Mitochondrial stress triggers a pro-survival response through epigenetic modifications of nuclear DNA.

Mitochondrial dysfunction represents an important cellular stressor and when intense and persistent cells must unleash an adaptive response to prevent their extinction. Furthermore, mitochondria can induce nuclear transcriptional changes and DNA methylation can modulate cellular responses to stress. We hypothesized that mitochondrial dysfunction could trigger an epigenetically mediated adaptive response through a distinct DNA methylation patterning. We studied cellular stress responses (i.e., apoptosis and autophagy) in mitochondrial dysfunction models. In addition, we explored nuclear DNA methylation in response to this stressor and its relevance in cell survival. Experiments in cultured human myoblasts revealed that intense mitochondrial dysfunction triggered a methylation-dependent pro-survival response. Assays done on mitochondrial disease patient tissues showed increased autophagy and enhanced DNA methylation of tumor suppressor genes and pathways involved in cell survival regulation. In conclusion, mitochondrial dysfunction leads to a "pro-survival" adaptive state that seems to be triggered by the differential methylation of nuclear genes.

Autophagy as an innate immunity response against pathogens: a Tango dance.

Intracellular infections as well as changes in the cell nutritional environment are main events that trigger cellular stress responses. One crucial cell response to stress conditions is autophagy. During the last 30 years, several scenarios involving autophagy induction or inhibition over the course of an intracellular invasion by pathogens have been uncovered. In this review, we will present how this knowledge was gained by studying different microorganisms. We intend to discuss how the cell, via autophagy, tries to repel these attacks with the objective of destroying the intruder, but also how some pathogens have developed strategies to subvert this. These two fates can be compared with a Tango, a dance originated in Buenos Aires, Argentina, in which the partner dancers are in close connection. One of them is the leader, embracing and involving the partner, but the follower may respond escaping from the leader. This joint dance is indeed highly synchronized and controlled, perfectly reflecting the interaction between autophagy and microorganism.

Biogenesis and Breakdown of Lipid Droplets in Pathological Conditions.

Lipid droplets (LD) have long been considered as mere fat drops; however, LD have lately been revealed to be ubiquitous, dynamic and to be present in diverse organelles in which they have a wide range of key functions. Although incompletely understood, the biogenesis of eukaryotic LD initiates with the synthesis of neutral lipids (NL) by enzymes located in the endoplasmic reticulum (ER). The accumulation of NL leads to their segregation into nanometric nuclei which then grow into lenses between the ER leaflets as they are further filled with NL. The lipid composition and interfacial tensions of both ER and the lenses modulate their shape which, together with specific ER proteins, determine the proneness of LD to bud from the ER toward the cytoplasm. The most important function of LD is the buffering of energy. But far beyond this, LD are actively integrated into physiological processes, such as lipid metabolism, control of protein homeostasis, sequestration of toxic lipid metabolic intermediates, protection from stress, and proliferation of tumours. Besides, LD may serve as platforms for pathogen replication and defense. To accomplish these functions, from biogenesis to breakdown, eukaryotic LD have developed mechanisms to travel within the cytoplasm and to establish contact with other organelles. When nutrient deprivation occurs, LD undergo breakdown (lipolysis), which begins with the LD-associated members of the perilipins family PLIN2 and PLIN3 chaperone-mediated autophagy degradation (CMA), a specific type of autophagy that selectively degrades a subset of cytosolic proteins in lysosomes. Indeed, PLINs CMA degradation is a prerequisite for further true lipolysis, which occurs via cytosolic lipases or by lysosome luminal lipases when autophagosomes engulf portions of LD and target them to lysosomes. LD play a crucial role in several pathophysiological processes. Increased accumulation of LD in non-adipose cells is commonly observed in numerous infectious diseases caused by intracellular pathogens including viral, bacterial, and parasite infections, and is gradually recognized as a prominent characteristic in a variety of cancers. This review discusses current evidence related to the modulation of LD biogenesis and breakdown caused by intracellular pathogens and cancer.

