Apicomplexans are important pathogens that cause severe infections in humans and animals. The biology and pathogeneses of these parasites have shown that proteins are intrinsically modulated during developmental transitions, physiological processes and disease progression. Also, proteins are integral components of parasite structural elements and organelles. Among apicomplexan parasites, Eimeria species are an important disease aetiology for economically important animals wherein identification and characterisation of proteins have been long-winded. Nonetheless, this review seeks to give a comprehensive overview of constitutively expressed Eimeria proteins. These molecules are discussed across developmental stages, organelles and sub-cellular components vis-à-vis their biological functions. In addition, hindsight and suggestions are offered with intention to summarise the existing trend of eimerian protein characterisation and to provide a baseline for future studies. Graphical Abstract

Continuous directed evolution of base editors (BEs) has been successful in bacteria cells, but not yet in mammalian cells. Here, we report the development of a Continuous Directed Evolution system in Mammalian cells (CDEM). CDEM enables the BE evolution in a full-length manner with Cas9 nickase. We harness CDEM to evolve the deaminases of cytosine base editor BE3 and adenine base editors, ABEmax and ABE8e. The evolved cytidine deaminase variants on BE4 architecture show not only narrowed editing windows, but also higher editing purity and low off-target activity without a trade-off in on-targeting activity. The evolved ABEmax and ABE8e variants exhibit narrowed or shifted editing windows to different extents, and lower off-target effects. The results illustrate that CDEM is a simple but powerful approach to continuously evolve BEs without size restriction in the mammalian environment, which is advantageous over continuous directed evolution system in bacteria cells.

Background: Protozoan parasite secretions can be triggered by various modified media and diverse physicochemical stressors. Equally, host-parasite interactions are known to co-opt the exchange and secretion of soluble biochemical components. Analysis of Eimeria falciformis sporozoite secretions in response to interaction with mouse intestinal epithelial cells (MIECs) may reveal parasite secretory motifs, protein composition and inflammatory activities of E. falciformis extracellular vesicles (EVs). Methods: Eimeria falciformis sporozoites were allowed to interact with inactivated MIECs. Parasite secretions were separated into EV and vesicle-free (VF) fractions by discontinuous centrifugation and ultracentrifugation. Secreted EVs were purified in an iodixanol density gradient medium and the protein composition of both EV and VF fractions were analyzed by liquid chromatoraphy-tandem mass spectroscopy. The inflammatory activities of E. falciformis sporozoite EV on MIECs were then investigated. Results: During the interaction of E. falciformis sporozoites with inactivated MIECs, the parasite secreted VF and vesicle-bound molecules. Eimeria falciformis vesicles are typical pathogenic protozoan EVs with a mean diameter of 264 ± 2 nm, and enclosed heat shock protein (Hsp) 70 as classical EV marker. Refractile body-associated aspartyl proteinase (or eimepsin), GAP45 and aminopeptidase were the main components of E. falciformis sporozoite EVs, while VF proteins include Hsp90, actin, Vps54 and kinases, among others. Proteomic data revealed that E. falciformis EV and VF proteins are aggregates of bioactive, antigenic and immunogenic molecules which act in concert for E. falciformis sporozoite motility, pathogenesis and survival. Moreover, in MIECs, E. falciformis EVs induced upregulation of gene expression and secretion of IL-1β, IL-6, IL-17, IL-18, MCP1 as well as pyroptosis-dependent caspase 11 and NLRP6 inflammasomes with the concomitant secretion of lactate dehydrogenase. Conclusions: Eimeria falciformis sporozoite interaction with MIECs triggered the secretion of immunogenic and antigenic proteins. In addition, E. falciformis sporozoite EVs constitute parasite-associated molecular pattern that induced inflammatory response and cell death. This study offers additional insight in the secretion and protein composition of E. falciformis secretomes as well as the proinflammatory functions of E. falciformis sporozoite EVs. Graphical

Background: Extracellular vesicles (EVs) are membranous structures that are formed during pathophysiology, host-parasite interactions and parasite motility. Typically, apicomplexan-infected host cells secrete EVs which traverse local and systemic strata of the host as the parasites develop. Methods: Extracellular vesicles were isolated from the caecum and serum of Eimeria falciformis-infected mice during oocyst ingestion (0 h post-infection [0 hpi]), merozont stages 1 and 2 (68 and 116 hpi), oocyst shedding (7 days post-infection [7 dpi]) and host recovery (10 dpi) and subsequently characterized and profiled by tandem mass tag (TMT). Results: With the progression of E. falciformis life stages, subpopulation of EVs bearing EV biomarkers, including CD9, CD82, heat shock protein 70 (HSP70) and major histocompatibility complex (MHC) molecules, increased. A total of 860 and 1024 differentially expressed proteins were identified in serum EVs (sEVs) and caecum EVs (cEVs), respectively. Identified immune-related molecules (such as cytokines, receptors, immunoglobins, complements, hormones, inflammasomes), ion exchange and cell death-associated proteins were significantly expressed, at least during the E. falciformis first and second merozont stages. Bioinformatics assessment indicated that sEV proteins were at all time points implicated in antigen processing and presentation as well as natural killer cell-mediated cytotoxicity (68 hpi), complement activation/blood coagulation (116 hpi/10 dpi) and catabolic activities (7 dpi). In contrast, cEV proteins were involved in catabolic process, ion transport and antigen presentation (68 and 116 hpi). Host response to E. falciformis infection was similar to intestinal bacterium at 7 dpi and cell adhesion and intercellular protein transport at 10 dpi. In both systems, ferroptosis and necroptosis were common across the parasite’s infectious cycle while apoptosis occurred at 68 hpi. Conclusion: The proteomic data indicate that E. falciformis infection co-opts cellular and humoral responses through EV secretions, and that, host cell death and ionic imbalance are associated with E. falciformis infection. This study offers additional insight into host-parasite interactions and host regulatory EV proteins as potential disease indicators or diagnostic molecules. Graphical