Host Target Cells Research Articles

'Candidatus Phytoplasma solani' Predicted Effector SAP11-like Alters Morphology of Transformed Arabidopsis Plants and Interacts with AtTCP2 and AtTCP4 Plant Transcription Factors.

Phytoplasmas are obligate intracellular pathogens that profoundly modify the development, physiology and behavior of their hosts by secreting effector proteins that disturb signal pathways and interactions both in plant and insect hosts. The characterization of effectors and their host-cell targets was performed for only a few phytoplasma species where it was shown that the SAP11 effector alters plant morphology by destabilizing plant transcription factors: TEOSINTE BRANCHED 1-CYCLOIDEA-PROLIFERATING CELL FACTOR (TCPs). To explore the possible role of the SAP11-like effector from 'Ca. P. solani', we used Arabidopsis thaliana as a model plant. The SAP11-like effector gene from 'Ca. P. solani' was introduced into arabidopsis by floral dip and transgenic lines were regenerated. In planta bimolecular fluorescence complementation (BIFC) assays in agroinfiltrated Nicotiana benthamiana leaf cells were conducted to detect interactions between SAP11-like and AtTCP2 and AtTCP4 using confocal microscopy. SAP11-like from 'Ca. P. solani' induced significant phenotypic changes in arabidopsis, including crinkled leaves with reduced size, lower biomass, more axillary branches, changes in root morphology, and crinkled and smaller siliques. The BIFC assays proved in planta interaction of SAP11-like effector with AtTCP2 and AtTCP4. To our knowledge, this is the first characterization of the interaction between the 'Ca. P. solani' effector and plant transcription factors, suggesting a potential mechanism of modulating plant development and induction of characteristic symptoms in 'Ca. P. solani'-infected plants.

What are the essential determinants of human papillomavirus carcinogenesis?

Human papillomavirus (HPV) infection is the leading viral cause of cancer. Over the past several decades, research on HPVs has provided remarkable insight into human cell biology and into the pathology of viral and non-viral cancers. The HPV E6 and E7 proteins engage host cellular proteins to establish an environment in infected cells that is conducive to virus replication. They rewire host cell signaling pathways to promote proliferation, inhibit differentiation, and limit cell death. The activity of the "high-risk" HPV E6 and E7 proteins is so potent that their dysregulated expression is sufficient to drive the initiation and maintenance of HPV-associated cancers. Consequently, intensive research efforts have aimed to identify the host cell targets of E6 and E7, in part with the idea that some or all of the virus-host interactions would be essential cancer drivers. These efforts have identified a large number of potential binding partners of each oncoprotein. However, over the same time period, parallel research has revealed that a relatively small number of genetic mutations drive carcinogenesis in most non-viral cancers. We therefore propose that a high-priority goal is to identify which of the many targets of E6 and E7 are critical drivers of HPV carcinogenesis. By identifying the cancer-driving targets of E6 and E7, it should be possible to better understand the distinct roles of other targets, perhaps in the viral life cycle, and to focus efforts to develop anti-cancer therapies on the subset of virus-host interactions for which therapeutic intervention would have the greatest impact.

Alpha-1 antitrypsin inhibits pertussis toxin

Pertussis (whooping cough) is a vaccine-preventable but re-emerging, highly infectious respiratory disease caused by Bordetella pertussis. There are currently no effective treatments for pertussis, complicating care for non-vaccinated individuals, especially newborns. Disease manifestations are predominantly caused by pertussis toxin (PT), a pivotal virulence factor classified as an ADP-ribosylating AB-type protein toxin. In this work, an unbiased approach using peptide libraries, bioassay-guided fractionation and mass spectrometry revealed α1-antitrypsin (α1AT) as a potent PT inhibitor. Biochemistry-, cell culture- and molecular modeling-based in vitro experimentation demonstrated that the α1AT mode of action is based on blocking PT-binding to the host target cell surface. In the infant mouse model of severe pertussis, α1AT expression was reduced upon infection. Further, systemic administration of α1AT significantly reduced B. pertussis-induced leukocytosis, which is a hallmark of infant infection and major risk factor for fatal pertussis. Taken together our data demonstrates that α1AT is a novel PT inhibitor and that further evaluation and development of α1AT as a therapeutic agent for pertussis is warranted. Importantly, purified α1AT is already in use clinically as an intravenous augmentation therapy for those with genetic α1AT deficiency and could be repurposed to clinical management of pertussis.

