Development of an Escherichia coli–Lactobacillus casei shuttle vector for heterologous protein expression in Lactobacillus casei

There is an increasing interest to develop various lactic acid bacteria (LAB) species as mucosal delivery vehicles, for which the development of a variety of cloning and expression systems for these bacteria is of primary importance. This study reports the complete nucleotide sequence of the cryptic plasmid pRCEID7.6 derived from the chicken probiotic LAB strain Lactobacillus casei TISTR1341. Sequence analysis and comparison showed that pRCEID7.6 is composed of nine putative open reading frames. The replicon origin of pRCEID7.6 consisted of untranslated origin of replication and translated replication protein B sequences. This region was used to construct Escherichia coli/L. casei shuttle vectors carrying erythromycin and chloramphenicol resistance genes as selective markers. Segregation and structural stability of the vectors in L. casei was sufficient for most genetic applications. The feasibility of this vector for heterologous protein expression in L. casei was determined by cloning in pRCEID-LC7.6, the gene encoding the nucleocapsid protein (NP), from the influenza A virus under the control of the homologous promoter from the lactate dehydrogenase gene. L. casei carrying this recombinant plasmid was shown to successfully express the NP protein. Therefore, this shuttle vector can be used for further study in the development of mucosal delivery vehicles.Peer Reviewe

Cloning and expression of a codon-optimized gene encoding the infl uenza A virus nucleocapsid protein in Lactobacillus casei

Lactic acid bacteria (LAB) species are envisioned as promising vehicles for the mucosal delivery of therapeutic and prophylactic molecules, including the development of oral vaccines. In this study, we report on the expression of a synthetic nucleocapsid (NP) gene of infl uenza A virus in Lactobacillus casei. The NP gene was re-designed based on the tRNA pool and the codon usage preference of L. casei BL23. The codon-optimized NP gene was then cloned and expressed in L. casei RCEID02 under the control of a constitutive promoter, that of the lactate dehydrogenase (ldh) gene. The synthetic NP gene was further expressed in L. casei EM116 under the control of an inducible promoter, that of the structural gene of nisin (nisA) from Lactococcus lactis. Based on Western blot analysis, the specifi c protein band of NP, with a molecular mass of 56.0 kDa, was clearly detected in both expression systems. Thus, our study demonstrates the success of expressing a codon-optimized infl uenza A viral gene in L. casei. The suitability of the recombinant LAB strains for immunization purposes is currently under evaluation. [Int Microbiol 2013; 16(2):93-101]Keywords: Lactobacillus casei; lactic acid bacteria; infl uenza A virus; viral nucleocapsid proteins; heterologous expression; codon usag

Sequencing and analysis of three plasmids from Lactobacillus casei TISTR1341 and development of plasmid-derived Escherichia coli-L. casei shuttle vectors

El pdf del artículo es la versión pre-print.-- et al.Pyrosequencing followed by conventional PCR and sequencing was used to determine the complete nucleotide sequence of three plasmids (pRCEID2.9, pRCEID3.2, and pRCEID13.9) from the Lactobacillus casei strain TISTR1341. The plasmid sequences were found to be almost identical, respectively, to those of pLA106, pLA105, and pLA103 from Lactobacillus acidophilus strain TK8912, suggesting that these strains may be related. Sequence analysis and comparison indicated that pRCEID2.9 replicates by a rolling circle (RC) mechanism, while pRCEID3.2 and pRCEID13.9 probably follow a theta-type mode of replication. Replicons of pRCEID2.9 and pRCEID13.9 were used to develop Escherichia coli/L. casei compatible shuttle vectors, which were stably maintained in different genetic backgrounds. Real-time quantitative PCR analysis showed copy numbers of around 4 and 15, respectively, for the pRCEID13.9- and pRCEID2.9-derived shuttle vectors per chromosome equivalent. The functionality of vector pRCEID-LC13.9 was proved by cloning and expressing in L. casei of a green fluorescent protein gene variant from Aequorea victoria under the control of the promoter from a homologous lactate dehydrogenase gene. The new vectors might complement those currently in use for the exploitation of L. casei as a cellular factory and in other biotechnological applications. © 2011 Springer-Verlag.This study was supported by grants from the Office of the Higher Education Commission through CHE Ph.D. Scholarship (to M.P.) and through the Higher Education Research Promotion and National Research University Project (to V.L.), the National Center for Genetics Engineering and Biotechnology (BIOTEC), and the Research and Diagnostic Center for Emerging Infectious Diseases (RCEID), Khon Kaen University, Thailand. Further support was obtained through a project from the Spanish Ministry of Science and Innovation (ref. AGL2007-61869-ALI) (to B.M.).Peer Reviewe

Lactic acid bacteria: an alternative platform for live vaccine development

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.Peer Reviewe

Evaluation of Nano-Wall Material for Production of Novel Lyophilized-Probiotic Product