Nanocarrier-enhanced intracellular delivery of benznidazole for treatment of Trypanosoma cruzi infection.

Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), an intracellular pathogen that causes significant morbidity and death among millions in the Americas from Canada to Argentina. Current therapy involves oral administration of the nitroimidazole benznidazole (BNZ), which has serious side effects that often necessitate cessation of treatment. To both avoid off-target side effects and reduce the necessary dosage of BNZ, we packaged the drug within poly(ethylene glycol)-block-poly(propylene sulfide) polymersomes (BNZ-PSs). We show that these vesicular nanocarriers enhanced intracellular delivery to phagocytic cells and tested this formulation in a mouse model of T. cruzi infection. BNZ-PS is not only nontoxic but also significantly more potent than free BNZ, effectively reducing parasitemia, intracellular infection, and tissue parasitosis at a 466-fold lower dose of BNZ. We conclude that BNZ-PS was superior to BNZ for treatment of T. cruzi infection in mice and that further modifications of this nanocarrier formulation could lead to a wide range of custom controlled delivery applications for improved treatment of Chagas disease in humans.

Precision Health for Chagas Disease: Integrating Parasite and Host Factors to Predict Outcome of Infection and Response to Therapy.

Chagas disease, caused by the infection with the protozoan parasite Trypanosoma cruzi, is clinically manifested in approximately one-third of infected people by inflammatory heart disease (cardiomyopathy) and, to a minor degree, gastrointestinal tract disorders (megaesophagus or megacolon). Chagas disease is a zoonosis transmitted among animals and people through the contact with triatomine bugs, which are found in much of the western hemisphere, including most countries of North, Central and South America, between parallels 45° north (Minneapolis, USA) and south (Chubut Province, Argentina). Despite much research on drug discovery for T. cruzi, there remain only two related agents in widespread use. Likewise, treatment is not always indicated due to the serious side effects of these drugs. On the other hand, the epidemiology and pathogenesis of Chagas disease are both highly complex, and much is known about both. However, it is still impossible to predict what will happen in an individual person infected with T. cruzi, because of the highly variability of parasite virulence and human susceptibility to infection, with no definitive molecular predictors of outcome from either side of the host-parasite equation. In this Minireview we briefly discuss the current state of T. cruzi infection and prognosis and look forward to the day when it will be possible to employ precision health to predict disease outcome and determine whether and when treatment of infection may be necessary.

Chronic Infections: A Possible Scenario for Autophagy and Senescence Cross-Talk.

Multiple tissues and systems in the organism undergo modifications during aging due to an accumulation of damaged proteins, lipids, and genetic material. To counteract this process, the cells are equipped with specific mechanisms, such as autophagy and senescence. Particularly, the immune system undergoes a process called immunosenescence, giving rise to a chronic inflammatory status of the organism, with a decreased ability to counteract antigens. The obvious result of this process is a reduced defence capacity. Currently, there is evidence that some pathogens are able to accelerate the immunosenescence process for their own benefit. Although to date numerous reports show the autophagy⁻senescence relationship, or the connection between pathogens with autophagy or senescence, the link between the three actors remains unexplored. In this review, we have summarized current knowledge about important issues related to aging, senescence, and autophagy.

The superfamily keeps growing: Identification in trypanosomatids of RibJ, the first riboflavin transporter family in protists.

BACKGROUND: Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems. CONCLUSIONS/SIGNIFICANCE: The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for trypanosomatids, and the structural/biochemical differences that RFVT/SLC52 and RibJ present, make the riboflavin transporter -and its downstream metabolism- a potential trypanocidal drug target.

Computer-guided drug repurposing: identification of trypanocidal activity of clofazimine, benidipine and saquinavir.

