2021 Volume 44 Issue 12 Pages 1886-1890
A lactic acid bacterial strain, Lactobacillus plantarum SN35N, which has been isolated from the pear, secretes negatively charged acidic exopolysaccharide (EPS) to outside cells. We have previously found that the SN35N-derived acidic EPS inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35) promoting inflammation. The aim of this study is to find other health benefits of EPS. EPS has been found to exhibit an inhibitory effect against the influenza virus (Alphainfluenzavirus Influenza A virus) and feline calicivirus (Vesivirus Feline calicivirus), which is recognized as a model of norovirus. Although more studies on the structure–function relationship of EPSs are needed, SN35N-derived EPS is a promising lead for developing not only anti-inflammatory agents, but also antiviral substances.
When administered in adequate amounts, living microorganisms that confer health benefits to the host are called “probiotics.”1) Lactic acid bacteria (LABs), as typical probiotics, have been used to produce fermented dairy products and other fermented foods. “LAB” is a generic name of non-pathogenic Gram-positive bacteria that generate one or two moles of lactic acid from one mole of sugar by fermentation.1,2) Some LAB strains have been reported to have potent health benefits with regard to immune modulation and lifestyle-related diseases.3–10)
LAB strains are roughly classified into two groups based on their isolation sources. One group consists of strains isolated from dairy products and intestines, and the other includes isolates from plant sources, such as vegetables, fruits, flowers, and medicinal herbs. The former and the latter are called animal- and plant-derived LABs, respectively.11–13) Because plant-derived LABs are generally more resistant to harsh environments than animal-derived LABs, we were interested in characteristics of plant-derived LABs.
We have already isolated and identified more than 1000 strains of LABs from plant sources and found that some of them are useful in preventive medicine, such as immune modulation, anti-obesity, and anti-inflammation medications.14–17) We have previously isolated some exopolysaccharide (EPS)-producing plant-derived LAB strains. These EPSs have been reported to inhibit the enzymatic activity of hyaluronidase (EC 3.2.1.36), which promotes inflammatory reactions.18–20) The hyaluronidase-inhibitory activity has been reported to correlate with histamine-release inhibition in inflammatory reactions through immunoglobulin E (IgE)-mediated mast cell degranulation.21–23) Therefore, EPSs that display hyaluronidase-inhibitory activity may have preventive and restorative effects against inflammatory diseases. In fact, EPS produced by the IJH-SONE68 strain has been demonstrated to prevent and improve picryl chloride-induced contact dermatitis in model mice.24)
The aim of this study is to investigate EPS to find other health-promoting functions. During the experiment, we found that negatively charged acidic EPS produced by the pear-derived Lactobacillus (Lb.) plantarum SN35N inhibits infection of influenza virus (Alphainfluenzavirus Influenza virus A (IFV A)) and feline calicivirus (Vesivirus Feline calicivirus (FCV)), as culturable surrogates for norovirus (Norovirus Norwalk virus), to the corresponding cultured cells. IFV A, which is the predominant virus in the seasonal flu,25) often causes pandemic outbreaks. Norovirus infection causes sudden severe vomiting and diarrhea if the outbreak occurs in public facilities.26) Because norovirus cannot be cultured in the laboratory, FCV is generally used as an alternative virus due to their relatedness and similarity in size.
Lb. plantarum SN35N and Lb. amylovorus PY45, which our research group isolated previously,18,20) were used as EPS-producing LAB strains.
De Man, Rogosa, and Sharpe (MRS) medium (Merck KGaA, Darmstadt, Germany) was used for the seed cultivation of LAB strains. A modified semi-defined medium (SDM), which consists of SDM27,28) supplemented with 0.2% (v/v) vitamin mixture and 0.1% (v/v) trace element solution instead of yeast nitrogen bases, was used to produce EPS.
Purification of EPS from a Culture Broth of LABsThe EPS from LAB strains was purified according to the previous report.20) Briefly, each seed culture was inoculated at 0.5% (v/v) into a fresh, modified SDM and incubated at 28 or 45 °C for 2 d to grow SN35N or PY45 strains, respectively. The cultured broth was boiled for 30 min, followed by adding a 100% (w/v) trichloroacetic acid (TCA) solution to the culture broth at a final concentration of 4% (v/v). The cell debris was precipitated by centrifugation, and the culture supernatant was collected. The acetone-precipitated crude EPS obtained by centrifugation was dried up and dissolved into a 50 mM Tris–HCl buffer (pH 8.0). After treatment with nuclease and protease, the EPS was precipitated by adding 3 volumes of ethanol and collected by centrifugation. The precipitant was dried up and dissolved into an appropriate volume of sterile water. Each EPS solution was dialyzed against sterile distilled water by using Amicon Ultra (MWCO = 10 kDa, Merck Millipore Ltd., Carrigtwohill, County Cork, Ireland) to obtain a purified EPS mixture.