Sulfated glycosaminoglycans are host epithelial cell targets of the Candida albicans toxin candidalysin.

Candidalysin, a cytolytic peptide produced by the fungal pathogen Candida albicans, is a key virulence factor. However, its host cell targets remain elusive. Here we performed a genome-wide loss-of-function CRISPR screen in the TR146 human oral epithelial cell line and identified that disruption of genes (XYLT2, B3GALT6 and B3GAT3) in glycosaminoglycan (GAG) biosynthesis conferred resistance to damage induced by candidalysin and live C. albicans. Surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin binds to sulfated GAGs, facilitating its enrichment on the host cell surface. Adding exogenous sulfated GAGs or the analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate also inhibited C. albicans invasion and fungal-induced epithelial cell cytokine production. In mice with vulvovaginal candidiasis, topical dextran sulfate administration reduced intravaginal tissue damage and inflammation. Collectively, sulfated GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage.

Label-Free Quantitative Proteomics Analysis of COVID-19 Vaccines by Nano LC-HRMS.

A nanoliter liquid chromatography-high resolution mass spectrometry-based method was developed for quantitative proteomics analysis of COVID-19 vaccines. It can be used for simultaneous qualitative and quantitative analysis of target proteins and host cell proteins (HCPs) in vaccine samples. This approach can directly provide protein information at the molecular level. Based on this, the proteomes of 15 batches of COVID-19 inactivated vaccine samples from two companies and 12 batches of COVID-19 recombinant protein vaccine samples from one company were successfully analyzed, which provided a significant amount of valuable information. Samples produced in different batches or by different companies can be systematically contrasted in this way, offering powerful supplements for existing quality standards. This strategy paves the way for profiling proteomics in complex samples and provides a novel perspective on the quality evaluation of bio-macromolecular drugs.

SARS-CoV-2 spike fusion peptide trans interaction with phosphatidylserine lipid triggers membrane fusion for viral entry.

The role of lipids in the host cell target membrane for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry is not clear. We do not know whether SARS-CoV-2 spike protein has any specificity in terms of lipid for membrane fusion reaction. Here, using in vitro reconstitution of membrane fusion assay and single-molecule fluorescence resonance energy transfer imaging of SARS-CoV-2 spike trimers on the surface of the virion, we have demonstrated that phosphatidylserine (PS) lipid plays a key role in SARS-CoV-2 spike-mediated membrane fusion reaction for entry. Membrane-externalized PS lipid strongly promotes spike-mediated membrane fusion and COVID-19 infection. Blocking externalized PS lipid with PS-binding protein or in the absence of PS, SARS-CoV-2 spike-mediated fusion is strongly inhibited. Therefore, PS is an important target for restricting viral entry and intervening spike, and PS interaction presents new targets for COVID-19 interventions.

On-Demand Elongation of Peptide Nanofibrils at Cellular Interfaces to Modulate Cell-Cell Interactions.

Natural cells can achieve specific cell-cell interactions by enriching nonspecific binding molecules on demand at intercellular contact faces, a pathway currently beyond synthetic capabilities. We are inspired to construct responsive peptide fibrils on cell surfaces, which elongate upon encountering target cells while maintaining a short length when contacting competing cells, as directed by a strand-displacement reaction arranged on target cell surfaces. With the display of ligands that bind to both target and competing cells, the contact-induced, region-selective fibril elongation selectively promotes host-target cell interactions via the accumulation of nonspecific ligands between matched cells. This approach is effective in guiding natural killer cells, the broad-spectrum effector lymphocytes, to eliminate specific cancer cells. In contrast to conventional methods relying on target cell-specific binding molecules for the desired cellular interactions, this dynamic scaffold-based approach would broaden the scope of cell combinations for manipulation and enhance the adjustability of cell behaviors for future applications.