In spite of remarkable advances in the knowledge on Trypanosoma cruzi biology, no medications to treat Chagas disease have been approved in the last 40 years and almost 8 million people remain infected. Since the public sector and non-profit organizations play a significant role in the research efforts on Chagas disease, it is important to implement research strategies that promote translation of basic research into the clinical practice. Recent international public-private initiatives address the potential of drug repositioning (i.e. finding second or further medical uses for known-medications) which can substantially improve the success at clinical trials and the innovation in the pharmaceutical field. In this work, we present the computer-aided identification of approved drugs clofazimine, benidipine and saquinavir as potential trypanocidal compounds and test their effects at biochemical as much as cellular level on different parasite stages. According to the obtained results, we discuss biopharmaceutical, toxicological and physiopathological criteria applied to decide to move clofazimine and benidipine into preclinical phase, in an acute model of infection. The article illustrates the potential of computer-guided drug repositioning to integrate and optimize drug discovery and preclinical development; it also proposes rational rules to select which among repositioned candidates should advance to investigational drug status and offers a new insight on clofazimine and benidipine as candidate treatments for Chagas disease. One Sentence Summary: We present the computer-guided drug repositioning of three approved drugs as potential new treatments for Chagas disease, integrating computer-aided drug screening and biochemical, cellular and preclinical tests.

High-throughput drug repositioning for the discovery of new treatments for Chagas disease.

Despite affecting around 8 million people worldwide and representing an economic burden above $7 billion/ year, currently approved medications to treat Chagas disease are still limited to two drugs, nifurtimox and benznidazole, which were developed more than 40 years ago and present important efficacy and safety limitations. Drug repositioning (i.e. finding second or further therapeutic indications for known drugs) has raised considerable interest within the international drug development community. There are many explanations to the current interest on drug repositioning including the possibility to partially circumvent clinical trials and the consequent saving in time and resources. It has been suggested as a particular attractive approach for the development of novel therapeutics for neglected diseases, which are usually driven by public or non-profit organizations. Here we review current computer-guided approaches to drug repositioning and reports on drug repositioning stories oriented to Chagas disease, with a focus on computer-guided drug repositioning campaigns.

Sequence variation in CYP51A from the Y strain of Trypanosoma cruzi alters its sensitivity to inhibition.

CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.

Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity.

Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.

Autophagy in protists.

Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles, and defense against parasitic invaders. During the last 10-20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target.

The autophagic pathway is a key component in the lysosomal dependent entry of Trypanosoma cruzi into the host cell.

The etiologic agent of Chagas disease, Trypanosoma cruzi, infects mammalian cells activating a signal transduction cascade that leads to the formation of its parasitophorous vacuole. Previous works have demonstrated the crucial role of lysosomes in the establishment of T. cruzi infection. In this work we have studied the possible relationship between this parasite and the host cell autophagy. We show, for the first time, that the vacuole containing T. cruzi (TcPV) is decorated by the host cell autophagic protein LC3. Furthermore, live cell imaging experiments indicate that autolysosomes are recruited to parasite entry sites. Interestingly, starvation or pharmacological induction of autophagy before infection significantly increased the number of infected cells whereas inhibitors of this pathway reduced the invasion. In addition, the absence of Atg5 or the reduced expression of Beclin 1 -- two proteins required at the initial steps of autophagosome formation -- limited parasite entry and reduced the association between TcPV and the classical lysosomal marker Lamp-1. These results indicate that mammalian autophagy is a key process that favors the colonization of T. cruzi in the host cell.

The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell.

