Subversion of a young phagosome: the survival strategies of intracellular pathogens. Microreview

Infection by intracellular pathogens remains a major cause of human diseases and death worldwide. The emergence of various resistant microbe strains associated to the problem of multidrug resistance emphasizes the urgency of finding new approaches to fight intracellular pathogens. Despite the severity of this health problem, it is surprising to realize that, until recently, little was known about the molecular mechanisms taking place at the subcellular level during host–pathogen interaction. Hopefully, a significant body of data has accumulated in the past few years and contributed to the birth of a new field at the frontier of microbiology and cell biology, cellular microbiology ( Falkow, 1999). This interest has widened our understanding of the strategies used by microorganisms to survive in mammalian cells. Recent reviews have described with details the survival strategies of intracellular pathogens within their host cells ( Finlay and Falkow, 1997; Sinai and Joiner, 1997; Aderem and Underhill, 1999; Dermine and Desjardins, 1999; Méresse et al., 1999a ) . Here, we will discuss mainly the mechanisms and molecules involved in phagolysosome biogenesis, and those used by intracellular pathogens to subvert the properties of host cell phagosomes and avoid the encounter with the harsh environment of phagolysosomes. We will also present some of the new approaches and technical developments that are likely to have a significant impact in our ability to decode and expose the subversive strategies of intracellular pathogens. Intracellular pathogens are internalized and sequestered within their host cells in membrane compartments originating from the plasma membrane. Despite its plurality, in order to simplify the description of this process, this internalization step is generally referred to as phagocytosis, leading to the formation of phagosomes ( Swanson and Baer, 1995). Phagosomes are pivotal organelles in the ability of mammalian cells, including professional and non-professional phagocytes, to restrict the establishment and spread of infectious diseases ( Rabinovitch, 1995). Understanding how phagosomes work is of prime importance to develop new approaches to fight intracellular pathogens. Newly formed phagosomes are immature organelles unable to kill and degrade microorganisms. In order to acquire and exert their microbicidal function, phagosomes must engage in a maturation process referred to as phagolysosome biogenesis ( Berón et al., 1995 ; Desjardins, 1995). This process has been more extensively described in macrophage cell lines fed with inert particles such as zymozan, latex beads or fixed Staphylococcus aureus, and more recently using different in vitro assays. Newly formed phagosomes display a composition similar to that of the plasma membrane from which they originate ( Lang et al., 1988 ). Rapidly after their formation, phagosomes modify their composition by recycling plasma membrane molecules ( Muller et al., 1983 ), including the FcII and mannose receptors, and by acquiring markers of the early endocytic pathway such as Rab5 and EEA1 ( Pitt et al., 1992a ; Desjardins et al., 1994a ; Scianimanico et al., 1999 ; Steele-Mortimer et al., 1999 ). The acquisition of early markers is believed to confer to phagosomes properties normally assigned to early endosomes, including the ability to fuse with other endocytic organelles ( Desjardins, 1995). Indeed, phagosomes have been shown to fuse sequentially with endosomes of increasing age or of increasing maturation level (early endosomes, late endosomes and lysosomes) both in cultured cells and in cell-free assays ( Desjardins et al., 1997 ; Jahraus et al., 1998 ). During this process, phagosomes acquire markers of late endosomes and lysosomes, including LAMP molecules, the proton pump ATPase, the cation-independent mannose-6-phosphate receptor (CI-MPR), MHC class II molecules, as well as various hydrolases including several members of the cathepsin family ( Pitt et al., 1992a ; Desjardins et al., 1994a ; Jahraus et al., 1994 ; Oh and Swanson, 1996; Claus et al., 1998 ; Ramachandra et al., 1999a ). Although the bulk of these hydrolases is assumed to be present in late endosomes and lysosomes, their sequential acquisition by phagosomes suggests that they are transferred to this organelle through sequential fusion with subsets of endosomes containing various levels of hydrolases ( Claus et al., 1998 ). For example, the bulk of cathepsin H activity has been found on early endosomes. Maturation of phagosomes is also accompanied by the acquisition of the membrane protein Nramp1, an ion transporter that confers to macrophages resistance to infection by unrelated pathogens including Mycobacteria, Salmonella and Leishmania ( Gruenheid et al., 1997 ). Changes in phospholipid composition is also observed during phagosome maturation ( Desjardins et al., 1994b ). While early phagosomes are enriched with phosphatidylcholine, late phagosomes are preferentially enriched with sphingomyelin. Altogether, these changes contribute to the formation of the phagolysosome, an acidic compartment