Glycosome turnover in Leishmania major is mediated by autophagy

Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite''s autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ∼20 glycosomes per cell, whereas amastigotes contain ∼10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ∼15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the turnover of glycosomes.     

ATG5 Is Essential for ATG8-Dependent Autophagy and Mitochondrial Homeostasis in Leishmania major

Macroautophagy has been shown to be important for the cellular remodelling required for Leishmania differentiation. We now demonstrate that L. major contains a functional ATG12-ATG5 conjugation system, which is required for ATG8-dependent autophagosome formation. Nascent autophagosomes were found commonly associated with the mitochondrion. L. major mutants lacking ATG5 (Δatg5) were viable as promastigotes but were unable to form autophagosomes, had morphological abnormalities including a much reduced flagellum, were less able to differentiate and had greatly reduced virulence to macrophages and mice. Analyses of the lipid metabolome of Δatg5 revealed marked elevation of phosphatidylethanolamines (PE) in comparison to wild type parasites. The Δatg5 mutants also had increased mitochondrial mass but reduced mitochondrial membrane potential and higher levels of reactive oxygen species. These findings indicate that the lack of ATG5 and autophagy leads to perturbation of the phospholipid balance in the mitochondrion, possibly through ablation of membrane use and conjugation of mitochondrial PE to ATG8 for autophagosome biogenesis, resulting in a dysfunctional mitochondrion with impaired oxidative ability and energy generation. The overall result of this is reduced virulence.     

Differential phagocytosis of Leishmania mexicana promastigotes and amastigotes by monocyte‐derived dendritic cells

Leishmania species are dimorphic protozoan parasites that live and replicate in the gut of sand flies as promastigotes or in mammalian hosts as amastigotes. Different immune cells, including DCs, and receptors differ in their involvement in phagocytosis of promastigotes and amastigotes and in recognition of different Leishmania species. In the case of L. mexicana, differences in phagocytosis of promastigotes and amastigotes by DCs and participation of C‐type lectin receptors (CLRs) have not been established. In the present study, flow cytometry and confocal microscopy were used to investigate the phagocytosis by monocyte‐derived dendritic cells (moDCs) of L. mexicana promastigotes and amastigotes in the presence or absence of immune serum during various periods of time. Blocking antibodies against mannose receptors and DC‐SIGN were used to explore the participation of these receptors in the phagocytosis of L. mexicana by moDC. The major differences in interactions of L. mexicana promastigotes and amastigotes with moDC were found to occur within the first 3 hr, during which phagocytosis of promastigotes predominated as compared with opsonization of promastigotes and amastigotes. However, after 6 hr of incubation, opsonized promastigotes were preferentially phagocytosed as compared with unopsonized promastigotes and amastigotes and after 24 hr of incubation there were no differences in the phagocytosis of promastigotes and amastigotes. Finally, after 3 hr incubation, DC‐SIGN was involved in the phagocytosis of promastigotes, but not of amastigotes.     

Localization and induction of the A2 virulence factor in Leishmania: evidence that A2 is a stress response protein

Leishmaniasis is a vector‐borne infectious disease with a wide range of pathologies depending on the species of Leishmania. Leishmania parasites are transmitted by the sand fly vector as promastigotes; within the mammalian host, Leishmania parasites differentiate into amastigotes and replicate in macrophages. The A2 protein from Leishmania donovani is expressed predominantly in amastigotes and therefore likely plays a role in survival in the mammalian host. In the present study, we have determined that the A2 protein colocalized with the Leishmania endoplasmic reticulum binding protein, BiP, was induced by stress and complexed with BiP following heat shock. The A2 gene in Leishmania major is a non‐expressed pseudogene, and we present evidence that ectopic expression of a transfected A2 gene in L. major enhanced its viability following heat shock. A2 may therefore play a role in protecting L. donovani from stress associated with infection in visceral organs, including the fever typically associated with visceral leishmaniasis. Interestingly, when comparing A2 protein localization, we also observed that the Leishmania secreted acid phosphatase SAcP protein was transported out of the parasite‐containing phagolysosome and was located throughout the macrophage cytoplasm in vesicles, providing the first example of a secreted Leishmania‐derived protein exiting the parasite‐containing phagolysosome.     

Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in Leishmania mexicana

In the past, ultrastructural investigations of Leishmania mexicana amastigotes revealed structures that were tentatively identified as autophagosomes. This study has now provided definitive data that autophagy occurs in the parasite during differentiation both to metacyclic promastigotes and to amastigotes, autophagosomes being particularly numerous during metacyclic to amastigote form transformation. Moreover, the results demonstrate that inhibiting two major lysosomal cysteine peptidases (CPA and CPB) or removing their genes not only interferes with the autophagy pathway but also prevents metacyclogenesis and transformation to amastigotes, thus adding support to the hypothesis that autophagy is required for cell differentiation. The study suggests that L. mexicana CPA and CPB perform similar roles to the aspartic peptidase PEP4 and the serine peptidase PRB1 in Saccharomyces cerevisiae. The results also provide an explanation for why L. mexicana CPA/CPB-deficient mutants transform to amastigotes very poorly and lack virulence in macrophages and mice.     

Distinct Roles in Autophagy and Importance in Infectivity of the Two ATG4 Cysteine Peptidases of Leishmania major

Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite.     

Evidence for Rosettes as an Unrecognized Stage in the Life Cycle of Leishmania Parasites

ABSTRACT. Leishmania parasites, which afflict 12 million people in 88 countries, exist as promastigotes transmitted by insect vectors and as amastigotes residing in mammalian macrophages. Promastigote cells arranged in rosettes have also been described but universally disregarded as a distinct stage in the life cycle. We present evidence that only rosettes of Leishmania major promastigotes express cell surface poly‐α2,8 N‐acetyl neuraminic acid (PSA) and PSA containing de‐N‐acetyl neuraminic acid (NeuPSA). Expression of rosette‐specific PSA antigens was mosaic, with individual promastigotes expressing PSA, NeuPSA or both. A 50 kDa protein was detected by Western blot analysis of a detergent‐insoluble cell fraction with both PSA and NeuPSA‐reactive antibodies. Frequencies of rosette formation as well as cell surface PSA/NeuPSA expression were temperature dependent. Rosettes also engaged in an unusual swarming behavior, congregating into extended clusters. Distinct structures resembling cellular fusion bodies were formed in and released from rosettes. The results indicate that rosettes are an unrecognized stage in the life cycle of Leishmania. We hypothesize that rosettes initiate mating in Leishmania during which PSA/NeuPSA expression plays an important role. Recognizing rosettes as a distinct form of the Leishmania life cycle opens new possibilities for treatment or prevention of disease and, possibly, in vitro genetic recombination without passage of cells through insect vectors.     

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.     

Developmental Life Cycle of Leishmania—Cultivation and Characterization of Cultured Extracellular Amastigotes1

ABSTRACT. The biochemistry and immunology of Leishmania promastigotes has been extensively studied; this is due primarily to the facility with which this stage, in contrast to the amastigotes stage, can be maintained in axenic culture. Several attempts to axenically culture lines of Leishmania amastigotes have been reported in the literature. This paper summarizes methods of adaptation (low pH, elevated temperature and culture medium) and characterization of several axenic lines of Leishmania amastigotes. Based on morphological, biological, immunological and biochemical evidence, these organisms appear to resemble amastigotes from infected macrophages or tissue. The axenically cultured amastigotes appear to be distinct from shocked (heat, serum deprivation, stressed) Leishmania promastigotes in the plethora of proteins synthesized, growth (multiplication) in culture, and developmental regulation observed. These data suggest that Leishmania organisms have a significant developmental response to certain signals (pH, temperature) mimicking their in vivo macrophage milieu. The response to other environmental parameters characteristic of the host-macrophage remain to be determined. These axenically cultured amastigotes should be of interest for further immunological, biochemical and developmental investigations of the disease-maintaining stage of this parasite.     

TMEM41B is a novel regulator of autophagy and lipid mobilization

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well‐characterized autophagy‐related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2‐ and DFCP1‐positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β‐oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER‐localized regulator of autophagosome biogenesis and lipid mobilization.     