Structural and Functional Insights into the Delivery Systems of Bacillus and Clostridial Binary Toxins.

Pathogenic Bacillus and clostridial (i.e., Clostridium and Clostridioides) bacteria express a diverse repertoire of effector proteins to promote disease. This includes production of binary toxins, which enter host epithelial cells and seriously damage the intestinal tracts of insects, animals, and humans. In particular, binary toxins form an AB-type complex composed of a catalytic subunit that is toxic (A) and an oligomeric cell-binding and delivery subunit (B), where upon delivery of A into the cytoplasm of the host cell it catalytically ADP-ribosylates actin and rapidly induces host cell death. In this review, binary toxins expressed by Bacillus thuringiensis, Clostridioides difficile, and Clostridium perfringens will be discussed, with particular focus placed upon the structural elucidations of their respective B subunits and how these findings help to deconvolute how toxic enzyme delivery into target host cells is achieved by these deadly bacteria.

Cascade-targeting polymeric particles eliminate intracellular C. neoformans in fungal infection therapy

C. neoformans, a life-threatening invasive fungal pathogen, can hijack the pulmonary macrophages as ‘Trojan horse’, leading to cryptococcal meningitis and recurrence. Combatting these elusive fungi has posed a long-standing challenge. Here, we report an inhaled cascade-targeting drug delivery platform that can sequentially target host cells and intracellular fungi. The delivery system involves encapsulating amphotericin B (AMB) into polymeric particles decorated with AMB, creating a unique surface pattern, denoted as APP@AMB. The surface topology of APP@AMB guides the efficient macrophages internalization and intracellular drugs accumulation. Following endocytosis, the surface-functionalized AMB specifically targets intracellular fungi by binding to ergosterol in the fungal membrane, as demonstrated through co-localization studies using confocal microscopy. Through on-site AMB delivery, APP@AMB displays superior efficacy in eliminating C. neoformans in the lungs and brain compared to free AMB following inhalation in infected mice. Additionally, APP@AMB significantly alleviates the nephrotoxicity associated with free AMB inhalation therapy. Thus, this biocompatible delivery system enabling host cells and intracellular fungi targeting in a cascade manner, provides a new avenue for the therapy of fungal infection.

The antiherpetic and anti-inflammatory activity of the frog-derived peptide Hylin-a1.

The high incidence of virus-related infections and the large diffusion of drug-resistant pathogens stimulate the search and identification of new antiviral agents with a broad spectrum of action. Antivirals can be designed to act on a single target by interfering with a specific step in the viral lifecycle. On the contrary, antiviral peptides (AVPs) are known for acting on a wide range of viruses, with a diversified mechanism of action targeting virus and/or host cell. In the present study, we evaluated the antiviral potential of the peptide Hylin-a1 secreted by the frog Hypsiobas albopunctatus against members of the Herpesviridae family. The inhibitory capacity of the peptide was evaluated in vitro by plaque assays in order to understand the possible mechanism of action. The results were also confirmed by real-time PCR and Western blot evaluating the expression of viral genes. Hylin-a1 acts to block the herpetic infection interfering at the early stages of both herpes simplex virus type 1 (HSV-1) and type 2 infection. Its mechanism is mainly directed on the membrane, probably by damaging the viral envelope. The same effect was also observed against HSV-1 strains resistant to acyclovir. The data presented in this study, such as the increased activity of the peptide when combined to acyclovir, a weak hemolytic profile, an anti-inflammatory effect, and a tolerable half-life in serum, indicates Hylin-a1 as a novel antiherpetic molecule with promising potential in the clinical setting.

