RESUMEN
S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography-pulse amperometric detector performed after ß-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow-derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow-derived dendritic cells from C-type lectin receptor-deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria.
Asunto(s)
Antígenos CD/metabolismo , Lactobacillus/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Cromatografía Líquida de Alta Presión , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Glicopéptidos/análisis , Glicopéptidos/química , Glicosilación , Inmunización , Interferón gamma/metabolismo , Lectinas Tipo C/deficiencia , Lectinas Tipo C/genética , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/química , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Polisacáridos/análisis , Polisacáridos/química , Células RAW 264.7 , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The development of new subunit vaccines has promoted the rational design of adjuvants able to induce a strong T-cell activation by targeting specific immune receptors. The S-layer is a (glyco)-proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea. Due to their ability to self-assemble in solution, they are attractive tools to be used as antigen/hapten carriers or adjuvants. Recently, we have demonstrated that S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 (SLP-8348) enhanced the LPS-induced response on macrophages in a Ca2+-dependent manner, but the receptors involved in these immunomodulatory properties remain unknown. Therefore, we aim to determine the C-type lectin receptors (CLRs) recognizing this bacterial surface glycoprotein as well as to investigate the role of glycans in both the immunogenicity and adjuvant capacity of SLP-8348. Here, using a mild periodate oxidation protocol, we showed that loss of SLP-8348 glycan integrity impairs the cell-mediated immune response against the protein. Moreover, our data indicate that the adjuvant capacity of SLP-8348 is also dependent of the biological activity of the SLP-8348 glycans. In order to evaluate the CLRs involved in the interaction with SLP-8348 an ELISA-based method using CLR-hFc fusion proteins showed that SLP-8348 interacts with different CLRs such as Mincle, SingR3, and hDC-SIGN. Using BMDCs derived from CLR-deficient mice, we show that SLP-8348 uptake is dependent of Mincle. Furthermore, we demonstrate that the SLP-8348-induced activation of BMDCs as well as its adjuvant capacity relies on the presence of Mincle and its signaling adaptor CARD9 on BMDCs, since SLP-8348-activated BMDCs from Mincle-/- or CARD9-/- mice were not capable to enhance OVA-specific response in CD4+ T cells purified from OT-II mice. These findings significantly contribute to the understanding of the role of glycans in the immunomodulation elicited by bacterial SLPs and generate a great opportunity in the search for new adjuvants derived from non-pathogenic microorganisms.
Asunto(s)
Factores Inmunológicos/inmunología , Lactobacillus/inmunología , Lectinas Tipo C/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas de la Membrana/inmunología , Animales , Humanos , Factores Inmunológicos/genética , Lactobacillus/genética , Lectinas Tipo C/genética , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Células RAW 264.7RESUMEN
Alphaviruses and flaviviruses are important human pathogens that include Chikungunya virus (CHIKV), Dengue virus (DENV), and Zika virus (ZIKV), which can cause diseases in humans ranging from arthralgia to hemorrhagic fevers and microcephaly. It was previously shown that treatment with surface layer (S-layer) protein, present on the bacterial cell-envelope of Lactobacillus acidophilus, is able to inhibit viral and bacterial infections by blocking the pathogen's interaction with DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), a trans-membrane protein that is a C-type calcium-dependent lectin. DC-SIGN is known to act as an attachment factor for several viruses including alphaviruses and flaviviruses. In the present study, we used alphaviruses as a model system to dissect the mechanism of S-layer inhibition. We first evaluated the protective effect of S-layer using 3T3 cells, either wild type or stably expressing DC-SIGN, and infecting with the alphaviruses Semliki Forest virus (SFV) and CHIKV and the flaviviruses ZIKV and DENV. DC-SIGN expression significantly enhanced infection by all four viruses. Treatment of the cells with S-layer prior to infection decreased infectivity of all viruses only in cells expressing DC-SIGN. In vitro ELISA experiments showed a direct interaction between S-layer and DC-SIGN; however, confocal microscopy and flow cytometry demonstrated that S-layer binding to the cells was independent of DC-SIGN expression. S-layer protein prevented SFV binding and internalization in DC-SIGN-expressing cells but had no effect on virus binding to DC-SIGN-negative cells. Inhibition of virus binding occurred in a time-dependent manner, with a significant reduction of infection requiring at least a 30-min pre-incubation of S-layer with DC-SIGN-expressing cells. These results suggest that S-layer has a different mechanism of action compared to mannan, a common DC-SIGN-binding compound that has an immediate effect in blocking viral infection. This difference could reflect slower kinetics of S-layer binding to the DC-SIGN present at the plasma membrane (PM). Alternatively, the S-layer/DC-SIGN interaction may trigger the activation of signaling pathways that are required for the inhibition of viral infection. Together our results add important information relevant to the potential use of L. acidophilus S-layer protein as an antiviral therapy.