifications, relatively low cost, and the highest level of safety possible. Experimentation using LAB of the genus Lactobacillus has become popular in recent years due to their ability to elicit strong systemic and mucosal immune responses. This article reviews Lactobacillus vaccine constructs, including Lactobacillus species, antigen expression, model organisms, and in vivo immune responses, with a primary focus on viral and bacterial antigens.
Aim: The present study aimed to investigate the effect of codon optimization on E6 recombinant protein production in Lactococcus lactis. Method: Here we define the construction of shuttle vector harboring wild-type and codon-optimized HPV16 E6 oncogene, with maximum number of infrequent codons exchanged with codons that are frequently used in Lactococcus lactis subsp. cremoris MG1363. Results: Hence, the codons encoding 159 amino acids were modified, in which a total of 91 codons were changed, resulting in approximately threefold increase in protein expression of recombinant E6 (rE6). Conclusion: Our data revealed that codon usage optimization according to L. lactis desired codon usage can dramatically increase the expression of HPV16 E6, suggesting that this strategy is a valuable approach for immunization through DNA vaccine.
The review centres on the human gastrointestinal tract; focusing first on the bacterial strass responses needed to overcome the physiochemical defences of the host, specifically how these stress survival strategies can be used as targets for alternative infection control strategies. The concluding part focuses on recent developments in molecular diagnostics; centring on the shifting paradigm from culture to molecular based diagnostics.
Given the increasing commercial and clinical relevance of probiotics, improving their stress tolerance profile and ability to overcome the physiochemical defences of the host is an important biological goal. Herein, I review the current state of the art in the design of engineered probiotic cultures, with a specific focus on their utility as therapeutics for the developing world; from the treatment of chronic and acute enteric infections, and their associated diarrhoeal complexes, to targeting HIV and application as novel mucosal vaccine delivery vehicles.
Despite remaining apparently frozen through the millennia, the genetic code is far more flexible than previously believed and can be extended and repurposed with relative ease.
Metagenomics provides a means of assessing the total genetic pool of all the microbes in a particular environment, in a culture-independent manner. It has revealed unprecedented diversity in microbial community composition, which is further reflected in the encoded functional diversity of the genomes, a large proportion of which consists of novel genes. Herein, we review both sequence-based and functional metagenomic methods to uncover novel genes and outline some of the associated problems of each type of approach, as well as potential solutions. Furthermore, we discuss the potential for metagenomic bio-therapeutic discovery, with a particular focus on the human gut microbiome and finally, we outline how the discovery of novel genes may be used to create bio-engineered probiotics.
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease, a granulomatous enteritis of cattle and other domesticated and wild ruminant species. Johne disease is prevalent worldwide and has a significant impact on the global agricultural economy. Current vaccines against Johne are insufficient in stemming its spread, and associated side-effects prevent their widespread use in control programs. Effective and safe vaccine strategies are needed. The main purpose of this paper is to propose and evaluate the development of a novel oral subunit-vaccine using a patho-biotechnological approach. This novel strategy, which harnesses patho-genetic elements from the intracellular pathogen Listeria monocytogenes, may provide a realistic route towards developing an effective next generation subunit vaccine against Johne's disease and paratuberculosis.
Traditional non-gastrointestinal vaccines can prevent effectively the invasion of pathogens; however, these vaccines are less effective against mucosal infections because there is not a sufficient immune response at the mucosa. Most pathogens invade via a mucosal pathway (oral, intranasal, or vaginal). ( 1) It is widely accepted that Lactobacillus species play a critical role as commensals in the gastrointestinal (GI) tract. ( 2) Their ability to survive in the digestive tract, their close association with the intestinal epithelium, their immunomodulatory properties and their safety even when consumed in large amounts make lactobacilli attractive candidates for live vehicles for the delivery of immunogens to the intestinal mucosa. ( 3) The oral or intranasal administration of Lactobacillus-based vaccines is a promising method to control mucosal infection because these vaccines could induce strong humoral and cellular immune responses both in the blood and at mucosal sites.