Host Cell Death and Modulation of Immune Response against Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a prevalent infectious disease affecting populations worldwide. A classic trait of TB pathology is the formation of granulomas, which wall off the pathogen, via the innate and adaptive immune systems. Some key players involved include tumor necrosis factor-alpha (TNF-α), foamy macrophages, type I interferons (IFNs), and reactive oxygen species, which may also show overlap with cell death pathways. Additionally, host cell death is a primary method for combating and controlling Mtb within the body, a process which is influenced by both host and bacterial factors. These cell death modalities have distinct molecular mechanisms and pathways. Programmed cell death (PCD), encompassing apoptosis and autophagy, typically confers a protective response against Mtb by containing the bacteria within dead macrophages, facilitating their phagocytosis by uninfected or neighboring cells, whereas necrotic cell death benefits the pathogen, leading to the release of bacteria extracellularly. Apoptosis is triggered via intrinsic and extrinsic caspase-dependent pathways as well as caspase-independent pathways. Necrosis is induced via various pathways, including necroptosis, pyroptosis, and ferroptosis. Given the pivotal role of host cell death pathways in host defense against Mtb, therapeutic agents targeting cell death signaling have been investigated for TB treatment. This review provides an overview of the diverse mechanisms underlying Mtb-induced host cell death, examining their implications for host immunity. Furthermore, it discusses the potential of targeting host cell death pathways as therapeutic and preventive strategies against Mtb infection.

A genome-scale screen identifies sulfated glycosaminoglycans as pivotal in epithelial cell damage by Candida albicans.

Candidalysin is a cytolytic peptide produced by the opportunistic fungal pathogen Candida albicans. This peptide is a key virulence factor in mouse models of mucosal and hematogenously disseminated candidiasis. Despite intense interest in the role of candidalysin in C. albicans pathogenicity, its host cell targets have remained elusive. To fill this knowledge gap, we performed a genome-wide loss-of-function CRISPR screen in a human oral epithelial cell line to identify specific host factors required for susceptibility to candidalysin-induced cellular damage. Among the top hits were XYLT2, B3GALT6 and B3GAT3, genes that function in glycosaminoglycan (GAG) biosynthesis. Deletion of these genes led to the absence of GAGs such as heparan sulfate on the epithelial cell surface and increased resistance to damage induced by both candidalysin and live C. albicans. Biophysical analyses including surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin physically binds to sulfated GAGs, facilitating its oligomerization or enrichment on the host cell surface. The addition of exogenous sulfated GAGs or the GAG analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate, but not non-sulfated dextran, also inhibited epithelial cell endocytosis of C. albicans and fungal-induced epithelial cell cytokine and chemokine production. In a murine model of vulvovaginal candidiasis, topical dextran sulfate administration reduced host tissue damage and decreased intravaginal IL-1β and neutrophil levels. Collectively, these data indicate that GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage.

Functional Divergence of the Paralog Salmonella Effector Proteins SopD and SopD2 and Their Contributions to Infection.

Salmonella enterica is a leading cause of bacterial food-borne illness in humans and is responsible for millions of cases annually. A critical strategy for the survival of this pathogen is the translocation of bacterial virulence factors termed effectors into host cells, which primarily function via protein-protein interactions with host proteins. The Salmonella genome encodes several paralogous effectors believed to have arisen from duplication events throughout the course of evolution. These paralogs can share structural similarities and enzymatic activities but have also demonstrated divergence in host cell targets or interaction partners and contributions to the intracellular lifecycle of Salmonella. The paralog effectors SopD and SopD2 share 63% amino acid sequence similarity and extensive structural homology yet have demonstrated divergence in secretion kinetics, intracellular localization, host targets, and roles in infection. SopD and SopD2 target host Rab GTPases, which represent critical regulators of intracellular trafficking that mediate diverse cellular functions. While SopD and SopD2 both manipulate Rab function, these paralogs display differences in Rab specificity, and the effectors have also evolved multiple mechanisms of action for GTPase manipulation. Here, we highlight this intriguing pair of paralog effectors in the context of host-pathogen interactions and discuss how this research has presented valuable insights into effector evolution.

