The Paradox of a Phagosomal Lifestyle: How Innate Host Cell-Leishmania amazonensis Interactions Lead to a Progressive Chronic Disease.

Intracellular phagosomal pathogens represent a formidable challenge for innate immune cells, as, paradoxically, these phagocytic cells can act as both host cells that support pathogen replication and, when properly activated, are the critical cells that mediate pathogen elimination. Infection by parasites of the Leishmania genus provides an excellent model organism to investigate this complex host-pathogen interaction. In this review we focus on the dynamics of Leishmania amazonensis infection and the host innate immune response, including the impact of the adaptive immune response on phagocytic host cell recruitment and activation. L. amazonensis infection represents an important public health problem in South America where, distinct from other Leishmania parasites, it has been associated with all three clinical forms of leishmaniasis in humans: cutaneous, muco-cutaneous and visceral. Experimental observations demonstrate that most experimental mouse strains are susceptible to L. amazonensis infection, including the C57BL/6 mouse, which is resistant to other species such as Leishmania major, Leishmania braziliensis and Leishmania infantum. In general, the CD4+ T helper (Th)1/Th2 paradigm does not sufficiently explain the progressive chronic disease established by L. amazonensis, as strong cell-mediated Th1 immunity, or a lack of Th2 immunity, does not provide protection as would be predicted. Recent findings in which the balance between Th1/Th2 immunity was found to influence permissive host cell availability via recruitment of inflammatory monocytes has also added to the complexity of the Th1/Th2 paradigm. In this review we discuss the roles played by innate cells starting from parasite recognition through to priming of the adaptive immune response. We highlight the relative importance of neutrophils, monocytes, dendritic cells and resident macrophages for the establishment and progressive nature of disease following L. amazonensis infection.

Plasmodium vivax Infection Alters Mitochondrial Metabolism in Human Monocytes.

Monocytes play an important role in the host defense against Plasmodium vivax as the main source of inflammatory cytokines and mitochondrial reactive oxygen species (mROS). Here, we show that monocyte metabolism is altered during human P. vivax malaria, with mitochondria playing a major function in this switch. The process involves a reprograming in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. P. vivax infection results in dysregulated mitochondrial gene expression and in altered membrane potential leading to mROS increase rather than ATP production. When monocytes were incubated with P. vivax-infected reticulocytes, mitochondria colocalized with phagolysosomes containing parasites representing an important source mROS. Importantly, the mitochondrial enzyme superoxide dismutase 2 (SOD2) is simultaneously induced in monocytes from malaria patients. Taken together, the monocyte metabolic reprograming with an increased mROS production may contribute to protective responses against P. vivax while triggering immunomodulatory mechanisms to circumvent tissue damage. IMPORTANCE Plasmodium vivax is the most widely distributed causative agent of human malaria. To achieve parasite control, the human immune system develops a substantial inflammatory response that is also responsible for the symptoms of the disease. Among the cells involved in this response, monocytes play an important role. Here, we show that monocyte metabolism is altered during malaria, with its mitochondria playing a major function in this switch. This change involves a reprograming process in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. The resulting altered mitochondrial membrane potential leads to an increase in mitochondrial reactive oxygen species rather than ATP. These data suggest that agents that change metabolism should be investigated and used with caution during malaria.

Leishmania donovani Metacyclic Promastigotes Impair Phagosome Properties in Inflammatory Monocytes.

Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes are readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli is efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidify poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan (LPG) was responsible for the inhibition of PV acidification, impairment of the NADPH oxidase assembly was independent of LPG and GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.

Sensing Host Arginine Is Essential for Leishmania Parasites' Intracellular Development.

Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.

Leishmania infection triggers hepcidin-mediated proteasomal degradation of Nramp1 to increase phagolysosomal iron availability.

