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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.

The role of CD4+ and CD8+ T lymphocytes in the development of a protective immune response against Cryptosporidium muris infection was studied by the reconstitution of severe combined immunodeficient (SCID) mice with well-defined populations of either naive or immune CD8+ or CD4+ T lymphocytes. Adoptive transfer of both naive and immune CD4+ T lymphocyte subpopulations protects SCID mice against cryptosporidiosis. Moreover, a significant biological impact of activated CD8+ T cells against gastric cryptosporidiosis was observed. The significant difference in the course and intensity of the infection in reconstituted SCID mice was found to be dependent on the protective function of both the CD4+ and CD8+ T-cell populations transferred. While SCID mice reconstituted with either immune or naive CD4+ or immune CD8+ T-cell subpopulations resolved the infection within 29, 37 and 51 days post-infection, respectively, those reconstituted with naive CD8+ T cells suffered from chronic infection similar to control SCID mice. Reconstitution with CD4+ T cells resulted in suppression of oocyst excretion and shortening of patent period in comparison with SCID mice reconstituted with CD8+ T cells. Thus, although CD4+ T cells are considered important in protective immunity, our results are the first to demonstrate the involvement of activated CD8+ T lymphocytes in the protection of mice against gastric cryptosporidiosis., (© 2011 Blackwell Publishing Ltd.)

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.

Human babesiosis is a malaria-like, tick-borne infectious disease with a global distribution. Babesiosis is caused by intraerythrocytic, apicomplexan parasites of the genus Babesia. In the United States, human babesiosis is caused by Babesia microti and Babesia duncani. Current treatment for babesiosis includes either the combination of atovaquone and azithromycin or the combination of clindamycin and quinine. However, the side effects of these agents and the resistance posed by these parasites call for alternative approaches for treating human babesiosis. Proteases play several roles in the context of parasitic lifestyle and regulate basic biological processes including cell death, cell progression, and cell migration. Using the SYBR Green-1 assay, we screened a protease inhibitor library that consisted of 160 compounds against B. duncani in vitro and identified 13 preliminary hits. Dose response assays of hit compounds against B. duncani and B. microti under in vitro conditions identified five effective inhibitors against parasite growth. Of these compounds, we chose ixazomib, a proteasome inhibitor as a potential drug for animal studies based on its lower IC 50 and a higher therapeutic index in comparison with other compounds. Our results suggest that Babesia proteasome may be an important drug target and that developing this class of drugs may be important to combat human babesiosis., (© 2024 The Author(s). Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists.)

Introduction: Anesthesiologists play an important role in the management of labor and delivery during acute malaria infection. The peripartum anesthesia considerations for such cases remain unclear., Findings: Important peripartum considerations include the severity of thrombocytopenia and coagulopathy, hemodynamic status and cardiac disease, and the likelihood of central nervous system (CNS) involvement. Several antimalarial drugs may interact with perioperative medications, causing hypoglycemia, methemoglobinemia, or QT prolongation. Labor should usually not be induced. Patient volume status should be optimized pre-induction, but fluids should be administered with caution given the risk of cerebral edema. In case of CNS involvement intracranial pressure should be maintained. Case reports describe the successful use of neuraxial anesthesia but this approach requires further confirmation of safety. Despite the risks accompanying airway management in pregnancy, in some cases, general anesthesia was preferred due to the chance of CNS infection and disease complications. Tight postoperative assessments of neurological and bleeding status are indicated regardless of the mode of delivery., Conclusions: Despite the prevalence of malaria, the perioperative risk and preferred mode of anesthesia for pregnant patients with acute malaria remain under-researched and outcome data are limited., (© 2024. The Author(s).)

