Endosome Sorting and Autophagy Are Essential for Differentiation and Virulence of Leishmania major

Cellular remodeling during differentiation is essential for lifecycle progression of many unicellular eukaryotic pathogens such as Leishmania, but the mechanisms involved are largely uncharacterized. The role of endosomal sorting in differentiation was analyzed in Leishmania major by overexpression of a dominant-negative ATPase, VPS4. VPS4E235Q accumulated in vesicles from the endocytic pathway, and the mutant L. major was deficient in endosome sorting. Mutant parasites failed to differentiate to the obligate infective metacyclic promastigote form. Furthermore, the autophagy pathway, monitored via the expression of autophagosome marker GFP-ATG8, and shown to normally peak during initiation of metacyclogenesis, was disrupted in the mutants. The defect in late endosome-autophagosome function in the VPS4E235Q parasites made them less able to withstand starvation than wild-type L. major. In addition, a L. major ATG4-deficient mutant was found also to be defective in the ability to differentiate. This finding, that transformation to the infective metacyclic form is dependent on late endosome function and, more directly, autophagy, makes L. major a good model for studying the roles of these processes in differentiation. Cellular remodeling during differentiation is essential for lifecycle progression of many unicellular eukaryotic pathogens such as Leishmania, but the mechanisms involved are largely uncharacterized. The role of endosomal sorting in differentiation was analyzed in Leishmania major by overexpression of a dominant-negative ATPase, VPS4. VPS4E235Q accumulated in vesicles from the endocytic pathway, and the mutant L. major was deficient in endosome sorting. Mutant parasites failed to differentiate to the obligate infective metacyclic promastigote form. Furthermore, the autophagy pathway, monitored via the expression of autophagosome marker GFP-ATG8, and shown to normally peak during initiation of metacyclogenesis, was disrupted in the mutants. The defect in late endosome-autophagosome function in the VPS4E235Q parasites made them less able to withstand starvation than wild-type L. major. In addition, a L. major ATG4-deficient mutant was found also to be defective in the ability to differentiate. This finding, that transformation to the infective metacyclic form is dependent on late endosome function and, more directly, autophagy, makes L. major a good model for studying the roles of these processes in differentiation. Cargo destined for the lysosome/vacuole compartment in eukaryotic cells requires sorting from the biosynthetic, secretory, and endocytic pathways. Transport of newly synthesized lysosomal proteins from the trans-Golgi network to the lysosomal compartment involves the delivery of cargo into the endosomal system, which serves as a sorting compartment as well as a collection site for endocytosed materials. The endocytic process, which is important for internalization of portions of the plasma membrane as well as extracellular fluids, involves a number of mechanisms (many of them receptor-mediated), including phagocytosis, macropinocytosis, caveolae, and clathrin-dependent or clathrin-independent endocytosis (1Conner S.D. Schmid S.L. Nature. 2003; 422: 37-44Crossref PubMed Scopus (3070) Google Scholar). Once internalized, receptor-bound ligands, solutes, and lipids are subject to complex intracellular trafficking pathways and can be recycled back to the plasma membrane or degraded in the lysosomal compartment. The endosomal system itself is complex, and several types of vacuoles have been identified, including late endosomes having a multivesicular aspect and called multivesicular bodies (MVB 2The abbreviations used are: MVB, multivesicular bodies; VPS, vacuolar protein sorting; ESCRT, endosomal sorting complexes required for transport; MVT, multivesicular tubule; GFP, green fluorescent protein; PE, phosphatidylethanolamine; EF1α, elongation factor 1α; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline. ; see Refs. 2Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1021) Google Scholar and references therein for a review). A screen for vacuolar protein sorting (Vps) mutants in yeast led to the discovery of the molecular machinery responsible for MVB formation and, in particular, the class E group of Vps mutants, which contained a large multilamellar cisternal compartment thought to represent an endosome unable to form intraluminal vesicles (3Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (680) Google Scholar, 4Rieder S.E. Banta L.M. Kohrer J.M. McCaffery J.M. Emr S.D. Mol. Biol. Cell. 1996; 7: 985-999Crossref PubMed Scopus (239) Google Scholar). Vps class E proteins are organized in several sub-complexes named “endosomal sorting complexes required for transport” (ESCRT) I, II, and III (5Babst M. Katzmann D.J. Snyder W.B. Wendland B. Emr S.D. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, 6Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Dev. Cell. 2002; 3: 271-282Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 7Conibear E. Mol. Cell. 2002; 10: 215-216Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 8Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1129) Google Scholar). The ESCRT proteins are recruited from the cytoplasm to the endosomal membrane where they function sequentially in the formation of MVB vesicles and the sorting of proteins into the MVB pathway. It has been proposed that a multimeric AAA-type ATPase, Vps4, binds to ESCRT III and catalyzes the disassembly of this complex in an ATP-dependent manner (9Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (625) Google Scholar). This Vps4-dependent dissociation of the ESCRT machinery is the final step of protein sorting into the MVB and is a prerequisite for vesicle formation (6Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Dev. Cell. 2002; 3: 271-282Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). The Vps4 homologue in mammals, SKD1, is involved in membrane transport through endosomes and overexpression of a dominant negative mutant, SKD1E235Q, resulted in the production of aberrant endosomes defective in membrane transport between late endosomes and lysosomes (10Yoshimori T. Yamagata F. Yamamoto A. Mizushima N. Kabeya Y. Nara A. Miwako I. Ohashi M. Ohsumi M. Ohsumi Y. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar). In the Leishmania, as in such as is a complex membrane network the an of the plasma membrane where the from the M. Mol. 2001; PubMed Scopus Google Scholar). This is an important of between the and is the site in the for endocytosis and and is responsible for such as of via endocytosis G. N. G. A. J. Biol. Full Text Full Text PDF PubMed Scopus Google and of that can the of Leishmania parasites is that promastigote form a lysosomal named the multivesicular J.M. Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar, F. M. J. 2000; PubMed Google Scholar, E. A. 2001; PubMed Scopus Google Scholar). This compartment has a and a in but the of lysosomes as the into but metacyclic form J.M. Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar). a of the Leishmania endosomal machinery have been the molecular such as G. N. A. EMBO J. 2003; PubMed Scopus Google and Mol. 2002; PubMed Scopus Google Scholar). has been shown that the is of a network of endosomes that the J.M. Mol. Biol. Cell. 2001; PubMed Scopus Google is them in the pathways in eukaryotic cells is autophagy, a that is important for protein and during differentiation and also as a starvation B. D.J. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The pathways of yeast and have been and, they have many they to in D.J. Scholar, F. D.J. Cell. 2002; PubMed Scopus Google Scholar). to the in is a compartment called the which is by a that the that are to be The of requires the of protein protein by and a protein to B. D.J. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, F. D.J. Cell. 2002; PubMed Scopus Google Scholar, D.J. 2001; PubMed Scopus Google Scholar). The autophagosome the to the lysosomal compartment for It in mammals, but in endosomal vesicles N. Ohsumi Y. T. 2002; PubMed Scopus Google Scholar, A. Mizushima N. Yamamoto A. Kabeya Y. Ohsumi Y. T. 2002; PubMed Scopus Google Scholar, T. Ohsumi Y. PubMed Scopus Google Scholar). in this the autophagosome requires the endosomal to the proposed for autophagy is a role in the during and this is B. D.J. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). have shown that differentiation is in autophagy mutants of N. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google A. M. B. 2003; PubMed Scopus Google and where autophagy to be important for A. 2002; PubMed Scopus Google Scholar). In this the function of in Leishmania major by the Vps4 A mutant accumulated the protein of the endocytic system and a defect in transport to the This is to has been in yeast and Vps4 mutants, a of role for this protein in MVB from the to L. VPS4E235Q in differentiation in and to a role for and the MVB compartment in these In addition, by the of in Leishmania as a marker and that these accumulated and in the VPS4E235Q mutant, have that the is involved the endosomal of an L. major mutant, which metacyclogenesis, that the of a and the differentiation defect of the mutant that major in by the A. J. Google from that been in for to the of of the this to and to to for and to for or L. major. the and in of L. major was by from L. major and and The was by and into Mol. 1996; PubMed Scopus Google was by of the and to used in this in a The as was by from and and into E. I. T. T. Mol. 2002; PubMed Scopus Google Scholar). The was named and contained the of A was by on as L. major was by from and and into to of a L. major Mutant and of was by from L. major and and and into A. 1996; PubMed Scopus Google to The was by and The was by and and into to The used for was by used for the of the was from by the the by a the A of parasites to was This was used for a of the from and for the the a was into the the and the of The was by and