Ghadimi Darab, Fölster-Holst Regina, Blömer Sophia, Ebsen Michael, Röcken Christoph, Uchiyama Jumpei, Matsuzaki Shigenobu, Bockelmann Wilhelm
Department of Microbiology and Biotechnology, Max Rubner-Institut, Hermann-Weigmann-Str 1, D-24103 Kiel, Germany.
Clinic of Dermatology, Venerology und Allergology, University Hospital Schleswig-Holstein, Schittenhelmstr. 7, D-24105 Kiel, Germany.
Exp Mol Pathol. 2025 Jun;142:104967. doi: 10.1016/j.yexmp.2025.104967. Epub 2025 Apr 25.
Under an inflamed-intestinal milieu, increased free sialic acids are associated with the overgrowth of some pathogenic bacterial strains. Recently, the protective immunomodulatory activity of gut bacteriophages (phages) has also been highlighted. However, the role of phages in triple reciprocal interactions between pathogenic bacteria, beneficial bacteria, and their host cell sialic acids has not been studied so far. We established a sialidase-explicit model in which beneficial and pathogenic bacteria interact through cross-feeding and competition for free sialic acid using a human triple co-culture cell model incorporating colonocytes (T84 cells), monocytes (THP-1 cells), and hepatocytes (Huh7 cells). Triple co-cultured cells were challenged with Gram-positive Bifidobacterium bifidum (B. bifidum) and Gram-negative Pseudomonas aeruginosa PAO1 (P. a PAO1) in the absence or presence of its KPP22 phage in two different cell culture mediums: 1) standard Dulbecco's Modified Eagle Medium (DMEM) and 2) DMEM with 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA). Changes in physiological, functional, and structural health markers of stimulated cocultured cells were evaluated. The concentrations of sialic acid and pro-inflammatory cytokines in the cell culture supernatants were quantified. P. a PAO1 triggered the release of interleukin 6 and 8 (IL-6 and IL-8), accompanied by increased levels of free sialic acid, reduced viability of co-cultured cells, and disrupted the integrity of the cellular monolayer. These disruptive effects were markedly attenuated by KPP22 phage and B. bifidum. In addition to well-documented differences in the structure and composition of the bacterial cell walls of Gram-negative pathogenic bacteria and bifidobacteria, two distinct factors seem to be pivotal in modulating the pathogen-host interface milieu: (i) the presence of phages and (ii) the utilization of free sialic acids secreted from host cells by bifidobacteria.
在肠道炎症环境下，游离唾液酸增加与某些致病菌株的过度生长有关。最近，肠道噬菌体（phages）的保护性免疫调节活性也受到了关注。然而，噬菌体在病原菌、有益菌及其宿主细胞唾液酸之间三重相互作用中的作用迄今尚未得到研究。我们建立了一个唾液酸酶明确的模型，在该模型中，有益菌和病原菌通过交叉喂养以及利用包含结肠上皮细胞（T84细胞）、单核细胞（THP-1细胞）和肝细胞（Huh7细胞）的人类三重共培养细胞模型对游离唾液酸进行竞争来相互作用。在两种不同的细胞培养基中，对三重共培养细胞进行革兰氏阳性双歧双歧杆菌（B. bifidum）和革兰氏阴性铜绿假单胞菌PAO1（P. a PAO1）的挑战，同时存在或不存在其KPP22噬菌体：1）标准的杜尔贝科改良伊格尔培养基（DMEM）和2）含有2,3-脱氢-2-脱氧-N-乙酰神经氨酸（DANA）的DMEM。评估刺激后共培养细胞的生理、功能和结构健康标志物的变化。对细胞培养上清液中唾液酸和促炎细胞因子的浓度进行定量。P. a PAO1引发白细胞介素6和8（IL-6和IL-8）的释放，同时游离唾液酸水平升高，共培养细胞活力降低，并破坏细胞单层的完整性。KPP22噬菌体和双歧双歧杆菌显著减弱了这些破坏作用。除了革兰氏阴性病原菌和双歧杆菌细胞壁结构和组成方面已充分记录的差异外，两个不同的因素似乎在调节病原体-宿主界面环境中起关键作用：（i）噬菌体的存在和（ii）双歧杆菌对宿主细胞分泌的游离唾液酸的利用。