displaying the harsh and lytic environment needed to kill, degrade and further process microorganism antigens for presentation at the cell surface (see Harding, 1995; Ramachandra et al., 1999b ). The molecules and mechanisms allowing (i) the sequential fusion of phagosomes with various endocytic organelles, (ii) the maturation of phagosomes and (iii) the maintenance of phagosomes and endosomes integrity during this process begin to be understood. The molecular machinery governing membrane interactions has been extensively studied in the past 10 years, including that of the endocytic apparatus (for a review see Novick and Zerial, 1997; Haas, 1998a; Gonzalez and Scheller, 1999; Pfeffer, 1999). Membrane interactions are regulated by Rab GTPases and their effectors, and by SNARE proteins which appear to act in conjunction to allow organelle movement, docking, fusion and fission ( McBride et al., 1999 ). A variety of Rab proteins and SNARE molecules have been identified on phagosomes, including Rab4, Rab5, Rab7 and Rab11 ( Desjardins et al., 1994a ; Mosleh et al., 1998 ; Cox et al. 2000 ), synaptobrevin I and II, and NSF ( Desjardins et al., 1997 ), as well as syntaxin 2, 3 and 4 ( Hackam et al., 1996 ). The Rab5 effectors EEA1 ( Scianimanico et al., 1999 ; Steele-Mortimer et al., 1999 ) and Rabaptin5 (M. Desjardins, unpublished observation) have also been identified on phagosomes. All the above proteins have been shown to be present on endosomes. This clearly indicates that mechanisms governing membrane interactions along the endocytic pathway apply to phagosome fusion. Using in vitro fusion assays, Stahl and colleagues (see Berón et al., 1995 ; Funato et al., 1997 ) as well as Griffiths and colleagues ( Jahraus et al., 1998 ) were able to demonstrate the involvement of small GTPases and SNARE molecules in endosome–phagosome fusion. The presence of certain of these proteins on the phagosome membrane allows to speculate about some of its specialized function. Rab5 and its effectors enable fusion with early endosomes ( Alvarez-Dominguez and Stahl, 1999), while Rab7 would allow phagosome fusion with late endocytic organelles ( Méresse et al., 1995 ). Although the early endosome localization of Rab5 has been challenged recently and extended to late endosomes and lysosomes ( Jahraus et al., 1998 ), several studies have confirmed the preferential association of this molecule to early endocytic and phagocytic compartments (see Deretic et al., 1997 ). The gradual loss of Rab5 and acquisition of Rab7 on phagosomes with time would favour fusion with late endosomes and drive the transformation of phagosomes towards phagolysosomes ( Desjardins et al., 1994a ; 1997). Retrieving of molecules from phagosomes ( Pitt et al., 1992b ) would be made possible by recycling processes enabled by Rab4 and Rab11 ( Cox et al. 2000 ), previously localized to recycling endosomes ( Ullrich et al., 1996 ). In addition to their essential role in fusion, rab proteins might also be involved in determining the specificity of the fusion event. Recently, the early endocytic marker EEA1, a Rab5 effector, has been shown to interact with components of the SNARE fusion machinery ( McBride et al., 1999 ). Because EEA1 is exclusively found on early endocytic organelles, and is a specific effector of Rab5, this suggests that the interaction with EEA1 would confer a directionality to the fusion reaction. The intense search for the identification of components regulating fusion along the endocytic pathway should help us to further understand the mechanistic of phagolysosome biogenesis. The study of phagolysosome biogenesis in macrophages has allowed us to describe a of interaction phagosomes and endocytic an the phagolysosome was believed to originate from the fusion of a formed phagosome with a in a fusion (see et al., ). that phagolysosome biogenesis is by of interactions phagosomes and endocytic organelles ( Desjardins et al., 1994a ). to the and ( Desjardins, 1995; and Desjardins, phagosomes and endosomes along and interact at were fusion assays confirmed the ability of phagosomes to along in a movement, towards the and ( et al., 1997 ). at the of the phagosome and endosome membrane fuse and the formation of a fusion which to allow the of the of these of by the fusion of both organelles, the fusion and organelles to engage in of fusion. this the and that the of phagosomes and endosomes to be ( Desjardins, 1995). in macrophage cell lines that endosomes containing particles of different were in able to to phagosomes their clearly that of both organelles was ( Desjardins et al., 1997 ). The mechanisms allowing membrane interaction and fusion are well that or for several and ( and of these be by changes in composition ( et al., ). as the composition of phagosomes is during maturation ( Desjardins et al., 1994b ). During the of the and process also as shown by the finding that late phagosomes were more early phagosomes in the of particles they from endosomes ( Desjardins et al., 1997 ). Because Rab proteins have been described as ( et al., ), they were as of the and fusion (see Recent studies a role for Rab and Rab5, in the of the and interactions endosomes and