[Show abstract][Hide abstract] ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.
For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.
Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.
[Show abstract][Hide abstract] ABSTRACT: The protozoan parasite Toxoplasma gondii develops within a parasitophorous vacuole (PV) in mammalian cells, from where it scavenges cholesterol. When cholesterol is present in excess in its environment, the parasite expulses this lipid into the PV or esterifies it for storage in lipid bodies. Here, we characterized a unique T. gondii homologue of mammalian lecithin:cholesterol acyltransferase (LCAT), a key enzyme that produces cholesteryl esters via transfer of acyl groups from phospholipids to the 3-OH of free cholesterol, leading to the removal of excess cholesterol from tissues. TgLCAT contains a motif characteristic of serine lipases AHSLG and the catalytic triad consisting of serine, aspartate and histidine [SDH] of LCAT enzymes. TgLCAT is secreted by the parasite, but unlike other LCAT enzymes, is cleaved into two proteolytic fragments that share the residues of the catalytic triad and that need to be reassembled to reconstitute enzymatic activity. TgLCAT uses phosphatidylcholine as substrate to form lysophosphatidylcholine that has the potential to disrupt membranes. The released fatty acid is transferred to cholesterol, although with a lower transesterification activity than mammalian LCAT. TgLCAT is stored in a subpopulation of dense granule secretory organelles, and following secretion, it localizes to the PV and parasite plasma membrane. LCAT-null parasites have impaired growth in vitro, reduced virulence in animals and exhibit delay in egress from host cells. Parasites overexpressing LCAT show increased virulence and faster egress. These observations demonstrate that TgLCAT influences the outcome of an infection, presumably by facilitating replication and egress depending on the parasite developmental stage.
Full-text · Article · Dec 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Author Summary
The opportunistic pathogen Toxoplasma gondii infects a large range of nucleated cells where it replicates intracellularly within a parasitophorous vacuole (PV) surrounded by a membrane (PVM). Parasites constitutively secrete dense-granule proteins (GRAs) both into and beyond the PV which participate in remodelling of the PVM, recruitment of host organelles, neutralization of the host cellular defences, and subversion of host cell functioning. In addition, the GRAs critically contribute to cyst wall formation, a process that critically ensures parasite persistence and transmission. To act as effector molecules, some of the GRAs must be translocated across the PVM. Within the related apicomplexan parasite P. falciparum, a repertoire of proteins exported beyond the PVM contain a motif cleaved by a specific protease, Plasmepsin V. Examination of the repertoire of GRAs in T. gondii revealed that some proteins exhibit such export-like motifs suggestive of protease involvement. In this study, we have functionally characterized the related aspartyl protease 5 (TgASP5) in both virulent and persistent T. gondii strains, and have investigated the phenotypic consequences of its deletion in the context of overall parasite biology, its intracellular niche, the infected host cells and the murine model. Our findings revealed fundamental roles of TgASP5 at the host-parasite interface.
[Show abstract][Hide abstract] ABSTRACT: In mammalian cells, the protozoan pathogen Toxoplasma resides in a nonfusiogenic vacuole that segregates it from host cell resources. How the parasite acquires nutrients and whether it is capable of internalizing host macromolecules have been long-standing mysteries. By exploiting a mutant of Toxoplasma lacking the cathepsin protease L, Dou et al. observed the accumulation of host cytosolic-derived proteins in a multivesicular post-Golgi compartment, which establishes the existence of a functional heterophagic pathway in Toxoplasma.
No preview · Article · Aug 2014 · Trends in Parasitology
[Show abstract][Hide abstract] ABSTRACT: Plasmodium falciparum (Pf) has a family of 11 Rab GTPases to regulate its vesicular transport. However, PfRab5B is unique in lacking a C-terminal geranyl-geranylation motif, while having N-terminal palmitoylation and myristoylation motifs. We show that the N-terminal glycine is required for PfRab5B myristoylation in vitro and when an N-terminal PfRab5B fragment possessing both acylation motifs is fused to GFP and expressed in transgenic P. falciparum parasites, the chimeric PfRab5B protein localizes to the plasma membrane. Upon substitution of the modified glycine by alanine the staining becomes diffuse and GFP is found in soluble subcellular fractions. Immuno-electron microscopy shows endogenous PfRab5B decorating the parasite's plasma and food vacuole membranes. Using reverse genetics rab5b couldn't be deleted from the haploid genome of asexual blood stage P. berghei parasites. The failure of PbRab5A or PbRab5C to complement for loss of PbRab5B function indicates non-overlapping roles for the three Plasmodium Rab5s, with PfRab5B involved in trafficking MSP1 to the food vacuole membrane and CK1 to the plasma membrane. We discuss similarities between Plasmodium Rab5B and Arabidopsis thaliana ARA6, a similarly unusual Rab5-like GTPase of plants.
