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

Abstract

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 particular type of interaction occurring between phagosomes and endocytic organelles. As an organelle, the phagolysosome was believed to originate from the fusion of a newly formed phagosome with a lysosome in a single, complete, fusion event (see Rabinowitz et al., 1992 ). Several observations indicated that phagolysosome biogenesis is instead driven by series of transient interactions between phagosomes and endocytic organelles ( Desjardins et al., 1994a ). According to the kiss and run hypothesis ( Desjardins, 1995; Storrie and Desjardins, 1996), phagosomes and endosomes move along cytoskeletal elements and interact at focal points were fusion events can occur. Cell-free assays confirmed the ability of phagosomes to move along microtubules in a bidirectional movement, towards the plus and minus ends ( Blocker et al., 1997 ). Once at close proximity, the outer leaflet of the phagosome and endosome membrane can fuse and induce the formation of a fusion pore, which expands enough to allow the exchange of the soluble content of these organelles. However, instead of being followed by the complete fusion of both organelles, the fusion pore closes and organelles separate, free to engage in another cycle of fusion. On this base, the kiss and run hypothesis predicted that the exchange of content between phagosomes and endosomes had to be size selective ( Desjardins, 1995). Studies performed in macrophage cell lines showed that endosomes containing gold particles of different sizes were in fact able to selectively transfer to phagosomes their smaller particles, clearly indicating that complete mixing of both organelles was not occurring ( Desjardins et al., 1997 ). The exact mechanisms allowing transient membrane interaction and fusion are not well known. Models predict that lipidic pores could expand irreversibly or remain open for several seconds and then close ( Monck and Fernandez, 1996). Closure of these lipidic pores could be caused by slight changes in lipid composition ( Nanavati et al., 1992 ). Interestingly, as mentioned above, the lipid composition of phagosomes is modified during maturation ( Desjardins et al., 1994b ). During maturation, the size-selective stringency of the kiss and run process also increases, as shown by the finding that late phagosomes were more restrictive than early phagosomes in the size of particles they can receive from endosomes ( Desjardins et al., 1997 ). Because Rab proteins have been described as ‘molecular switches’ ( Bourne et al., 1990 ), they were highlighted as potential regulators of the kiss and run fusion (see Fig. 1, lower panel). Recent studies support a role for Rab proteins, and particularly Rab5, in the regulation of the transient kiss and run interactions occurring between endosomes and phagosomes. First, the Rab5 GTPase activity acts as a timer that determines the frequency of membrane docking/fusion events ( Rybin et al., 1996 ). Second, Rab5 accumulates at focal points on endosome membranes prior to fusion ( Roberts et al., 1999 ). Third, activation of Rab5 stimulates the recruitment of fusion effectors to endosome membranes ( Horiuchi et al., 1997 ). Fourth, expression of an active mutant of Rab5, unable to hydrolyse GTP, leads to the formation of giant endosomes, probably through uncontrolled complete fusions between these organelles ( Stenmark et al., 1994 ). The possible involvement of some of the other Rab proteins present on phagosomes in the kiss and run process cannot be ruled out. The finding that Rab3 modulates the levels of neurotransmitter release, a biological system where ‘kiss and run’ type of interactions between neurotransmitter-containing vesicles and the plasma membrane have been shown to occur, suggests that rab proteins might indeed be involved in the regulation of transient fusion events ( Alvarez de Toledo et al., 1993 ; Albillos et al., 1997 ; Geppert and Sudhof, 1998). (Top panel) Intracellular trafficking of various microorganisms within their mammalian hosts. Although they originate from the plasma membrane, phagosomes containing different pathogens eventually display markers of early endosomes, late endosomes/lysosomes, or even organelles of the biosynthetic apparatus. By escaping the harsh environment of phagolysosomes, pathogens find a cosy niche within the host cell, in which they can replicate safely. Recently, mechanisms and molecules involved in the subversion of young phagosomes by their resident pathogens have been identified. Some of these mechanisms include active modulation of the phagosome compostition, or direct interference with key molecules regulating phagolysosome biogenesis. Although presented as being in the same early endosome-like compartment, pathogens like Mycobacteria, Salmonella and Leishmania reside in compartments that may differ in the relative abundance of certain markers of early and late endosomes. This indicates that there might be as many modified