Advances in protease inhibition-based chemotherapy: A decade of insights from Malaria research.

Over the last decade, research on the most studied parasite, Plasmodium falciparum, has disclosed significant findings in protease research. Detailed descriptions of the individual roles of protease isoenzymes from various protease classes encoded by the parasite genome have been elucidated, along with their functional and biochemical characterizations. These insights have enabled the development of innovative chemotherapy using low molecular weight inhibitors targeting specific molecular sites. Progress has been made in understanding the proteolytic cascade associated with the apical complex, particularly the roles of aspartyl proteases plasmepsins IX and X as master regulators. Additionally, advancements in direct and alternative methods of proteasome inhibition and expression regulation have been achieved. Research on digestive/food vacuole-associated proteases, with a focus on essential metalloproteases, has also seen significant developments. The rise of extensive genomic datasets and functional genomic tools for other parasitic organisms now allows these approaches to be applied to the study and treatment of other, less known parasitic diseases, aiming to uncover specific biological mechanisms and develop innovative, less toxic chemotherapies.

Chelation of Mitochondrial Iron as an Antiparasitic Strategy.

Iron, as an essential micronutrient, plays a crucial role in host-pathogen interactions. In order to limit the growth of the pathogen, a common strategy of innate immunity includes withdrawing available iron to interfere with the cellular processes of the microorganism. Against that, unicellular parasites have developed powerful strategies to scavenge iron, despite the effort of the host. Iron-sequestering compounds, such as the approved and potent chelator deferoxamine (DFO), are considered a viable option for therapeutic intervention. Since iron is heavily utilized in the mitochondrion, targeting iron chelators in this organelle could constitute an effective therapeutic strategy. This work presents mitochondrially targeted DFO, mitoDFO, as a candidate against a range of unicellular parasites with promising in vitro efficiency. Intracellular Leishmania infection can be cleared by this compound, and experimentation with Trypanosoma brucei 427 elucidates its possible mode of action. The compound not only affects iron homeostasis but also alters the physiochemical properties of the inner mitochondrial membrane, resulting in a loss of function. Furthermore, investigating the virulence factors of pathogenic yeasts confirms that mitoDFO is a viable candidate for therapeutic intervention against a wide spectrum of microbe-associated diseases.

Establishment of a stable transfection and gene targeting system in Babesia divergens.

Babesia divergens is an emerging tick-borne pathogen considered as the principal causative agent of bovine babesiosis in Europe with a notable zoonotic risk to human health. Despite its increasing impact, considerable gaps persist in our understanding of the molecular interactions between this parasite and its hosts. In this study, we address the current limitation of functional genomic tools in B. divergens and introduce a stable transfection system specific to this parasite. We define the parameters for a drug selection system hdhfr-WR99210 and evaluate different transfection protocols for highly efficient generation of transgenic parasites expressing GFP. We proved that plasmid delivery into bovine erythrocytes prior to their infection is the most optimal transfection approach for B. divergens, providing novel evidence of Babesia parasites' ability to spontaneously uptake external DNA from erythrocytes cytoplasm. Furthermore, we validated the bidirectional and symmetrical activity of ef-tgtp promoter, enabling simultaneous expression of external genes. Lastly, we generated a B. divergens knockout line by targeting a 6-cys-e gene locus. The observed dispensability of this gene in intraerythrocytic parasite development makes it a suitable recipient locus for further transgenic application. The platform for genetic manipulations presented herein serves as the initial step towards developing advanced functional genomic tools enabling the discovery of B. divergens molecules involved in host-vector-pathogen interactions.

Insight Into the Dynamics of the Ixodes ricinus Nymphal Midgut Proteome.

Ticks are ectoparasites that feed on blood and have an impressive ability to consume and process enormous amounts of host blood, allowing extremely long periods of starvation between blood meals. The central role in the parasitic lifestyle of ticks is played by the midgut. This organ efficiently stores and digests ingested blood and serves as the primary interface for the transmission of tick-borne pathogens. In this study, we used a label-free quantitative approach to perform a novel dynamic proteomic analysis of the midgut of Ixodesricinus nymphs, covering their development from unfed to pre-molt stages. We identified 1534 I. ricinus-specific proteins with a relatively low proportion of host proteins. This proteome dataset, which was carefully examined by manual scrutiny, allowed precise annotation of proteins important for blood meal processing and their dynamic changes during nymphal ontogeny. We focused on midgut molecules related to lipid hydrolysis, storage, and transport, opening a yet unexplored avenue for studying lipid metabolism in ticks. Further dynamic profiling of the tick's multi-enzyme digestive network, protease inhibitors, enzymes involved in redox homeostasis and detoxification, antimicrobial peptides, and proteins responsible for midgut colonization by Borrelia spirochetes promises to uncover new targets for targeting tick nymphs, the most critical life stage for transmission the pathogens that cause tick-borne diseases.

