Souza Renata F S, Jardin Julien, Cauty Chantal, Rault Lucie, Bouchard Damien S, Bermúdez-Humarán Luis G, Langella Philippe, Monedero Vicente, Seyffert Núbia, Azevedo Vasco, Le Loir Yves, Even Sergine
INRA, UMR 1253 STLO, Rennes, France.
Agrocampus Ouest, UMR1253 STLO, Rennes, France.
PLoS One. 2017 Mar 21;12(3):e0174060. doi: 10.1371/journal.pone.0174060. eCollection 2017.
Probiotics have been considered as a promising strategy to prevent various diseases in both humans and animals. This approach has gained interest in recent years as a potential means to control bovine mastitis. In a previous study, we found that several L. casei strains, including BL23, were able to inhibit the internalization of S. aureus, a major etiologic agent of mastitis, into bovine mammary epithelial cells (bMEC). This antagonism required a direct contact between L. casei and bMEC or S. aureus, suggesting the inhibition relied on interactions between L. casei cell surface components and bMEC. In this study, we have investigated the impact of some candidates which likely influence bacteria host cell interactions. We have shown that L. casei BL23 fbpA retained its inhibitory potential, indicating that L. casei BL23 antagonism did not rely (solely) on competition between S. aureus and L. casei fibronectin-binding proteins for adhesion to bMEC. We have then investigated the impact of four sortase mutants, srtA1, srtA2, srtC1 and srtC2, and a double mutant (srtA1-srtA2) on L. casei BL23 inhibitory potential. Sortases are responsible for the anchoring on the bacterial cell wall of LPXTG-proteins, which reportedly play an important role in bacteria-host cell interaction. All the srt mutants tested presented a reduced inhibition capacity, the most pronounced effect being observed with the srtA2 mutant. A lower internalization capacity of L. casei srtA2 into bMEC was also observed. This was associated with several changes at the surface of L. casei BL23 srtA2 compared to the wild type (wt) strain, including altered abundance of some LPXTG- and moonlighting proteins, and modifications of cell wall structure. These results strongly support the role of sortase A2 in L. casei BL23 inhibition against S. aureus internalization. Deciphering the contribution of the cell surface components altered in srtA2 strain in the inhibition will require further investigation.
益生菌已被视为预防人类和动物各种疾病的一种有前景的策略。近年来，这种方法作为控制牛乳腺炎的一种潜在手段受到了关注。在之前的一项研究中，我们发现包括BL23在内的几种干酪乳杆菌菌株能够抑制乳腺炎的主要病原体金黄色葡萄球菌内化到牛乳腺上皮细胞（bMEC）中。这种拮抗作用需要干酪乳杆菌与bMEC或金黄色葡萄球菌直接接触，这表明抑制作用依赖于干酪乳杆菌细胞表面成分与bMEC之间的相互作用。在本研究中，我们研究了一些可能影响细菌与宿主细胞相互作用的候选因素的影响。我们已经表明，干酪乳杆菌BL23 fbpA保留了其抑制潜力，这表明干酪乳杆菌BL23的拮抗作用并不（仅仅）依赖于金黄色葡萄球菌和干酪乳杆菌纤连蛋白结合蛋白之间对bMEC黏附的竞争。然后，我们研究了四种分选酶突变体srtA1、srtA2、srtC1和srtC2以及一个双突变体（srtA1 - srtA2）对干酪乳杆菌BL23抑制潜力的影响。分选酶负责将LPXTG蛋白锚定在细菌细胞壁上，据报道LPXTG蛋白在细菌与宿主细胞相互作用中起重要作用。所有测试的srt突变体的抑制能力均降低，其中srtA2突变体的效果最为明显。还观察到干酪乳杆菌srtA2内化到bMEC中的能力较低。这与干酪乳杆菌BL23 srtA2与野生型（wt）菌株相比表面的几个变化有关，包括一些LPXTG和兼性蛋白丰度的改变以及细胞壁结构的修饰。这些结果有力地支持了分选酶A2在干酪乳杆菌BL23抑制金黄色葡萄球菌内化中的作用。进一步研究将需要阐明srtA2菌株中改变的细胞表面成分在抑制中的贡献。