The neutral and acidic EPSs were further separated by column chromatography using the anion exchange resin (TOYOPEARL DEAE-650M, Tosoh Bioscience, Tokyo, Japan) as described previously.29) After dialysis against sterile distilled water using Amicon Ultra (MWCO = 10 kDa), each EPS sample was lyophilized and stored at 4 °C until use. The SN35N-derived acidic EPS has been recently demonstrated to be composed of glucose, galactose, and mannose at a molecular ratio of 15.0 : 5.7 : 1.0, and its molecular weight was estimated approximately 250 kDa.20)
Antiviral AssayAn antiviral assay was performed as described previously.30) MDCK (+) cells (canine kidney-derived cells, described in the previous literature)31) and CRFK cells (feline kidney-derived cells) were infected with IFV A/Udorn/72 (H3N2) and FCV F9 strains, respectively.
For the assay, a 0.1 volume of virus and a 0.9 volume of 1% (w/v) solution of the purified EPS were mixed and incubated for 3 min at room temperature. The reaction was then serially diluted tenfold with Dulbecco’s modified Eagle medium (DMEM) and inoculated to cells in a 96-well culture plate. The cells were incubated in DMEM until cytopathic effects developed, and the 50% endpoint of virus infection was determined via the Behrens–Karber method to calculate the 50% tissue culture infectious dose (TCID50). Sterile water was used instead of the EPS solution for a negative control. The fucoidan derived from Laminaria japonica (Carbosynth Ltd., Compton, Berkshire, U.K.) was used as a reference polysaccharide. Twenty micrograms per milliltter of trypsin was included in DMEM for the assay of IFV.
Hemagglutination Inhibition AssayThe following procedure was used to perform the hemagglutination of sheep red blood cells (RBCs) caused by the influenza virus. Sheep RBCs were prepared by the centrifugation of sheep preserved blood (Japan Lamb Ltd., Hiroshima, Japan) at 600 × g for 15 min at 4 °C. The RBCs were resuspended in phosphate-buffered saline (PBS, pH 7.2) and centrifuged again. After washing three times, the RBCs were resuspended in PBS to a final concentration of 0.5% (v/v).
A hemagglutination inhibition assay was done on a U-bottom microtiter plate. First, a 50 µL mixture of 0.1 µg/mL inactivated IFV A (H3N2) antigen (HyTest Ltd., Turku, Finland) and the desired concentrations of EPS was added to each well. Then, a 50 µL portion of the 0.5% (v/v) RBC suspension was added to each well. A hemagglutination-positive control well was prepared without EPS. After 90 min of incubation at room temperature (r.t.), the concentration at which the RBCs appeared cloudy at the bottom of the well was defined as hemagglutination positive, whereas non-agglutinated RBCs made a small red button in the well.
Neuraminidase-Inhibition AssayThe inhibitory activities against the neuraminidase of EPS samples were evaluated using an EnzyChrom Neuraminidase Assay Kit (BioAssay Systems, Hayward, CA, U.S.A.) with purified neuraminidase enzymes (from Arthrobacter ureafaciens, Nacalai Tesque, Inc., Kyoto, Japan). The assay was conducted by a colorimetric procedure in accordance with the manufacturer’s protocol.
The SN35N-derived EPS was preliminarily incubated with IFV or FCV for 3 min at r.t. before exposure to infected cells. This EPS strain significantly inactivated the infectivity of IFV to less than the detection limit by more than a 5-log reduction (Fig. 1). It also significantly inactivated FCV by more than a 3-log reduction. On the other hand, the EPS derived from Lb. amylovorus PY4518) did not suppress IFV or FCV infection (Fig. 1). The EPS solutions did not show any cytotoxicity at the concentrations used in the test, demonstrating that the SN35N EPS can fully inactivate IFV and FCV, a surrogate of Norwalk virus from the Norovirus genus. On the other hand, no inhibitory effects were observed against either virus on fucoidan.
An SN35N- or PY45-derived EPS was incubated with IFV or FCV for 3 min at room temperature, and the remaining infectivity was measured using the TCID50 method. The average infectivities of three or four independent experiments are shown in the graphs, and error bars indicate standard deviations. The detection limit of the assay is indicated by dotted lines. * p < 0.05 by the Mann–Whitney U-test, in comparison with a control sample.
