LDL receptor and pathogen processes: Functions beyond normal lipids.

Although the role of the LDL receptor concerning lipids is well known, its role in various viral and parasitic infections, and in regulating the inflammatory response is poorly understood. Several infectious agents use the LDL receptor as a port of entry, and others depend on it for their cycle of infection. In this review, we focus on the discovery, structure, and normal function of the LDL receptor, as well as its role in a selection of infections. The LDL receptor plays an important role in certain infections and is a potential target for treatment deserving further research.

Fucose Ameliorates Tritrichomonas sp.-Associated Illness in Antibiotic-Treated Muc2-/- Mice.

The mucus layer in the intestine plays a critical role in regulation of host-microbe interactions and maintaining homeostasis. Disruptions of the mucus layer due to genetic, environmental, or immune factors may lead to inflammatory bowel diseases (IBD). IBD frequently are accompanied with infections, and therefore are treated with antibiotics. Hence, it is important to evaluate risks of antibiotic treatment in individuals with vulnerable gut barrier and chronic inflammation. Mice with a knockout of the Muc2 gene, encoding the main glycoprotein component of the mucus, demonstrate a close contact of the microbes with the gut epithelium which leads to chronic inflammation resembling IBD. Here we demonstrate that the Muc2-/- mice harboring a gut protozoan infection Tritrichomonas sp. are susceptible to an antibiotic-induced depletion of the bacterial microbiota. Suppression of the protozoan infection with efficient metronidazole dosage or L-fucose administration resulted in amelioration of an illness observed in antibiotic-treated Muc2-/- mice. Fucose is a monosaccharide presented abundantly in gut glycoproteins, including Mucin2, and is known to be involved in host-microbe interactions, in particular in microbe adhesion. We suppose that further investigation of the role of fucose in protozoan adhesion to host cells may be of great value.

Pathomechanisms in the Kidneys in Selected Protozoan Parasitic Infections.

Leishmaniasis, malaria, toxoplasmosis, and acanthamoebiasis are protozoan parasitic infections. They remain important contributors to the development of kidney disease, which is associated with increased patients' morbidity and mortality. Kidney injury mechanisms are not fully understood in protozoan parasitic diseases, bringing major difficulties to specific therapeutic interventions. The aim of this review is to present the biochemical and molecular mechanisms in kidneys infected with Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Acanthamoeba spp. We present available mechanisms of an immune response, oxidative stress, apoptosis process, hypoxia, biomarkers of renal injury in the serum or urine, and the histopathological changes of kidneys infected with the selected parasites. Pathomechanisms of Leishmania spp. and Plasmodium spp. infections have been deeply investigated, while Toxoplasma gondii and Acanthamoeba spp. infections in the kidneys are not well known yet. Deeper knowledge of kidney involvement in leishmaniasis and malaria by presenting their mechanisms provides insight into how to create novel and effective treatments. Additionally, the presented work shows gaps in the pathophysiology of renal toxoplasmosis and acanthamoebiasis, which need further research.

Mass mortality in endangered fan mussels Pinna nobilis (Linnaeus 1758) caused by co-infection of Haplosporidium pinnae and multiple Vibrio infection in Çanakkale Strait, Turkey.

PURPOSE: Pinna nobilis (fan mussel) is one of the most important endemic bivalve molluscs in the Mediterranean and mass mortality events were observed in these mussels in recent years. In this study, we report mass mortalities caused by Haplosporidium pinnae, which has been spreading in the Mediterranean for 3 years, and reached the Çanakkale Strait, which is the entrance of the Marmara and the Black Sea. MATERIAL AND METHODS: Field observations during sampling and subsequent histopathological, biochemical, genetic, and microbiological analyses were carried out. RESULTS: These analyses showed that H. pinnae infection spread among the natural beds of P. nobilis, causing severe tissue damage and oxidative stress. Our phylogenetic analyses suggested that the parasite spread through the Mediterranean much faster than thought. The results showed that vibriosis originating from Vibrio coralliilyticus, Vibrio tubiashii, Vibrio mediterranei, and Vibrio hispanicus, acted together with H. pinnae in infected individuals and caused death. CONCLUSION: It is highly probable that the spread of H. pinnae to the Sea of Marmara and the Black Sea may occur earlier than expected, and it was concluded that mass deaths were caused by co-infection with H. pinnae and a geographically specific marine pathogen that can infect P. nobilis populations.

