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Tick-transmitted Babesia are a major global veterinary threat and an emerging risk to humans. Unlike their Plasmodium relatives, these erythrocyte-infecting Apicomplexa have been largely overlooked and lack specific treatment. Selective targeting of the Babesia proteasome holds promise for drug development. In this study, we screened a library of peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B for their activity against Babesia . Several of these compounds showed activity against both the asexual and sexual blood stages of Plasmodium falciparum . These compounds inactivate β5 proteasome subunit activity in the lysates of Babesia divergens and Babesia microti in the low nanomolar range. Several compounds were tested with the purified B. divergens proteasome and showed IC 50 values comparable to carfilzomib, an approved anticancer proteasome inhibitor. They also inhibited B. divergens growth in bovine erythrocyte cultures with solid EC 50 values, but importantly, they appeared less toxic to human cells than carfilzomib. These compounds therefore offer a wider therapeutic window and provide new insights into the development of small proteasome inhibitors as selective drugs for babesiosis., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

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.

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.

Ticks possess components of a primordial complement system that presumably play a role in the interaction of the tick immune system with tick-borne pathogens and affect their transmission. Here we characterized a novel complement component, tagged as IrC2/Bf, from the hard tick Ixodes ricinus, the principal vector of Lyme disease in Europe. IrC2/Bf is a multi-domain molecule composed of 5-7 CCP modules, varied by alternative splicing, followed by a von Willebrand factor A domain and a C-terminal trypsin-like domain. The primary structure and molecular architecture of IrC2/Bf displays the closest homology to the C3-complement component convertases described in horseshoe crabs. The irc2/bf gene is mainly expressed in the tick fat body associated with the trachea and, as determined by western blotting, the protein is present in low amounts in tick hemolymph. Expression of irc2/bf mRNA was significantly up-regulated in response to the intra-hemocoelic injection of the yeast Candida albicans and all tested Borrelia sp. strains (B. burgdorferi NE5264, B. burgdorferi CB26, B. garinii MSLB, B. afzelii CB43), but was not affected by injection of model Gram-negative and Gram-positive bacteria or the aseptic injection control. In-line with these results, RNAi-mediated silencing of irc2/bf inhibited phagocytosis of B. afzelii and C. albicans but not the other bacteria. Tissue expression profiles, specific responses to microbial challenges, and patterns of phagocytic phenotypes upon RNAi silencing observed for IrC2/Bf match well with the previously reported characteristics of I. ricinus C3-related molecule 1 (IrC3-1). Therefore we presume that IrC2/Bf functions as a convertase in the same complement activation pathway protecting ticks against yeast and Borrelia infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Helminths and bacteria are major players in the mammalian gut ecosystem and each influences the host immune system and health. Declines in helminth prevalence and bacterial diversity appear to play a role in the dramatic rise of immune mediated inflammatory diseases (IMIDs) in western populations. Helminths are potent modulators of immune system and their reintroduction is a promising therapeutic avenue for IMIDs. However, the introduction of helminths represents a disturbance for the host and it is important to understand the impact of helminth reintroduction on the host, including the immune system and gut microbiome. We tested the impact of a benign tapeworm, Hymenolepis diminuta, in a rat model system. We find that H. diminuta infection results in increased interleukin 10 gene expression in the beginning of the prepatent period, consistent with induction of a type 2 immune response. We also find induction of humoral immunity during the patent period, shown here by increased IgA in feces. Further, we see an immuno-modulatory effect in the small intestine and spleen in patent period, as measured by reductions in tissue immune cells. We observed shifts in microbiota community composition during the patent period (beta-diversity) in response to H. diminuta infection. However, these compositional changes appear to be minor; they occur within families and genera common to both treatment groups. There was no change in alpha diversity. Hymenolepis diminuta is a promising model for helminth therapy because it establishes long-term, stable colonization in rats and modulates the immune system without causing bacterial dysbiosis. These results suggest that the goal of engineering a therapeutic helminth that can safely manipulate the mammalian immune system without disrupting the rest of the gut ecosystem is in reach.

