Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines

BACKGROUND: The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization. RESULTS: Bacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy. CONCLUSION: Lactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can be used as live oral vaccines to immunize broilers against infectious diseases

Analysis of expressed sequence tags from Trypanosoma cruzi amastigotes

A total of 880 expressed sequence tags (EST) originated from clones randomly selected from a Trypanosoma cruzi amastigote cDNA library have been analyzed. Of these, 40% (355 ESTs) have been identified by similarity to sequences in public databases and classified according to functional categorization of their putative products. About 11% of the mRNAs expressed in amastigotes are related to the translational machinery, and a large number of them (9% of the total number of clones in the library) encode ribosomal proteins. A comparative analysis with a previous study, where clones from the same library were selected using sera from patients with Chagas disease, revealed that ribosomal proteins also represent the largest class of antigen coding genes expressed in amastigotes (54% of all immunoselected clones). However, although more than thirty classes of ribosomal proteins were identified by EST analysis, the results of the immunoscreening indicated that only a particular subset of them contains major antigenic determinants recognized by antibodies from Chagas disease patients

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

Repeat-Driven Generation of Antigenic Diversity in a Major Human Pathogen, Trypanosoma cruzi.

Trypanosoma cruzi, a zoonotic kinetoplastid protozoan parasite, is the causative agent of American trypanosomiasis (Chagas disease). Having a very plastic, repetitive and complex genome, the parasite displays a highly diverse repertoire of surface molecules, with pivotal roles in cell invasion, immune evasion and pathogenesis. Before 2016, the complexity of the genomic regions containing these genes impaired the assembly of a genome at chromosomal level, making it impossible to study the structure and function of the several thousand repetitive genes encoding the surface molecules of the parasite. We here describe the genome assembly of the Sylvio X10/1 genome sequence, which since 2016 has been used as a reference genome sequence for T. cruzi clade I (TcI), produced using high coverage PacBio single-molecule sequencing. It was used to analyze deep Illumina sequence data from 34 T. cruzi TcI isolates and clones from different geographic locations, sample sources and clinical outcomes. Resolution of the surface molecule gene distribution showed the unusual duality in the organization of the parasite genome, a synteny of the core genomic region with related protozoa flanked by unique and highly plastic multigene family clusters encoding surface antigens. The presence of abundant interspersed retrotransposons in these multigene family clusters suggests that these elements are involved in a recombination mechanism for the generation of antigenic variation and evasion of the host immune response on these TcI strains. The comparative genomic analysis of the cohort of TcI strains revealed multiple cases of such recombination events involving surface molecule genes and has provided new insights into T. cruzi population structure

Identification to the species level of <it>Lactobacillus </it>isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling

Abstract Background The accurate identification of Lactobacillus and other co-isolated bacteria during microbial ecological studies of ecosystems such as the human or animal intestinal tracts and food products is a hard task by phenotypic methods requiring additional tests such as protein and/or lipids profiling. Results Bacteria isolated in different probiotic prospecting studies, using de Man, Rogosa and Sharpe medium (MRS), were typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR products. The set of enzymes chosen differentiates most species of Lactobacillus genus and also co-isolated bacteria such as Enterococcus, Streptococcus, Weissella, Staphylococcus, and Escherichia species. The in silico predictions of restriction patterns generated by the Lactobacillus shorter spacers digested with 11 restriction enzymes with 6 bp specificities allowed us to distinguish almost all isolates at the species level but not at the subspecies one. Simultaneous theoretical digestions of the three spacers (long, medium and short) with the same set of enzymes provided more complex patterns and allowed us to distinguish the species without purifying and cloning of PCR products. Conclusion Lactobacillus isolates and several other strains of bacteria co-isolated on MRS medium from gastrointestinal ecosystem and fermented food products could be identified using DNA fingerprints generated by restriction endonucleases. The methodology based on amplified ribosomal DNA restriction analysis (ARDRA) is easier, faster and more accurate than the current methodologies based on fermentation profiles, used in most laboratories for the purpose of identification of these bacteria in different prospecting studies.</p

PCR amplification of rRNA spacer regions was done and the shorter amplicon was excised from an agarose gel after electrophoresis at 100 V for 45 min

<p><b>Copyright information:</b></p><p>Taken from "Identification to the species level of isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling"</p><p>BMC Microbiology 2005;5():15-15.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1079852.</p><p>Copyright © 2005 Moreira et al; licensee BioMed Central Ltd.</p> Gel purified DNA was cleaved with 11 enzymes and fragments separated by electrophoresis as before. Plus and minus signs mean positive or negative cleavage of PCR amplicon. GibcoBRL 1 kb DNA ladder was used for sizing DNA fragments and molecular masses are shown at right in bp

PCR amplification of rRNA spacer regions was done and amplicons were cleaved with 11 restriction enzymes and fragments separated by agarose gel electrophoresis at 100 V for 45 min

<p><b>Copyright information:</b></p><p>Taken from "Identification to the species level of isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling"</p><p>BMC Microbiology 2005;5():15-15.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1079852.</p><p>Copyright © 2005 Moreira et al; licensee BioMed Central Ltd.</p> Plus and minus signs mean positive or negative cleavage of long, medium or short PCR amplicon, respectively. GibcoBRL 1 kb DNA ladder was used for sizing DNA fragments and molecular masses are shown at right in bp

Genetic transformation of novel isolates of chicken bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines-3

<p><b>Copyright information:</b></p><p>Taken from "Genetic transformation of novel isolates of chicken bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines"</p><p>BMC Biotechnology 2006;6():2-2.</p><p>Published online 5 Jan 2006</p><p>PMCID:PMC1360068.</p><p>Copyright © 2006 Mota et al; licensee BioMed Central Ltd.</p>ium azide, and photographed under a laser scanning microscope (LSM) at scale of 54,8 × 54,8 μm. (C) Represents merged images of (A,B), and (F) represents merged images of (D,E)

Genetic transformation of novel isolates of chicken bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines-2

<p><b>Copyright information:</b></p><p>Taken from "Genetic transformation of novel isolates of chicken bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines"</p><p>BMC Biotechnology 2006;6():2-2.</p><p>Published online 5 Jan 2006</p><p>PMCID:PMC1360068.</p><p>Copyright © 2006 Mota et al; licensee BioMed Central Ltd.</p>striction fragments onto pCR2.1-TOPO vector. The genes AM and mut2 were also PCR amplified from other plasmids and used as selective marker and reporter gene, respectively