Miyoshi Anderson, Bermúdez-Humarán Luis G, Ribeiro Luciana A, Le Loir Yves, Oliveira Sérgio C, Langella Philippe, Azevedo Vasco
Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brasil.
Microb Cell Fact. 2006 Mar 23;5:14. doi: 10.1186/1475-2859-5-14.
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 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.
Only 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.
We 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.
流产布鲁氏菌是一种兼性胞内病原体，主要感染牛和人类。目前的疫苗依赖于减毒活的流产布鲁氏菌菌株，这些菌株可能会恢复其致病状态，因此在人体中使用并不完全安全。因此，开发以食品级乳酸菌乳酸乳球菌作为抗原递送载体的黏膜活疫苗，是一种有吸引力的替代方法，也是一种针对流产布鲁氏菌更安全的疫苗接种策略。在此，我们报告了对乳酸乳球菌菌株进行基因改造，使其在细胞内或分泌这两个细胞位置产生流产布鲁氏菌GroEL热休克蛋白（一种候选抗原）。
GroEL仅以分泌形式在乳酸乳球菌中稳定产生，这表明GroEL蛋白在该细菌细胞内产生时具有有害作用。在诱导的乳球菌培养物的上清液部分仅检测到微量的成熟GroEL，并且GroEL前体仍堆积在细胞部分中。尝试提高分泌产量，但即使将GroEL与合成前肽融合，该抗原的分泌也没有改善。
我们发现乳酸乳球菌能够产生并将稳定形式的GroEL分泌到细胞外培养基中。尽管GroEL的分泌效率较低，这表明该抗原与乳酸乳球菌的细胞膜相互作用，但分泌似乎是实现产量和蛋白质产量的最佳方式，无论细胞位置如何。分泌GroEL的乳酸乳球菌菌株具有体内免疫的潜力。