phagosomes. the Rab5 activity as a that the of membrane ( et al., 1996 ). Rab5 at on endosome to fusion ( et al., 1999 ). of Rab5 the of fusion effectors to endosome ( et al., 1997 ). of an of Rab5, unable to to the formation of endosomes, through these organelles ( et al., 1994 ). The possible involvement of some of the other Rab proteins present on phagosomes in the and process be The finding that the levels of a and of interactions and the plasma membrane have been shown to suggests that rab proteins might be involved in the of fusion ( et al., ; et al., 1997 ; and Intracellular of various microorganisms within their mammalian Although they originate from the plasma phagosomes containing different pathogens display markers of early endosomes, late or organelles of the the harsh environment of pathogens a within the host in which they Recently, mechanisms and molecules involved in the of phagosomes by their pathogens have been of these mechanisms of the phagosome or with molecules regulating phagolysosome biogenesis. Although as in the early pathogens Mycobacteria, Salmonella and Leishmania in compartments that in the of certain markers of early and late endosomes. This indicates that might be as intracellular as are pathogens host cells. of the and The and that fusion and fission phagosomes and endosomes during the process of phagolysosomes biogenesis. Rab GTPases such as Rab5 are to have a role in the of this of the Rab family act as molecular by their In its Rab5 to the membrane a of effectors EEA1 and the Rab5 effectors on the membrane in molecular which also EEA1 with a of the syntaxin for endocytic organelles fusion (see McBride et al., 1999 ). In this EEA1 as a and with the of syntaxin membrane in order to the of a fusion of Rab5 by a the molecule from the the fusion and the fission of the This would of phagosomes and endosomes (see Desjardins et al., 1997 ), the for recycling This process would further allow phagosomes to acquire molecules from the endocytic pathway and phagolysosomes displaying the needed to their role as Because EEA1 is present on early endocytic and phagocytic organelles, this would confer a directionality to the fusion event. molecules to the role in the maturation process are to be and interactions have several (i) allow organelles to their the of their the for recycling processes and the of (ii) restrict the ability of pathogens to and the endocytic (iii) of membrane molecules has the further of allowing the gradual transformation of organelles observed along the endocytic and phagocytic for and have been from various including interaction ( and Desjardins et al., 1994a ; interaction ( et al., 1995 ; et al., 1999 ) and ( et al., ; et al., 1997 ; et al., 1999 ). In the of the fusion formed cell and the plasma membrane for several allowing the fusion of the with the plasma membrane . fusion have also been to ( 1995). The of the and fusion in these processes remains to be and within their mammalian host cells, intracellular pathogens must to avoid the harsh environment of phagolysosomes. by finding in various intracellular (see Sinai and Joiner, 1997). have at the compartments in which different pathogens Using a small of these were shown to display of early endocytic organelles, organelles of the apparatus or the (see In to these intracellular compartments have the lytic environment for the and of pathogens. pathogens in these new the and needed for their survival and the identification of these of the is to understand how pathogens to The process of some of the of the compartment in which pathogens are to within host cells. This has been shown by the that the of inert particles internalized by their along the phagolysosome pathway ( Oh and Swanson, 1996; and . are involved in leading to different mechanisms of internalization such as phagocytosis, or ( Swanson and Baer, 1995; et al., 1998 ). and have shown recently that phagocytosis, which in a in is by and small GTPases involved in In phagocytosis, by the small a The or mechanisms of used during have major on the of the phagosomes ( et al., 1994 ). the are which host cells by and inert which cells by a process the involved have been identified While inert particles have been shown to be internalized in early compartments displaying a composition similar to that of the plasma membrane ( Lang et al., 1988 ), to the formation of a that of the host membrane proteins ( and and 1997). to be these are several unrelated including Mycobacteria, and which appear to for a certain time in compartments to the early of the endocytic inert these pathogens have found to the phagosome from its towards phagolysosomes. indicates that they by the composition of their phagosomes or by with some of the molecules involved in phagolysosome biogenesis. the more we about the compartments in which pathogens the more it that will be the mechanisms the of microorganisms within their Using a of and it was that the of Leishmania its a that the surface of the to fusion at the of infection ( Desjardins and 1997; Dermine et al., 2000 ) (see Indeed, has been shown to be an involved in a variety of allowing survival in their ( and and The by which Leishmania to the properties