Abstract
BackgroundBrucella abortus is a facultative intracellular pathogen that mainly infects cattle and humans. Current vaccines rely on live attenuated strains of B. abortus, which can revert to their pathogenic status and thus are not totally safe for use in humans. Therefore, the development of mucosal live vaccines using the food-grade lactic acid bacterium, Lactococcus lactis, as an antigen delivery vector, is an attractive alternative and a safer vaccination strategy against B. abortus. Here, we report the construction of L. lactis strains genetically modified to produce B. abortus GroEL heat-shock protein, a candidate antigen, in two cellular locations, intracellular or secreted.ResultsOnly the secreted form of GroEL was stably produced in L. lactis, suggesting a detrimental effect of GroEL protein when intracellularly produced in this bacterium. Only trace amounts of mature GroEL were detected in the supernatant fraction of induced lactococcal cultures, and the GroEL precursor remained stacked in the cell fraction. Attempts to raise the secretion yields were made, but even when GroEL was fused to a synthetic propeptide, secretion of this antigen was not improved.ConclusionWe found that L. lactis is able to produce, and to secrete, a stable form of GroEL into the extracellular medium. Despite the low secretion efficiency of GroEL, which suggest that this antigen interacts with the cell envelope of L. lactis, secretion seems to be the best way to achieve both production and protein yields, regardless of cellular location. The L. lactis strain secreting GroEL has potential for in vivo immunization.
Highlights
Brucella abortus is a facultative intracellular pathogen that mainly infects cattle and humans
Construction of recombinant L. lactis strains to produce either cytoplasmic or secreted forms of GroEL As the protective response depends on the antigen, the delivery system and the location of the antigen [25,35,36], we evaluated the impact of B. abortus GroEL production by L. lactis in two different cellular locations, intracellular and secreted
Two expression vectors were initially constructed, pCYT:groEL and pSEC:groEL for cytoplasmic and secreted GroEL production, respectively. These plasmids are derived from two broad-host-range expression vectors, pCYT:Nuc and pSEC:Nuc (Table 1; [34]); pCYT:Nuc harbors a transcriptional fusion between the ribosome-binding site (RBSusp45) of the usp45 gene [37] and the DNA sequence encoding the mature part of the staphylococcal nuclease, NucB [38] (Table 1), and pSEC:Nuc harbors a transcriptional fusion between RBSusp45 and the DNA sequence encoding the signal peptide (SPusp45) of Usp45 plus nucB (Table 1)
Summary
Brucella abortus is a facultative intracellular pathogen that mainly infects cattle and humans. Current vaccines rely on live attenuated strains of B. abortus, which can revert to their pathogenic status and are not totally safe for use in humans. Current strategies for the development of new vaccines against B. abortus have been based on the identification of immunodominant antigens able to elicit a cellular immune response [6,7,8,9], which is required to resist the intracellular location of this pathogen [10,11,12]. Cattle and mice infected with B. abortus exhibit an immune response to GroEL [8,16,17] Taken together, these observations suggest the potential of GroEL as a candidate antigen for the development of a brucellosis vaccine
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