What are two mechanisms by which phagocytes kill bacteria once they are taken up into a phagosome?
Phagocytes employ a range of mechanisms to kill bacteria once they are engulfed into a phagosome. Two important mechanisms are:
1. Reactive Oxygen Species (ROS) production: Phagocytes, such as neutrophils and macrophages, produce reactive oxygen species (ROS) to kill bacteria. This occurs through an enzyme system called the NADPH oxidase complex, which generates superoxide radicals (O2-) from molecular oxygen. These radicals can be converted into other ROS like hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). These ROS molecules help in killing bacteria by causing oxidative damage to bacterial membranes, proteins, and DNA.
To determine the production of ROS by phagocytes, a common laboratory technique is to use a dihydrorhodamine-123 fluorescent dye. This dye enters cells and is converted into a fluorescent compound by ROS. The fluorescence intensity can be measured using flow cytometry or fluorescence microscopy. A higher fluorescence signal indicates increased production of ROS.
2. Acidification and degradative enzymes: Phagocytes acidify the phagosome by pumping protons (H+) into it using the vacuolar-type H+-ATPase enzyme. This acidic environment activates lysosomal enzymes, such as proteases and nucleases, which degrade bacterial components. These enzymes break down proteins, nucleic acids, carbohydrates, and lipids, ultimately leading to the destruction of bacteria.
To assess the acidification and degradative activity of phagocytes, a common approach is to use acidotropic dyes like LysoTracker or LysoSensor. These dyes accumulate in acidic compartments, such as lysosomes and acidic phagosomes, and emit fluorescence. By measuring the fluorescence intensity, one can determine the acidification level and evaluate the activity of degradative enzymes.
In conclusion, the two mechanisms by which phagocytes kill bacteria once engulfed into a phagosome are the production of reactive oxygen species (ROS) and the acidification and degradative activity facilitated by enzymatic action. These mechanisms play important roles in the innate immune response to bacterial infections.