DPPD (Rv0061) is a difficult to express protein of Mycobacterium tuberculosis that elicits strong and specific delayed type hypersensitivity reactions in humans infected with M. tuberculosis. Therefore e DPPD is a molecule that can improve the specificity of the tuberculin skin test, which is widely used as an aid for the diagnosis of tuberculosis. However, a pitfall of our initial studies was that the DPPD molecule used to perform the skin tests was engineered as fusion molecule with another Mycobacterium protein. This approach was used because no expression of DPPD could be achieved either as a single molecule or as a fusion protein using a variety of commercially available expression systems. Here, we report the production and purification of rDPPD using a synthetic gene engineered to contain E. coli codon bias. The gene was cloned into pET14b expression vector, which was subsequently used to transform Rosetta 2(DE3) pLysS or BL-21(DE3)pLysS host cells. The recombinant protein was over-expressed after induction with IPTG and its purification was easily achieved at levels of 5 - 10 mg/l of bacterial broth cultures. The purified protein was confirmed to be DPPD by Mass Spectroscopy sequencing analysis. Moreover, purified rDPPD stimulated peripheral blood mononuclear cells of PPD positive blood donors to produce high levels of IFN-γ, thus confirming that this molecule is biologically active. Because of the DPPD gene is restricted to the tuberculosis-complex organisms of Mycobacterium genus, this highly purified molecule should be useful for the identification of individuals sensitized with tubercle bacilli.
Oral vaccines that elicit a mucosal immune response may be effective against human immunodeficiency virus type 1 (HIV-1) because
its transmission occurs mainly at the mucosa. The aim of this study was to construct recombinant Lactobacillus for oral delivery of oral vaccines against HIV-1 and to evaluate their immunogenicity. A recombinant Lactobacillus acidophilus strain expressing the HIV-1 Gag on the bacterial cell surface was established by fusion with the signal peptide and anchor
motif of a mucus binding protein (Mub) from L. acidophilus with or without coexpression of Salmonella enterica serovar Typhimurium flagellin (FliC) fused to a different Mub signal peptide and anchor. Using HEK293 cells engineered to
express Toll-like receptor 5 (TLR5), the biological activity of FliC on the bacterial cell surfaces was determined. The surface-exposed
flagellin retained its TLR5-stimulating activity, suggesting that the recombinant strain with Gag and FliC dual display might
provide a different immunopotency than the strain expressing only Gag. The immunological properties of the recombinant strains
were assessed by coculture with human myeloid dendritic cells (DCs). The heterologous antigens on the cell surface affected
maturation and cytokine responses of DCs. Acquired immune responses were also investigated by intragastric immunization of
mice. The enzyme-linked immunosorbent spot assay showed induction of gamma interferon-producing cells at local mucosa after
immunization of mice with the Gag-producing strain. Meanwhile, the immunization with L. acidophilus displaying both Gag and FliC resulted in an increase of Gag-specific IgA-secreting cells. These results suggested that the
Gag-displaying L. acidophilus elicited specific immune responses and the coexistence of FliC conferred an adjuvant effect on local IgA production.
Lactobacillus plantarum LMG P-26358 isolated from a soft French artisanal cheese produces a potent class IIa bacteriocin with 100% homology to plantaricin 423 and bacteriocidal activity against Listeria innocua and Listeria monocytogenes. The bacteriocin was found to be highly stable at temperatures as high as 100°C and pH ranges from 1-10. While this relatively narrow spectrum bacteriocin also exhibited antimicrobial activity against species of enterococci, it did not inhibit dairy starters including lactococci and lactobacilli when tested by well diffusion assay (WDA). In order to test the suitability of Lb. plantarum LMG P-26358 as an anti-listerial adjunct with nisin-producing lactococci, laboratory-scale cheeses were manufactured. Results indicated that combining Lb. plantarum LMG P-26358 (at 108 colony forming units (cfu)/ml) with a nisin producer is an effective strategy to eliminate the biological indicator strain, L. innocua. Moreover, industrial-scale cheeses also demonstrated that Lb. plantarum LMG P-26358 was much more effective than the nisin producer alone for protection against the indicator. MALDI-TOF mass spectrometry confirmed the presence of plantaricin 423 and nisin in the appropriate cheeses over an 18 week ripening period. A spray-dried fermentate of Lb. plantarum LMG P-26358 also demonstrated potent anti-listerial activity in vitro using L. innocua. Overall, the results suggest that Lb. plantarum LMG P-26358 is a suitable adjunct for use with nisin-producing cultures to improve the safety and quality of dairy products.
Mycobacterium avium subsp. paratuberculosis (MAP) infection causes in ruminants a regional chronic enteritis that is increasingly being recognized as a significant problem affecting animal health, farming and the food industry due to the high prevalence of the disease and to recent research data strengthening the link between the pathogen and human inflammatory bowel disease (IBD). Control of the infection through hygiene-management measures and test and culling of positive animals has to date not produced the expected results and thus a new focus on vaccination against this pathogen is necessary. This review summarizes all vaccination studies of cattle, sheep or goats reporting production, epidemiological or pathogenetic effects of vaccination published before January 2010 and that provide data amenable to statistical analyses. The meta analysis run on the selected data, allowed us to conclude that most studies included in this review reported that vaccination against MAP is a valuable tool in reducing microbial contamination risks of this pathogen and reducing or delaying production losses and pathogenetic effects but also that it did not fully prevent infection. However, the majority of MAP vaccines were very similar and rudimentary and thus there is room for improvement in vaccine types and formulations.