intracellular niches as there are pathogens infecting host cells. (Bottom panel) Biogenesis of phagolysosomes: the kiss and run hypothesis. The kiss and run model proposes that multiple fusion (kiss) and fission (run) events occur between phagosomes and endosomes during the process of phagolysosomes biogenesis. Rab GTPases such as Rab5 are thought to have a role in the regulation of this process. Molecules of the Rab family can act as molecular switches by changing their nucleotide binding state. In its GTP-bound form, Rab5 actively recruits to the membrane a series of effectors like EEA1 and the complex Rabaptin-5/Rabex-5. These Rab5 effectors assemble on the membrane in high molecular weight oligomers, which also contain NSF. EEA1 directly interacts with a member of the t-SNARE family, syntaxin 13, required for endocytic organelles fusion (see McBride et al., 1999 ). In this model, EEA1 acts as a docking protein, and together with NSF, mediates the local activation of syntaxin 13 upon membrane tethering in order to co-ordinate the assembly of a fusion pore. Then, hydrolysis of Rab5 catalysed by a cytoplasmic GAP releases the molecule from the membrane, thereby terminating the fusion event, and promoting the subsequent fission of the organelles. This would permit limited exchange (size selective) of content between phagosomes and endosomes without complete intermixing (see Desjardins et al., 1997 ), limiting the need for large-scale recycling processes. This process would further allow phagosomes to gradually acquire molecules from the endocytic pathway and transform into phagolysosomes displaying all the characteristics needed to fulfil their role as degradative organelles. Because EEA1 is only present on early endocytic and phagocytic organelles, this would confer a directionality to the fusion event. Other molecules susceptible to play the same role later in the maturation process are still to be identified. Kiss and run interactions have several advantages. (i) They allow organelles to exchange their lumenal contents without the complete mixing of their membranes, thereby minimizing the need for large-scale recycling processes and preventing the generation of aberrant gigantic organelles. (ii) They restrict the ability of pathogens to invade and colonize the whole endocytic pathway. (iii) Limited exchange of membrane molecules has the further potential of allowing the gradual transformation of organelles observed along the maturing endocytic and phagocytic pathways. Evidence for ‘kiss and run’ exchanges have been obtained from various biological systems including phagosome–endosome interaction ( Wang and Goren, 1987; Desjardins et al., 1994a ; 1997), endosome–endosome interaction ( Berthiaume et al., 1995 ; Roberts et al., 1999 ) and exocytosis ( Alvarez de Toledo et al., 1993 ; Albillos et al., 1997 ; Alés et al., 1999 ). In the case of exocytosis, the fusion pore formed between mast cell granules and the plasma membrane could stay open for several seconds, allowing the complete transmitter release without full fusion of the vesicle with the plasma membrane . Transient fusion events between Golgi tubules have also been proposed to occur ( Weidman, 1995). The significance of the kiss and run fusion in these biological processes remains to be firmly established. To successfully invade and replicate within their mammalian host cells, intracellular pathogens must find ways to avoid the harsh environment of phagolysosomes. They do so by finding safe haven in various intracellular niches (see Sinai and Joiner, 1997). Several laboratories have devoted efforts at characterizing the compartments in which different pathogens reside. Using a relatively small set of markers, these niches were shown to display characteristics of early endocytic organelles, organelles of the biosynthetic apparatus or the nucleus (see Fig. 1, upper panel). In contrast to phagolysosomes, these intracellular compartments do not have the lytic environment required for the killing and degradation of pathogens. Instead, pathogens find in these new environments the nutrients and conditions needed for their survival and replication. Beyond the mere identification of these compartments, one of the most important challenges is to understand how pathogens manage to reach them. The process of phagocytosis itself determines some of the characteristics of the first compartment in which pathogens are going to reside within host cells. This has nicely been shown by the observation that the biochemical nature of inert particles internalized by phagocytosis influence their trafficking along the phagolysosome pathway ( Oh and Swanson, 1996; de Chastellier and Thilo, 1997) . Several receptors are involved in phagocytosis leading to widely different mechanisms of internalization such as coiling phagocytosis, zipper phagocytosis or trigger phagocytosis ( Swanson and Baer, 1995; Rittig et al., 1998 ). Caron and Hall (1998) have shown recently that Fc γ-induced phagocytosis, which results in a respiratory burst in