Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes - which were destroyed - differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation – a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the mechanisms involved in the recognition and fusion of PVs.
Veras PS, Moulia C, Dauguet C, Tunis CT, Thibon M, et al. (1995) Entry and survival of Leishmania amazonensis amastigotes within phagolysosome-like vacuoles that shelter Coxiella burnetii in Chinese hamster ovary cells. Infect Immun 163: 3502–3506.
[3]
Rabinovitch M, Veras PS (1996) Cohabitation of Leishmania amazonensis and Coxiella burnetii. Trends Microbiol 4: 158–161. doi: 10.1016/0966-842X(96)10027-5
[4]
de Chastellier C, Thibon M, Rabinovitch M (1999) Construction of chimeric phagosomes that shelter Mycobacterium avium and Coxiella burnetii (phase II) in doubly infected mouse macrophages: an ultrastructural study. Eur J Cell Biol 78: 580–592. doi: 10.1016/S0171-9335(99)80024-7
[5]
Andreoli WK, Taniwaki NN, Mortara RA (2006) Survival of Trypanosoma cruzi metacyclic trypomastigotes within Coxiella burnetii vacuoles: differentiation and replication within an acidic milieu. Microbes Infect 8: 172–182. doi: 10.1016/j.micinf.2005.06.013
[6]
Alexander J, Satoskar AR, Russell DG (1999) Leishmania species, models of intracellular parasitism. J Cell Sci 112: 2993–3002.
[7]
Alexander J, Vickerman K (1975) Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexicana infected macrophages. J Protozool 22: 502–508. doi: 10.1111/j.1550-7408.1975.tb05219.x
[8]
Berman JD, Fioretti TB, Dwyer DM (1981) In vivo and in vitro localization of Leishmania within macrophage phagolysosomes: use of colloidal gold as a lysosomal label. J Protozool 28: 239–242. doi: 10.1111/j.1550-7408.1981.tb02839.x
[9]
Shepherd VL, Stahl PD, Bernd P, Rabinovitch M (1983) Receptor-mediated entry of β-glucuronidase into the parasitophorous vacuoles of macrophages infected with Leishmania mexicana amazonensis. J Exp Med 157: 1471–1482. doi: 10.1084/jem.157.5.1471
[10]
Antoine JC, Prina E, Lang T, Courret N (1998) The biogenesis and properties of the parasitophorous vacuoles that harbour Leishmania in murine macrophages. Trends Microbiol 7: 392–401. doi: 10.1016/S0966-842X(98)01324-9
[11]
Duclos S, Diez R, Garin J, Papadopoulou B, Descoteaux A, et al. (2000) Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages. J Cell Sci 113: 3531–3541.
[12]
Courret N, Frehel C, Gouhier N, Pouchelet M, Prina E, et al. (2002) Biogenesis of Leishmania-harbouring parasitophorous vacuoles following phagocytosis of the metacyclic promastigote or amastigote stages of the parasite. J Cell Sci 115: 2303–2316.
[13]
Lippuner C, Paape D, Paterou A, Brand J, Richardson M, et al. (2009) Real-time imaging of Leishmania mexicana-infected early phagosomes: a study using primary macrophages generated from green fluorescent protein-Rab5 transgenic mice. FASEB J 23: 483–491. doi: 10.1096/fj.08-108712
[14]
Antoine JC, Prina E, Jouanne C, Bongrand P (1990) Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH. Infect Immun 58: 779–787.
[15]
Veras PS, de Chastellier C, Rabinovitch M (1992) Transfer of zymosan (yeast cell walls) to the parasitophorous vacuoles of macrophages infected with Leishmania amazonensis. J Exp Med 176: 639–646. doi: 10.1084/jem.176.3.639
[16]
Russell DG, Xu S, Chakraborty P (1992) Intracellular trafficking and the parasitophorous vacuole of Leishmania mexicana-infected macrophages. J Cell Sci 103: 1193–1210.
[17]
Collins HL, Schaible UE, Ernst JD, Russell DG (1997) Transfer of phagocytosed particles to the parasitophorous vacuole of Leishmania mexicana is a transient phenomenon preceding the acquisition of annexin I by the phagosome. J Cell Sci 110: 191–200.
[18]
Schaible UE, Schlesinger PH, Steinberg TH, Mangel WF, Kobayashi T, et al. (1999) Parasitophorous vacuoles of Leishmania mexicana acquire macromolecules from the host cell cytosol via two independent routes. J Cell Sci 112: 681–693.
[19]
Veras PS, Topilko A, Gouhier N, Moreau MF, Rabinovitch M, et al. (1996) Fusion of Leishmania amazonensis parasitophorous vacuoles with phagosomes containing zymosan particles: cinemicrographic and ultrastructural observations. Braz J Med Biol Res 29: 1009–1018.