The growth rate of Mycobacterium avium subsp. paratuberculosis was assessed by different methods in 7H9 medium supplemented with OADC (oleic acid, albumin, dextrose, catalase), Tween 80,
and mycobactin J. Generation times and maximum specific growth rates were determined by wet weight, turbidometric measurement,
viable count, and quantitative PCR (ParaTB-Kuanti; F57 gene) for 8 M. avium subsp. paratuberculosis strains (K10, 2E, 316F, 81, 445, 764, 22G, and OVICAP 49). Strain-to-strain differences were observed in growth curves and
calculated parameters. The quantification methods gave different results for each strain at specific time points. Generation
times ranged from an average of 1.4 days for viable count and qPCR to approximately 10 days for wet weight and turbidometry.
The wet-weight, turbidometry, and ParaTB-Kuanti qPCR methods correlated best with each other. Generally, viability has been
assessed by viable count as a reference method; however, due to M. avium subsp. paratuberculosis clumping problems and the presence of noncultivable M. avium subsp. paratuberculosis cells, we conclude that qPCR of a single-copy gene may be used reliably for rapid estimation of M. avium subsp. paratuberculosis bacterial numbers in a sample.
The draft genome sequence of Lactobacillus salivarius GJ-24 isolated from the feces of healthy adults was determined. Its properties, including milk fermentation activity and
bacteriocin production, suggest its potential uses as a probiotic lactic acid bacterium and start culture for dairy products.
Biomedical and biotechnological research relies on processes leading to the successful expression and production of key biological products. High-quality proteins are required for many purposes, including protein structural and functional studies. Protein expression is the culmination of multistep processes involving regulation at the level of transcription, mRNA turnover, protein translation, and post-translational modifications leading to the formation of a stable product. Although significant strides have been achieved over the past decade, advances toward integrating genomic and proteomic information are essential, and until such time, many target genes and their products may not be fully realized. Thus, the focus of this review is to provide some experimental support and a brief overview of how codon usage bias has evolved relative to regulating gene expression levels.
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne disease, a granulomatous enteritis of cattle and other domesticated and wild ruminant species. Johne disease is prevalent worldwide and has a significant impact on the global agricultural economy. Current vaccines against Johne are insufficient in stemming its spread, and associated side-effects prevent their widespread use in control programs. Effective and safe vaccine strategies are needed. The main purpose of this paper is to propose and evaluate the development of a novel oral subunit-vaccine using a patho-biotechnological approach. This novel strategy, which harnesses patho-genetic elements from the intracellular pathogen Listeria monocytogenes, may provide a realistic route towards developing an effective next generation subunit vaccine against Johne disease and paratuberculosis.
In spite of extensive research on the effect of mutation and selection on codon usage, a general model of codon usage bias due to mutational bias has been lacking. Because most amino acids allow synonymous GC content changing substitutions in the third codon position, the overall GC bias of a genome or genomic region is highly correlated with GC3, a measure of third position GC content. For individual amino acids as well, G/C ending codons usage generally increases with increasing GC bias and decreases with increasing AT bias. Arginine and leucine, amino acids that allow GC-changing synonymous substitutions in the first and third codon positions, have codons which may be expected to show different usage patterns.
In analyzing codon usage bias in hundreds of prokaryotic and plant genomes and in human genes, we find that two G-ending codons, AGG (arginine) and TTG (leucine), unlike all other G/C-ending codons, show overall usage that decreases with increasing GC bias, contrary to the usual expectation that G/C-ending codon usage should increase with increasing genomic GC bias. Moreover, the usage of some codons appears nonlinear, even nonmonotone, as a function of GC bias. To explain these observations, we propose a continuous-time Markov chain model of GC-biased synonymous substitution. This model correctly predicts the qualitative usage patterns of all codons, including nonlinear codon usage in isoleucine, arginine and leucine. The model accounts for 72%, 64% and 52% of the observed variability of codon usage in prokaryotes, plants and human respectively. When codons are grouped based on common GC content, 87%, 80% and 68% of the variation in usage is explained for prokaryotes, plants and human respectively.
The model clarifies the sometimes-counterintuitive effects that GC mutational bias can have on codon usage, quantifies the influence of GC mutational bias and provides a natural null model relative to which other influences on codon bias may be measured.
