microorganisms Article Streptococcus Thermophilus UASt-09 Upregulates Goblet Cell Activity in Colonic Epithelial Cells to a Greater Degree than other Probiotic Strains Madhur D. Shastri 1,2,*, Wai Chin Chong 3, Ravichandra Vemuri 1,4 , Christopher J. Martoni 5, Santosh Adhikari 1, Harinder Bhullar 1, Dale Kunde 1, Stephen G. Tristram 1 and Rajaraman D. Eri 1,* 1 School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston 7250, Australia; [email protected] (R.V.); [email protected] (S.A.); [email protected] (H.B.); [email protected] (D.K.); [email protected] (S.G.T.) 2 School of Pharmacy and Pharmacology, University of Tasmania, Hobart 7005, Australia 3 Department of Molecular and Translational Science, Monash University, Clayton 3800, Australia; [email protected] 4 Department of Pathology, Section of Comparative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA 5 UAS Laboratories, Madison, Windsor, WI 53598, USA; [email protected] * Correspondence: [email protected] (M.D.S.); [email protected] (R.D.E.); Tel.: +61-363-245-467 (M.D.S.); +04-30-029-656 (R.D.E.); Fax: +61-363-245-555 (R.D.E.) Received: 29 September 2020; Accepted: 6 November 2020; Published: 9 November 2020 Abstract: Probiotics have been widely used in maintaining gastrointestinal health, despite their actual mechanism remaining obscure. There are several hypotheses behind the beneficial effects of probiotics including the regulation of intestinal barrier function and improvement in immune responses in the gastrointestinal system. Multiple probiotics have been introduced in the market as effective dietary supplements in improving gastrointestinal integrity, but there are no or few studies that demonstrate their underlying mechanism. In the current study, we investigated and compared the efficacy of four probiotics (based on different bacterial species) in refining gastrointestinal health by improving mucus biosynthesis and intestinal immune response under in-vitro conditions. By analyzing the gene expression of mucus biosynthesis and intestinal immune response markers, we found that probiotic Streptococcus thermophilus UASt-09 showed promising potential in refining mucosal barrier and gastrointestinal health in human colonic epithelial cells, as compared to other commercial probiotics. Keywords: probiotics; Mucin-2; goblet cells; intestinal peptides; immunomodulation 1. Introduction Microbiota are found in many parts of the human body, of which the gastrointestinal tract contains the largest and most diverse collection [1]. It plays a pivotal role in digestion, nutrient absorption and removal of waste and microbial products. In addition to the microbial population, the gastrointestinal tract contains various cells carrying out individual roles [1]. These include enterocytes that absorb nutrients, Paneth cells secreting antimicrobial proteins, enteroendocrine cells involved in the production of multiple gastrointestinal hormones and microfold cells that sample pathogen antigens for intestinal immune system, while goblet cells produce mucin glycoprotein to form the outermost mucus layer on intestinal epithelial cells [2]. The gastrointestinal tract contains over 1000 microbial species identified so far and is constantly challenged by a myriad of commensal and/or pathogenic microbes daily [2]. As a protective measure, goblet cells produce a layer of mucin glycoprotein that act as a first line Microorganisms 2020, 8, 1758; doi:10.3390/microorganisms8111758 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 1758 2 of 15 defense against harmful pathogens reaching the intestinal epithelial cells [3]. The mucus consists of 2 distinct layers: the outer loose layer (~100 um thick) that can be easily removed by gently suctioning, and the inner adherent layer (~50 um thick) that can be removed only by scraping [4]. The outer layer is comprised of attachment sites for microbes and nutrients that favor the colonization and growth of intestinal commensal flora [5]. Whereas, the inner mucus layer acts as a filter to separate the bacteria on the outer layer from invading into the intestinal epithelial cells [5,6]. Mucin glycoproteins are highly glycosylated and rich in serine and threonine [7]. So far, there are more than 20 different types of mucin glycoproteins that have been identified and named from mucin (MUC)-1 to MUC20 according to the order of their discovery [8]. Among them, MUC2 is the major secretory mucin glycoprotein produced by the goblet cells with a molecular mass of 540 kDa and consisting of 5179 amino acids [8]. It has a