Internalization of Gram-Negative Bacteria into Osteocyte-like Cells Is a Host Cell-Mediated Process
Location
Philadelphia, PA
Start Date
9-5-2018 1:00 PM
Description
Introduction: Chronic osteomyelitis, an infection of bone with many etiological origins associated with a diverse pool of infectious microorganisms, is often a polymicrobial infection with Staphylococcus aureus as the predominant species with an assortment of other bacteria including Gram-negative species. As it has been proposed that bacterial internalization is a critical step in the progression of chronic osteomyelitis, we propose the hypothesis that Gram-negative bacteria can be internalized by osteocytes via a host cell-mediated process.
Methods: To test this hypothesis, we employed an internalization assay and two osteocyte-like cell lines, MLO-A5 and MLO-Y4, with the former being the less differentiated of the two cell lines. The cells were pre-treated with endocytosis inhibitor drugs (methyl-β-cyclodextrin and chloroquine), and exposed to the Gram-negative bacteria, Serratia marscesens and Proteus mirabilis. After the internalization protocol, extracellular bacteria were killed using an antibiotic solution containing gentamicin and ciprofloxacin. Finally, the cells were lysed to release internalized bacteria and the resultant colonies were counted.
Results: Exposure to chloroquine and methyl-β-cyclodextrin had no appreciable impact on cell viability for the osteocyte-like cell lines or bacteria under the conditions tested. Both drugs successfully reduced the number of internalized bacteria compared to the controls. Chloroquine reduced internalization of both bacterial species by greater than 50% in each cell line. Methyl-β-cyclodextrin reduced internalization of both species by greater than 90% in MLO-A5 cells, and by 60 - 70% in MLO-Y4 cells.
Discussion: These data support the proposed cellular mechanism for chronic osteomyelitis, that internalization of Gram-negative bacteria is a host cell-mediated process that can be blocked at the level of the cell membrane.
Embargo Period
5-30-2018
Internalization of Gram-Negative Bacteria into Osteocyte-like Cells Is a Host Cell-Mediated Process
Philadelphia, PA
Introduction: Chronic osteomyelitis, an infection of bone with many etiological origins associated with a diverse pool of infectious microorganisms, is often a polymicrobial infection with Staphylococcus aureus as the predominant species with an assortment of other bacteria including Gram-negative species. As it has been proposed that bacterial internalization is a critical step in the progression of chronic osteomyelitis, we propose the hypothesis that Gram-negative bacteria can be internalized by osteocytes via a host cell-mediated process.
Methods: To test this hypothesis, we employed an internalization assay and two osteocyte-like cell lines, MLO-A5 and MLO-Y4, with the former being the less differentiated of the two cell lines. The cells were pre-treated with endocytosis inhibitor drugs (methyl-β-cyclodextrin and chloroquine), and exposed to the Gram-negative bacteria, Serratia marscesens and Proteus mirabilis. After the internalization protocol, extracellular bacteria were killed using an antibiotic solution containing gentamicin and ciprofloxacin. Finally, the cells were lysed to release internalized bacteria and the resultant colonies were counted.
Results: Exposure to chloroquine and methyl-β-cyclodextrin had no appreciable impact on cell viability for the osteocyte-like cell lines or bacteria under the conditions tested. Both drugs successfully reduced the number of internalized bacteria compared to the controls. Chloroquine reduced internalization of both bacterial species by greater than 50% in each cell line. Methyl-β-cyclodextrin reduced internalization of both species by greater than 90% in MLO-A5 cells, and by 60 - 70% in MLO-Y4 cells.
Discussion: These data support the proposed cellular mechanism for chronic osteomyelitis, that internalization of Gram-negative bacteria is a host cell-mediated process that can be blocked at the level of the cell membrane.