Abstract
BackgroundThere 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.ResultsWe 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.ConclusionWe 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.
Highlights
There is a growing awareness of the importance of intracellular events in determining the outcome of infectious disease
Granule-phagosome fusion is an integral part of phagosome maturation and a requirement for killing of ingested microorganisms
Bacteria made magnetic can be opsonized and bacterial aggregates can be removed by gentle centrifugation
Summary
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. Phagocytosis and killing of microorganisms by phagocytes form an essential part of our innate immune system. Phagocytosis is important for killing of microorganisms, and as a link between innate and acquired immunity by enhancing antigen presentation by dendritic cells [2,3]. Granule-phagosome fusion is an integral part of phagosome maturation and a requirement for killing of ingested microorganisms. Streptococcus pyogenes bacteria of the M1 serotype can survive phagocytosis by neutrophils [6], and have been shown to interfere with the fusion of azurophilic granules with the phagosome [7]
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