Natural Clerodendrum-derived tick repellent: learning from Nepali culture.

Ticks attaching to ear canals of humans and animals are the cause of otoacariasis, common in rural areas of Nepal. The plant Clerodendrum viscosum is used in multiple indigenous systems of medicine by ethnic communities in the Indo-Nepali-Malaysian region. Visiting the Chitwan National Park, we learned that in indigenous medicine, flower extract of C. viscosum is utilized to treat digestive disorders and extracts from leaves as tick repellent to prevent ticks from invading or to remove them from the ear canal. The objective of our study was to provide support to indigenous medicine by characterizing the in vivo effect of leave extracts on ticks under laboratory conditions and its phytochemical composition. We collected plant parts of C. viscosum (leaves and flowers) and mango (Mangifera indica) leaves at the Chitwan National Park, previously associated with repellent activity to characterize their effect on Ixodes ricinus ticks by in vivo bioassays. A Q-ToF high-resolution analysis (HPLC-ESI-QToF) was conducted to elucidate phenolic compounds with potential repellent activity. Clerodendrum viscosum and M. indica leaf extracts had the highest tick repellent efficacy (%E = 80-100%) with significant differences when compared to C. viscosum flowers extracts (%E = 20-60%) and phosphate-buffered saline. Phytochemicals with tick repellent function as caffeic acid, fumaric acid and p-coumaric acid glucoside were identified in C. viscosum leaf extracts by HPLC-ESI-QToF, but not in non-repellent flower extracts. These results support the Nepali indigenous medicine application of C. viscosum leaf extracts to repel ticks. Additional research is needed for the development of natural and green repellent formulations to reduce the risks associated with ticks resistant to acaricides.

Degrade to survive: the intricate world of piroplasmid proteases.

Piroplasmids of the genera Babesia, Theileria, and Cytauxzoon are tick-transmitted parasites with a high impact on animals and humans. They have complex life cycles in their definitive arthropod and intermediate vertebrate hosts involving numerous processes, including invasion of, and egress from, host cells, parasite growth, transformation, and migration. Like other parasitic protozoa, piroplasmids are equipped with different types of protease to fulfill many of such essential processes. Blockade of some key proteases, using inhibitors or antibodies, hinders piroplasmid growth, highlighting their potential usefulness in drug therapies and vaccine development. A better understanding of the functional significance of these enzymes will contribute to the development of improved control measures for the devastating animal and human diseases caused by these pathogens.

Blood-feeding adaptations and virome assessment of the poultry red mite Dermanyssus gallinae guided by RNA-seq.

Dermanyssus gallinae is a blood-feeding mite that parasitises wild birds and farmed poultry. Its remarkably swift processing of blood, together with the capacity to blood-feed during most developmental stages, makes this mite a highly debilitating pest. To identify specific adaptations to digestion of a haemoglobin-rich diet, we constructed and compared transcriptomes from starved and blood-fed stages of the parasite and identified midgut-enriched transcripts. We noted that midgut transcripts encoding cysteine proteases were upregulated with a blood meal. Mapping the full proteolytic apparatus, we noted a reduction in the suite of cysteine proteases, missing homologues for Cathepsin B and C. We have further identified and phylogenetically analysed three distinct transcripts encoding vitellogenins that facilitate the reproductive capacity of the mites. We also fully mapped transcripts for haem biosynthesis and the ferritin-based system of iron storage and inter-tissue trafficking. Additionally, we identified transcripts encoding proteins implicated in immune signalling (Toll and IMD pathways) and activity (defensins and thioester-containing proteins), RNAi, and ion channelling (with targets for commercial acaricides such as Fluralaner, Fipronil, and Ivermectin). Viral sequences were filtered from the Illumina reads and we described, in part, the RNA-virome of D. gallinae with identification of a novel virus, Red mite quaranjavirus 1.