Natural resistance-associated macrophage protein 1 (Nramp1) was originally discovered as a genetic determinant of resistance against multiple intracellular pathogens, including Leishmania. It encodes a transmembrane protein of the phago-endosomal compartments, where it functions as an iron transporter. But the mechanism by which Nramp1 controls host-pathogen dynamics and determines final outcome of an infection is yet to be fully deciphered. Whether the expression of Nramp1 is altered in response to a pathogen attack is also unknown. To address these, Nramp1 status was examined in Leishmania major-infected murine macrophages. We observed that at 12 hrs post infection, there was drastic lowering of Nramp1 level accompanied by increased phagolysosomal iron content and enhanced intracellular parasite growth. Leishmania infection-induced Nramp1 downregulation was caused by ubiquitin-proteasome degradation pathway, which in turn was found to be mediated by the iron-regulatory peptide hormone hepcidin. Blocking of Nramp1 degradation with proteasome inhibitor or transcriptional agonist of hepcidin resulted in depletion of phagolysosomal iron pool that led to significant reduction of intracellular parasite burden. Interestingly, Nramp1 level was restored to normalcy after 30 hrs of infection with a concomitant drop in phagolysosomal iron, which is suggestive of a host counteractive response to deprive the pathogen of this essential micronutrient. Taken together, our study implicates Nramp1 as a central player in the host-pathogen battle for phagolysosomal iron. We also report Nramp1 as a novel target for hepcidin, and this 'hepcidin-Nramp1' axis may have a broader role in regulating macrophage iron homeostasis.

DNA flowerstructure co-localizes with human pathogens in infected macrophages.

Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology.

Host transcriptomic signature as alternative test-of-cure in visceral leishmaniasis patients co-infected with HIV.

BACKGROUND: Visceral leishmaniasis (VL) treatment in HIV patients very often fails and is followed by high relapse and case-fatality rates. Hence, treatment efficacy assessment is imperative but based on invasive organ aspiration for parasite detection. In the search of a less-invasive alternative and because the host immune response is pivotal for treatment outcome in immunocompromised VL patients, we studied changes in the whole blood transcriptional profile of VL-HIV patients during treatment. METHODS: Embedded in a clinical trial in Northwest Ethiopia, RNA-Seq was performed on whole blood samples of 28 VL-HIV patients before and after completion of a 29-day treatment regimen of AmBisome or AmBisome/miltefosine. Pathway analyses were combined with a machine learning approach to establish a clinically-useful 4-gene set. FINDINGS: Distinct signatures of differentially expressed genes between D0 and D29 were identified for patients who failed treatment and were successfully treated. Pathway analyses in the latter highlighted a downregulation of genes associated with host cellular activity and immunity, and upregulation of antimicrobial peptide activity in phagolysosomes. No signs of disease remission nor pathway enrichment were observed in treatment failure patients. Next, we identified a 4-gene pre-post signature (PRSS33, IL10, SLFN14, HRH4) that could accurately discriminate treatment outcome at end of treatment (D29), displaying an average area-under-the-ROC-curve of 0.95 (CI: 0.75-1.00). INTERPRETATION: A simple blood-based signature thus holds significant promise to facilitate treatment efficacy monitoring and provide an alternative test-of-cure to guide patient management in VL-HIV patients. FUNDING: Project funding was provided by the AfricoLeish project, supported by the European Union Seventh Framework Programme (EU FP7).

The host cell secretory pathway mediates the export of Leishmania virulence factors out of the parasitophorous vacuole.

To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV.

The arginine sensing and transport binding sites are distinct in the human pathogen Leishmania.

The intracellular protozoan parasite Leishmania donovani causes human visceral leishmaniasis. Intracellular L. donovani that proliferate inside macrophage phagolysosomes compete with the host for arginine, creating a situation that endangers parasite survival. Parasites have a sensor that upon arginine deficiency activates an Arginine Deprivation Response (ADR). L. donovani transport arginine via a high-affinity transporter (LdAAP3) that is rapidly up-regulated by ADR in intracellular amastigotes. To date, the sensor and its ligand have not been identified. Here, we show that the conserved amidino group at the distal cap of the arginine side chain is the ligand that activates ADR, in both promastigotes and intracellular amastigotes, and that arginine sensing and transport binding sites are distinct in L. donovani. Finally, upon addition of arginine and analogues to deprived cells, the amidino ligand activates rapid degradation of LdAAP3. This study provides the first identification of an intra-molecular ligand of a sensor that acts during infection.

Autophagy: A necessary process during the Trypanosoma cruzi life-cycle.

Autophagy is a well-conserved process of self-digestion of intracellular components. T. cruzi is a protozoan parasite with a complex life-cycle that involves insect vectors and mammalian hosts. Like other eukaryotic organisms, T. cruzi possesses an autophagic pathway that is activated during metacyclogenesis, the process that generates the infective forms of parasites. In addition, it has been demonstrated that mammalian autophagy has a role during host cell invasion by T. cruzi, and that T. cruzi can modulate this process to its own benefit. This review describes the latest findings concerning the participation of autophagy in both the T. cruzi differentiation processes and during the interaction of parasites within the host cells. Data to date suggest parasite autophagy is important for parasite survival and differentiation, which offers interesting prospects for therapeutic strategies. Additionally, the interruption of mammalian autophagy reduces the parasite infectivity, interfering with the intracellular cycle of T. cruzi inside the host. However, the impact on other stages of development, such as the intracellular replication of parasites is still not clearly understood. Further studies in this matter are necessaries to define the integral effect of autophagy on T. cruzi infection with both in vitro and in vivo approaches.

