황색포도알균의 감염에 따른 세포 내에서의 균의 증식과 Matrix Metalloproteinase (MMP)의 역할 ; Roles of Matrix Metalloproteinases on Intracellular Staphylococcus aureus Growth in Bronchial Epithelial Cell

Background: Staphylococcus aureus frequently colonizes and infects hospitalized patients. Respiratory infections with Staphylococcus aureus are common in patients with compromised airway defenses. However the mechanisms of S. aureus invasion from colonization to the epithelium are unclear. Cell invasion by S. aureus would require destruction of the extracellular matrix, which is believed to be the result of increased matrix metalloproteinases (MMP) activity. Methods: In this study, respiratory epithelial cells were infected with S. aureus. After removing the extracellular bacteria by washing, the internalized bacteria in the cells were assessed by counting the colonized forming units (CFUs). The cell adhesion proteins, dysadherin and E-cadherin, were evaluated by Western blotting. The MMPs in the bacterial invasion were evaluated by pretreating the cells with GM6001, a MMP inhibitor. Results: The internalization of S. aureus was found to be both time and dose dependent, and the increase in MMP 2 and 9 activity was also dependent on the incubation time and the initial amount of bacterial inoculation. The invasion of S. aureus was attenuated by GM6001 after 12 hours incubation with a multiply of infection (MOI)=50. The expression of dysadherin, a membrane protein, was increased in a time and dose dependent manner, while the expression of E-cadherin was decreased. Conclusion: MMPs may mediate the invasion of S. aureus into epithelial cells. (Tuberc Respir Dis 2008;64:22-27) Key Words: Staphylococcus aureus, Epithelial cell, Matrix metalloproteinases ; 연구배경: 본 연구에서는 황색포도알균이 숙주세포 내 로 침입하여 증식하는 정도를 관찰하고자 한다. 이때 세 포 외 바탕 단백질의 변화가 수반될 것으로 가설을 설정하 고, 이러한 변화에 영향을 미칠 것으로 생각되는 matrix metalloproteinase (MMP)의 발현과 역할에 대해 연구하 고자 하였다. 방 법: 황색포도알균은 106∼107 CFU/ml을 105개의 기 관지상피세포인 BEAS-2B 세포에 2시간 동안 침입시킨다. 이후 세척으로 세포 밖에 있는 황색포도알균을 제거한 후, BEAS-2B 세포를 다양한 시간 동안(4, 6, 8, 12 시간) 배양 한 후 황색포도알균의 집락수(CFU/ml)를 측정하였고, 단 백질을 분리하여 세포 외 바탕단백질의 발현 정도와 MMP 의 활성도를 측정하였다. 또한 MMP 억제제인 GM6001을 전처치한 후 황색포도알균을 세포에 침입시킨 후 세포 내 에서의 균의 집락수 및 세포 외 ...