Babesia, Theileria, Plasmodium and Hemoglobin.

The Propagation of Plasmodium spp. and Babesia/Theileria spp. vertebrate blood stages relies on the mediated acquisition of nutrients available within the host's red blood cell (RBC). The cellular processes of uptake, trafficking and metabolic processing of host RBC proteins are thus crucial for the intraerythrocytic development of these parasites. In contrast to malarial Plasmodia, the molecular mechanisms of uptake and processing of the major RBC cytoplasmic protein hemoglobin remain widely unexplored in intraerythrocytic Babesia/Theileria species. In the paper, we thus provide an updated comparison of the intraerythrocytic stage feeding mechanisms of these two distantly related groups of parasitic Apicomplexa. As the associated metabolic pathways including proteolytic degradation and networks facilitating heme homeostasis represent attractive targets for diverse antimalarials, and alterations in these pathways underpin several mechanisms of malaria drug resistance, our ambition is to highlight some fundamental differences resulting in different implications for parasite management with the potential for novel interventions against Babesia/Theileria infections.

Plasmepsin-like Aspartyl Proteases in Babesia.

Apicomplexan genomes encode multiple pepsin-family aspartyl proteases (APs) that phylogenetically cluster to six independent clades (A to F). Such diversification has been powered by the function-driven evolution of the ancestral apicomplexan AP gene and is associated with the adaptation of various apicomplexan species to different strategies of host infection and transmission through various invertebrate vectors. To estimate the potential roles of Babesia APs, we performed qRT-PCR-based expressional profiling of Babesia microti APs (BmASP2, 3, 5, 6), which revealed the dynamically changing mRNA levels and indicated the specific roles of individual BmASP isoenzymes throughout the life cycle of this parasite. To expand on the current knowledge on piroplasmid APs, we searched the EuPathDB and NCBI GenBank databases to identify and phylogenetically analyse the complete sets of APs encoded by the genomes of selected Babesia and Theileria species. Our results clearly determine the potential roles of identified APs by their phylogenetic relation to their homologues of known function-Plasmodium falciparum plasmepsins (PfPM I-X) and Toxoplasma gondii aspartyl proteases (TgASP1-7). Due to the analogies with plasmodial plasmepsins, piroplasmid APs represent valuable enzymatic targets that are druggable by small molecule inhibitors-candidate molecules for the yet-missing specific therapy for babesiosis.

Mialostatin, a Novel Midgut Cystatin from Ixodes ricinus Ticks: Crystal Structure and Regulation of Host Blood Digestion.

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

Protease Inhibition-An Established Strategy to Combat Infectious Diseases.

Therapeutic agents with novel mechanisms of action are urgently needed to counter the emergence of drug-resistant infections. Several decades of research into proteases of disease agents have revealed enzymes well suited for target-based drug development. Among them are the three recently validated proteolytic targets: proteasomes of the malarial parasite Plasmodium falciparum, aspartyl proteases of P. falciparum (plasmepsins) and the Sars-CoV-2 viral proteases. Despite some unfulfilled expectations over previous decades, the three reviewed targets clearly demonstrate that selective protease inhibitors provide effective therapeutic solutions for the two most impacting infectious diseases nowadays-malaria and COVID-19.

Comparison of the hemolysis machinery in two evolutionarily distant blood-feeding arthropod vectors of human diseases.

Host blood protein digestion plays a pivotal role in the ontogeny and reproduction of hematophagous vectors. The gut of hematophagous arthropods stores and slowly digests host blood and represents the primary gateway for transmitted pathogens. The initial step in blood degradation is induced lysis of host red blood cells (hemolysis), which releases hemoglobin for subsequent processing by digestive proteolytic enzymes. The activity cycles and characteristics of hemolysis in vectors are poorly understood. Hence, we investigated hemolysis in two evolutionarily distant blood-feeding arthropods: The mosquito Culex pipiens and the soft tick Argas persicus, both of which are important human and veterinary disease vectors. Hemolysis in both species was cyclical after blood meal ingestion. Maximum digestion occurs under slightly alkaline conditions in females. Hemolytic activity appears to be of lipoid origin in C. pipiens and enzymatic activity (proteolytic) in A. persicus. We have assessed the effect of pH, incubation time, and temperature on hemolytic activity and the hemolysin. The susceptibility of red blood cells from different hosts to the hemolysin and the effect of metabolic inhibition of hemolytic activity were assessed. We conclude that in C. pipiens and A. persicus midgut hemolysins control the amplitude of blood lysis step to guarantee an efficient blood digestion.