Lyophilization is one of the most used methods for bacterial preservation. In this process, the cryoprotectant not only largely decreases cellular damage but also plays an important part in the conservation of viability during freeze-drying. This study investigated using cryoprotectant and a mixture of the cryoprotectant to maintain probiotic activity. Seven probiotic strains were considered: (Limosilactobacillus reuteri KUKPS6103; Lacticaseibacillus rhamnosus KUKPS6007; Lacticaseibacillus paracasei KUKPS6201; Lactobacillus acidophilus KUKPS6107; Ligilactobacillus salivarius KUKPS6202; Bacillus coagulans KPSTF02; Saccharomyces cerevisiae subsp. boulardii KUKPS6005) for the production of a multi-strain probiotic and the complex medium for the lyophilized synbiotic production. Cholesterol removal, antioxidant activity, biofilm formation and gamma aminobutyric acid (GABA) production of the probiotic strains were analyzed. The most biofilm formation occurred in L. reuteri KUKPS6103 and the least in B. coagulans KPSTF02. The multi-strain probiotic had the highest cholesterol removal. All the probiotic strains had GABA production that matched the standard of γ-aminobutyric acid. The lyophilized synbiotic product containing complex medium as a cryoprotectant and wall material retained a high viability of 7.53 × 108 CFU/g (8.89 log CFU/g) after 8 weeks of storage. We found that the survival rate of the multi-strain probiotic after freeze-drying was 15.37% in the presence of complex medium that was used as high performing wall material. Our findings provided a new type of wall material that is safer and more effective and, can be extensively applied in relevant food applications

Cloning and expression of enterovirus 71 capsid protein 1 in a probiotic Bifidobacterium pseudocatenulatum

This study investigated cloning and expression of enterovirus 71 viral capsid protein 1 (EV71‐VP1) in Bifidobacterium pseudocatenulatum (B. pseudocatenulatum) M115. To achieve this, a codon‐optimized gene coding for EV71‐VP1 was analysed, designed, synthesized and cloned into a plasmid vector flanked by a transcriptional promoter and terminator sequences. The promoter was based on that of P919, a constitutive promoter of the gene encoding the large ribosomal protein of B. bifidum BGN4, while the terminator was based on that of the peptidase N gene of Lactococcus lactis. The construct was amplified in Escherichia coli XL1‐blue and then transferred into B. pseudocatenulatum M115 by electrotransformation. Western blot analysis revealed that the EV71‐VP1 was intracellularly expressed in B. pseudocatenulatum M115 under the control of the selected heterologous promoter. In addition, plasmid stability analysis showed the construct was maintained stably for more than 160 generations, enough for most future applications. The results derived from this study open the possibility to utilize the bacterium carrying a specific expression plasmid as cell factory for the production of proteins with high commercial and health‐promoting value.This study was supported by the Office of the HigherEducation Commission, the College of Medicine andPublic Health, and Ubon Ratchathani University, and theThailand Research Fund (TRF Grant No. MRG5680081 toMP), the Higher Education Research Promotion andNational Research University Project of Thailand to VL,and the Spanish Ministry of Economy and Competitive-ness (Project No. AGL-2014-57820-R to BM).Peer reviewe

Secretion of M2e:HBc fusion protein by Lactobacillus casei using Cwh signal peptide

The ability to serve as a delivery vehicle for various interesting biomolecules makes lactic acid bacteria (LAB) very useful in several applications. In the medical field, recombinant LAB expressing pathogenic antigens at different cellular locations have been used to elicit both mucosal and systemic immune responses. Expression-secretion vectors (ESVs) with a signal peptide (SP) are pivotal for protein expression and secretion. In this study, the genome sequence of Lactobacillus casei ATCC334 was explored for new SPs using bioinformatics tools. Three new SPs of the proteins Cwh, SurA and SP6565 were identified and used to construct an ESV based on our Escherichia coli-L. casei shuttle vector, pRCEID-LC13.9. Functional testing of these constructs with the green fluorescence protein (GFP) gene showed that they could secrete the GFP. The construct with CwhSP showed the highest GFP secretion. Consequently, CwhSP was selected to develop an ESV construct carrying a synthetic gene encoding the extracellular domain of the matrix 2 protein fused with the hepatitis B core antigen (M2e:HBc). This ESV was shown to efficiently express and secrete the M2e:HBc fusion protein. The identified SPs and the developed ESVs can be exploited for expression and secretion of homologous and heterologous proteins in L. casei.This study was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, Thailand and by the invitation research grant (I57301) from the Faculty of Medicine, Khon Kaen University.Peer Reviewe

Cloning and expression of a codon-optimized gene encoding the influenza A virus nucleocapsid protein in Lactobacillus casei