Repurposing Auranofin and Evaluation of a New Gold(I) Compound for the Search of Treatment of Human and Cattle Parasitic Diseases: From Protozoa to Helminth Infections.

Neglected parasitic diseases remain a major public health issue worldwide, especially in tropical and subtropical areas. Human parasite diversity is very large, ranging from protozoa to worms. In most cases, more effective and new drugs are urgently needed. Previous studies indicated that the gold(I) drug auranofin (Ridaura®) is effective against several parasites. Among new gold(I) complexes, the phosphole-containing gold(I) complex {1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl (abbreviated as GoPI) is an irreversible inhibitor of both purified human glutathione and thioredoxin reductases. GoPI-sugar is a novel 1-thio-ß-d-glucopyranose 2,3,4,6-tetraacetato-S-derivative that is a chimera of the structures of GoPI and auranofin, designed to improve stability and bioavailability of GoPI. These metal-ligand complexes are of particular interest because of their combined abilities to irreversibly target the essential dithiol/selenol catalytic pair of selenium-dependent thioredoxin reductase activity, and to kill cells from breast and brain tumors. In this work, screening of various parasites-protozoans, trematodes, and nematodes-was undertaken to determine the in vitro killing activity of GoPI-sugar compared to auranofin. GoPI-sugar was found to efficiently kill intramacrophagic Leishmania donovani amastigotes and adult filarial and trematode worms.

Clearing or subverting the enemy: Role of autophagy in protozoan infections.

The protozoan parasites are evolutionarily divergent, unicellular eukaryotic pathogens representing one of the essential sources of parasitic diseases. These parasites significantly affect the economy and cause public health burdens globally. Protozoan parasites share many cellular features and pathways with their respective host cells. This includes autophagy, a process responsible for self-degradation of the cell's components. There is conservation of the central structural and functional machinery for autophagy in most of the eukaryotic phyla, however, Plasmodium and Toxoplasma possess a decreased number of recognizable autophagy-related proteins (ATG). Plasmodium noticeably lacks clear orthologs of the initiating kinase ATG1/ULK1/2, and both Plasmodium and Toxoplasma lack proteins involved in the nucleation of autophagosomes. These organisms have essential apicoplast, a plastid-like non-photosynthetic organelle, which is an adaptation that is used in penetrating the host cell. Furthermore, available evidence suggests that Leishmania, an intracellular protozoan parasite, induces autophagy in macrophages. The autophagic pathway in Trypanosoma cruzi is activated during metacyclogenesis, a process responsible for the infective forms of parasites. Therefore, numerous pathogens have developed strategies to impair the autophagic mechanism in phagocytes. Regulating autophagy is essential to maintain cellular health as adjustments in the autophagy pathway have been linked to the progression of several physiological and pathological conditions in humans. In this review, we report current advances in autophagy in parasites and their host cells, focusing on the ramifications of these studies in the design of potential anti-protozoan therapeutics.

Host Invasion by Pathogenic Amoebae: Epithelial Disruption by Parasite Proteins.

The epithelium represents the first and most extensive line of defence against pathogens, toxins and pollutant agents in humans. In general, pathogens have developed strategies to overcome this barrier and use it as an entrance to the organism. Entamoeba histolytica, Naegleriafowleri and Acanthamoeba spp. are amoebae mainly responsible for intestinal dysentery, meningoencephalitis and keratitis, respectively. These amoebae cause significant morbidity and mortality rates. Thus, the identification, characterization and validation of molecules participating in host-parasite interactions can provide attractive targets to timely intervene disease progress. In this work, we present a compendium of the parasite adhesins, lectins, proteases, hydrolases, kinases, and others, that participate in key pathogenic events. Special focus is made for the analysis of assorted molecules and mechanisms involved in the interaction of the parasites with epithelial surface receptors, changes in epithelial junctional markers, implications on the barrier function, among others. This review allows the assessment of initial host-pathogen interaction, to correlate it to the potential of parasite invasion.

Intravital microscopy: Imaging host-parasite interactions in the brain.