of their host phagosomes understood. Recent studies that in the phagosome membrane to modify its properties mainly by the formation of an in properties ( et al., 1995 ). a would to an and or the in increasing the for fusion fusion. of the of within macrophages that these are able to the to phagosomes of the a molecule for phagosome fusion with late endocytic organelles ( Scianimanico et al., 1999 ). a are to phagolysosomes. they appear to within an early compartment for at a few which with the compartment the of the that phagolysosome ( et al., ). clearly that Leishmania to avoid the harsh environment of phagolysosomes for a certain of time of Leishmania within macrophages in its which a and the on its after the phagosomes the early endocytic pathway fuse with late endosomes or This of fusion is by the presence of on the surface and its possible the phagosome membrane. Leishmania of the environment of phagosomes to their transformation During this process, the needed for the survival within phagolysosomes. The to transformation is also accompanied by the and of which the fusion and allow phagolysosome biogenesis to this are to survive and within phagolysosomes. phagosomes, are to that from those previously in the the other of The and in the mammalian host the of ( and This transformation is accompanied by the of which in the survival of Leishmania within phagolysosomes ( et al., 1998 ). The to transformation is also accompanied by the of are observed in phagolysosomes. Indeed, of from the surface of Leishmania to the of the fusion with endocytic organelles allowing phagosome maturation to that have the molecules to the harsh environment of phagolysosomes. of these proteins are likely to new for the of strategies to fight (see in Scianimanico et al., 1999 ). The work of the of in allowed us to demonstrate that the intracellular of in cells this to compartments displaying after phagosomes fuse with early endosomes and in compartments displaying EEA1, an early marker ( al., ; 1999). phagosomes display the late markers Rab7 or the mannose-6-phosphate and to fuse with lysosomes ( al., ) . they and that the fusion of early phagosomes with organelles of the which be LAMP are within a compartment for LAMP and the markers and the protein The mechanisms and molecules allowing to the fusion of phagosomes with have been The presence of the marker on phagosomes that mechanisms to might be involved ( al., Recent that for and members of a new are to compartments are present within phagosomes unable to fusion with lysosomes ( et al., 1998 ). The and phagosome properties is the presence of in suggests that of effector molecules to the host might be involved in intracellular ( et al. 2000 ). studies should allow us to are the molecules the fusion of early phagosomes with the of molecules to phagosomes. its host cells through a specialized of referred to as ( This in phagosomes that to and fuse with lysosomes ( and Rapidly after phagosomes with and and appear as with ( Swanson and 1995). phagosomes display with ( Swanson and 1995). display of the markers of the endocytic apparatus that maturation of these towards phagolysosomes is early after Recently, a family of involved in have been identified (see and and of the was shown to an membrane protein for fusion ( and 1997; et al., 1998 ). Because are of they by the cell fusion ( et al., 1998 ; et al., 1998 ). The of these proteins on phagosomes is Although and appear to within compartments displaying or the biogenesis of these phagosomes is clearly In to the which the phagocytic pathway for a certain of the of markers on phagosomes suggests that from the phagocytic pathway after internalization for this The by during its through the phagocytic apparatus is understood. in intracellular compartments referred to as are and interact with endocytic organelles as shown by the of the late markers and the ( et al., 1996 ; et al., 1999 ). other studies the of early and late endocytic markers in or the ( et al., 1997 ). A few after their formation, the pathway and with containing ( et al., 1996 ). A few containing several to fuse This process to of proteins of the family and their in the membrane. Although the study of the of within host cells is by the of to the using proteins have shown that might be involved in fusion ( et al., 1999 ). it that is involved in the fusion of with studies will be needed to the about or the at some in the endocytic to which proteins are for the of fusion with lysosomes, and the fusion of the with and the fusion. The to which phagosomes fuse with endocytic organelles is studies that phagosomes Salmonella were ( and and while that within phagosomes that have with lysosomes ( et al., ; Oh et al., 1996 ). of their fusion phagosomes and display molecules of the endocytic A study on early of infection by has allowed us to the of of various markers to the phagosome ( Steele-Mortimer et al., 1999 ). EEA1, a Rab5 effector, was present on early phagosomes and during maturation of the The receptor the of and with a The acquisition of the proton pump and clearly while early markers were present on phagosomes. phagosomes are of the and of on this receptor ( Méresse et al., 