Mucosal Immunology and Biology Research Center, Division of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital.
Mucosal Immunology and Biology Research Center, Division of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital; Department of Pediatrics, Harvard Medical School.
J Vis Exp. 2024 Feb 9(204). doi: 10.3791/66426.
The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.
人类肠道病原体志贺氏菌每年导致数百万人感染，对儿科患者造成长期生长影响，是全球腹泻死亡的主要原因。病原体通过胃肠道并感染结肠上皮细胞，导致水样或血性腹泻。由于抗生素耐药性惊人增加和目前缺乏批准的疫苗，因此标准化的研究方案对于研究这种强大的病原体至关重要。在这里，提出了使用体外分析结肠上皮细胞中细菌粘附、侵袭和细胞内复制来研究志贺氏菌分子发病机制的方法。在感染分析之前，通过在琼脂平板上吸收刚果红染料来验证志贺氏菌菌落的毒力表型。在细菌培养过程中也可以考虑补充实验室培养基，以模拟体内条件。然后，将细菌细胞按照标准化方案用于组织培养板中的结肠上皮细胞感染，感染倍数固定，并进行适应性分析以研究感染的各个阶段。对于粘附测定，将志贺氏菌细胞在低营养水平下孵育，以促进细菌与上皮细胞的接触。对于侵袭和细胞内复制测定，应用庆大霉素进行不同时间间隔的处理，以消除细胞外细菌，并能够评估侵袭和/或细胞内复制率。所有感染方案通过连续稀释感染的上皮细胞裂解物并将细菌集落形成单位相对于刚果红琼脂平板上的感染滴度进行平板计数来定量计算粘附、侵袭和/或细胞内的细菌。总之，这些方案能够独立表征和比较志贺氏菌感染上皮细胞的各个阶段，以成功研究这种病原体。