Development of an automated image analysis protocol for quantification of intracellular forms of Leishmania spp.

Leishmania parasites cause a set of neglected tropical diseases with considerable public health impact, the leishmaniases, which are often fatal if left untreated. Since current treatments for the leishmaniases exhibit high toxicity, low efficacy and prohibitive prices, many laboratories throughout the world are engaged in research for the discovery of novel chemotherapeutics. This entails the necessity of screening large numbers of compounds against the clinically relevant form of the parasite, the obligatory intracellular amastigote, a procedure that in many laboratories is still carried out by manual inspection. To overcome this well-known bottleneck in Leishmania drug development, several studies have recently attempted to automate this process. Here we implemented an image-based high content triage assay for Leishmania which has the added advantages of using primary macrophages instead of macrophage cell lines and of enabling identification of active compounds against parasite species developing both in small individual phagolysosomes (such as L. infantum) and in large communal vacuoles (such as L. amazonensis). The automated image analysis protocol is made available for IN Cell Analyzer systems, and, importantly, also for the open-source CellProfiler software, in this way extending its implementation to any laboratory involved in drug development as well as in other aspects of Leishmania research requiring analysis of in vitro infected macrophages.

Phagosome proteomics to study Leishmania's intracellular niche in macrophages.

Intracellular pathogens invade their host cells and replicate within specialized compartments. In turn, the host cell initiates a defensive response trying to kill the invasive agent. As a consequence, intracellular lifestyle implies morphological and physiological changes in both pathogen and host cell. Leishmania spp. are medically important intracellular protozoan parasites that are internalized by professional phagocytes such as macrophages, and reside within the parasitophorous vacuole inhibiting their microbicidal activity. Whereas the proteome of the extracellular promastigote form and the intracellular amastigote form have been extensively studied, the constituents of Leishmania's intracellular niche, an endolysosomal compartment, are not fully deciphered. In this review we discuss protocols to purify such compartments by means of an illustrating example to highlight generally relevant considerations and innovative aspects that allow purification of not only the intracellular parasites but also the phagosomes that harbor them and analyze the latter by gel free proteomics.

LC3-associated phagocytosis in microbial pathogenesis.

Phagocytosis is essential for uptake and elimination of pathogenic microorganisms. Autophagy is a highly conserved mechanism for incorporation of cellular constituents to replenish nutrients by degradation. Recently, parts of the autophagy machinery - above all microtubule-associated protein 1 light chain 3 (LC3) - were found to be specifically recruited to phagosomal membranes resulting in phagosome-lysosome fusion and efficient degradation of internalized cargo in a process termed LC3-associated phagocytosis (LAP). Many pathogenic bacterial, fungal and parasitic microorganisms reside within LAP-targeted single-membrane phagosomes or vacuoles after infection of host cells. In this minireview we describe the state of knowledge on the interaction of pathogens with LAP or LAP-like pathways and report on various pathogens that have evolved strategies to circumvent degradation in LAP compartments.

Rab22a controls MHC-I intracellular trafficking and antigen cross-presentation by dendritic cells.

Cross-presentation by MHC class I molecules allows the detection of exogenous antigens by CD8+ T lymphocytes. This process is crucial to initiate cytotoxic immune responses against many pathogens (i.e., Toxoplasma gondii) and tumors. To achieve efficient cross-presentation, dendritic cells (DCs) have specialized endocytic pathways; however, the molecular effectors involved are poorly understood. In this work, we identify the small GTPase Rab22a as a key regulator of MHC-I trafficking and antigen cross-presentation by DCs. Our results demonstrate that Rab22a is recruited to DC endosomes and phagosomes, as well as to the vacuole containing T. gondii parasites. The silencing of Rab22a expression did not affect the uptake of exogenous antigens or parasite invasion, but it drastically reduced the intracellular pool and the recycling of MHC-I molecules. The knockdown of Rab22a also hampered the cross-presentation of soluble, particulate and T. gondii-associated antigens, but not the endogenous MHC-I antigen presentation through the classical secretory pathway. Our findings provide compelling evidence that Rab22a plays a central role in the MHC-I endocytic trafficking, which is crucial for efficient cross-presentation by DCs.