Intravital fluorescence microscopy (IVM) is a powerful technique for imaging multiple organs, including the brain of living mice and rats. It enables the direct visualisation of cells in situ providing a real-life view of biological processes that in vitro systems cannot. In addition, to the technological advances in microscopy over the last decade, there have been supporting innovations in data storage and analytical packages that enable the visualisation and analysis of large data sets. Here, we review the advantages and limitations of techniques predominantly used for brain IVM, including thinned skull windows, open skull cortical windows, and a miniaturised optical system based on microendoscopic probes that can be inserted into deep tissues. Further, we explore the relevance of these techniques for the field of parasitology. Several protozoan infections are associated with neurological symptoms including Plasmodium spp., Toxoplasma spp., and Trypanosoma spp. IVM has led to crucial findings on these parasite species, which are discussed in detail in this review.

A genome-wide analysis of coatomer protein (COP) subunits of apicomplexan parasites and their evolutionary relationships.

BACKGROUND: Protein secretion is an essential process in all eukaryotes including organisms belonging to the phylum Apicomplexa, which includes many intracellular parasites. The apicomplexan parasites possess a specialized collection of secretory organelles that release a number of proteins to facilitate the invasion of host cells and some of these proteins also participate in immune evasion. Like in other eukaryotes, these parasites possess a series of membrane-bound compartments, namely the endoplasmic reticulum (ER), the intermediate compartments (IC) or vesicular tubular clusters (VTS) and Golgi complex through which proteins pass in a sequential and vectorial fashion. Two sets of proteins; COPI and COPII are important for directing the sequential transfer of material between the ER and Golgi complex. RESULTS: Here, using in silico approaches, we identify the components of COPI and COPII complexes in the genome of apicomplexan organisms. The results showed that the COPI and COPII protein complexes are conserved in most apicomplexan genomes with few exceptions. Diversity among the components of COPI and COPII complexes in apicomplexan is either due to the absence of a subunit or due to the difference in the number of protein domains. For example, the COPI epsilon subunit and COPII sec13 subunit is absent in Babesia bovis, Theileria parva, and Theileria annulata genomes. Phylogenetic and domain analyses for all the proteins of COPI and COPII complexes was performed to predict their evolutionary relationship and functional significance. CONCLUSIONS: The study thus provides insights into the apicomplexan COPI and COPII coating machinery, which is crucial for parasites secretory network needed for the invasion of host cells.

PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection.

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.

Activity-Based Protein Profiling for the Study of Parasite Biology.

Parasites exist within most ecological niches, often transitioning through biologically and chemically complex host environments over the course of their parasitic life cycles. While the development of technologies for genetic engineering has revolutionised the field of functional genomics, parasites have historically been less amenable to such modification. In light of this, parasitologists have often been at the forefront of adopting new small-molecule technologies, repurposing drugs into biological tools and probes. Over the last decade, activity-based protein profiling (ABPP) has evolved into a powerful and versatile chemical proteomic platform for characterising the function of enzymes. Central to ABPP is the use of activity-based probes (ABPs), which covalently modify the active sites of enzyme classes ranging from serine hydrolases to glycosidases. The application of ABPP to cellular systems has contributed vastly to our knowledge on the fundamental biology of a diverse range of organisms and has facilitated the identification of potential drug targets in many pathogens. In this chapter, we provide a comprehensive review on the different forms of ABPP that have been successfully applied to parasite systems, and highlight key biological insights that have been enabled through their application.

Protozoan Parasite Auxotrophies and Metabolic Dependencies.

Diseases caused by protozoan parasites have a major impact on world health. These early branching eukaryotes cause significant morbidity and mortality in humans and livestock. During evolution, protozoan parasites have evolved toward complex life cycles in multiple host organisms with different nutritional resources. The conservation of functional metabolic pathways required for these successive environments is therefore a prerequisite for parasitic lifestyle. Nevertheless, parasitism drives genome evolution toward gene loss and metabolic dependencies (including strict auxotrophy), especially for obligatory intracellular parasites. In this chapter, we will compare and contrast how protozoan parasites have perfected this metabolic adaptation by focusing on specific auxotrophic pathways and scavenging strategies used by clinically relevant apicomplexan and trypanosomatid parasites to access host's nutritional resources. We will further see how these metabolic dependencies have in turn been exploited for therapeutic purposes against these human pathogens.

Long-term affected flat oyster (Ostrea edulis) haemocytes show differential gene expression profiles from naïve oysters in response to Bonamia ostreae.