[20]
Real F, Pouchelet M, Rabinovitch M (2008) Leishmania (L.) amazonensis: fusion between parasitophorous vacuoles in infected bone-marrow derived mouse macrophages. Exp Parasitol 119: 15–23. doi: 10.1016/j.exppara.2007.12.013
[21]
Chang KP, Dwyer DM (1978) Leishmania donovani. Hamster macrophage interactions in vitro: cell entry, intracellular survival, and multiplication of amastigotes. J Exp Med 147: 515–530. doi: 10.1084/jem.147.2.515
[22]
Castro R, Scott K, Jordan T, Evans B, Craig J, et al. (2006) The ultrastructure of the parasitophorous vacuole formed by Leishmania major. J Parasitol 92: 1162–1170. doi: 10.1645/GE-841R.1
[23]
K？rner U, Fuss V, Steigerwald J, Moll H (2006) Biogenesis of Leishmania major-harboring vacuoles in murine dendritic cells. Infect Immun 74: 1305–1312. doi: 10.1128/IAI.74.2.1305-1312.2006
[24]
Sp？th GF, Schlesinger P, Schreiber R, Beverley SM (2009) A novel role for Stat1 in phagosome acidification and natural host resistance to intracellular infection by Leishmania major. PLoS Pathog 5: e1000381. doi: 10.1371/journal.ppat.1000381
[25]
Sp？th GF, Beverley SM (2001) A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation. Exp Parasitol 99: 97–103. doi: 10.1006/expr.2001.4656
[26]
Zamboni DS, Rabinovitch M (2003) Nitric oxide partially controls Coxiella burnetii phase II infection in mouse primary macrophages. Infect Immun 71: 1225–1233. doi: 10.1128/IAI.71.3.1225-1233.2003
[27]
Peters C, Stierhof YD, Ilg T (1997) Proteophosphoglycan secreted by Leishmania mexicana amastigotes causes vacuole formation in macrophages. Infect Immun 65: 783–786.
[28]
Scianimanico S, Desrosiers M, Dermine JF, Méresse S, Descoteaux A, et al. (1999) Impaired recruitment of the small GTPase rab7 correlates with the inhibition of phagosome maturation by Leishmania donovani promastigotes. Cell Microbiol 1: 19–32. doi: 10.1046/j.1462-5822.1999.00002.x
[29]
Vinet AF, Fukuda M, Turco SJ, Descoteaux A (2009) The Leishmania donovani lipophosphoglycan excludes the vesicular proton-ATPase from phagosomes by impairing the recruitment of synaptotagmin V. PLoS Pathog 5: e1000628. doi: 10.1371/journal.ppat.1000628
[30]
Silverman JM, Clos J, de'Oliveira CC, Shirvani O, Fang Y, et al. (2010) An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages. J Cell Sci 123: 842–852. doi: 10.1242/jcs.056465
[31]
Ndjamen B, Kang BH, Hatsuzawa K, Kima PE (2010) Leishmania parasitophorous vacuoles interact continuously with the host cell's endoplasmic reticulum; parasitophorous vacuoles are hybrid compartments. Cell Microbiol. doi:10.1111/j.1462-5822.2010.01483.x.
[32]
Duclos S, Desjardins M (2000) Subversion of a young phagosome: the survival strategies of intracellular pathogens. Cell Microbiol 2: 365–377. doi: 10.1046/j.1462-5822.2000.00066.x
[33]
Kima PE, Dunn W (2005) Exploiting calnexin expression on phagosomes to isolate eishmania parasitophorous vacuoles. Microb Pathog 38: 139–145. doi: 10.1016/j.micpath.2004.11.003
[34]
Cortázar TM, Hernández J, Echeverry MC, Camacho M (2006) Role of the parasitophorous vacuole of murine macrophages infected with Leishmania amazonensis in molecule acquisition. Biomedica 26: 26–37.
[35]
Oates PJ, Touster O (1980) In vitro fusion of Acanthamoeba phagolysosomes. III. Evidence that cyclic nucleotides and vacuole subpopulations respectively control the rate and the extent of vacuole fusion in Acanthamoeba homogenates. J Cell Biol 85: 804–810. doi: 10.1083/jcb.85.3.804
[36]
Mayer A (2002) Membrane fusion in eukaryotic cells. Annu Rev Cell Dev Biol 18: 289–314. doi: 10.1146/annurev.cellbio.18.032202.114809
[37]
Haluska CK, Riske KA, Marchi-Artzner V, Lehn JM, Lipowsky R, et al. (2006) Time scales of membrane fusion revealed by direct imaging of vesicle fusion with high temporal resolution. Proc Natl Acad Sci U S A 103: 15841–15846. doi: 10.1073/pnas.0602766103
[38]
Südhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323: 474–477. doi: 10.1126/science.1161748
[39]
Scott P, Sher A (1986) A spectrum in the susceptibility of leishmanial strains to intracellular killing by murine macrophages. J Immunol 136: 1461–1466.
[40]
Alpuche-Aranda CM, Racoosin EL, Swanson JA, Miller SI (1994) Salmonella stimulate macrophage macropinocytosis and persist within spacious phagosomes. J Exp Med 179: 601–608. doi: 10.1084/jem.179.2.601
[41]
Akopyants NS, Kimblin N, Secundino N, Patrick R, Peters N, et al. (2009) Demonstration of genetic exchange during cyclical development of Leishmania in the sand fly vector. Science 324: 265–268. doi: 10.1126/science.1169464
[42]
Bastrenta B, Mita N, Buitrago R, Vargas F, Flores M, et al. (2003) Human mixed infections of Leishmania spp. And Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme. Mem Inst Oswaldo Cruz 98: 255–264. doi: 10.1590/S0074-02762003000200015
[43]
Lang T, Lecoeur H, Prina E (2009) Imaging Leishmania development in their host cells. Trends Parasitol 25: 464–473. doi: 10.1016/j.pt.2009.07.006
