Decreased rotavirus infection of MA104 cells via probiotic extract binding to Hsc70 and β3 integrin receptors

Univ. Sci. 23 (2): 219-239, 2018. doi: 10.11144/Javeriana.SC23-2.drio Bogotá original article Decreased rotavirus infection of MA104 cells via probiotic extract binding to Hsc70 and ß3 integrin receptors Sandra Patricia Salas-Cárdenas1, Nury Nathalia Olaya-Galán1, Karem Fernández1, Fernando Velez2, Carlos Arturo Guerrero3, Maria Fernanda Gutiérrez1, * Edited by Juan Carlos Salcedo-Reyes () 1. Laboratorio de Virología, Grupo de Enfermedades Infecciosas, Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá – Colombia 2. Departamento de Matemáticas, Facultad de Ciencias, Universidad El Bosque, Bogotá – Colombia 3. Laboratorio de Biología Molecular del Virus, Facultad de Medicina, Universidad Nacional de Colombia, Bogotá – Colombia. * Received: 30-07-2017 Accepted: 20-05-2018 Published on line: 31-07-2018 Citation: Salas-Cárdenas SP, Olaya-Galán NN, Fernández K, Velez F, Guerrero CA, Gutiérrez MF. Decreased rotavirus infection of MA104 cells via probiotic extract binding to Hsc70 and β3 integrin receptors, Universitas Scientiarum, 23 (2): 219-239, 2018. doi: 10.11144/Javeriana.SC23-2.drio Abstract Probiotic bacteria are microorganisms beneficial to human health, useful to improving biological conditions. Thanks to probiotic bacteria the symptoms of viral infections can be alleviated. Different mechanisms whereby probiotic bacteria exert they antiviral effect have been proposed. The aim of this study was to determine whether probiotic bacteria extracts bind to receptors of host cells susceptible of rotavirus (RV) infection. To accomplish this objective, four probiotic bacterial strains of Lactobacillus spp. and Bifidobacterium spp. were tested. Probiotic extracts were obtained after bacterial growth, cell lysis and centrifugation. Obtained probiotic extracts were used in assays to interfere with adhesion and penetration of a RV strain in the mammal cell line MA104. Furthermore, the interaction between probiotic extracts and MA104 cell receptors was evaluated by co-immunoprecipitation assays using anti-β3-integrins and anti-Hsc70 antibodies. All four probiotic, protein-rich, extracts reduced RV infections in MA104 cells, suggesting a successful antiviral activity mediated by these probiotic extracts. All probiotic extracts significantly exerted their antiviral activity by interfering with RV adhesion on MA104 cell receptors, with proteins in probiotic extracts competitively interacting with cell surface receptors necessary to RV infection. Co-immunoprecipitation assay results showed that proteins in probiotic extracts were able to bind to β3-integrinsand Hsc70, which are two cellular receptors required to viral infection. The most significant contribution of this study is an insight into the mechanisms of probiotic antiviral activity, thus expanding current probiotics fundamental knowledge. Keywords: Probiotics; Antiviral effect; Rotavirus; Hsc70; integrin β3 Funding: COLCIENCIAS through grant 629 2009. Electronic supplementary material: N.A. Introduction Probiotics are live microbial organisms that, when administered in adequate amounts, are beneficial to their host’s health. Probiotic bacteria are known to inhabit several places of the host organism and are part of the intestinal Universitas Scientiarum, Journal of the Faculty of Sciences, Pontificia Universidad Javeriana, is licensed under the Creative Commons Attribution 4.0 International Public License 220 Decreased rotavirus infection by probiotics microbiota in humans. Probiotics have the capacity to prevent and improve health conditions, such as infections caused by several pathogens, including viruses [1-4]. Several mechanisms whereby probiotic bacteria are beneficial to their hosts have been proposed. First, probiotic bacteria can prevent the attachment of some pathogens via colonization of the intestinal epithelial surface. Secondly, it has been observed that probiotics have the capacity of binding to cellular receptors on the surface of mature enterocytes, thereby interfering with pathogen penetration processes into these cells. Thirdly, probiotics can also elicit the activation of the intracellular mechanisms of transcription, translation, and stimulate the host’s immune response, thus strengthening its defenses against various pathogens, including viruses [3, 4]. Rotaviruses (RVs) are considered as one of the most important viral pathogens leading to diarrhea in children worldwide. Although vaccines have proven effective in decreasing the strength of RV-induced diarrhea [5, 6], researchers continue seeking for alternative ways to prevent this disease [3, 7-9]. The mechanism of cell infection of several RV strains has been hypothesized to be mediated by sialic acid residues on the cell membrane, acting as receptors. However, RV infection also requires co-receptors, such as integrins (i.e., α2β1, αvβ3, αxβ2, and α4β1) and thermal shock proteins (i.e., hsc70) [10-16]. Four probiotic bacterial strains that decrease RV infection have been identified via in vitro assays [17, 18]. This finding raises the question whether the observed positive effect of these probiotic bacteria is achieved by blocking specific host cell receptors necessary to viral adhesion and penetration. The aim of the present study was to determine if probiotic bacteria extracts could decrease RV infection by binding to host cell receptors necessary for RV entry. Materials and methods Probiotic bacteria, cell line, and virus strain Probiotic bacteria of the species Lactobacillus casei, Lactobacillus fermentum, Bifidobacterium adolescentis, and Bifidobacterium bifidum, previously studied by Fernandez et al. [17] and Olaya et al. [18], were used for the experimental approaches in the present study. Bacteria were grown on MRS broth under anaerobic conditions until exponential growth was obtained (between 8-10 hours depending on the strain).Cultures at a cellular concentration of 108 CFU/mL were used for the development of the experiments as reported elsewhere [19]. Rhesus monkey kidney-derived MA104 cells were used to replicate the virus and to evaluate bacterial extracts. MA104 cells were grown in Dulbecco’s Universitas Scientiarum Vol. 23 (2): 219-239 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 221 Salas-Cárdenas et al. modified Eagle’s medium (DMEM) supplemented with 4 % fetal bovine serum (FBS), 2 mM L-glutamine, and an antibiotic/antimicotic cocktail (comprising 10 000 µg/mL penicillin, 10 000 µg/mL streptomycin, and 25 µg/mL Fungizone at a 100X concentration). Cells were harvested using phosphate buffer solution (PBS) with trypsin (0.25 %) at 37 ◦ C until they detached from the culture flask. The assays were performed in 24-well plates, with a cell confluence of 90 %. Antiviral assays were performed with the Rhesus rotavirus (RRV) strain. Prior to MA104 infection, viral particles were activated with 10 µg/mL of trypsin to cleavage capsid proteins. All viral in (...truncated)