[Show abstract][Hide abstract] ABSTRACT: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
[Show abstract][Hide abstract] ABSTRACT: Plasmodium parasites successfully colonize different habitats within mammals and mosquitoes, and adaptation to various environments is accompanied by changes in their organelle composition and size. Previously, we observed that during hepatocyte infection, Plasmodium discards organelles involved in invasion and expands those implicated in biosynthetic pathways. We hypothesized that this process is regulated by autophagy. Plasmodium spp. possess a rudimentary set of known autophagy-related proteins that includes the ortholog of yeast Atg8. In this study, we analyzed the activity of the ATG8-conjugation pathway over the course of the lifecycle of Plasmodium falciparum and during the liver stage of Plasmodium berghei. We engineered a transgenic P. falciparum strain expressing mCherry-PfATG8. These transgenic parasites expressed mCherry-PfATG8 in human hepatocytes and erythrocytes, and in the midgut and salivary glands of Anopheles mosquitoes. In all observed stages, mCherry-PfATG8 was localized to tubular structures. Our EM and colocalization studies done in P. berghei showed the association of PbATG8 on the limiting membranes of the endosymbiont-derived plastid-like organelle known as the apicoplast. Interestingly, during parasite replication in hepatocytes, the association of PbATG8 with the apicoplast increases as this organelle expands in size. PbATG3, PbATG7 and PbATG8 are cotranscribed in all parasitic stages. Molecular analysis of PbATG8 and PbATG3 revealed a novel mechanism of interaction compared with that observed for other orthologs. This is further supported by the inability of Plasmodium ATG8 to functionally complement atg8Δ yeast or localize to autophagosomes in starved mammalian cells. Altogether, these data suggests a unique role for the ATG8-conjugation system in Plasmodium parasites.
[Show abstract][Hide abstract] ABSTRACT: Like any obligate intracellular pathogen, the parasite Toxoplasma gondii has lost its capacity for living independently of another organism. Toxoplasma lacks many genes that encode for entire metabolic pathways and has, in return, expanded genes that promote nutrient scavenging to meet its basic metabolic requirements. Although sequestrated in a parasitophorous vacuole and thus insulated from the nutrient-rich host cytosol and organelles by a membrane, T. gondii has evolved efficient strategies to acquire essential metabolites from mammalian cells. This review explores the natural auxotrophies and nutrient scavenging activities of the parasite, emphasizing unique transport systems and salvage pathways. We describe the mechanisms deployed by Toxoplasma to modify its parasitophorous vacuole to gain access to host cytosolic molecules and to hijack host organelles to retrieve their nutrient content. From a therapeutic perspective, we survey the different possibilities to starve T. gondii by nutrient depletion or disruption of salvage pathways.
No preview · Article · Oct 2013 · International journal for parasitology
[Show abstract][Hide abstract] ABSTRACT: Apicomplexa are some of the most widespread and poorly controlled pathogens in the world. The metabolism of lipids in these parasites, which include Plasmodium spp., Toxoplasma gondii and Cryptosporidium spp., is essential for the production of infectious progeny and pathogen persistence in their mammalian hosts. Metabolic maps of apicomplexan lipid syntheses reveal auxotrophies for many lipid species, which force these parasites to meet their high demand for lipids through networks of both synthesis and scavenging. Here, I review the unique lipid biosynthetic enzymes and lipid transporter systems of Apicomplexa, focusing on isoprenoids, sphingolipids and cholesterol, and highlight promising chemotherapeutic targets in the lipid synthetic and salvage pathways.
No preview · Article · Oct 2013 · Nature Reviews Microbiology
[Show abstract][Hide abstract] ABSTRACT: Control of parasite replication exerted by MHC class I restricted CD8+ T-cells in the liver is critical for vaccination-induced protection against malaria. While many intracellular pathogens subvert the MHC class I presentation machinery, its functionality in the course of malaria replication in hepatocytes has not been characterized. Using experimental systems based on specific identification, isolation and analysis of human hepatocytes infected with P. berghei ANKA GFP or P. falciparum 3D7 GFP sporozoites we demonstrated that molecular components of the MHC class I pathway exhibit largely unaltered expression in malaria-infected hepatocytes until very late stages of parasite development. Furthermore, infected cells showed no obvious defects in their capacity to upregulate expression of different molecular components of the MHC class I machinery in response to pro-inflammatory lymphokines or trigger direct activation of allo-specific or peptide-specific human CD8+ T-cells. We further demonstrate that ectopic expression of circumsporozoite protein does not alter expression of critical genes of the MHC class I pathway and its response to pro-inflammatory cytokines. In addition, we identified supra-cellular structures, which arose at late stages of parasite replication, possessed the characteristic morphology of merosomes and exhibited nearly complete loss of surface MHC class I expression. These data have multiple implications for our understanding of natural T-cell immunity against malaria and may promote development of novel, efficient anti-malaria vaccines overcoming immune escape of the parasite in the liver.