1999b ). that phagosomes interact with early endosomes, with late Méresse and confirmed that infection of cells by the in the of phagosomes of a compartment enriched in and in ( Méresse et al., 1999b ). further that the small Rab7 regulated this Altogether, these that Salmonella with the ability of phagosomes to phagolysosomes. A indicates that a protein within the might be involved in the of fusion ( et al., 1999 ). This protein to act after its in the through a The of in the has been identified The finding that in the fusion of phagosomes with endosomes in an in vitro fusion suggests that it interact with a of the fusion Recent studies have clearly that Salmonella proteins to the by interact with and the of For example, and with and and to of the host and the internalization process in cells ( and 1999; et al., 1999 ). In might also interact with Rab5 to fusion of the phagosomes with early endosomes and with the pathway leading to phagolysosomes ( et al., 2000 ). in the past few years has contributed to the of the machinery involved in phagolysosome biogenesis. The molecular components allowing phagosomes to and along to and to other organelles, and to fuse in a sequential with subsets of endosomes are of phagosome molecules to the of those used as maturation markers is an step towards the understanding of the mechanisms governing this organelle properties ( Haas, this is a of the involved in phagosome function. Indeed, of phagosome indicates that of proteins might phagosomes ( et al., 1995 ; Scianimanico et al., 1997 ). to or more of these proteins might with the of phagosomes. microorganisms have a of to subvert phagosomes from their of phagolysosomes. The few organelle markers at our are to how pathogens the phagosome machinery in order to transformation of this at a more of markers are will be needed to within their host cells and understand the mechanisms allowing their establishment in have recently been and used to study phagosomes and a of this Aderem and colleagues have a of phagosome components that will help to the in phagosome proteins in various ( et al., 1999 ). The of an by on organelle by has allowed us to to the association and of molecules from the of phagosomes ( Scianimanico et al., 1999 ; Steele-Mortimer et al., 1999 ). In to the is and to of of organelles, of this be used to and organelles, endosomes or phagosomes, in the cell ( et al., 1997 ). from our that organelle be used to phagosomes containing microorganisms and Desjardins, unpublished In the past few years, the field of has contributed to a new is to and study proteins of a cell or This new (for the by the ( et al., 1996 ), is likely to an of as proteins of the cell understand phagosome function, we have the of the proteins associated to this organelle ( Desjardins et al., 1994b ; et al., 1995 ; Scianimanico et al., 1997 ). The emergence of on the of new to and and of proteins from (see technical in allows the of of proteins in a in a ( and approaches have been to a level that allows for the of proteins from small and the to their in using ( and 1997). Using such a we have been able to proteins present on phagosomes. this has allowed us to a protein of this organelle have been identified protein more and Desjardins, in and that will allow us to changes in of proteins on a This will be made on the allowing to and their phagosomes of interest with our We that this will allow us to are the specific components of phagosomes containing various and contribute to our understanding of the molecular mechanisms pathogens to the of phagolysosomes. Recently, and colleagues used a on phagosomes to that a of the preferentially to the membrane of phagosomes containing ( et al., 1999 ). The of this study that a on phagosomes that interaction with endocytic Although phagosomes fuse with early endosomes ( et al., 1995 ) this to the level of fusion with lysosomes ( and et al., 1994 ), and their to a significant of late endosome and markers ( et al., 1996 ). Hopefully, approaches to the will be to help the molecules involved in the of phagosome function. The phagosome proteins from macrophages are to their using The is on a The of phagosomes is by the that of protein are associated to this organelle a have been identified (see Desjardins et al., 1994b ). The identification of the phagosome proteins is likely to the molecular mechanisms governing phagosome function. The the of a leading to the identification of proteins by to a protein is and The protein is with an that after the and of different This of is on a of an on the The is by an which molecules to are are by a are by the time it for to from the ion to the The is using to of proteins from on the to the protein or In this the protein was identified as cathepsin an present in phagosomes. indicates the of on the In some host–pathogen interaction is a at determining will of the The of phagosomes allows their transformation in a of organelles able to a variety of have to modify this compartment and of some of its to a environment for their The of phagosome and study of their will help us to this as The to for technical and and for of the This work was by from the of and from is the of a from is a from