A mechanism of self-lipid endocytosis mediated by the receptor Mincle.

Cellular lipid uptake (through endocytosis) is a basic physiological process. Dysregulation of this process underlies the pathogenesis of diseases such as atherosclerosis, obesity, diabetes, and cancer. However, to date, only some mechanisms of lipid endocytosis have been discovered. Here, we show a previously unknown mechanism of lipid cargo uptake into cells mediated by the receptor Mincle. We found that the receptor Mincle, previously shown to be a pattern recognition receptor of the innate immune system, tightly binds a range of self-lipids. Moreover, we revealed the minimal molecular motif in lipids that is sufficient for Mincle recognition. Superresolution microscopy showed that Mincle forms vesicles in cytoplasm and colocalizes with added fluorescent lipids in endothelial cells but does not colocalize with either clathrin or caveolin-1, and the added lipids were predominantly incorporated in vesicles that expressed Mincle. Using a model of ganglioside GM3 uptake in brain vessel endothelial cells, we show that the knockout of Mincle led to a dramatic decrease in lipid endocytosis. Taken together, our results have revealed a fundamental lipid endocytosis pathway, which we call Mincle-mediated endocytosis (MiME), and indicate a prospective target for the treatment of disorders of lipid metabolism, which are rapidly increasing in prevalence.

Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19.

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.

Receptor Mincle promotes skin allergies and is capable of recognizing cholesterol sulfate.

Sterile (noninfected) inflammation underlies the pathogenesis of many widespread diseases, such as allergies and autoimmune diseases. The evolutionarily conserved innate immune system is considered to play a key role in tissue injury recognition and the subsequent development of sterile inflammation; however, the underlying molecular mechanisms are not yet completely understood. Here, we show that cholesterol sulfate, a molecule present in relatively high concentrations in the epithelial layer of barrier tissues, is selectively recognized by Mincle (Clec4e), a C-type lectin receptor of the innate immune system that is strongly up-regulated in response to skin damage. Mincle activation by cholesterol sulfate causes the secretion of a range of proinflammatory mediators, and s.c. injection of cholesterol sulfate results in a Mincle-mediated induction of a severe local inflammatory response. In addition, our study reveals a role of Mincle as a driving component in the pathogenesis of allergic skin inflammation. In a well-established model of allergic contact dermatitis, the absence of Mincle leads to a significant suppression of the magnitude of the skin inflammatory response as assessed by changes in ear thickness, myeloid cell infiltration, and cytokine and chemokine secretion. Taken together, our results provide a deeper understanding of the fundamental mechanisms underlying sterile inflammation.

Diagnostic Value of IgA Antibody Measurement in Tick-Borne Spotted Fever (Astrakhan Rickettsial Fever).

Tick-borne spotted fevers caused by Rickettsia occur worldwide. The symptoms of this bacterial infection are similar to those of viral infection, and thus, diagnostic accuracy has special clinical importance. One of the commonly used methods for the diagnosis of tick-borne spotted fever is enzyme-linked immunosorbent assay (ELISA), which is based on estimation of the presence of specific IgM antibodies in blood. However, IgA analysis has not been used for the diagnosis of rickettsial diseases thus far. We investigated the diagnostic value of IgA antibody determination using patient sera collected in the Astrakhan region of Russia, where an isolated site of Astrakhan rickettsial fever (ARF) caused by Rickettsia conorii subsp. caspia is located. Our investigation was performed on serum samples collected from 185 patients diagnosed with Astrakhan rickettsial fever from May to October 2019. Western blot analysis revealed that specific IgA antibodies, as well as IgM antibodies, from patient sera bind to high-molecular-weight pathogen proteins with similar masses. The obtained data show that the determination of IgM alone allows for serological confirmation of diagnosis in only 46.5% of cases but that the determination of both IgM and IgA increases this rate to 66.5%. Taken together, the findings show an important diagnostic value of IgA evaluation for tick-borne spotted fever rickettsiosis. IMPORTANCE Tick-borne spotted fevers caused by Rickettsia occur worldwide. The symptoms of this bacterial infection are similar to the symptoms of viral infection, and thus, diagnostic accuracy has special clinical importance. The most serious spotted fever group rickettsiosis is Rocky Mountain fever in the United States, which is caused by Rickettsia rickettsii, and disease complications can lead to hemiparesis, blindness, or amputation. Rickettsia conorii subsp. caspia causes a rickettsial spotted fever named Astrakhan rickettsial fever (ARF). One of the commonly used methods for the diagnosis of tick-borne spotted fevers is ELISA, which is based on estimation of the presence of specific IgM antibodies in blood, though IgA has not been used for the diagnosis of rickettsial diseases thus far. In this study, we showed that both IgA and IgM should be analyzed in the blood serum samples of patients to significantly enhance the accuracy of diagnostics of tick-borne spotted fever rickettsiosis.

Integrated into Environmental Biofilm Chromobacterium vaccinii Survives Winter with Support of Bacterial Community.

Chromobacterium species are common in tropical and subtropical zones in environmental samples according to numerous studies. Here, we describe an environmental case of resident Chromobacterium vaccinii in biofilms associated with Carex spp. roots in Moscow region, Russia (warm-summer humid continental climate zone). We performed broad characterization of individual properties as well as surrounding context for better understanding of the premise of C. vaccinii survival during the winter season. Genome properties of isolated strains propose some insights into adaptation to habit and biofilm mode of life, including social cheaters carrying ΔluxR mutation. Isolated C. vaccinii differs from previously described strains in some biochemical properties and some basic characteristics like fatty acid composition as well as unique genome features. Despite potential to modulate membrane fluidity and presence of several genes responsible for cold shock response, isolated C. vaccinii did not survive during exposure to 4 °C, while in the complex biofilm sample, it was safely preserved for at least half a year in vitro at 4 °C. The surrounding bacterial community within the same biofilm with C. vaccinii represented a series of psychrophilic bacterial species, which may share resistance to low temperatures with other species within biofilm and provide C. vaccinii an opportunity to survive during the cold winter season.