Design, synthesis, and in vitro evaluation of aza-peptide aldehydes and ketones as novel and selective protease inhibitors.

Aza-peptide aldehydes and ketones are a new class of reversible protease inhibitors that are specific for the proteasome and clan CD cysteine proteases. We designed and synthesised aza-Leu derivatives that were specific for the chymotrypsin-like active site of the proteasome, aza-Asp derivatives that were effective inhibitors of caspases-3 and -6, and aza-Asn derivatives that inhibited S. mansoni and I. ricinus legumains. The crystal structure of caspase-3 in complex with our caspase-specific aza-peptide methyl ketone inhibitor with an aza-Asp residue at P1 revealed a covalent linkage between the inhibitor carbonyl carbon and the active site cysteinyl sulphur. Aza-peptide aldehydes and ketones showed no cross-reactivity towards cathepsin B or chymotrypsin. The initial in vitro selectivity of these inhibitors makes them suitable candidates for further development into therapeutic agents to potentially treat multiple myeloma, neurodegenerative diseases, and parasitic infections.

Babesia Life Cycle - When Phylogeny Meets Biology.

Although Babesia represents an important worldwide veterinary threat and an emerging risk to humans, this parasite has been poorly studied as compared to Plasmodium, its malaria-causing relative. In fact, Babesia employs highly specific survival strategies during its intraerythrocytic development and its intricate journey through the tick vector. This review introduces a substantially extended molecular phylogeny of the order Piroplasmida, challenging previous taxonomic classifications. The intriguing developmental proficiencies of Babesia are highlighted and compared with those of other haemoparasitic Apicomplexa. Molecular mechanisms associated with distinctive events in the Babesia life cycle are emphasized as potential targets for the development of Babesia-specific treatments.

The Complexity of Piroplasms Life Cycles.

Although apicomplexan parasites of the group Piroplasmida represent commonly identified global risks to both animals and humans, detailed knowledge of their life cycles is surprisingly limited. Such a discrepancy results from incomplete literature reports, nomenclature disunity and recently, from large numbers of newly described species. This review intends to collate and summarize current knowledge with respect to piroplasm phylogeny. Moreover, it provides a comprehensive view of developmental events of Babesia, Theileria, and Cytauxzoon representative species, focusing on uniform consensus of three consecutive phases: (i) schizogony and merogony, asexual multiplication in blood cells of the vertebrate host; (ii) gamogony, sexual reproduction inside the tick midgut, later followed by invasion of kinetes into the tick internal tissues; and (iii) sporogony, asexual proliferation in tick salivary glands resulting in the formation of sporozoites. However, many fundamental differences in this general consensus occur and this review identifies variables that should be analyzed prior to further development of specific anti-piroplasm strategies, including the attractive targeting of life cycle stages of Babesia or Theileria tick vectors.

Validation of Babesia proteasome as a drug target.

Babesiosis is a tick-transmitted zoonosis caused by apicomplexan parasites of the genus Babesia. Treatment of this emerging malaria-related disease has relied on antimalarial drugs and antibiotics. The proteasome of Plasmodium, the causative agent of malaria, has recently been validated as a target for anti-malarial drug development and therefore, in this study, we investigated the effect of epoxyketone (carfilzomib, ONX-0914 and epoxomicin) and boronic acid (bortezomib and ixazomib) proteasome inhibitors on the growth and survival of Babesia. Testing the compounds against Babesia divergens ex vivo revealed suppressive effects on parasite growth with activity that was higher than the cytotoxic effects on a non-transformed mouse macrophage cell line. Furthermore, we showed that the most-effective compound, carfilzomib, significantly reduces parasite multiplication in a Babesia microti infected mouse model without noticeable adverse effects. In addition, treatment with carfilzomib lead to an ex vivo and in vivo decrease in proteasome activity and accumulation of polyubiquitinated proteins compared to untreated control. Overall, our results demonstrate that the Babesia proteasome is a valid target for drug development and warrants the design of potent and selective B. divergens proteasome inhibitors for the treatment of babesiosis.

