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.


CONCLUSIONS
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.
Mota
Rodrigo M
RM
Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. rodrigo_m_mota@yahoo.com.br
Moreira
João Luiz S
JL
Souza
Marcelo R
MR
Horta
M Fátima
MF
Teixeira
Santuza M R
SM
Neumann
Elisabeth
E
Nicoli
Jacques R
JR
Nunes
Alvaro C
AC
D000284:Administration, Oral; D000818:Animals; D001426:Bacterial Proteins / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology; D001715:Bird Diseases / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control; D002645:Chickens / Q000276:immunology / Q000382:microbiology*; D024861:Genetic Enhancement / Q000379:methods; D007778:Lactobacillus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D019936:Probiotics / Q000008:administration & dosage*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000276:immunology / Q000378:metabolism; D014169:Transformation, Bacterial / Q000235:genetics
D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D011994:Recombinant Proteins