macrophages, is mediated by Cdc42 and Rac, two small GTPases involved in actin organization. In contrast, complement-induced phagocytosis, mediated by the small GTPase Rho does not provoke a burst. The receptors or mechanisms of entry used during phagocytosis undoubtedly have major consequences on the biochemical nature of the nascent phagosomes ( Small et al., 1994 ). Perhaps the two extreme examples are Toxoplasma gondii, which invade host cells by active penetration, and inert particles, which enter cells by a receptor-mediated process (although the receptors involved have not been identified so far). While inert particles have been shown to be internalized in early compartments displaying a biochemical composition similar to that of the plasma membrane ( Lang et al., 1988 ), T. gondii appears to induce the formation of a vacuole that excludes most of the host membrane proteins ( de Carvahlo and de Souza, 1989; Mordue and Sibley, 1997). Between what seems to be these two extremes are several unrelated pathogens, including Mycobacteria, Salmonella, Leishmania, and Brucella, which appear to transit for a certain time in compartments related to the early stages of the endocytic pathway. However, unlike inert particles, these pathogens have found ways to divert the young phagosome from its usual trafficking towards phagolysosomes. Increasing evidence indicates that they do so by actively modulating the composition of their phagosomes or by interfering with some of the key molecules involved in phagolysosome biogenesis. However, the more we learn about the compartments in which pathogens reside, the more obvious it seems that there will be no unifying model describing the mechanisms underlying the trafficking of microorganisms within their hosts. Using a combination of genetic and biochemical approaches, it was demonstrated that the promastigote form of Leishmania uses its lipophosphoglycan (LPG), a glycolipid that covers the whole surface of the parasite, to inhibit phagosome–endosome fusion at the onset of infection ( Desjardins and Descoteaux, 1997; Dermine et al., 2000 ) (see Fig. 2). Indeed, LPG has been shown to be an important virulence factor involved in a wide variety of functions allowing parasite survival in their hosts ( Turco and Descoteaux, 1992; Beverley and Turco, 1998). The way by which Leishmania parasites use LPG to alter the properties of their host phagosomes is, however, poorly understood. Recent studies suggest that LPG could insert itself in the phagosome membrane to modify its properties mainly by preventing the formation of an inverted hexagonal structure, resulting in reduced fusogenic properties ( Miao et al., 1995 ). As a consequence, LPG would give rise to an effective ‘steric repulsion’ between phagosomal and endosomal membranes or reduce the negative curvature strain in bilayers, increasing the energy barrier for forming highly curved fusion intermediates, thereby preventing fusion. Analysis of the trafficking of promastigotes within macrophages indicated that these parasites are able to impair the translocation to phagosomes of the GTPase Rab7, a key molecule for phagosome fusion with late endocytic organelles ( Scianimanico et al., 1999 ). As a result, promastigotes are not directed to mature phagolysosomes. Instead, they appear to stay within an early endosome-like compartment for at least a few hours, which contrasts with the compartment housing the transformed amastigote form of the parasite that displays typical phagolysosome characteristics ( Russell et al., 1992 ). These observations clearly indicate that Leishmania parasites need to avoid the harsh environment of phagolysosomes for a certain period of time only. Survival strategy of Leishmania donovani within macrophages. L. donovani infects macrophages in its promastigote form, which displays a flagellum and the lipophosphoglycan (LPG) on its surface. Early after infection, the L. donovani-containing phagosomes intersect the early endocytic pathway but do not fuse with late endosomes or lysosomes. This partial inhibition of fusion is mediated by the presence of LPG on the parasite surface and its possible insertion into the phagosome membrane. Leishmania promastigotes take advantage of the hospitable environment of ‘early’ phagosomes to initiate their transformation into amastigotes. During this process, the parasite synthezises ‘survival proteins’ needed for the subsequent amastigote survival within phagolysosomes. The promastigote to amastigote transformation is also accompanied by the downregulation and disappearance of LPG, which lift the phagosome-late endosome–lysosome fusion inhibition and allow phagolysosome biogenesis to take place. At this point, amastigotes are fully competent to survive and proliferate within phagolysosomes. Inside phagosomes, promastigotes are exposed to conditions that differ greatly from those previously encountered in the sandfly, the other vector of Leishmania. The elevated temperature and decreased phagosomal pH encountered in the mammalian host trigger