[Show abstract][Hide abstract] ABSTRACT: Amino acid utilization is important for the growth of the erythrocytic stages of the human malaria parasite Plasmodium falciparum, however the molecular mechanism that permits survival of the parasite during conditions of limiting amino acid supply is poorly understood. We provide data here suggesting that an autophagy pathway functions in P. falciparum despite the absence of a typical lysosome for digestion of the autophagosomes. It involves PfATG8, which has a C-terminal glycine which is absolutely required for association of the protein with autophagosomes. Amino acid starvation provoked increased colocalization between PfATG8- and PfRAB7-labeled vesicles and acidification of the colabeled structures consistent with PfRAB7-mediated maturation of PfATG8-positive autophagosomes; this is a rapid process facilitating parasite survival. Immuno-electron microscopic analyses detected PfRAB7 and PfATG8 on double-membrane-bound vesicles and also near or within the parasite's food vacuole, consistent with autophagosomes fusing with the endosomal system before being routed to the food vacuole for digestion. In nonstarved parasites, PfATG8, but not PfRAB7, was found on the intact apicoplast membrane and on apicoplast-targeted vesicles and apicoplast remnants when the formation of the organelle was disrupted; a localization also requiring the C-terminal glycine. These findings suggest that in addition to a classical role in autophagy, which involves the PfRAB7-endosomal system and food vacuole, PfATG8 is associated with apicoplast-targeted vesicles and the mature apicoplast, and as such contributes to apicoplast formation and maintenance. Thus, PfATG8 may be unique in having such a second role in addition to the formation of autophagosomes required for classical autophagy.
[Show abstract][Hide abstract] ABSTRACT: The bacterium Chlamydia trachomatis and the protozoan parasite Toxoplasma gondii are the causative agents of chlamydiosis and toxoplasmosis in humans, respectively. Both micro-organisms are obligate intracellular pathogens and notorious for extensively modifying the cytoskeletal architecture and the endomembrane system of their host cells to establish productive infections. This review highlights the similar tactics developed by these two pathogens to manipulate their host cell despite their genetic unrelatedness. By using an in vitro cell culture model whereby single fibroblasts are infected by C. trachomatis and T. gondii simultaneously, thus setting up an intracellular competition, we demonstrate that the solutions to the problem of intracellular survival deployed by the parasite and the bacterium may represent an example of convergent evolution, driven by the necessity to acquire nutrients in a hostile environment. This article is protected by copyright. All rights reserved.
Preview · Article · Jul 2013 · Pathogens and Disease
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidium species are waterborne apicomplexan parasites that cause diarrheal disease worldwide. Although the mechanisms underlying
Cryptosporidium-host cell interactions are not well understood, mucin-like glycoproteins of the parasite are known to mediate attachment
and invasion in vitro. We identified C. parvum Clec (CpClec), a novel mucin-like glycoprotein that contains a C-type lectin domain (CTLD) and has orthologs in C. hominis and C. muris. CTLD-containing proteins are ligand-binding proteins that function in adhesion and signaling and are present in a wide range
of organisms, from humans to viruses. However, this is the first report of a CTLD-containing protein in protozoa and in Apicomplexa. CpClec is predicted to be a type 1 membrane protein, with a CTLD, an O-glycosylated mucin-like domain, a transmembrane domain,
and a cytoplasmic tail containing a YXXϕ sorting motif. The predicted structure of CpClec displays several characteristics of canonical CTLD-containing proteins, including a long loop region hydrophobic core
associated with calcium-dependent glycan binding as well as predicted calcium- and glycan-binding sites. CpClec expression during C. parvum infection in vitro is maximal at 48 h postinfection, suggesting that it is developmentally regulated. The 120-kDa mass of native CpClec is greater than predicted, most likely due to O-glycosylation. CpClec is localized to the surface of the apical region and to dense granules of sporozoites and merozoites. Taken together,
these findings, along with the known functions of C. parvum mucin-like glycoproteins and of CTLD-containing proteins, strongly implicate a significant role for CpClec in Cryptosporidium-host cell interactions.