Mycobacterium avium subsp. paratuberculosis (MAP) causes a chronic gastroenteritis affecting many species. Johne's disease is one of the most widespread and economically important disease of ruminants. Since 1992 and the opening of the European market, the exposure and the transmission of MAP in cattle herds considerably increased. Improvements in diagnostic strategies for Ireland and elsewhere are urgently required. In total, 290 cattle from seven Irish herds with either a history or a strong likelihood of paratuberculosis infection were selected by a veterinary team over 2 years. Faecal samples (290) were collected and screened for MAP by a conventional culture method and two PCR assays. In order to further evaluate the usefulness of molecular testing, a nested PCR was also assessed.
M. paratuberculosis was isolated and cultured from 23 faecal samples (7.9%) on solid medium. From a molecular perspective, 105 faecal samples (36%) were PCR positive for MAP specific DNA. A complete correlation (100%) was observed between the results of both molecular targets (IS900 and ISMAP02). Sensitivity was increased by ~10% with the inclusion of a nested PCR for ISMAP02 (29 further samples were positive). When culturing and PCR were retrospectively compared, every culture positive faecal sample also yielded a PCR positive result for both targets. Alternatively, however not every PCR positive sample (n = 105, 36%) produced a corresponding culture isolate. Interestingly though when analysed collectively at the herd level, the correlation between culture and PCR results was 100% (ie every herd which recorded at least 1 early PCR +ve result later yielded culture positive samples within that herd).
PCR on bovine faecal samples is a fast reliable test and should be applied routinely when screening for MAP within herds suspected of paratuberculosis. Nested PCR increases the threshold limit of detection for MAP DNA by approximately 10% but proved to be problematic in this study. Although slow and impractical, culturing is still regarded as one of the most reliable methods for detecting MAP among infected cattle.
Lactobacillus salivarius is a homofermentative lactic acid bacterium and is frequently isolated from mucosal surfaces of healthy humans. L. salivarius CECT 5713, a strain isolated simultaneously from breast milk and infant feces of a healthy mother-infant pair, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several in vitro and in vivo assays. Here, we report its complete and annotated genome sequence. Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Mycobacterium avium subsp. paratuberculosis is a pathogen which causes a debilitating chronic enteritis in ruminants. Unfortunately, the mechanisms that control M. avium subsp. paratuberculosis persistence during infection are poorly understood and the key steps for developing Johne's disease remain elusive. A proteomic analysis approach, based on one dimensional polyacrylamide gel electrophoresis (SDS-PAGE) followed by LC-MS/MS, was used to identify and characterize the cell wall associated proteins of M. avium subsp. paratuberculosis K10 and an cell surface enzymatic shaving method was used to determine the surface-exposed proteins. 309 different proteins were identified, which included 101 proteins previously annotated as hypothetical or conserved hypothetical. 38 proteins were identified as surface-exposed by trypsin treatment. To categorize and analyze these proteomic data on the proteins identified within cell wall of M. avium subsp. paratuberculosis K10, a rational bioinformatic approach was followed. The analyses of the 309 cell wall proteins provided theoretical molecular mass and pI distributions and determined that 18 proteins are shared with the cell surface-exposed proteome. In short, a comprehensive profile of the M. avium subsp. paratuberculosis K10 cell wall subproteome was created. The resulting proteomic profile might become the foundation for the design of new preventive, diagnostic and therapeutic strategies against mycobacterial diseases in general and M. avium subsp. paratuberculosis in particular.
Recent studies have suggested that the thermodynamic stability of mRNA secondary structure near the start codon can regulate translation efficiency in Escherichia coli, and that translation is more efficient the less stable the secondary structure. We survey the complete genomes of 340 species for signals of reduced mRNA secondary structure near the start codon. Our analysis includes bacteria, archaea, fungi, plants, insects, fishes, birds, and mammals. We find that nearly all species show evidence for reduced mRNA stability near the start codon. The reduction in stability generally increases with increasing genomic GC content. In prokaryotes, the reduction also increases with decreasing optimal growth temperature. Within genomes, there is variation in the stability among genes, and this variation correlates with gene GC content, codon bias, and gene expression level. For birds and mammals, however, we do not find a genome-wide trend of reduced mRNA stability near the start codon. Yet the most GC rich genes in these organisms do show such a signal. We conclude that reduced stability of the mRNA secondary structure near the start codon is a universal feature of all cellular life. We suggest that the origin of this reduction is selection for efficient recognition of the start codon by initiator-tRNA.
The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition and enable to solve an ever-growing number of high resolution structures. In the past, many skilful optimization steps were required to achieve sufficient expression of functional membrane proteins. Optimization was performed individually for every membrane protein, but provided insight about commonly encountered bottlenecks and, more importantly, general guidelines how to alleviate cellular limitations during microbial membrane protein expression. Lately, system-wide analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous protein production and their use in microbial membrane protein expression has grown in popularity during the past months.
