Abstract

Background. Group B Streptococcus (GBS) is a Gram-positive and commensal bacterium of the human intestine and colonizer of the genitourinary tract that is the main bacterial agent associated with sepsis, meningitis, and pneumonia in newborns. GBS may present as early onset disease (EOD) or late-onset disease (LOD). It can be prevented by intrapartum antibiotic prophylaxis (IAP), thus avoiding only EOD-type infections. LOD-type infections continue to cause morbidity and mortality. The Surface Immunogenic Protein (SIP) of GBS is present in all its serotypes and is highly conserved. It could be a potential target of GBS vaccine. Lactococcus lactis is widely used in heterologous gene expression and vaccine development. In a preclinical model, a L. lactis that secretes SIP generated a protective humoral and cellular immune response and is an ideal candidate for oral immunization in women. Thus, a strain of Lactococcus lactis that has the sip gene integrated into its genome would be a great advance in the development of an oral vaccine against Streptococcus agalactiae. Methods. Two strategies were used: double crossover homologous recombination based on a suicide vector and gene editing by CRISPR-Cas9. In the first strategy, the pMBsacB plasmid—a thermosensitive plasmid vector cloned with the sacB gene—was used for sucrose counterselection. Second, with CRISPR-Cas9, two cuts were generated to excise the upp gene and then the sip gene was inserted into the L. lactis genome by homologous recombination. Results. We designed two strategies for excision of the L. lactis upp gene and insertion of the sip gene: homologous recombination or by CRISPR-Cas9. The excision of the upp gene generates resistance to 5-fluorouracil, thus serving as a selection marker for mutants. Conclusion. The strategies designed to obtain a knock-in of the sip gene in L. lactis by cleaving the upp gene are functional and sufficient to obtain a potential vaccine for GBS.