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Endocytosis of Salmonella typhimurium 395 MS and MR10 by HeLa cells
Abstract:
Monolayer of HeLa cells were examined for their ability to endocytose Salmonella typhimurium 395 MS (wild) and MR10 (chemotype RD). Monolayers treated with the glycolytic inhibitors iodoacetic acid (IAA) or N-ethylmaleimide (NEM) or the respiratory inhibitor sodium azide (NaN3) or cytochalasin B (CB) were incubated with S. typhimurium. The numbers of cell-associated (intracellular plus cell-membrane attached extracellular) and intracellular bacteria were determined by viable counts, together with the HeLa cell ATP levels. IAA and NEM at concentrations 10(-4)M and 10(-3)M decreased significantly the number of intracellular MR10 and the cellular ATP levels, but did not influence significantly the total number of cell-associated bacteria except for 10(-3)M IAA which slightly increased the association. On the other hand, NaN3 at concentrations 10(-4)M and 10(-3)M did not affect the number of associated or intracellular bacteria, or the cellular ATP levels. CB at concentrations of 5, 10 and 20 microgram/ml increased the number of associated bacteria, decreased the number of intracellular bacteria and caused a small decrease in cellular ATP levels. Thus, HeLa cells may internalize S. typhimurium by an energy-requiring, glycolysis-dependent process. CB had a dose-dependent inhibitory effect on the internalization without influencing significantly the HeLa cell ATP levels. This indicates that CB might affect the internalization process by some means other than decreasing the ATP content.
Insights
HeLa cells internalize Salmonella typhimurium through an energy-dependent process relying on glycolysis. Inhibitors like iodoacetic acid and N-ethylmaleimide blocked bacterial entry by depleting cellular ATP, while cytochalasin B affected internalization independently of ATP levels.
Area of Science:
- Cell Biology
- Microbiology
- Biochemistry
Background:
- Salmonella typhimurium is a pathogen that invades host cells.
- Understanding the mechanisms of bacterial internalization is crucial for developing therapeutic strategies.
Purpose of the Study:
- To investigate the energy requirements and pathways involved in HeLa cell endocytosis of Salmonella typhimurium.
- To determine the role of glycolysis and cellular ATP levels in bacterial internalization.
Main Methods:
- HeLa cell monolayers were treated with various metabolic inhibitors: iodoacetic acid (IAA), N-ethylmaleimide (NEM), sodium azide (NaN3), and cytochalasin B (CB).
- Cells were incubated with Salmonella typhimurium (wild-type and chemotype RD).
- Bacterial association and internalization were quantified using viable counts, and HeLa cell ATP levels were measured.
Main Results:
- IAA and NEM significantly reduced intracellular Salmonella and cellular ATP levels, indicating glycolysis dependence.
- Sodium azide did not affect bacterial internalization or ATP levels, suggesting the respiratory chain is not the primary energy source.
- Cytochalasin B inhibited internalization in a dose-dependent manner, decreasing intracellular bacteria without significantly altering ATP levels.
Conclusions:
- HeLa cell internalization of Salmonella typhimurium is an energy-requiring process dependent on glycolysis.
- Cytochalasin B inhibits Salmonella internalization through a mechanism distinct from ATP depletion, possibly by interfering with cytoskeletal rearrangements.