European flat oyster (Ostrea edulis) production has suffered a severe decline due to bonamiosis. The responsible parasite enters in oyster haemocytes, causing an acute inflammatory response frequently leading to death. We used an immune-enriched oligo-microarray to understand the haemocyte response to Bonamia ostreae by comparing expression profiles between naïve (NS) and long-term affected (AS) populations along a time series (1 d, 30 d, 90 d). AS showed a much higher response just after challenge, which might be indicative of selection for resistance. No regulated genes were detected at 30 d in both populations while a notable reactivation was observed at 90 d, suggesting parasite latency during infection. Genes related to extracellular matrix and protease inhibitors, up-regulated in AS, and those related to histones, down-regulated in NS, might play an important role along the infection. Twenty-four candidate genes related to resistance should be further validated for selection programs aimed to control bonamiosis.

Comparative sphingolipidomics of disease-causing trypanosomatids reveal unique lifecycle- and taxonomy-specific lipid chemistries.

Trypanosomatids are parasitic protozoa which cause a spectrum of diseases, including trypanosomiasis and leishmaniasis, affecting millions of humans and animals worldwide. The surface of most protozoan parasites is heavily decorated with lipids and lipid-anchored molecules, forming protective barriers and acting as virulence factors during infection. Sphingolipids (SP) are major components of eukaryotic biomembranes, which play important roles in structural integrity, energy homeostasis and signaling. However, the precise chemical composition of SP in pathogens as well as their biochemical pathways and functions remain poorly characterized. Here, we present the first system-scale analyses of SP found in a panel of 7 trypanosomatids, including Leishmania donovani, Trypanosoma brucei and Trypanosoma cruzi. We characterized the structure of aminoethylphosphonate-containing ceramides, which are found exclusively in stercorarian Trypanosoma. Employing the sensitive and semi-quantitative sphingolipidomics approach that we developed, we report the detection of over 300 molecular species of SP, and identified unique metabolic signatures which serve as discriminants of the pathogens based on their taxonomy and lifecycle stages. The deep sphingolipidome presented here is an important biochemical and technological resource for future works to dissect SP metabolism and functions in these medically and agriculturally relevant systems.

Dynamic protein S-palmitoylation mediates parasite life cycle progression and diverse mechanisms of virulence.

Eukaryotic parasites possess complex life cycles and utilize an assortment of molecular mechanisms to overcome physical barriers, suppress and/or bypass the host immune response, including invading host cells where they can replicate in a protected intracellular niche. Protein S-palmitoylation is a dynamic post-translational modification in which the fatty acid palmitate is covalently linked to cysteine residues on proteins by the enzyme palmitoyl acyltransferase (PAT) and can be removed by lysosomal palmitoyl-protein thioesterase (PPT) or cytosolic acyl-protein thioesterase (APT). In addition to anchoring proteins to intracellular membranes, functions of dynamic palmitoylation include - targeting proteins to specific intracellular compartments via trafficking pathways, regulating the cycling of proteins between membranes, modulating protein function and regulating protein stability. Recent studies in the eukaryotic parasites - Plasmodium falciparum, Toxoplasma gondii, Trypanosoma brucei, Cryptococcus neoformans and Giardia lamblia - have identified large families of PATs and palmitoylated proteins. Many palmitoylated proteins are important for diverse aspects of pathogenesis, including differentiation into infective life cycle stages, biogenesis and tethering of secretory organelles, assembling the machinery powering motility and targeting virulence factors to the plasma membrane. This review aims to summarize our current knowledge of palmitoylation in eukaryotic parasites, highlighting five exemplary mechanisms of parasite virulence dependent on palmitoylation.

Opioid and Opiate Immunoregulatory Processes.

The discovery of the ability of the nervous system to communicate through "public" circuits with other systems of the body is attributed to Ernst and Berta Scharrer, who described the neurosecretory process in 1928. Indeed, the immune system has been identified as another important neuroendocrine target tissue. Opioid peptides are involved in this communication (i.e., neuroimmune) and with that of autoimmunoregulation (communication between immunocytes). The significance of opioid neuropeptide involvement with the immune system is ascertained from the presence of novel δ, µ., and κ receptors on inflammatory cells that result in modulation of cellular activity after activation, as well as the presence of specific enzymatic degradation and regulation processes. In contrast to the relatively uniform antinociceptive action of opiate and opioid signal molecules in neural tissues, the presence of naturally occurring morphine in plasma and a novel µ3 opiate-specific receptor on inflammatory cells adds to the growing knowledge that opioid and opiate signal molecules may have antagonistic actions in select tissues. In examining various disorders (e.g., human immunodeficiency virus, substance abuse, parasitism, and the diffuse inflammatory response associated with surgery) evidence has also been found for the involvement of opiate/opioid signaling in prominent mechanisms. In addition, the presence of similar mechanisms in man and organisms 500 million years divergent in evolution bespeaks the importance of this family of signal molecules. The present review provides an overview of recent advances in the field of opiate and opioid immunoregulatory processes and speculates as to their significance in diverse biological systems.