This review covers the most prominent solutions and pitfalls in expression of eukaryotic membrane proteins using microbial hosts (prokaryotes, yeasts), highlights skilful applications of our basic understanding to improve membrane protein production. Omics technologies provide new concepts to engineer microbial hosts for membrane protein production.
The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes. In the nisin-producing L. lactis strain NZ9700, the specific beta-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, beta-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no beta-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more beta-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The beta-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.
In eubacteria, stalled ribosomes are rescued by a conserved quality-control mechanism involving transfer-messenger RNA (tmRNA) and its protein partner, SmpB. Mimicking a tRNA, tmRNA enters stalled ribosomes, adds Ala to the nascent polypeptide, and serves as a template to encode a short peptide that tags the nascent protein for destruction. To further characterize the tagging process, we developed two genetic selections that link tmRNA activity to cell death. These negative selections can be used to identify inhibitors of tagging or to identify mutations in key residues essential for ribosome rescue. Little is known about which ribosomal elements are specifically required for tmRNA activity. Using these selections, we isolated rRNA mutations that block the rescue of ribosomes stalled at rare Arg codons or at the inefficient termination signal Pro-opal. We found that deletion of A1150 in the 16S rRNA blocked tagging regardless of the stalling sequence, suggesting that it inhibits tmRNA activity directly. The C889U mutation in 23S rRNA, however, lowered tagging levels at Pro-opal and rare Arg codons, but not at the 3' end of an mRNA lacking a stop codon. We concluded that the C889U mutation does not inhibit tmRNA activity per se but interferes with an upstream step intermediate between stalling and tagging. C889 is found in the A-site finger, where it interacts with the S13 protein in the small subunit (forming intersubunit bridge B1a).
Herein we review the most recent advances in probiotic research and applications with particular emphasis on the novel concept of patho-biotechnology: the application of pathogen-derived (ex vivo and in vivo) stress survival strategies for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications.
Different synonymous codons are favored by natural selection for translation efficiency and accuracy in different organisms. The rules governing the identities of favored codons in different organisms remain obscure. In fact, it is not known whether such rules exist or whether favored codons are chosen randomly in evolution in a process akin to a series of frozen accidents. Here, we study this question by identifying for the first time the favored codons in 675 bacteria, 52 archea, and 10 fungi. We use a number of tests to show that the identified codons are indeed likely to be favored and find that across all studied organisms the identity of favored codons tracks the GC content of the genomes. Once the effect of the genomic GC content on selectively favored codon choice is taken into account, additional universal amino acid specific rules governing the identity of favored codons become apparent. Our results provide for the first time a clear set of rules governing the evolution of selectively favored codon usage. Based on these results, we describe a putative scenario for how evolutionary shifts in the identity of selectively favored codons can occur without even temporary weakening of natural selection for codon bias.
Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate how, we engineered
a synthetic library of 154 genes that varied randomly at synonymous sites, but all encoded the same green fluorescent protein
(GFP). When expressed in Escherichia coli, GFP protein levels varied 250-fold across the library. GFP messenger RNA (mRNA) levels, mRNA degradation patterns, and bacterial
growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near
the ribosomal binding site explained more than half the variation in protein levels. In our analysis, mRNA folding and associated
rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias
influences global translation efficiency and cellular fitness.
The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes. In the nisin-producing L. lactis strain NZ9700, the specific beta-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, beta-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no beta-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more beta-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The beta-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.
The usage of alternative synonymous codons in Mycobacterium tuberculosis (and M. bovis) genes has been investigated. This species is a member of the high-G+C Gram-positive bacteria, with a genomic G+C content around 65 mol%. This G+C-richness is reflected in a strong bias towards C- and G-ending codons for every amino acid: overall, the G+C content at the third positions of codons is 83%. However, there is significant variation in codon usage patterns among genes, which appears to be associated with gene expression level. From the variation among genes, putative optimal codons were identified for 15 amino acids. The degree of bias towards optimal codons in an M. tuberculosis gene is correlated with that in homologues from Escherichia coli and Bacillus subtilis. The set of selectively favoured codons seems to be quite highly conserved between M. tuberculosis and another high-G+C Gram-positive bacterium, Corynebacterium glutamicum, even though the genome and overall codon usage of the latter are much less G+C-rich.
During DNA sequence analysis of cosmid L373 from the Mycobacterium leprae genome, an open reading frame of 1.4 kb encoding a protein with some homology to the immunodominant 34-kDa protein of Mycobacterium paratuberculosis, but lacking significant serological activity, was detected. The DNA sequence predicted a signal peptide with a modified lipoprotein consensus sequence, but the protein proved to be devoid of lipid attachment.
The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively
investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted
biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented
in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin
nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited toL. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (β-glucuronidase) or a
model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice.
