High-Throughput, Single-Cell Analysis of Macrophage Interactions with Fluorescently LabeledBacillus anthracisSpores

Abstract

The engulfment of Bacillus anthracis spores by macrophages is an important step in the pathogenesis of inhalational anthrax. However, from a quantitative standpoint, the magnitude to which macrophages interact with and engulf spores remains poorly understood, in part due to inherent limitations associated with commonly used assays. To analyze phagocytosis of spores by RAW264.7 macrophage-like cells in a highthroughput, nonsubjective manner, we labeled B. anthracis Sterne 7702 spores prior to infection with an Alexa Fluor 488 amine-reactive dye in a manner that did not alter their germination, growth kinetics, and heat resistance. Using flow cytometry, large numbers of cells exposed to labeled spores were screened to concurrently discriminate infected from uninfected cells and surface-associated from internalized spores. These experiments revealed that spore uptake was not uniform, but instead, highly heterogeneous and characterized by subpopulations of infected and uninfected cells, as well as considerable variation in the number of spores associated with individual cells. Flow cytometry analysis of infections demonstrated that spore uptake was independent of the presence or absence of fetal bovine serum, a germinant that, while routinely used in vitro, complicates the interpretation of the outcome of infections. Two commonly used macrophage cell lines, RAW264.7 and J774A.1 cells, were compared, revealing significant disparity between these two models in the rates of phagocytosis of labeled spores. These studies provide the experimental framework for investigating mechanisms of spore phagocytosis, as well as quantitatively evaluating strategies for interfering with macrophage binding and uptake of spores.

Tracking bacterial infection of macrophages using a novel red-emission pH sensor

“…While most of the studies focused on late stages of bacteria-macrophage interaction [9, 35], we used the pH sensor M to monitor the early entry event of bacteria into macrophage and the bacteria-host interaction during phagocytosis process. Although the studies with B. anthracis spores suggested that a considerable percentage of spores can be killed soon after up-took into macrophage vacuoles [35-37], our study using single cell qRT-PCR suggested that bacteria are mostly intact 120 min after infection.…”

Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

“…To evaluate this issue, the recovery of viable, intracellular B. anthracis was compared subsequent to uptake by RAW264.7 cells in the absence or presence of FBS (10%), using the gentamicin protection assay [11,21,46,47]. These studies indicated that there were not significant differences in intracellular CFU after 5 min post-infection (Figure 6).…”

“…As quality control, spore preparations were tested for both heat resistance and the capacity to germinate, as described [46]. …”

“…Previously, 0.5% trypan blue was demonstrated to completely quench the fluorescence emission of Alexa Fluor 488-labeled spores bound to the surface of mammalian cells, while having no affect the fluorescence emission of internalized spores [46]. From these data, the percentage of cells with internalized B. anthracis was calculated by dividing the number of viable cells with greater than background auto-fluorescence by the total number of viable cells.…”

Testing Nucleoside Analogues as Inhibitors of Bacillus anthracis Spore Germination In Vitro and in Macrophage Cell Culture

“…The timing of cell death is variable, but most cells have entered necrosis at 5 h postinfection. This variability is probably due to nonuniformity in spore intake by macrophages, which leads to heterogeneous cell killing (33).…”