into E. I. T. T. Mol. 2002; PubMed Scopus Google by the to the L. major wild-type of the and the and of and metacyclic to was a from J. Google Scholar). parasites in an of the in The of by of promastigote and was to in in which cells from in in and late or a of for in by and the in The of by and intracellular and as was by the number of and the number of intracellular parasites in and in and to the The number of L. major was by of of wild-type or L. major by and the of cells as well as the number of these of cells for the formation of in or in to starvation from by and in in and for to of L. major during starvation was by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium as for Leishmania J. PubMed Scopus Google Scholar). in as and for to a final of and the was a cells in during the of and The as a of the for the cells to the of the cells and in a a the of the was to an on which the to the was as Mol. PubMed Scopus Google Scholar). The used and Mol. PubMed Scopus Google E. J. 2001; PubMed Scopus Google and The system was used to of L. major by and in the cells a in for and in and for in and for cells in for in and for cells in of and of was for and for a in the of in for in PBS, and to A cells in of of A was and cells for in and as for as S.D. from and during starvation for A of was used as the of of L. major and of a L. major was by the L. proteins the yeast Vps4 as a of yeast and and proteins to the and to a in the of the protein in of L. major from and as well as a protein of The in during in was an of the protein in the metacyclic form of the the function of the protein in L. a mutant was This Leishmania mutant is to the yeast mutant M. PubMed Scopus Google and the mutant (10Yoshimori T. Yamagata F. Yamamoto A. Mizushima N. Kabeya Y. Nara A. Miwako I. Ohashi M. Ohsumi M. Ohsumi Y. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google and the was to a defect in (9Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (625) Google Scholar). L. major and The wild-type and normally as in but the mutant a from the to wild-type parasites of the of in but into a than wild-type or this in an number of VPS4E235Q and and see VPS4E235Q in The of an for the expression of the to expression of This have shown that the defect was more the mutant was in of in and this of VPS4E235Q protein the In cells the withstand of to or on that the the marker to the overexpression of was shown to be to the cells as the normally in the of of and a defect expression and the on from from and of wild-type L. major and The and protein and is shown on the of and VPS4E235Q in The has been the is shown in the of or VPS4E235Q endocytosed and A is shown in and the is the are late of and major of and VPS4E235Q and expression of the proteins and was a than the expression of was to Leishmania is that the cells the protein to for this cells from these by a the a to the of (10Yoshimori T. Yamagata F. Yamamoto A. Mizushima N. Kabeya Y. Nara A. Miwako I. Ohashi M. Ohsumi M. Ohsumi Y. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar, N. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google and Vps4 in yeast (9Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (625) Google Scholar). the was in and and to the class E compartment of which is of (3Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (680) Google Scholar). The of these in Leishmania in the of the the of the endocytic pathways in the G. N. A. EMBO J. 2003; PubMed Scopus Google Scholar, Mol. 2002; PubMed Scopus Google Scholar). the compartment in which accumulated in Leishmania, of endosomal which of proteins in endosomes of M. Mol. PubMed Scopus Google was to but was which been shown to in Leishmania Mol. 2002; PubMed Scopus Google accumulated in the to vesicles late to the for to be internalized, A a in the of the This that the compartment in which is of the endosomal system or This is in a late endosomal as for (9Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (625) Google Scholar, T. Yamagata F. Yamamoto A. Mizushima N. Kabeya Y. Nara A. Miwako I. Ohashi M. Ohsumi M. Ohsumi Y. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar, N. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar, M. I. J. PubMed Scopus Google Scholar). Transport of Cargo to the Leishmania in the VPS4E235Q was used to the of transport to the lysosomal compartment in the promastigote This fluorescent endocytic marker is to the vacuolar membrane in yeast Emr S.D. J. Biol. PubMed Scopus Google and has been used to class E S.E. Banta L.M. Kohrer J.M. McCaffery J.M. Emr S.D. Mol. Biol. Cell. 1996; 7: 985-999Crossref PubMed Scopus (239) Google Scholar). has been used in Leishmania as a marker for the in the promastigote form of the J.M. Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar). used in a the was found to be the and the of the a of in the was in the as well as the In largely in the in the but also the was in used fluorescent as a endocytic are to be through the endosomal system to the of Leishmania E. A. 2001; PubMed Scopus Google Scholar). was normally to the in the accumulated in the in the mutant cells that is a defect in transport to the or that be an of that in the VPS4E235Q Furthermore, the that the is in the in the This that the of transport are in VPS4E235Q to into the is for the and of Leishmania in the J. J. PubMed Google Scholar). the was to of during in was in this the and metacyclic promastigote of L. including PubMed Scopus Google A. J. Google expression of proteins Mol. PubMed Scopus Google and E. J. 2001; PubMed Scopus Google to J. Google and to A. J. Google Scholar). of these wild-type and cells in late of in into the metacyclic form. In the VPS4E235Q failed to have metacyclic form and proteins or and to by to the the the having was that a of the cells The mutant was found to be less and in wild-type parasites the or the the number of the was found to be the that been found the was cells that the of A. J. Google was to an infective and that the cells failed to differentiate mutants are less infective to in and of number of parasites S.D. of The shown are of of from VPS4E235Q Mutant a in membrane trafficking by has been proposed to be involved in the late of the in A. Mizushima N. Yamamoto A. Kabeya Y. Ohsumi Y. T. 2002; PubMed Scopus Google and also in autophagy in yeast T. Ohsumi Y. PubMed Scopus Google Scholar). of yeast proteins that have been in the involved in autophagosome and can be in L. including and several of proteins M. D.J. PubMed Scopus Google Scholar). A. J. and G. in The L. major the to yeast proteins was used in this has been a marker for in yeast and cells D.J. Scholar, J. D.J. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). to formation of in L. major In L. major was the cytoplasm in cells in be as in the cytoplasm the of the in the of during the number of was during the This that the number of cells as well as the number of from to in wild-type and the into differentiation into the metacyclic the number of and the of cells a of cells in a which was as the by The of in L. major be by the of or of formation in yeast and cells D.J. to the the of in autophagy in Leishmania, was into and to parasites in in was found the cytoplasm a cells contained in to the more contained analyzed for production of the a to that of parasites In more in the mutants in and the number of was also found to be than in the is in yeast and cells as a starvation B. D.J. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, D.J. 2001; PubMed Scopus Google Scholar). found that the of in the cells of L. major be by in for In the mutants, which a number of than the in the number of the several in to and to the membrane Y. T. T. Y. Y. N. Mizushima N. I. E. Ohsumi M. T. Ohsumi Y. Nature. 2000; PubMed Scopus Google Scholar). used on from and cells to the form the the of during progression from to of or cells for in In the mutant a of the form in and This is the by of the in the by than the that the mutant is more to than the the of during starvation of the number of which was in the that the have an autophagy in which are and which makes the cells more to Mutant a in is a of autophagy in the differentiation process, a mutant of which is of in the of L. major M. D.J. PubMed Scopus Google Scholar). A. J. and G. in is a involved in and is for autophagosome function J. D.J. J. Biol. 2001; PubMed Scopus Google Scholar, T. Y. Kabeya Y. Mizushima N. T. Ohsumi M. T. T. Ohsumi Y. J. Biol. 2000; PubMed Scopus Google Scholar). L. major mutant by in which the of the and the of the by and of a a to the of the wild-type which was in the In a of was of the and and but as they are shown for The ability of L. major mutants to form was by them that they the The of and the number of in was than for wild-type cells a of and In addition, the number of during of as wild-type on of wild-type and that the mutant an of the form of to the form This is an of the form of in the mutant, by starvation was on the also less able to withstand starvation than the wild-type cells that the accumulated GFP-ATG8, as are unable to the autophagy pathway, which in an autophagy This be to wild-type by of a of in the mutant The Mutant to into the the ability of to differentiate into metacyclic the as found to be defective in expression of metacyclic proteins and and more to by between the cells and was by of wild-type and cells by of between the mutant these to in the mutant that are defective in In this have the of L. major MVB by dominant-negative mutants of which MVB sorting In Vps4 is involved in the disassembly of the ESCRT III complex from the endosomal and the mutant from the late endosomal membrane (9Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (625) Google Scholar). In L. was to a which the late endosomal marker A the of the between the and the was found the This is having a role in L. major as in yeast and and this is by the of of the ESCRT III complex in the of L. major It has been proposed for the mutants that these endosomes represent a between late endosomes and lysosomes M. Y. Nara A. T. M. J. 2003; PubMed Scopus Google Scholar). are