The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
Quorum sensing in lactic acid bacteria (LAB) involves peptides that are directly sensed by membrane-located histidine kinases, after which the signal is transmitted to an intracellular response regulator. This regulator in turn activates transcription of target genes, that commonly include the structural gene for the inducer molecule. The two-component signal-transduction machinery has proven to be indispensable for transcription activation and production of several autoinducers found in LAB, which are predominantly bacteriocins or bacteriocin-like peptides. In the nisin autoregulation process in Lactococcus lactis the NisK protein acts as the sensor for nisin and the NisR protein as the response regulator, activating transcription of target genes. The cis-acting elements for NisR were identified as the nisA and nisF promoter fragments and these were further analysed for inducibility. Based on this knowledge efficient nisin-controlled expression (NICE) systems were developed for several different lactic acid bacteria. A promising application of the NICE system is the development of autolytic starter lactococci, which will lyse in an early stage during cheese ripening thereby facilitating the release of intracellular enzymes which can contribute to flavour formation.
Bovine paratuberculosis is a highly prevalent chronic infection of the small intestine in cattle, caused by Mycobacterium avium subspecies paratuberculosis. Current control strategies based on test-and-cull and biosecurity measures do not suffice in lowering the prevalence of paratuberculosis in an adequate manner. Therefore, control programmes are in need of an effective vaccine, but at the moment no vaccine is registered for use in cattle in the European Union. This review provides a brief overview of the microbiology, epidemiology and immunology of bovine paratuberculosis, and focuses on recent advances in the development of vaccines against paratuberculosis.
Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis of the small intestine in domestic and wild ruminants, causes substantial losses to livestock industry. Control of this disease is seriously hampered by the lack of adequate diagnostic tools, vaccines and therapies. In this study, we have evaluated the vaccine potential of two MAP proteins, i.e. MAP0586c and MAP4308c, previously identified by postgenomic and immunoproteomic analysis of MAP secretome as novel serodiagnostic antigens. Immunizations of BALB/c and C57BL/6 mice with plasmid DNA encoding MAP0586c and MAP4308c induced strong Th1 type immune responses to both antigens, whereas antibody responses were only induced upon immunization with DNA encoding MAP4308c. Homologous boosting of DNA vaccinated mice with recombinant protein resulted in strong antibody responses against both proteins. Using synthetic overlapping peptides, immunodominant H-2(d) and H-2(b) restricted Th1 T cell epitopes were identified. Finally, MAP infected mice generated strong MAP0586c-specific T cell responses and MAP0586c DNA vaccination could protect BALB/c but not C57BL/6 mice against MAP challenge mice to the same extent as the Mycobacterium bovis BCG vaccine, indicating that this putative transglycosylase is an interesting vaccine candidate that warrants further investigation.
Investigations were undertaken into optimizing the expression of Cestode parasite vaccine antigens in the bacterium, Escherichia coli to levels sufficient for mass production. A strategy to genetically engineer the antigens and improve their expression in E. coli was investigated. Plasmid constructs encoding truncated parasite antigens were prepared, leading to removal of N and C-terminal hydrophobic domains of the antigens. This approach was found to be an effective strategy for improving expression of the TSOL18 recombinant antigen of Taenia solium in E. coli. Clear demonstration that plasmid construct modification can be used to significantly improve heterologous expression in E. coli was shown for the EG95 antigen of Echinococcus granulosus. Removal of hydrophobic stretches of amino acids from the N and C termini of EG95 by genetic manipulation led to a substantial change in expression of the protein from an insoluble to a soluble form. The data demonstrate that the occurrence of hydrophobic regions in the antigens are a major feature that hindered their expression in E. coli. It was also shown that retaining a minimal protein domain (a single fibronectin type III domain) led to high level expression of functional protein that is antigenic and host protective. Two truncated antigens were combined from two species of parasite (EG95NC⁻ from E. granulosus and Tm18N⁻ from Taenia multiceps) and expressed as a single hybrid antigen in E. coli. The hybrid antigens were expressed at a high level and retained antigenicity of their respective components, thereby simplifying production of a multi-antigen vaccine. The findings are expected to have an impact on the preparation of recombinant Cestode vaccine antigens using E. coli, by increasing their utility and making them more amenable to large-scale production.