The influenza virus has a membrane fusion glycoprotein, called hemagglutinin, which causes a virus to bind to target cells through interaction with sialic acid. Hemagglutination assay was used to evaluate this virus binding to facilitating host cells to determine whether LAB-derived EPS inhibits the virus–target cell interaction. In the presence of 50 ng/mL INF A antigen, the sheep RBCs were significantly agglutinated (Fig. 2). A control EPS, which does not have antiviral activity but inhibits IFV A antigen-mediated hemagglutination, showed inhibition activity in a dose-dependent manner. However, SN35N EPS could not inhibit hemagglutination.
EPS concentrations decreased from left to right wells. After incubation for 90 min at r.t., the concentration where the RBCs appeared cloudy at the bottom of the well was defined as hemagglutination positive, whereas non-agglutinated RBCs made a small red button in the well. In the presence of IFV A antigens, the RBCs were certainly agglutinated (the left two wells of the second panel). A control EPS showed the inhibition activity in a dose-dependent manner, but SN35N EPS did not.
The influenza virus’s membrane fusion enzyme, neuraminidase, also known as sialidase, plays several important roles in infection: 1) promoting the release of virus from infected cells, 2) helping the virus itself to spread to uninfected cells nearby, and 3) preventing the aggregation of the virus. We confirmed whether the SN35N EPS affected the activity of the neuraminidase; however, no inhibition was observed (data not shown).
The relationship between bioactivity and the chemical structure of EPS is not elucidated at this time. Therefore, more information with regard to the relationship of EPSs from other LABs is needed to clarify the problem. It has been reported that the antimicrobial properties of the LAB strain, including antiviral effects, result from its immune-modulating property.32) However, EPS produced by animal-derived Lb. delbrueckii ssp. bulgaricus OLL1073R-1 has been reported to exhibit anti-IFV activity by augmenting the activity of natural killer cells.33,34) Unlike in the case of the OLL1073R-1 strain, the antiviral effect of the SN35N-derived EPS was observed in vitro, suggesting that EPS may bind to the virus surface directly without hemagglutinin and neuraminidase, which are important for IFV infection, and may inhibit the binding of the virus to host cells. Therefore, it might be predicted that EPS interacts non-specifically with unknown targets of the virus, with the result that adhesion of the virus particle to the target cell may be inhibited.
In contrast to the hyaluronidase inhibition, the antiviral activity of EPSs seems not to be widely preserved in EPSs. In fact, the PY45-derived EPS has no effect on virus infection (Fig. 1). We also analyzed the antiviral activity of EPSs obtained from other LABs and sulfated polysaccharide fucoidan, but no remarkable effects were observed (data not shown). Spirulan, which is a specific sulfated acidic polysaccharide composed of some saccharides, sulfate, and calcium, is extracted from a blue–green alga, Spirulina platensis. Hayashi et al. showed that spirulan inhibits the replication of several enveloped viruses by interfering with the interaction between viruses and host cells; however, chelation of the calcium ion with sulfate groups results in the loss of antiviral activity.35) Generally, virus infection is initiated by a virus–host cell binding mediated by a virus surface glycoprotein, such as hemagglutinin proteins, and glycan receptors on the surface of the host cell. The viral glycan receptors contain sialic acid and thus have a negative charge, whereas hemagglutinin proteins tend to charge positively. This virus–host cell binding might play an important role in not only viral attachment but also its entry into host cells.36–38) Therefore, the negatively charged acidic polysaccharides seem to be predicted to interact with the positively charged receptors, resulting in the inhibition of virus attachment and invasion of host cells. In fact, a sulfated acidic EPS, heparan sulfate, has also been found to bind with the virus surface glycoprotein and to inhibit the interaction between virus and cognate receptor competitively.39) However, no antiviral activities were observed on fucoidan in this study, in spite of its sulfate residue. Therefore, the antiviral activity found on SN35N-derived EPS is expected to be due to its unique structural characteristics rather than only its acidic properties. Our previous report showed that the SN35N strain has four EPS-biosynthesizing gene clusters on its chromosomal DNA, and another one is located on the pSN35N-3 plasmid.20) Interestingly, although the EPS productivity of the plasmid pSN35N-3-cured mutant (SN35N-Δp3) was drastically decreased (from 48.2 to 3.3 mg/L), our preparatory experiments indicated that the EPS obtained from SN35N-Δp3 is more effective against IFV infection (data not shown). This result suggests that the EPS clusters on chromosomal DNA are more important in the anti-viral activity but not in the production of EPS.
This study reports inhibitory effects observed in the Lb. plantarum SN35N-derived EPS on virus infection of host cells. Although more studies on the structural characterization and structure–function relationship of EPSs are needed, EPSs show promise for developing not only anti-inflammatory agents but also antiviral substances.
We are grateful to Marie Mizuguchi and Nasrin Sultana for their technical assistance in the Sugiyama laboratory. We also thank the Analysis Center of Life Science, Hiroshima University, for the use of their facilities.
The authors declare no conflict of interest.