a of VPS4E235Q endosomal and the marker that in the VPS4E235Q mutant endocytosed cargo the the and expression of in the mutant that major in in the mutants. The in lysosomal transport is to the for yeast and mutants (9Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (625) Google Scholar, M. Y. Nara A. T. M. J. 2003; PubMed Scopus Google Scholar). This in L. a in cells that many of metacyclic that they a defect in The was that the VPS4E235Q cells on is complex and in the to the A. Y. PubMed Scopus Google is that this is of proteins such as and and in the of of the of this to differentiate into the metacyclic form was and of the mutant parasites in In yeast and late endosomal membrane trafficking by have been shown to be important for the function of the A. Mizushima N. Yamamoto A. Kabeya Y. Ohsumi Y. T. 2002; PubMed Scopus Google Scholar, T. Ohsumi Y. PubMed Scopus Google Scholar). In Leishmania, autophagy, monitored by the of was during the differentiation of Leishmania to the infective metacyclic promastigote form It also be in by the cells and a that autophagy in yeast and the of the be by the of autophagy and D.J. In VPS4E235Q the number of cells and them was than in the in and they a that is that production of Furthermore, the form of was found to be in VPS4E235Q cells than in wild-type This that the VPS4E235Q cells accumulated to a defect in the ability of the to the compartment. In wild-type the to the in metacyclic parasites that the differentiation between and the infective metacyclic which by many of and is dependent on endosome function and The of the in cells requires a MVB compartment as by the defect in autophagy in the Vps4 mutant A. Mizushima N. Yamamoto A. Kabeya Y. Ohsumi Y. T. 2002; PubMed Scopus Google Scholar). the of the of the endosomal compartment in the in yeast is less F. D.J. Cell. 2002; PubMed Scopus Google Scholar, F. T. D.J. Mol. Biol. Cell. PubMed Scopus Google Scholar). have shown that in Leishmania, the VPS4E235Q cells a defect in autophagy, and that to in the that in this the the endosomal network in to for the of autophagy in differentiation of the was by the of the mutants. is essential for autophagy in yeast and J. D.J. J. Biol. 2001; PubMed Scopus Google Scholar, T. Y. Kabeya Y. Mizushima N. T. Ohsumi M. T. T. Ohsumi Y. J. Biol. 2000; PubMed Scopus Google is a that is involved in the of the membrane of by the of the protein to In where is of the mutant the ability to and to a defect in autophagy Y. T. T. Y. Y. N. Mizushima N. I. E. Ohsumi M. T. Ohsumi Y. Nature. 2000; PubMed Scopus Google Scholar). are in but and roles have been J. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, G. J. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). that the mutant of of the L. major and has a defect in autophagy, as by the ability of the mutants to withstand It to that be involved in to late a that is for progression of autophagy in yeast Y. T. T. Y. Y. N. Mizushima N. I. E. Ohsumi M. T. Ohsumi Y. Nature. 2000; PubMed Scopus Google Scholar). The mutant is also in ability to differentiate into the metacyclic is a between the machinery and the differentiation It is that for of VPS4E235Q and the on This a role between endosome sorting and autophagy in the of differentiation. The VPS4E235Q cells more to starvation than the and, the the number of in this It is to that on in shown that autophagy was in a mutant of this T. Ohsumi Y. PubMed Scopus Google Scholar). mutants to be in ability to the formation of but these The of the VPS4E235Q cells to starvation be a of several the VPS4E235Q defective in the transport of endocytosed cargo to the and ability to and from the be the itself to be the lysosomal is and in the in VPS4E235Q cells in the final of the autophagy in VPS4E235Q cells the parasites more to starvation to a ability of the parasites to as or This defect is to be found the mutant was in the role of autophagy in the of Leishmania during and are of autophagy in yeast and cells B. D.J. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, D.J. 2001; PubMed Scopus Google Scholar). In autophagy has been proposed to be differentiation B. D.J. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google where This was by but a of such a role of processes in differentiation have N. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. B. 2003; PubMed Scopus Google Scholar, A. 2002; PubMed Scopus Google Scholar). Leishmania an in of where is a and is this that they differentiate from promastigote to the infective metacyclic form. an number of in the of differentiation autophagy normally during differentiation and, as this has is for autophagy made have on the role of autophagy during have shown for the that autophagy has an important role in the differentiation and of of a roles for autophagy from and remodeling to but role in differentiation is Full of the molecular mechanisms of autophagy, which are largely from unicellular to but in through can from the of Leishmania, which has and can be are to for the of to for the of and and to the for L. major