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

Development of a PCR assay and pyrosequencing for identification of important human fish-borne trematodes and its potential use for detection in fecal specimens

BACKGROUND: Small liver and minute intestinal flukes are highly prevalent in Southeast Asia. Definitive diagnosis of parasite infection is usually achieved parasitologically by finding the fluke eggs in feces. However, their eggs are difficult to differentiate morphologically in fecal samples, even for experienced technicians. The present study developed a PCR assay coupled with DNA pyrosequencing for identification of the fish-borne trematodes (FBT), Opisthorchis viverrini, Clonorchis sinensis, Haplorchis taichui, H. pumilio and Stellantchasmus falcatus, and to evaluate potential detection in fecal specimens, and identification and differentiation of cercarial and metacercarial stages. METHODS: Primers targeting the partial 28S large subunit ribosomal RNA gene were designed and about 46–47 nucleotides were selected as the target region for species identification by a PCR assay coupled with a pyrosequencing technique. RESULTS: The nucleotide variations at 24 positions, which is sufficient for the identification of the five species of FBT were selected. The method could identify O. viverrini and C. sinensis eggs in feces, cercarial and metacercarial stages of O. viverrini, and metacercarial stage of H. pumilio and H. taichui. The detection limit was as little as a single O. viverrini or C. sinensis egg artificially inoculated in 100 mg of non-infected fecal sample (equivalent to 10 eggs per gram), indicating highly sensitivity. The method was found to be superior to the traditional microscopy method and was more rapid than Sanger DNA sequencing. CONCLUSIONS: DNA pyrosequencing-based identification is a valuable tool for differentiating O. viverrini and other Opisthorchis-like eggs, and can be applied to epidemiological studies and for molecular taxonomic investigation of FBT in endemic areas

Comparative Proteomics of Activated THP-1 Cells Infected with Mycobacterium tuberculosis Identifies Putative Clearance Biomarkers for Tuberculosis Treatment.

CAPRISA, 2015.Abstract available in pdf

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

CHaraCTEriZaTiON OF β-TuBuliN CdNa FrOM a BENZiMidaZOlE rESiSTaNT STRONGYLOIDES STERCORALIS iSOlaTE

Abstract. Previous reports have indicated that benzimidazole resistance in gastrointestinal nematodes is linked to a mutation in the β-tubulin codons 167 and 200. In our study, total RNA was isolated from an albendazole-resistant (ABZR) Strongyloides stercoralis filariform larval isolate, followed by reverse transcription PCR that was amplified using primers designed according to the alignment of the β-tubulin mRNA of S. stercoralis (GenBank accession No. AY 898942). The cDNA sequence of the β-tubulin gene of ABZR S. stercoralis larvae revealed 100% identity with the sequence of S. stercoralis (AY 898942). The polymorphisms at codons 167 and 200 encoded phenylalanine (Phe). The resistance mechanisms for benzimidazole in S. stercoralis were discussed in the light of these results