Isolation of bacteria-containing phagosomes by magnetic selection

Abstract

Background: There is a growing awareness of the importance of intracellular events in determining the outcome of infectious disease. To improve the understanding of such events, like phagosome maturation, we set out to develop a versatile technique for phagosome isolation that is rapid and widely applicable to different pathogens.

Results: We developed two different protocols to isolate phagosomes containing dead or live bacteria modified with small magnetic particles, in conjunction with a synchronized phagocytosis protocol and nitrogen cavitation. For dead bacteria, we performed analysis of the phagosome samples by microscopy and immunoblot, and demonstrated the appearance of maturation markers on isolated phagosomes.

Conclusion: We have presented detailed protocols for phagosome isolation, which can be adapted for use with different cell types and prey. The versatility and simplicity of the approach allow better control of phagosome isolation, the parameters of which are critical in studies of host-bacteria interaction and phagosome maturation.

Figure 1

Overview of method. 1. "Magnetic bacteria" are prepared by covalently attaching very small magnetite particles to the surface of the bacteria. This can be done in large batches. If dead bacteria are used the finished product may be stored for several weeks at 4°C. 2. Synchronized phagocytosis of the magnetic bacteria is achieved through a 30-s centrifugation of a mixture of phagocytic cells and magnetic bacteria. This step may be repeated after resuspension to increase the interaction efficiency. Simultaneous phagocytosis of multiple samples can be performed using multi-channel pipettes in conjunction with either test tubes or microtiter plates. 3. After completed presentation, free bacteria are washed away. Following an optional chase period, the suspension is then put on ice and pooled, and the buffer changed to an isotonic sucrose solution containing protease inhibitors and DNAse. The resulting suspension is put in a bomb cylinder and subjected to nitrogen cavitation (300 psi, 10 min) to disrupt the phagocytic cells. 4. Aliquots of cell lysate are put into microtiter wells. Phagosomes are retrieved magnetically using a magnetic rod. Each well is probed several times to increase yield. 5. Phagosome integrity is determined using direct fluorescent staining of a phagosome membrane marker and antibodies recognizing free or partially free bacteria. 6. Isolated phagosomes are analyzed using immunofluorescence microscopy, flow cytometry, or immunoblot. Steps 2–5 can be achieved in less than 1 h.

Figure 2

Attachment of superparamagnetic particles to the surface of bacteria. Panel A shows the two principles for covalent linkage of magnetic particles to bacteria. Top: glutaraldehyde can be used to create a pentyl bridge between amino group-exposing particles and the bacteria. Bottom: carbodiimide activation of carboxy group-exposing particles can be used to create peptide bonds with the bacteria. Panel B demonstrates the visualization of magnetite particles (arrowheads). Upper images: phase contrast and differential interference contrast, respectively; scale bar 1 μm. Lower image: electron micrograph depicting a bacterium (S. pyogenes) with attached magnetite particles; scale bar 0.5 μm. Panel C illustrates that, using the carbodiimide protocol, the attachment of particles (arrowhead) does not compromise the viability of bacteria, as determined by a BacLight Live-Dead kit (green = live, red = dead); scale bar 1 μm.

Figure 3

Magnetic purification. A shows intact HL-60 cells after phagocytosis of bacteria. B shows the same material after nitrogen cavitation, and C shows what can be retrieved from such a sample by one magnetic purification step (mostly phagosomes and free bacteria); scale bar 10 μm.

Figure 4

Phagosome integrity. In A, discrimination of free bacteria from intact and broken phagosomes is illustrated. Alexa 488-labeled annexin V (green) stains phagosomal membrane and Cy3-labeled anti-human Fab fragments (red) stains damaged phagosomes and free opsonized bacteria. Arrows indicate free bacteria, arrowheads intact phagosomes, and the double arrowhead shows a partial phagosome; scale bar 10 μm. B shows quantification of phagosomes from three separate experiments ± SEM.

Figure 5

Phagosome analysis. Analysis of 15-min phagosomes, purified using the protocol for heat-killed bacteria. In A, immunofluorescence analysis of phagosomes is exemplified. Arrows show free bacteria, arrowheads intact phagosomes, double arrowheads broken phagosomes, and the dotted circle represents an intact phagosome negative for CD63 staining; scale bar 10 μm. Panel B shows Western blot analysis. Phagosomes (5·10⁶) and varying amounts of whole-cell lysates (4·10⁴, 1·10⁵, 2·10⁵, and 4·10⁵) were probed with an antibody against myeloperoxidase (MPO). Two mature-form fragments (MPO_large and MPO_small) and a precursor form (proMPO) can be detected.