Full-text · Article · Jul 2013 · Infection and immunity
[Show abstract][Hide abstract] ABSTRACT: The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear as the PV avoids fusion with host organelles. In this study, we have explored the host Golgi-PV interaction and evaluated the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during an infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous mini-stacks, which surround the PV, and hijacks host Golgi-derived vesicles within the PV. These vesicles, marked with Rab14, Rab30 or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus, our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.
Full-text · Article · Apr 2013 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: Malaria is characterized by cyclical fevers and high levels of inflammation, and while an early inflammatory response contributes to parasite clearance, excessive and persistent inflammation can lead to severe forms of the disease. Here, we show that Plasmodium falciparum-infected erythrocytes contain uric acid precipitates in the cytoplasm of the parasitophorous vacuole, which are released when erythrocytes rupture. Uric acid precipitates are highly inflammatory molecules that are considered a danger signal for innate immunity and are the causative agent in gout. We determined that P. falciparum-derived uric acid precipitates induce maturation of human dendritic cells, increasing the expression of cell surface co-stimulatory molecules such as CD80 and CD86, while decreasing human leukocyte antigen-DR expression. In accordance with this, uric acid accounts for a significant proportion of the total stimulatory activity induced by parasite-infected erythrocytes. Moreover, the identification of uric acid precipitates in P. falciparum- and P. vivax-infected erythrocytes obtained directly from malaria patients underscores the in vivo and clinical relevance of our findings. Altogether, our data implicate uric acid precipitates as a potentially important contributor to the innate immune response to Plasmodium infection and may provide a novel target for adjunct therapies.
[Show abstract][Hide abstract] ABSTRACT: Lipid bodies are eukaryotic structures for temporary storage of neutral lipids such as acylglycerols and steryl esters. Fatty acyl-CoA and cholesterol are two substrates for cholesteryl ester (CE) synthesis via the ACAT reaction. The intracellular parasite Toxoplasma gondii is incapable of sterol synthesis and unremittingly scavenges cholesterol from mammalian host cells. We previously demonstrated that the parasite expresses a cholesteryl ester-synthesizing enzyme, TgACAT1. In this article, we identified and characterized a second ACAT-like enzyme, TgACAT2, which shares 56% identity with TgACAT1. Both enzymes are endoplasmic reticulum-associated and contribute to CE formation for storage in lipid bodies. While TgACAT1 preferentially utilizes palmitoyl-CoA, TgACAT2 has broader fatty acid specificity and produces more CE. Genetic ablation of each individual ACAT results in parasite growth impairment whereas dual ablation of ACAT1 and ACAT2 is not tolerated by Toxoplasma. ΔACAT1 and ΔACAT2 parasites have reduced CE levels, fewer lipid bodies, and accumulate free cholesterol, which causes injurious membrane effects. Mutant parasites are particularly vulnerable to ACAT inhibitors. This study underlines the important physiological role of ACAT enzymes to store cholesterol in a sterol-auxotrophic organism such as Toxoplasma, and furthermore opens up possibilities of exploiting TgACAT as targets for the development of antitoxoplasmosis drugs.
No preview · Article · Feb 2013 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Apicomplexans are obligate intracellular parasites that actively penetrate their host cells to create an intracellular niche for replication. Commitment to invasion is thought to be mediated by the rhoptries, specialized apical secretory organelles that inject a protein complex into the host cell to form a tight-junction for parasite entry. Little is known about the molecular factors that govern rhoptry biogenesis, their subcellular organization at the apical end of the parasite and subsequent release of this organelle during invasion. We have identified a Toxoplasma palmitoyl acyltransferase, TgDHHC7, which localizes to the rhoptries. Strikingly, conditional knockdown of TgDHHC7 results in dispersed rhoptries that fail to organize at the apical end of the parasite and are instead scattered throughout the cell. While the morphology and content of these rhoptries appears normal, failure to tether at the apex results in a complete block in host cell invasion. In contrast, attachment and egress are unaffected in the knockdown, demonstrating that the rhoptries are not required for these processes. We show that rhoptry targeting of TgDHHC7 requires a short, highly conserved C-terminal region while a large, divergent N-terminal domain is dispensable for both targeting and function. Additionally, a point mutant lacking a key residue predicted to be critical for enzyme activity fails to rescue apical rhoptry tethering, strongly suggesting that tethering of the organelle is dependent upon TgDHHC7 palmitoylation activity. We tie the importance of this activity to the palmitoylated Armadillo Repeats-Only (TgARO) rhoptry protein by showing that conditional knockdown of TgARO recapitulates the dispersed rhoptry phenotype of TgDHHC7 knockdown. The unexpected finding that apicomplexans have exploited protein palmitoylation for apical organelle tethering yields new insight into the biogenesis and function of rhoptries and may provide new avenues for therapeutic intervention against Toxoplasma and related apicomplexan parasites.