the differentiation of promastigotes into non-motile amastigotes ( Zilberstein and Shapira, 1994). This transformation is accompanied by the expression of virulence determinants, which undoubtedly play key roles in the subsequent survival of Leishmania within phagolysosomes ( Antoine et al., 1998 ). The promastigote to amastigote transformation is also accompanied by the downregulation of LPG, explaining why amastigotes are observed in phagolysosomes. Indeed, disappearance of LPG from the surface of transforming Leishmania probably leads to the lift of the fusion inhibition with endocytic organelles allowing phagosome maturation to resume. At that point, amastigotes have synthesized all the molecules required to sustain the harsh environment of phagolysosomes. Identification of these ‘survival’ proteins are likely to offer new targets for the development of therapeutic strategies to fight leishmaniases (see model in Scianimanico et al., 1999 ). The elegant work of the group of Gorvel in Marseille allowed us to demonstrate that the intracellular trafficking of Brucella in HeLa cells leads this pathogen to compartments displaying autophagosome/endoplasmic reticulum (ER)-like characteristics. Early after infection, Brucella-containing phagosomes fuse with early endosomes and reside in compartments displaying EEA1, an early endosomal marker ( Pizarro-Cerdáet al., 1998a ; 1999). These phagosomes do not display the late endosomal markers Rab7 or the mannose-6-phosphate receptors and do not seem to fuse with lysosomes ( Pizarro-Cerdáet al., 1998b ) . However, they gradually accumulate LAMP1 and LAMP2 suggesting that Brucella can induce the fusion of early phagosomes with organelles of the biosynthetic pathway, which may be carrying newly synthesized LAMP molecules. At approximately 1 h post inoculation, bacteria are located within a compartment positive for LAMP and the (ER) markers sec61beta and the protein disulphide isomerase. The mechanisms and molecules allowing Brucella to induce the fusion of phagosomes with ER elements have not been identified. The presence of the autophagosomal marker monodansylcadaverin on Brucella-containing phagosomes suggests, however, that mechanisms related to autophagy might be involved ( Pizarro-Cerdáet al., 1998a , b). Recent findings indicate that Brucella abortus mutants for bvr-R and bvr-S, members of a new two-components regulatory system, are not directed to ER-like compartments but are rather present within phagosomes unable to inhibit fusion with lysosomes ( Sola-Landa et al., 1998 ). The way bvr-R and bvr-S products modulate phagosome properties is not known. However, the presence of type IV secretion systems in Brucella suggests that translocation of effector molecules to the host cytoplasm might be involved in Brucella intracellular trafficking ( Foulongne et al. 2000 ). Further studies should allow us to identify what are the molecules enabling the fusion of early phagosomes with ER elements or, alternatively, the recruitment of ER molecules to phagosomes. Legionella pneumophilia enters its host cells through a specialized form of phagocytosis referred to as coiling phagocytosis ( Horwitz, 1984). This pathogen resides in phagosomes that fail to acidify and fuse with lysosomes ( Horwitz, 1983a; Horwitz and Maxfield, 1984). Rapidly after entry, phagosomes associate with smooth vesicles, mitochondria and ER, and appear as vacuoles covered with ribosomes ( Horwitz, 1983b; Swanson and Isberg, 1995). These replicative phagosomes display similarities with autophagic vacuoles ( Swanson and Isberg, 1995). They do not display any of the usual markers of the endocytic apparatus indicating that maturation of these vacuoles towards phagolysosomes is inhibited early after phagocytosis. Recently, a family of genes involved in bacterial virulence have been identified (see Kirby and Isberg, 1998; Segal and Shuman, 1998). One of these, the dotA gene was shown to encode an inner membrane protein required for phagosome–lysosome fusion inhibition ( Roy and Isberg, 1997; Roy et al., 1998 ). Because icm/dot complexes are capable of forming pores into eukaryotic membranes, they may function by secreting macromolecules into the cell cytoplasm preventing phagosome–lysosome fusion ( Kirby et al., 1998 ; Vogel et al., 1998 ). The target of these proteins on phagosomes is not known. Although Legionella and Brucella appear to reside within compartments displaying ER or autophagosome features, the biogenesis of these phagosomes is clearly not identical. In contrast to the Brucella phagosome, which follows the phagocytic pathway for a certain period of time, the absence of endocytic/phagocytic markers on Legionella-containing phagosomes suggests that diversion from the phagocytic pathway occurs rapidly after internalization for this pathogen. The benefit gained by Brucella during its transit through the phagocytic apparatus is not fully understood. Chlamydia resides in intracellular compartments referred to as inclusions. Inclusions are not acidified and do not i