The application of the phagocytic receptor agonists in cancer immunotherapy was studied. Agonists (laminarin, molecules with terminal mannose, N-Formyl-methioninyl-leucyl-phenylalanine) were firmly anchored to the tumor cell surface. When particular agonists of phagocytic receptors were used together with LPS (Toll-like receptor agonist), high synergy causing tumour shrinkage and a temporary or permanent disappearance was observed. Methods of anchoring phagocytic receptor agonists (charge interactions, anchoring based on hydrophobic chains, covalent bonds) and various regimes of phagocytic agonist/LPS mixture applications were tested to achieve maximum therapeutic effect. Combinations of mannan/LPS and f-MLF/LPS (hydrophobic anchors) in appropriate (pulse) regimes resulted in an 80% and 60% recovery for mice, respectively. We propose that substantial synergy between agonists of phagocytic and Toll-like receptors (TLR) is based on two events. The TLR ligand induces early and massive inflammatory infiltration of tumors. The effect of this cell infiltrate is directed towards tumor cells, bearing agonists of phagocytic receptors on their surface. The result of these processes was effective killing of tumor cells. This novel approach represents exploitation of innate immunity mechanisms for treating cancer.

Ticks are hematophagous arachnids transmitting a wide variety of pathogens including viruses, bacteria, and protozoans to their vertebrate hosts. The tick vector competence has to be intimately linked to the ability of transmitted pathogens to evade tick defense mechanisms encountered on their route through the tick body comprising midgut, hemolymph, salivary glands or ovaries. Tick innate immunity is, like in other invertebrates, based on an orchestrated action of humoral and cellular immune responses. The direct antimicrobial defense in ticks is accomplished by a variety of small molecules such as defensins, lysozymes or by tick-specific antimicrobial compounds such as microplusin/hebraein or 5.3-kDa family proteins. Phagocytosis of the invading microbes by tick hemocytes is likely mediated by the primordial complement-like system composed of thioester-containing proteins, fibrinogen-related lectins and convertase-like factors. Moreover, an important role in survival of the ingested microbes seems to be played by host proteins and redox balance maintenance in the tick midgut. Here, we summarize recent knowledge about the major components of tick immune system and focus on their interaction with the relevant tick-transmitted pathogens, represented by spirochetes (Borrelia), rickettsiae (Anaplasma), and protozoans (Babesia). Availability of the tick genomic database and feasibility of functional genomics based on RNA interference greatly contribute to the understanding of molecular and cellular interplay at the tick-pathogen interface and may provide new targets for blocking the transmission of tick pathogens.

Gastric cryptosporidia only inhabit the glandular part of the stomach of all age categories of their hosts and can cause chronic life-long infections independent of a host's immune status. The immune response in the stomach mucosa during the primary infection and re-infection with Cryptosporidium muris (TS03 and CB03) in immunocompetent BALB/c mice was characterized using flow cytometry analysis and measurement of IFN-gamma and IL10 by enzyme-linked immunosorbent assays (ELISA). Significantly, elevated migration of T lymphocytes (more than 1,000-fold), especially CD8+ T lymphocytes, to the stomach mucosa occurred during primary infection and persisted for more than 2 months after its resolution. The ex vivo cultures of splenocytes revealed very low levels of IFN-gamma production during the course of the primary infection (0.5 ng/ml), whereas in the following re-exposure to the parasites, the concentration of IFN-gamma rapidly increased 22-fold. Although the two parasite strains that were tested were genetically distinct, they yielded similar results in the induction of cellular immune responses, suggesting that these patterns are not unique to a single parasite strain. These results imply that the CD8+ T lymphocytes are involved in the immune response to gastric cryptosporidiosis and could play an important role in the elimination of C. muris infection in mice.