1814K.A. JoinerJ. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/97/04/1814/04$2.00Volume 99, Number 8, April 1997, 1814–1817Perspectives Series: Host/Pathogen InteractionsFor more than two decades, the standard paradigm for discuss-ing membrane traffic in pathogen-infected cells has centered around fusion of the microbe-containing vacuole with lyso-somes, and upon vacuole acidification. Organisms have been placed in one of three relatively invariant categories: patho-gens residing in vacuoles which fuse with lysosomes and acid-ify, those that readily disrupt the vacuolar membrane and sub-sequently replicate in the host cell cytosol, and microbes residing in vacuoles which neither fuse with lysosomes nor acidify. The nature of membrane traffic was thought to be dic-tated almost exclusively by the requirement of the pathogen to avoid killing by the low pH and the degradative enzymes present in lysosomes.It is increasingly clear that this paradigm is oversimplified (1, 2). It neither appropriately reflects the selectivity and plas-ticity of membrane traffic within cells, nor does it give ade-quate weight to membrane traffic events which may be largely of importance for nutrient acquisition or induction of gene ex-pression by the intracellular organism. This perspective will describe membrane traffic in pathogen-infected cells in the context of more recent advances in the cell biology of vesicular traffic, and with the above considerations in mind.Phagosome biogenesis and maturation: relationship to pathogen vacuolesCurrent understanding of the fate of an inert particle internal-ized by phagocytosis is to rapidly proceed through an extensive series of membrane fusion and budding/remodeling events which eventually deliver the particle to a phagolysosome. Af-ter internalization, modification of the phagosome membrane occurs rapidly in a manner which is dependent upon the nature of the particle itself and upon the host cell type. In phagocytic cells, plasma membrane proteins are rapidly removed, and fu-sion with early endosomes occurs within several minutes. Even proteins localized predominantly to late endosomes can be added to steady state within as short a period as 5 min, and can be largely removed after an additional 5–10 min of incubation.Hence, experiments which do not look at early time points may substantially oversimplify the membrane traffic events in-volved in phagosome maturation, whether with pathogens or with inert particles.With the development of new integral membrane protein markers of the endocytic cascade, it is also clear that the mor-phology of cellular compartments is more complex than appre-ciated previously. For example, certain macrophages contain extensive tubuloreticular compartments bearing a high concentration of lysosome-associated membrane proteins (LAMPs). 1 Phagosomes containing inert particles in mouse peritoneal macrophages may fuse predominantly with these tubular lysosomes, rather than with high density, electron dense, mannose-6-phosphate receptor (M6PR)–negative ter-minal lysosomes with pH 4.5–5.0. Therefore, previous mor-phologic studies analyzing delivery of electron dense markers from terminal lysosomes to pathogen vacuoles may not reflect normal phagosome-lysosome traffic as determined with more recently developed reagents and approaches.Vacuole docking and fusion: mechanisms for control of fusion of pathogen-containing vacuolesThe SNARE hypothesis is now widely recognized and serves as a useful framework for analysis of fusion events surround-ing pathogen-containing vacuoles. Since this hypothesis and the data supporting it, have been reviewed extensively (3),they will be stated here in only the briefest and most general of terms. Intracellular transport vesicles contain transmembrane proteins (Vesicle SNAP receptors or V-SNARES) which bind with high affinity and specificity to a complimentary set of transmembrane proteins on the target membrane (Target SNAP receptors or T-SNARES) with which they will ulti-mately fuse. Cytosolic proteins ( N -ethylmaleimide sensitive factor or NSF, and soluble NSF attachment proteins or SNAPs) associate with the SNARES to assemble a 20s com-plex which is necessary to trigger fusion between the vesicle and target membrane. Additional proteins are required for ini-tial vesicle budding (Coat proteins or COPs and ADP ribosy-lation factors or ARFs) and for activating vesicles for subse-quent docking and fusion (Rabs).In the context of the SNARE model, pathogen vacuoles which display selective or absent fusion with host cell vesicular compartments must either contain a microbial inhibitor which prevents assembly or function of the 20s docking and fusion complex, or must lack the V-SNARES or associated compo-nents (such as Rabs) necessary for vacuole activation, docking,and fusion. Additional proteins, including annexins, host cell cytoskeletal components, and the myristoylated, alanine rich C-kinase substrate (MARCKS) are also likely to participate in vacuole biogenesis or maturation.Address correspondence to Keith A. Joiner, M.D., LCI 808, Section of Infectious Diseases, Yale University School of Medicine, 333 Ce-dar Street, P.O. Box 208022, New Haven, CT 06520-8022. Phone: 203-785-4140; FAX: 203-785-3864; E-mail: Keith_Joiner@qm.yale.eduReceived for publication 27 February 1997 and accepted for publi-cation 13 March 1997.1. Abbreviations used in this paper: ER, endoplasmic reticulum;LAMPs, lysosome-associated membrane proteins. Membrane-Protein Traffic in Pathogen-infected CellsKeith A. JoinerSection of Infectious Diseases, Yale University School of Medicine, New Haven, Connecticut 06520-8022
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