Biogenesis of Autophagosome in Trichomonas vaginalis during Macroautophagy Induced by Rapamycin‐treatment and Iron or Glucose Starvation Conditions

Autophagy is an adaptive response for cell survival in which cytoplasmic components and organelles are degraded in bulk under normal and stress conditions. Trichomonas vaginalis is a parasite highly adaptable to stress conditions such as iron (IR) and glucose restriction (GR). Autophagy can be traced by detecting a key autophagy protein (Atg8) anchored to the autophagosome membrane by a lipid moiety. Our goal was to perform a morphological and cellular study of autophagy in T. vaginalis under GR, IR, and Rapamycin (Rapa) treatment using TvAtg8 as a putative autophagy marker. We cloned tvatg8a and tvatg8b and expressed and purified rTvAtg8a and rTvAtg8b to produce specific polyclonal antibodies. Autophagy vesicles were detected by indirect immunofluorescence assays and confirmed by ultrastructural analysis. The biogenesis of autophagosomes was detected, showing intact cytosolic cargo. TvAtg8 was detected as puncta signal with the anti‐rTvAtg8b antibody that recognized soluble and lipid‐associated TvAtg8b by Western blot assays in lysates from stress‐inducing conditions. The TvAtg8b signal co‐localized with the CytoID and lysotracker labeling (autolysosomes) that accumulated after E‐64d treatment in GR parasites. Our data suggest that autophagy induced by starvation in T. vaginalis results in the formation of autophagosomes for which TvAtg8b could be a putative autophagy marker.     

Interplay of Trypanosome Lytic Factor and innate immune cells in the resolution of cutaneous Leishmania infection

Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein (HDL) complex that, through the cation channel-forming protein apolipoprotein L-1 (APOL1), provides innate immunity to select kinetoplastid parasites. The immunoprotective effects of TLF have been extensively investigated in the context of its interaction with the extracellular protozoan Trypanosoma brucei brucei, to which it confers sterile immunity. We previously showed that TLF could act against an intracellular pathogen Leishmania, and here we dissected the role of TLF and its synergy with host-immune cells. Leishmania major is transmitted by Phlebotomine sand flies, which deposit the parasite intradermally into mammalian hosts, where neutrophils are the predominant phagocytes recruited to the site of infection. Once in the host, the parasites are phagocytosed and shed their surface glycoconjugates during differentiation to the mammalian-resident amastigote stage. Our data show that mice producing TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major, the infective, fly-transmitted stage. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. In vitro we find that only metacyclic promastigotes co-incubated with TLF in an acidic milieu were lysed. However, amastigotes were not killed by TLF at any pH. These findings correlated with binding experiments, revealing that labeled TLF binds specifically to the surface of metacyclic promastigotes, but not to amastigotes. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates LPG and/or PPG (lpg1^- and lpg5A^-/lpg5B^- respectively) whose absence mimics the amastigote surface, were resistant to TLF-mediated lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon it kills the parasite in the acidic phagosome of phagocytes. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs several hours after phagocytosis by immune cells, creating a relatively short-lived but effective window for TLF to act against Leishmania.     

Autophagosomes and lipid droplets: no longer just chewing the fat

Autophagosomes are organelles capable of sequestering and degrading diverse cytoplasmic cargo for nutritional and quality control purposes. Targeted are also lipid droplets (LDs), the cytoplasmic stores of neutral lipids. In this issue of The EMBO Journal, Shpilka et al ( 2015 ) show that the relationship between LDs and autophagosomes is far more intricate and that LDs regulate autophagosome biogenesis.     

Studies on the antileishmanial properties of the antimicrobial peptides temporin A,B and 1Sa

Given the paucity and toxicity of available drugs for leishmaniasis, coupled with the advent of drug resistance, the discovery of new therapies for this neglected tropical disease is recognised as being of the utmost urgency. As such antimicrobial peptides (AMPs) have been proposed as promising compounds against the causative Leishmania species, insect vector‐borne protozoan parasites. Here the AMP temporins A, B and 1Sa have been synthesised and screened for activity against Leishmania mexicana insect stage promastigotes and mammalian stage amastigotes, a significant cause of human cutaneous disease. In contrast to previous studies with other species the activity of these AMPs against L. mexicana amastigotes was low. This suggests that amastigotes from different Leishmania species display varying susceptibility to peptides from the temporin family, perhaps indicating differences in their surface structure, the proposed target of these AMPs. In contrast, insect stage L. mexicana promastigotes were sensitive to two of the screened temporins which clearly demonstrates the importance of screening AMPs against both forms of the parasite. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Activity of 1-aryl-4-(naphthalimidoalkyl) piperazine derivatives against Leishmania major and Leishmania mexicana