Avirulent strains of Toxoplasma gondii infect macrophages by active invasion from the phagosome.

Unlike most intracellular pathogens that gain access into host cells through endocytic pathways, Toxoplasma gondii initiates infection at the cell surface by active penetration through a moving junction and subsequent formation of a parasitophorous vacuole. Here, we describe a noncanonical pathway for T. gondii infection of macrophages, in which parasites are initially internalized through phagocytosis, and then actively invade from within a phagosomal compartment to form a parasitophorous vacuole. This phagosome to vacuole invasion (PTVI) pathway may represent an intermediary link between the endocytic and the penetrative routes for host cell entry by intracellular pathogens. The PTVI pathway is preferentially used by avirulent strains of T. gondii and confers an infectious advantage over virulent strains for macrophage tropism.

CD4+ T Cells: guardians of the phagosome.

CD4(+) T cells are key cells of the adaptive immune system that use T cell antigen receptors to recognize peptides that are generated in endosomes or phagosomes and displayed on the host cell surface bound to major histocompatibility complex molecules. These T cells participate in immune responses that protect hosts from microbes such as Mycobacterium tuberculosis, Cryptococcus neoformans, Leishmania major, and Salmonella enterica, which have evolved to live in the phagosomes of macrophages and dendritic cells. Here, we review studies indicating that CD4(+) T cells control phagosomal infections asymptomatically in most individuals by secreting cytokines that activate the microbicidal activities of infected phagocytes but in a way that inhibits the pathogen but does not eliminate it. Indeed, we make the case that localized, controlled, persistent infection is necessary to maintain large numbers of CD4(+) effector T cells in a state of activation needed to eradicate systemic and more pathogenic forms of the infection. Finally, we posit that current vaccines for phagosomal infections fail because they do not produce this "periodic reminder" form of CD4(+) T cell-mediated immune control.

Targeted extracellular signal-regulated kinase activation mediated by Leishmania amazonensis requires MP1 scaffold.

Leishmania amazonensis infection promotes alteration of host cellular signaling and intracellular parasite survival, but specific mechanisms are poorly understood. We previously demonstrated that L. amazonensis infection of dendritic cells (DC) activated extracellular signal-regulated kinase (ERK), an MAP-kinase kinase kinase, leading to altered DC maturation and non-healing cutaneous leishmaniasis. Studies using growth factors and cell lines have shown that targeted, robust, intracellular phosphorylation of ERK1/2 from phagolysosomes required recruitment and association with scaffolding proteins, including p14/MP1 and MORG1, on the surface of late endosomes. Based on the intracellular localization of L. amazonensis within a parasitophorous vacuole with late endosome characteristics, we speculated that scaffolding proteins would be important for intracellular parasite-mediated ERK signaling. Our findings demonstrate that MP1, MORG1, and ERK all co-localized on the surface of parasite-containing LAMP2-positive phagolysosomes. Infection of MEK1 mutant fibroblasts unable to bind MP1 demonstrated dramatically reduced ERK1/2 phosphorylation following L. amazonensis infection but not following positive control EGF treatment. This novel mechanism for localization of intracellular L. amazonensis-mediated ERK1/2 phosphorylation required the endosomal scaffold protein MP1 and localized to L. amazonensis parasitophorous vacuoles. Understanding how L. amazonensis parasites hijack host cell scaffold proteins to modulate signaling cascades provides targets for antiprotozoal drug development.

Leishmania promastigotes: building a safe niche within macrophages.

Upon their internalization by macrophages, Leishmania promastigotes inhibit phagolysosome biogenesis. The main factor responsible for this inhibition is the promastigote surface glycolipid lipophosphoglycan (LPG). This glycolipid has a profound impact on the phagosome, causing periphagosomal accumulation of F-actin and disruption of phagosomal lipid microdomains. Functionally, this LPG-mediated inhibition of phagosome maturation is characterized by an impaired assembly of the NADPH oxidase and the exclusion of the vesicular proton-ATPase from phagosomes. In this chapter, we review the current knowledge concerning the nature of the intra-macrophage compartment in which Leishmania donovani promastigotes establish infection. We also describe how LPG enables this parasite to remodel the parasitophorous vacuole.