A quantitative description of the relationship between protein expression levels and open reading frame (ORF) nucleotide sequences is important for understanding natural systems, designing synthetic systems, and optimizing heterologous expression. Codon identity, mRNA secondary structure, and nucleotide composition within ORFs markedly influence expression levels. Bioinformatic analysis of ORF sequences in 816 bacterial genomes revealed that these features show distinct regional trends. To investigate their effects on protein expression, we designed 285 synthetic genes and determined corresponding expression levels in vitro using Escherichia coli extracts. We developed a mathematical function, parameterized using this synthetic gene data set, which enables computation of protein expression levels from ORF nucleotide sequences. In addition to its practical application in the design of heterologous expression systems, this equation provides mechanistic insight into the factors that control translation efficiency. We found that expression is strongly dependent on the presence of high AU content and low secondary structure in the ORF 5' region. Choice of high-frequency codons contributes to a lesser extent. The 3' terminal AU content makes modest, but detectable contributions. We present a model for the effect of these factors on the three phases of ribosomal function: initiation, elongation, and termination.
Induction of protective immunity against pathogenic microbes, including Bacillus anthracis, requires efficient vaccines that potentiate antibody avidity and increase T-cell longevity. We recently reported that the delivery of targeted B. anthracis protective antigen (PA) genetically fused to a DC-binding peptide (DCpep) by Lactobacillus acidophilus induced mucosal and systemic immunity against B. anthracis challenge in mice.
Improvement of this oral vaccine strategy was attempted by use of the high copy and genetically stable q-replicating vector, pTRKH2, for expression of the targeted PA fusion protein in Lactobacillus gasseri, a common human commensal microbe, to vaccinate animals against anthrax Sterne infection.
Oral application of L. gasseri expressing the PA-DCpep fusion proteins elicited robust PA-neutralizing antibody and T-cell mediated immune responses against anthrax Sterne challenge, resulting in complete animal survival. Collectively, this improved expression vaccine strategy reduced the number of inoculations and length of the boosting period, leading to animal protection via efficacious bacterial adjuvanticity and safe oral delivery of this vaccine to mucosal immune cells, including dendritic cells.
Lactobacillus-based delivery offers tremendous practical advantages. Recombinant antigens such as PA would not require chemical coupling agents, and the recombinant bacteria can be administered orally where upon both mucosal and systemic immune responses are elicited.
How are the economics of synthetic biology likely to develop in the coming years?
Paratuberculosis or Johne's disease is a chronic gastric disease of ruminants. For this disease there is no effective treatment or preventive measure available. 16.8 kDa protein is an immunogenic protein of Mycobacterium avium paratuberculosis and can be an ideal candidate for developing a DNA vaccine construct. In present study a bicistronic DNA vaccine construct pIR16.8/IFN was developed using eukaryotic vector pIRES 6.1. Two genes MPT (expressing 16.8 kDa protein) and murine IFNgamma were cloned, expressed and immunoreactivity was studied in murine model. Immunoreactivity was also compared with monocistronic construct pIR16.8 expressing 16.8 kDa protein. Both pIR16.8 and pIR16.8/IFN showed eukaryotic expression of respective proteins in BHK21 cells. The expressed proteins also showed immunoreactivity when reacted with hyperimmune sera raised against recombinant 16.8 kDa protein in western blot assay and immunofluorence assay. Both constructs were used as DNA vaccine in murine model and immunogenecity was studied by DTH, lymphocyte proliferation assay and NO determination. DTH reaction was significantly high in pIR16.8/IFN than pIR16.8 group, similarly lymphocyte proliferation and NO release was higher in pIR16.8/IFN group than pIR16.8 group. This indicated T cell epitopic nature of 16.8 kDa protein. The study also showed that co-expression of IFNgamma with mycobacterial gene can enhance immunogenecity of DNA vaccine and can be used as immunoadjuvant.
A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold.
Johne's disease or paratuberculosis in cattle is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although the stages of infection have been well described, very few virulence factors of MAP have been studied in detail. We aimed to study the localization and immunogenicity of members of the polymorphic PPE protein family which is unique to Mycobacteria and has been linked to virulence in Mycobacterium tuberculosis (Mtb). The presence of PPE proteins in the cell wall was investigated by enzymatic digest of surface exposed proteins of live MAP bacteria and analysis by LC-MS/MS. Polyclonal antisera were generated against a recombinant fragment of one PPE protein and a synthetic peptide of the other to confirm their surface exposure. Sera from naturally infected cows were investigated for the presence of specific antibodies against the recombinant PPE protein. Two PPE proteins, Map3420c and Map1506, were detected by mass spectrometry and confirmed to be surface exposed on live MAP cells by immunohistochemistry. The sera from naturally infected animals contained specific antibodies against recombinantly expressed Map3420c as demonstrated by western blotting. These findings show the in vitro expression of two PPE proteins. Additionally the surface exposure and immunogenicity of PPE proteins of MAP was demonstrated.