A series of 1-aryl-4-(phthalimidoalkyl) piperazines and 1-aryl-4-(naphthalimidoalkyl) piperazines were retrieved from a proprietary library based on their high structural similarity to haloperidol, an antipsychotic with antiparasitic activity, and assessed as potential antileishmanial scaffolds. Selected compounds were tested for antileishmanial activity against promastigotes of Leishmania major and Leishmania mexicana in dose-response assays. Two of the 1-aryl-4-(naphthalimidoalkyl) piperazines (compounds 10 and 11) were active against promastigotes of both Leishmania species without being toxic to human fibroblasts. Their activity was found to correlate with the length of their alkyl chains. Further analyses showed that compound 11 was also active against intracellular amastigotes of both Leishmania species. In promastigotes of both Leishmania species, compound 11 induced collapse of the mitochondrial electrochemical potential and increased the intracellular Ca²⁺ concentration. Therefore, it may serve as a promising lead compound for the development of novel antiparasitic drugs.     

ATG24 Represses Autophagy and Differentiation and Is Essential for Homeostasy of the Flagellar Pocket in Trypanosoma brucei

We have previously identified homologs for nearly half of the approximately 30 known yeast Atg’s in the genome database of the human sleeping sickness parasite Trypanosoma brucei. So far, only a few of these homologs have their role in autophagy experimentally confirmed. Among the candidates was the ortholog of Atg24 that is involved in pexophagy in yeast. In T. brucei, the peroxisome-like organelles named glycosomes harbor core metabolic processes, especially glycolysis. In the autotrophic yeast, autophagy is essential for adaptation to different nutritional environments by participating in the renewal of the peroxisome population. We hypothesized that autophagic turnover of the parasite’s glycosomes plays a role in differentiation during its life cycle, which demands adaptation to different host environments and associated dramatic changes in nutritional conditions. We therefore characterized T. brucei ATG24, the T. brucei ortholog of yeast Atg24 and mammalian SNX4, and found it to have a regulatory role in autophagy and differentiation as well as endocytic trafficking. ATG24 partially localized on endocytic membranes where it was recruited via PI3-kinase III/VPS34. ATG24 silencing severely impaired receptor-mediated endocytosis of transferrin, but not adsorptive uptake of a lectin, and caused a major enlargement of the flagellar pocket. ATG24 silencing approximately doubled the number of autophagosomes, suggesting a role in repressing autophagy, and strongly accelerated differentiation, in accordance with a role of autophagy in parasite differentiation. Overexpression of the two isoforms of T. brucei ATG8 fused to GFP slowed down differentiation, possibly by a dominant-negative effect. This was overcome by ATG24 depletion, further supporting its regulatory role.     

Accumulation of autophagosomes in Semliki Forest virus-infected cells is dependent on expression of the viral glycoproteins

Autophagy is a cellular process that sequesters cargo in double-membraned vesicles termed autophagosomes and delivers this cargo to lysosomes to be degraded. It is enhanced during nutrient starvation to increase the rate of amino acid turnover. Diverse roles for autophagy have been reported for viral infections, including the assembly of viral replication complexes on autophagic membranes and protection of host cells from cell death. Here, we show that autophagosomes accumulate in Semliki Forest virus (SFV)-infected cells. Despite this, disruption of autophagy had no effect on the viral replication rate or formation of viral replication complexes. Also, viral proteins rarely colocalized with autophagosome markers, suggesting that SFV did not utilize autophagic membranes for its replication. Further, we found that SFV infection, unlike nutrient starvation, did not inactivate the constitutive negative regulator of autophagosome formation, mammalian target of rapamycin, suggesting that SFV-dependent accumulation of autophagosomes was not a result of enhanced autophagosome formation. In starved cells, addition of NH(4)Cl, an inhibitor of lysosomal acidification, caused a dramatic accumulation of starvation-induced autophagosomes, while in SFV-infected cells, NH(4)Cl did not further increase levels of autophagosomes. These results suggest that accumulation of autophagosomes in SFV-infected cells is due to an inhibition of autophagosome degradation rather than enhanced rates of autophagosome formation. Finally, we show that the accumulation of autophagosomes in SFV-infected cells is dependent on the expression of the viral glycoprotein spike complex.     

Exocyst Subcomplex Functions in Autophagosome Biogenesis by Regulating Atg9 Trafficking

During autophagy, double-membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of exocyst, an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy. Our findings indicate that not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst, which are dispensable for autophagic function, do not associate with the autophagy specific subcomplex of exocyst.