The etiologic agent of Q fever Coxiella burnetii, is an intracellular obligate parasite that develops large vacuoles with phagolysosomal characteristics, containing multiple replicating bacteria. We have previously shown that Phase II C. burnetii replicative vacuoles generated after 24-48 h post infection are decorated with the autophagic protein LC3. The aim of the present study was to examine, at earlier stages of infection, the distribution and roles of the small GTPases Rab5 and Rab7, markers of early and late endosomes respectively, as well as of the protein LC3 on C. burnetii trafficking. Our results indicate that: (i) Coxiella phagosomes (Cph) acquire the two Rab proteins sequentially during infection; (ii) overexpression of a dominant negative mutant form of Rab5, but not of Rab7, impaired Coxiella entry, whereas both Rab5 and Rab7 dominant negative mutants inhibited vacuole formation; (iii) Cph colocalized with the protein LC3 as early as 5 min after infection; acquisition of this protein appeared to be a bacterially driven process, because it was inhibited by the bacteriostatic antibiotic chloramphenicol and (iv) C. burnetii delayed the arrival of the typical lysosomal protease cathepsin D to the Cph, which delay is further increased by starvation-induced autophagy. Based on our results we propose that C. burnetii transits through the normal endo/phagocytic pathway but actively interacts with autophagosomes at early times after infection. This intersection with the autophagic pathway delays fusion with the lysosomal compartment possibly favouring the intracellular differentiation and survival of the bacteria.

The two faces of autophagy: Coxiella and Mycobacterium.

In the world of pathogen-host cell interactions, the autophagic pathway has been recently described as a component of the innate immune response against intracellular microorganisms. Indeed, some bacterial survival mechanisms are hampered when this process is activated. Mycobacterium tuberculosis infection of macrophages, for example, is impaired upon autophagy induction and the bacterial phagosomes are redirected to autophagosomes. On the other hand, pathogens like Coxiella burnetii are benefited by this cellular response and subvert the autophagy process resulting in a more efficient replication. We study at the molecular level these two different faces of the autophagy process in pathogen life in order to elucidate the intricate routes modulated by the microorganisms as survival strategies.

Changes in phosphomannosyl ligands correlate with cation-dependent mannose-6-phosphate receptors in rat liver during perinatal development.

The co-existence of two mannose-6-phosphate receptors (CD-MPR and CI-MPR) in most cell types is still a dilemma to be resolved. In this study, some parameters were measured to explore lysosomal apparatus evolution in rat liver during perinatal development, and establish a possible involvement of CD- and/or CI-MPR in lysosome maturation. Activity of four acid hydrolases was measured in the whole organ at different ages and it was found that N-acetyl-beta-D-glucosaminidase (NAG), beta-galactosidase, and beta-glucuronidase change during development, reaching a peak at the 10th day after birth. These results correlated with the expression and binding properties of CD-MPR previously reported. We also used a method that recognizes phosphomannosylated ligands by using purified biotinylated CI-MPR as a probe, and found that the highest concentrations of ligands also appear around the 10th day. Binding assays were also carried out, incubating endogenous NAG from 10-day-old and adult rats with membranes from their respective ages, and the results indicated that cation-dependent mannose-6-phosphate receptor (CD-MPR) has more impact on trafficking of the enzyme at the 10th day after birth. We concluded that lysosome maturation in the rat liver occurs around the 10th day after birth, and that the CD-MPR may participate in that event.

Expression and binding properties of the two mannose-6-phosphate receptors differ during perinatal development in rat liver.

Mammalian tissues express both cation-dependent (CD-MPR) and cation-independent (CI-MPR) mannose-6-phosphate receptors, which mediate the targeting of acid hydrolases to lysosomes. The coexistence of the two receptors in all cell types and tissues is still poorly understood. To determine whether these receptors might play a role in maturation, we studied their expression and binding properties in rat liver during perinatal development. CI-MPR expression decreases progressively from 18-day fetuses to adults, whereas the CD-MPR showed a transient decrease in newborn and at the 5th day after birth. Immunostaining of the tissues showed that both receptors localize to hepatocytes at all the ages and, additionally, the CD-MPR was reactive in megakaryocytes at early stages. Binding assays showed differences in the B(max) and K(D) values between the ages studied. These results demonstrate that both receptors change differentially during perinatal development, suggesting that they play distinct roles during organ maturation.