Lactic acid bacteria (LAB) species are envisioned as promising vehicles for the mucosal delivery of therapeutic and prophylactic molecules, including the development of oral vaccines. In this study, we report on the expression of a synthetic nucleocapsid (NP) gene of influenza A virus in Lactobacillus casei. The NP gene was re-designed based on the tRNA pool and the codon usage preference of L. casei BL23. The codon-optimized NP gene was then cloned and expressed in L. casei RCEID02 under the control of a constitutive promoter, that of the lactate dehydrogenase (ldh) gene. The synthetic NP gene was further expressed in L. casei EM116 under the control of an inducible promoter, that of the structural gene of nisin (nisA) from Lactococcus lactis. Based on Western blot analysis, the specific protein band of NP, with a molecular mass of 56.0 kDa, was clearly detected in both expression systems. Thus, our study demonstrates the success of expressing a codon-optimized influenza A viral gene in L. casei. The suitability of the recombinant LAB strains for immunization purposes is currently under evaluation.This study was supported by the Higher Education Research Promotion and the National Research University Project of Thailand, Office of the Higher Education Commission, and by The National Center for Genetic Engineering and Biotechnology, Thailand.Peer Reviewe

Antibiotic resistance-susceptibility profiles of enterococcus faecalis and streptococcus spp. from the human vagina, and genome analysis of the genetic basis of intrinsic and acquired resistances

The spread of antibiotic resistance is a major public health concern worldwide. Commensal bacteria from the human genitourinary tract can act as reservoirs of resistance genes playing a role in their transfer to pathogens. In this study, the minimum inhibitory concentration of 16 antibiotics to 15 isolates from the human vagina, identified as Enterococcus faecalis, Streptococcus anginosus, and Streptococcus salivarius, was determined. Eight isolates were considered resistant to tetracycline, five to clindamycin and quinupristin-dalfopristin, and four to rifampicin. To investigate the presence of antimicrobial resistance genes, PCR analysis was performed in all isolates, and five were subjected to whole-genome sequencing analysis. PCR reactions identified tet(M) in all tetracycline-resistant E. faecalis isolates, while both tet(M) and tet(L) were found in tetracycline-resistant S. anginosus isolates. The tet(M) gene in E. faecalis VA02-2 was carried within an entire copy of the transposon Tn916. In S. anginosus VA01-10AN and VA01-14AN, the tet(M) and tet(L) genes were found contiguous with one another and flanked by genes encoding DNA mobilization and plasmid replication proteins. Amplification and sequencing suggested the lsaA gene to be complete in all E. faecalis isolates resistant to clindamycin and quinupristin-dalfopristin, while the gene contain mutations rendering to a non-functional LsaA in susceptible isolates. These results were subsequently confirmed by genome analysis of clindamycin and quinupristindalfopristin resistant and susceptible E. faecalis strains. Although a clinical breakpoint to kanamycin for S. salivarius has yet to be established, S. salivarius VA08-2AN showed an MIC to this antibiotic of 128 µg mL−1 . However, genes involved in kanamycin resistance were not identified. Under the assayed conditions, neither tet(L) nor tet(M) from either E. faecalis or S. anginosus was transferred by conjugation to recipient strains of E. faecalis, Lactococcus lactis, or Lactobacillus plantarum. Nonetheless, the tet(L) gene from S. anginosus VA01-10AN was amplified by PCR, and cloned and expressed in Escherichia coli, to which it provided a resistance of 48–64 µg mL−1 to tetracycline. Our results expand the knowledge of the antibiotic resistance-susceptibility profiles of vaginal bacteria and provide the genetic basis of their intrinsic and acquired resistanceThis study was supported by a grant of the Royal Golden Jubilee (RGJ) Ph.D. Program from the Thailand Research Fund (TRF), Thailand, Ref. PHD/0133/2558, and a project from Asturias Principality, Spain, Ref. IDI/2018/000114.Peer reviewe

Antibiotic Susceptibility Profiles of Lactic Acid Bacteria from the Human Vagina and Genetic Basis of Acquired Resistances

© 2020 by the authors.Lactic acid bacteria can act as reservoirs of antibiotic resistance genes that can be ultimately transferred to pathogens. The present work reports on the minimum inhibitory concentration (MIC) of 16 antibiotics to 25 LAB isolates of five Lactobacillus and one Bifidobacterium species from the human vagina. Acquired resistances were detected to kanamycin, streptomycin, chloramphenicol, gentamicin, and ampicillin. A PCR analysis of lactobacilli failed to identify genetic determinants involved in any of these resistances. Surprisingly, a tet(W) gene was detected by PCR in two Bifidobacterium bifidum strains, although they proved to be tetracycline-susceptible. In agreement with the PCR results, no acquired genes were identified in the genome of any of the Lactobacillus spp. strains sequenced. A genome analysis of B. bifidum VA07-1AN showed an insertion of two guanines in the middle of tet(W) interrupting the open reading frame. By growing the strain in the presence of tetracycline, stable tetracycline-resistant variants were obtained. An amino acid substitution in the ribosomal protein S12 (K43R) was further identified as the most likely cause of VA07-1AN being streptomycin resistance. The results of this work expand our knowledge of the resistance profiles of vaginal LAB and provide evidence for the genetic basis of some acquired resistances.This study was supported by projects from the Royal Golden Jubilee (RGJ)-PhD Program Grant of the Thailand Research Fund (TRF), Thailand (Ref. PHD/0133/2558), and Asturias Principality, Spain (Ref. IDI/2018/000114).Peer reviewe