In the News story “The bacteria fight back” (Special Section on Drug Resistance, 18 July, p. [356][1]), G. Taubes describes the ongoing war between bacteria and antibiotics, which the bacteria appear to be winning. Against this backdrop, scientists are struggling to uncover viable therapeutic
Within Escherichia coli and other species, a clear codon bias exists among the 61 amino acid codons found within the population of mRNA molecules, and the level of cognate tRNA appears directly proportional to the frequency of codon usage. Given this situation, one would predict translational problems with an abundant mRNA species containing an excess of rare low tRNA codons. Such a situation might arise after the initiation of transcription of a cloned heterologous gene in the E. coli host. Recent studies suggest clusters of AGG/AGA, CUA, AUA, CGA or CCC codons can reduce both the quantity and quality of the synthesized protein. In addition, it is likely that an excess of any of these codons, even without clusters, could create translational problems.
Conventional methods of engineering recombinant DNA make use of restriction enzymes to cut molecules apart at specific nucleotide sequences and ligases to rejoin the parts. A significant limitation of this technology is that restriction enzymes are sequence dependent and these recognition sequences appear more or less randomly in DNA. That is, restriction enzymes cut where recognition sites are located and not necessarily at optimal positions along the gene for purposes of genetic engineering. The polymerase chain reaction (PCR) has made possible a sequence-independent engineering method that is referred to as “gene splicing by overlap extension” or “SOE.” This technology is especially useful in complicated constructions that require precise recombination points—such as joining two coding sequences in frame—and it also provides a straightforward way of performing site-directed mutagenesis.
We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.
Missense substitutions and processivity errors in the translation of heterologous proteins are expected to occur at higher frequencies than the corresponding errors of normal translation. The resulting error-containing products may overload chaperone systems. Likewise, there may be a risk of an immunogenic response to heterologous proteins introduced into vertebrates. Recent work has been carried out on the mechanisms by which such errors arise and on their occurrence in cloned, heterologous gene products.
Many 'economic' studies of livestock diseases in Great Britain have been carried out over time. Most studies have considered just one or two diseases and used a different methodology and valuation base from other studies, hampering any comparative assessment of the economic impact of diseases. A standardized methodology was applied to the estimation of the direct costs to livestock production of some 30 endemic diseases/conditions of farm animals in Great Britain. This involved identification of the livestock populations at risk, estimation of the annual incidence of each disease in these populations, identification of the range and incidence of physical effects of each disease on production, valuation of the physical effects of each disease and estimation of the financial value of output losses/resource wastage due to a disease and the costs of specific treatment and prevention measures. The wider economic impacts of disease (such as the implications for human health, animal welfare and markets) were not included in the assessments. Using this standardized methodology with common financial values, a simple spreadsheet model was constructed for each disease. Given the paucity of appropriate disease data for economic assessment, 'low' and 'high' values were used to reflect uncertainties surrounding key disease parameters. Preliminary estimates of the value of disease output losses/resource wastage, treatment and prevention costs are presented for each disease. Despite the limitations of the spreadsheet models and of the estimates derived from them, we conclude that the models represent a useful start in developing a system for the comparative economic assessment of livestock diseases in Great Britain.
The poor stability of membrane proteins in detergent solution is one of the main technical barriers to their structural and functional characterization. Here we describe a solution to this problem for diacylglycerol kinase (DGK), an integral membrane protein from Escherichia coli. Twelve enhanced stability mutants of DGK were obtained using a simple screen. Four of the mutations were combined to create a quadruple mutant that had improved stability in a wide range of detergents. In n-octylglucoside, the wild-type DGK had a thermal inactivation half-life of 6 min at 55 degrees C, while the quadruple mutant displayed a half-life of 35 min at 80 degrees C. In addition, the quadruple mutant had improved thermodynamic stability. Our approach should be applicable to other membrane proteins that can be conveniently assayed.
A set of 102 peptides with affinity for the class I MHC HLA-A0201 molecule was subjected to three-dimensional quantitative structure-affinity relationship (3D QSAR) studies using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). A test set of 50 peptides was used to determine the predictive value of the models. The CoMFA models gave q(2) and r(2)pred below 0.5. The best CoMSIA model has q(2) = 0.542 and r(2)pred = 0.679, and includes hydrophobic, steric, and H-bond donor fields. The hydrophobic interactions play a dominant role in peptide-MHC molecule binding. CoMSIA coefficient contour maps were used to analyze the structural features of the peptides accounting for the affinity in terms of the three positively contributing physicochemical properties: local hydrophobicity, steric bulk and hydrogen-bond-donor ability.
In most bacteria, synonymous codons are not used with equal frequencies. Different factors have been proposed to contribute to codon usage preference, including translational selection, GC composition, strand-specific mutational bias, amino acid conservation, protein hydropathy, transcriptional selection and even RNA stability. The review discusses these factors and their contribution to bias in synonymous codon usage in bacterial genomes.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.