Single engineered microbial species cannot always conduct complex transformations, while complex, incompletely defined microbial consortia have heretofore been suited to a limited range of tasks. As biodesigners bridge this gap with intentionally designed microbial communities, they will, intentionally or otherwise, build communities that embody particular ideas about what microbial communities can and should be. Here, we suggest that metaphors-ideas about what microbial communities are like-are therefore important tools for designing synthetic consortia-based bioreactors. We identify a range of metaphors currently employed in peer-reviewed microbiome research articles, characterizing each through its potential structural implications and distinctive imagery. We present this metaphor catalogue in the interest of, first, making metaphors visible as design choices, second, enabling deliberate experimentation with them towards expanding the potential design space of the field, and third, encouraging reflection on the goals and values they embed., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Ticks carry a diverse community of microorganisms including non-pathogenic symbionts, commensals, and pathogens, such as viruses, bacteria, protozoans, and fungi. The assessment of tick-borne microorganisms (TBM) in tortoises and their ticks is essential to understand their eco-epidemiology, and to map and monitor potential pathogens to humans and other animals. The aim of this study was to characterize the diversity of microorganisms found in ticks collected from the spur-thighed tortoise (Testudo graeca) in North Africa and Anatolia. Ticks feeding on wild T. graeca were collected, and pathogens were screened by polymerase chain reaction using group-specific primers. In total, 131 adult Hyalomma aegyptium ticks were collected from 92 T. graeca in Morocco (n = 48), Tunisia (n = 2), Algeria (n = 70), and Turkey (n = 11). Bacteria and protozoa detected included Hemolivia mauritanica (22.9%), Midichloria mitochondrii (11.4%), relapsing-fever borreliae (8.4%), Ehrlichia spp. (7.6%), Rickettsia spp. (3.4%), Borrelia burgdorferi s.l. (0.9%), Francisella spp. (0.9%), and Wolbachia spp. (0.8%). The characterization of Rickettsia included R. sibirica mongolitimonae (Algeria), R. aeschlimannii (Turkey), and R.africae (Morocco). Hemolivia mauritanica and Ehrlichia spp. prevalence varied significantly with the sampling region/country. We did not detect significant associations in microorganism presence within ticks, nor between microorganism presence and tick mitochondrial DNA haplogroups. This is the first report of Francisella persica-like, relapsing fever borreliae, M. mitochondrii, and Wolbachia spp. in H. aegyptium ticks collected from wild hosts from the South and Eastern Mediterranean region, and of R. sibirica mongolitimonae and R. africae in H. aegyptium from Algeria and Morocco, respectively. Given that T. graeca is a common species in commercial and non-commercial pet trade, the evaluation of the role of this species and its ticks as hosts for TBM is particularly relevant for public health., (© 2021 Wiley-VCH GmbH.)

The main objective of this study was to determine which biochemical blood parameters can serve as indicators of Zn or Zn/Cd burden and tapeworm infection. This study was performed on 44 Wistar male rats during a 6-week period, when rats were or were not fed a zinc/cadmium rich diet and were or were not infected with tapeworms (Hymenolepis diminuta). Total protein, albumin, urea, glucose, triacylglycerols, non-esterified fatty acids, cholesterol, alkaline phosphatase, aspartate aminotransferase, uric acid, Mg, Ca, P and Zn levels were analysed. Control rats with tapeworm infection had significantly higher (p ≤ 0.05) total protein, urea and phosphorus concentrations than did rats unaffected by any experimental factor. Rats given overdoses of zinc lactate exhibited significantly lower glucose levels than did the other rats, especially those infected with tapeworms. Low glucose level in uninfected rats indicate a Zn overdose; high doses of zinc lactate likely decrease levels of glucose via cortisol, which is released during stress. Rats fed the Zn/Cd hyperaccumulating plant Arabidopsis halleri and infected with tapeworms had significantly higher (p ≤ 0.01) cholesterol and urea levels but lower zinc, triacylglycerol, and alkaline phosphatase levels than did rats fed the same diet but free of tapeworms. The increase of alkaline phosphatase level in uninfected rats may indicate both Zn/Cd burden and rat liver damage. Overall, this study not only supports the theory that H. diminuta can serve as a promising model for helminth therapy of the host mammal but also confirmed that this tapeworm is capable to protect somehow the host organism from the harmful effects of heavy metals., (© 2021 Wiley-VCH GmbH.)

Inhibitors of apoptosis (IAPs) are regulators of cell death and may play a role in the salivary glands of ticks during blood-feeding. We cloned the open reading frame (ORF) sequence of the IAP gene in Rhipicephalus haemaphysaloides (RhIAP). The RhIAP ORF of 1887 bp encodes a predicted protein of 607 amino acids, which contains three baculovirus IAP repeat domains and a RING finger motif. A real-time PCR assay showed that RhIAP mRNA was expressed in all the tick developmental stages (eggs, larvae, nymphs, and adults) and in all tissues examined (midgut, ovary, salivary glands, fat body, and hemolymph). Western blot showed that the protein level of RhIAP in salivary glands increased during tick blood-feeding and decreased towards the end of tick engorgement. RhIAP gene silencing in vitro experiments with salivary glands demonstrated that RhIAP could be effectively knocked down within 48 h after dsRNA treatment, and as a consequence, salivary glands displayed apoptotic morphology. RhIAP gene silencing also inhibited tick blood-feeding and decreased the engorgement rate. These data suggest that RhIAP might be a suitable RNAi target for tick control.