Far beyond Phagocytosis: Phagocyte-Derived Extracellular Traps Act Efficiently against Protozoan Parasites In Vitro and In Vivo.

Professional mononuclear phagocytes such as polymorphonuclear neutrophils (PMN), monocytes, and macrophages are considered as the first line of defence against invasive pathogens. The formation of extracellular traps (ETs) by activated mononuclear phagocytes is meanwhile well accepted as an effector mechanism of the early host innate immune response acting against microbial infections. Recent investigations showed evidence that ETosis is a widely spread effector mechanism in vertebrates and invertebrates being utilized to entrap and kill bacteria, fungi, viruses, and protozoan parasites. ETs are released in response to intact protozoan parasites or to parasite-specific antigens in a controlled cell death process. Released ETs consist of nuclear DNA as backbone adorned with histones, antimicrobial peptides, and phagocyte-specific granular enzymes thereby producing a sticky extracellular matrix capable of entrapping and killing pathogens. This review summarizes recent data on protozoa-induced ETosis. Special attention will be given to molecular mechanisms of protozoa-induced ETosis and on its consequences for the parasites successful reproduction and life cycle accomplishment.

Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites.

The malaria-causing Plasmodium parasites are transmitted to vertebrates by mosquitoes. To support their growth and replication, these intracellular parasites, which belong to the phylum Apicomplexa, have developed mechanisms to exploit their hosts. These mechanisms include expropriation of small metabolites from infected host cells, such as purine nucleotides and amino acids. Heretofore, no evidence suggested that transfer RNAs (tRNAs) could also be exploited. We identified an unusual gene in Apicomplexa with a coding sequence for membrane-docking and structure-specific tRNA binding. This Apicomplexa protein-designated tRip (tRNA import protein)-is anchored to the parasite plasma membrane and directs import of exogenous tRNAs. In the absence of tRip, the fitness of the parasite stage that multiplies in the blood is significantly reduced, indicating that the parasite may need host tRNAs to sustain its own translation and/or as regulatory RNAs. Plasmodium is thus the first example, to our knowledge, of a cell importing exogenous tRNAs, suggesting a remarkable adaptation of this parasite to extend its reach into host cell biology.

Three-dimensional structures in the design of therapeutics targeting parasitic protozoa: reflections on the past, present and future.

Parasitic protozoa cause a range of diseases which threaten billions of human beings. They are responsible for tremendous mortality and morbidity in the least-developed areas of the world. Presented here is an overview of the evolution over the last three to four decades of structure-guided design of inhibitors, leads and drug candidates aiming at targets from parasitic protozoa. Target selection is a crucial and multi-faceted aspect of structure-guided drug design. The major impact of advances in molecular biology, genome sequencing and high-throughput screening is touched upon. The most advanced crystallographic techniques, including XFEL, have already been applied to structure determinations of drug targets from parasitic protozoa. Even cryo-electron microscopy is contributing to our understanding of the mode of binding of inhibitors to parasite ribosomes. A number of projects have been selected to illustrate how structural information has assisted in arriving at promising compounds that are currently being evaluated by pharmacological, pharmacodynamic and safety tests to assess their suitability as pharmaceutical agents. Structure-guided approaches are also applied to incorporate properties into compounds such that they are less likely to become the victim of resistance mechanisms. A great increase in the number of novel antiparasitic compounds will be needed in the future. These should then be combined into various multi-compound therapeutics to circumvent the diverse resistance mechanisms that render single-compound, or even multi-compound, drugs ineffective. The future should also see (i) an increase in the number of projects with a tight integration of structural biology, medicinal chemistry, parasitology and pharmaceutical sciences; (ii) the education of more `medicinal structural biologists' who are familiar with the properties that compounds need to have for a high probability of success in the later steps of the drug-development process; and (iii) the expansion of drug-development capabilities in middle- and low-income countries.