Bacterial Pathogenesis: Assessment of Intracellular Positioning of Pathogen-Containing Vacuoles During Infection.

Intracellular bacterial pathogens implement a diverse array of strategies to target host cells and establish infection. For vacuolar pathogens, the process of pathogen-containing vacuole movement within host cells, termed intracellular trafficking, is central to both pathogen survival and infection progression. Typically a process mediated by secreted virulence factors that manipulate the host cytoskeletal machinery, internalized pathogen-containing vacuoles traffic to the site of replication to establish a unique replicative niche, and if applicable, traffic back toward the host cell periphery for cell-to-cell spread. As such, the intracellular positioning of pathogen-containing vacuoles represents a fundamental measure of infection progression. Here, we describe a fluorescence microscopy-based method to quantitatively assess bacterial intracellular positioning, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This experimental approach can be modified to study infection in diverse host cell types, and with a broad array of pathogens. The system can also be adapted to examine the kinetics of infection, identify secreted virulence factors that mediate host trafficking, investigate host factors that are targeted by the pathogen for trafficking, and assess functional domains within a virulence factor responsible for mediating the phenotype. Collectively, these tools can provide fundamental insight into the pathogenesis of a diverse array of intracellular bacterial pathogens, and new host factors that are hijacked to mediate infection. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture and preparation of host cells Alternate Protocol: Culture and preparation of host cells to assess host factor contribution to bacterial positioning Basic Protocol 2: Infection of epithelial cells with S. Typhimurium Basic Protocol 3: Fluorescence staining for analysis of bacterial positioning Basic Protocol 4: Fluorescence microscopy analysis of bacterial positioning.

Agonists or positive allosteric modulators of α7 nicotinic acetylcholine receptor prevent interaction of SARS-Cov-2 receptor-binding domain with astrocytoma cells

SARS-Cov-2, the virus causing COVID-19, penetrates host target cells via the receptor of angiotensin-converting enzyme 2 (ACE2). Disrupting the virus interaction with ACE2 affords a plausible mechanism for prevention of cell penetration and inhibiting dissemination of the virus. Our studies demonstrate that ACE2 interaction with the receptor binding domain of SARS-Cov-2 spike protein (RBD) can be impaired by modulating the α7 nicotinic acetylcholine receptor (α7 nAChR) contiguous with ACE2. U373 cells of human astrocytoma origin were shown to bind both ACE2-specific antibody and recombinant RBD in Cell-ELISA. ACE2 was found to interact with α7 nAChR in U373 cell lysates studied by Sandwich ELISA. Our studies demonstrate that inhibition of RBD binding to ACE2-expressing U373 cells were defined with α7 nAChR agonists choline and PNU282987, but not a competitive antagonist methyllicaconitine (MLA). Additionally, the type 2 positive allosteric modulator (PAM2) PNU120596 and hydroxyurea (HU) also inhibited the binding. Our studies demonstrate that activation of α7 AChRs has efficacy in inhibiting the SARS-Cov-2 interaction with the ACE2 receptor and in such a way can prevent virus target cell penetration. These studies also help to clarify the consistent efficacy and positive outcomes for utilizing HU in treating COVID-19.

Hypertension Increases Susceptibility to Experimental Malaria in Mice.