Novel Structural Mechanism of Allosteric Regulation of Aspartic Peptidases via an Evolutionarily Conserved Exosite.

Pepsin-family aspartic peptidases are biosynthesized as inactive zymogens in which the propeptide blocks the active site until its proteolytic removal upon enzyme activation. Here, we describe a novel dual regulatory function for the propeptide using a set of crystal structures of the parasite cathepsin D IrCD1. In the IrCD1 zymogen, intramolecular autoinhibition by the intact propeptide is mediated by an evolutionarily conserved exosite on the enzyme core. After activation, the mature enzyme employs the same exosite to rebind a small fragment derived from the cleaved propeptide. This fragment functions as an effective natural inhibitor of mature IrCD1 that operates in a pH-dependent manner through a unique allosteric inhibition mechanism. The study uncovers the propeptide-binding exosite as a target for the regulation of pepsin-family aspartic peptidases and defines the structural requirements for exosite inhibition.

Multiple legumain isoenzymes in ticks.

By searching nucleotide databases for the North American Lyme disease vector, Ixodes scapularis, we have complemented the previously characterized European Ixodes ricinus legumain IrAE1 with a full set of nine analogous genes (isae1-9). Six of these were PCR confirmed as genes present in all tick genomes tested. The absolute mRNA copy number examined by quantitative (q)PCR enabled expression profiling and an absolute comparison of mRNA levels for individual I. scapularis (Is)AEs in tick tissues. Four IsAEs (1, 2, 4, 9) were expressed solely in the gut and thus are proposed to be involved in host blood digestion. Expression qPCR profiling over developmental stages confirmed IsAE1, the direct analogue of previously characterized I. ricinus IrAE1, as the principle legumain transcript in partially engorged females, and demonstrated its strong regulation by on-host feeding in larvae, nymphs and females. In contrast, IsAE2 was the predominant gut legumain in unfed nymphs, unfed females and males. In-silico, IsAE1 and IsAE2 protein three-dimensional structural models displayed minimal differences in overall proenzyme structures, even in comparison with recently resolved crystal structures of mammalian prolegumain. Three functional studies were performed in I. ricinus with IsAE1/IsAE2 analogues: double IrAE1/IrAE2 RNA interference silencing, feeding of ticks on IrAE1+IrAE2 immunized hosts and in vitro membrane tick feeding on blood containing a legumain-specific inhibitor. The latter experiment led to reduced weights of fully engorged ticks and limited oviposition, and indicated the potential of legumain inhibitors for novel anti-tick interventions.

Parasite Cathepsin D-Like Peptidases and Their Relevance as Therapeutic Targets.

Inhibition of aspartic cathepsin D-like peptidases (APDs) has been often discussed as an antiparasite intervention strategy. APDs have been considered as virulence factors of Trypanosoma cruzi and Leishmania spp., and have been demonstrated to have important roles in protein trafficking mechanisms of apicomplexan parasites. APDs also initiate blood digestion as components of multienzyme proteolytic complexes in malaria, platyhelminths, nematodes, and ticks. Increasing DNA and RNA sequencing data indicate that parasites express multiple APD isoenzymes of various functions that can now be specifically evaluated using new functional-genomic and biochemical tools, from which we can further assess the potential of APDs as targets for novel effective intervention strategies against parasitic diseases that still pose an alarming threat to mankind.

Acquisition of exogenous haem is essential for tick reproduction.

Haem and iron homeostasis in most eukaryotic cells is based on a balanced flux between haem biosynthesis and haem oxygenase-mediated degradation. Unlike most eukaryotes, ticks possess an incomplete haem biosynthetic pathway and, together with other (non-haematophagous) mites, lack a gene encoding haem oxygenase. We demonstrated, by membrane feeding, that ticks do not acquire bioavailable iron from haemoglobin-derived haem. However, ticks require dietary haemoglobin as an exogenous source of haem since, feeding with haemoglobin-depleted serum led to aborted embryogenesis. Supplementation of serum with haemoglobin fully restored egg fertility. Surprisingly, haemoglobin could be completely substituted by serum proteins for the provision of amino-acids in vitellogenesis. Acquired haem is distributed by haemolymph carrier protein(s) and sequestered by vitellins in the developing oocytes. This work extends, substantially, current knowledge of haem auxotrophy in ticks and underscores the importance of haem and iron metabolism as rational targets for anti-tick interventions.