Global prevalence of hypertension is on the rise, burdening healthcare, especially in developing countries where infectious diseases, such as malaria, are also rampant. Whether hypertension could predispose or increase susceptibility to malaria, however, has not been extensively explored. Previously, we reported that hypertension is associated with abnormal red blood cell (RBC) physiology and anemia. Since RBC are target host cells for malarial parasite, Plasmodium, we hypothesized that hypertensive patients with abnormal RBC physiology are at greater risk or susceptibility to Plasmodium infection. To test this hypothesis, normotensive (BPN/3J) and hypertensive (BPH/2J) mice were characterized for their RBC physiology and subsequently infected with Plasmodium yoelii (P. yoelii), a murine-specific non-lethal strain. When compared to BPN mice, BPH mice displayed microcytic anemia with RBC highly resistant to osmotic hemolysis. Further, BPH RBC exhibited greater membrane rigidity and an altered lipid composition, as evidenced by higher levels of phospholipids and saturated fatty acid, such as stearate (C18:0), along with lower levels of polyunsaturated fatty acid like arachidonate (C20:4). Moreover, BPH mice had significantly greater circulating Ter119+ CD71+ reticulocytes, or immature RBC, prone to P. yoelii infection. Upon infection with P. yoelii, BPH mice experienced significant body weight loss accompanied by sustained parasitemia, indices of anemia, and substantial increase in systemic pro-inflammatory mediators, compared to BPN mice, indicating that BPH mice were incompetent to clear P. yoelii infection. Collectively, these data demonstrate that aberrant RBC physiology observed in hypertensive BPH mice contributes to an increased susceptibility to P. yoelii infection and malaria-associated pathology.

Effect of Polyphenols on Inflammation Induced by Membrane Vesicles from Staphylococcus aureus.

Staphylococcus aureus, a bacterium found on human skin, produces toxins and various virulence factors that can lead to skin infections such as atopic dermatitis. These toxins and virulence factors are carried in membrane vesicles (MVs), composed of the bacterium's own cell membranes, and are expected to reach host target cells in a concentrated form, inducing inflammation. This study investigated the effects of two polyphenols, (-)-epigallocatechin gallate (EGCG) and nobiletin (NOL), on the expression of S. aureus virulence factors and the inflammation induced by MVs. The study found that EGCG alone decreased the production of Staphylococcal Enterotoxin A (SEA), while both EGCG and NOL reduced biofilm formation and the expression of virulence factor-related genes. When S. aureus was cultured in a broth supplemented with these polyphenols, the resulting MVs showed a reduction in SEA content and several cargo proteins. These MVs also exhibited decreased levels of inflammation-related gene expression in immortalized human keratinocytes. These results suggest that EGCG and NOL are expected to inhibit inflammation in the skin by altering the properties of MVs derived from S. aureus.

Triple-fusion protein (TriFu): A potent, targeted, enzyme-like inhibitor of all three complement activation pathways

The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several invitro/exvivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel invitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.

Targeting host-specific metabolic pathways-opportunities and challenges for anti-infective therapy.

Microorganisms can takeover critical metabolic pathways in host cells to fuel their replication. This interaction provides an opportunity to target host metabolic pathways, in addition to the pathogen-specific ones, in the development of antimicrobials. Host-directed therapy (HDT) is an emerging strategy of anti-infective therapy, which targets host cell metabolism utilized by facultative and obligate intracellular pathogens for entry, replication, egress or persistence of infected host cells. This review provides an overview of the host lipid metabolism and links it to the challenges in the development of HDTs for viral and bacterial infections, where pathogens are using important for the host lipid enzymes, or producing their own analogous of lecithin-cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL) thus interfering with the human host's lipid metabolism.

Efficient Expression in Leishmania tarentolae (LEXSY) of the Receptor-Binding Domain of the SARS-CoV-2 S-Protein and the Acetylcholine-Binding Protein from Lymnaea stagnalis.

Leishmania tarentolae (LEXSY) system is an inexpensive and effective expression approach for various research and medical purposes. The stated advantages of this system are the possibility of obtaining the soluble product in the cytoplasm, a high probability of correct protein folding with a full range of post-translational modifications (including uniform glycosylation), and the possibility of expressing multi-subunit proteins. In this paper, a LEXSY expression system has been employed for obtaining the receptor binding domain (RBD) of the spike-protein of the SARS-CoV-2 virus and the homopentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis. RBD is actively used to obtain antibodies against the virus and in various scientific studies on the molecular mechanisms of the interaction of the virus with host cell targets. AChBP represents an excellent structural model of the ligand-binding extracellular domain of all subtypes of nicotinic acetylcholine receptors (nAChRs). Both products were obtained in a soluble glycosylated form, and their structural and functional characteristics were compared with those previously described.