highly glycosylated central tandem repeat domain rich in proline, serine and threonine, allowing the monomer to get linked by other proteins having a cysteine-rich domain, such as von Willebrand factor [9]. With such characteristics, MUC2 mucin can easily undergo dimerization or oligomerization with other proteins, hence creating a gel-like semi-permeable network [10]. However, this mucosal barrier integrity is affected by the quantity and quality of the mucin glycoprotein produced by goblet cells and highly influenced by internal factors including bile salts, microbial by-products, hormones, and cytokines [11]. Besides MUC2 mucin glycoproteins, intestinal goblet cells also produce a myriad of intestinal peptides including Trefoil Factor 3 (TFF3), Resistin-like Molecule β (RELMβ), Fc-Gamma Binding Protein (FCGBP), and Anterior Gradient Homolog 2 (AGR2), and their specific functions are summarized in Table1. Table 1. Goblet cell-secreted components and their specific roles in colon physiology. Goblet Cell Secreted Components Functions Reference Major component involved in the formation Mucin 2 (MUC2) of mucus layer that acts as the first [4] host-defense barrier Stabilization and crosslinking of the MUC2 Fc-Gamma Binding Protein (FCGBP) [6] mucin barrier to the inner mucin layer Associated in mucosal protection and Trefoil Factor 3 (TFF3) [12] healing process Involved in mucosal immune defense [13] against microbial infection. Resistin-like Molecule β (RELMβ) Promote spatial segregation of the [14] microbiota and the colonic epithelium Anterior Gradient Homolog 2 (AGR2) Essential in mucin biosynthesis [15] One of the common disorders involving the malfunction of the mucus layer is inflammatory bowel disease (IBD) [16]. IBD is an idiopathic inflammatory disorder, where the intestinal tract undergoes chronic or relapsing inflammation, leading to either ulcerative colitis or Crohn’s disease. While the actual mechanism behind IBD remains obscure, increasing studies have highlighted the involvement of gut microbiota in the pathophysiology of IBD [17–22]. For instance, Nishino and colleagues reported elevated levels of Proteobacteria like Escherichia, Ruminococcus, and Cetobacterium and reduction in Firmicutes and Bacteroidetes, such as genera Faecalibacterium and Roseburia, in endoscopic samples of patients diagnosed with IBD compared to a non-IBD control population [17]. Moreover, recent studies revealed that IBD patients have relatively lower spatial microbiota heterogeneity compared to healthy individuals [19,22]. Therefore, studies recommend the use of probiotics as a counter-measure for the restoration of gut microbiota/homeostasis in order to alleviate the risk of IBD [23–25]. It has been proposed that probiotics can restore the intestinal tract homeostasis either by disrupting pathogen signaling via the inhibition of quorum sensing [26], production of antimicrobial compounds that can inhibit pathogens [27], improving mucosal integrity against pathogens [28], or by competing for and reducing the adherence site for pathogens [29]. In the human gastrointestinal tract, Lactobacillus, Bifidobacterium, and Streptococcus subsp. have been widely trialed as probiotics in relieving diarrhea, Microorganisms 2020, 8, 1758 3 of 15 irritable bowel syndrome, and IBD [30–32]. While there are many studies that reported the beneficial effects of probiotics and their contribution to longevity and digestive health, there are few studies that directly evaluate the efficacy of different probiotic strains in MUC2 mucin biosynthesis. The current study tested multiple probiotic strains (obtained from diverse origins) and aimed to evaluate their efficacy in promoting gut mucosal health by assessing MUC2 mucin synthesis, increment in antimicrobial peptides such as TFF3, RELMβ, and other essential goblet cell-associated factors like AGR2 and FCGBP. Our study demonstrated that a Streptococcus thermophilus UASt-09 probiotic strain was superior to other tested probiotics in orchestrating a goblet cell activation program and a specific immune signature. 2. Materials and Methods 2.1. Bacterial Strains and Origins Bacterial strains utilized in the study are summarized in Table2. All other probiotic strains were obtained from UAS Laboratories, Windsor, WI, USA. The strains were cultured on De Man Rogosa agar supplemented with 0.05% (w/v) of L-cysteine (MRS-C), except UASt-09, which was cultured on M17 agar supplemented with 10% (w/v) lactose. All strains were incubated at 37 ◦C for 48 h under anaerobic conditions. Table 2. Probiotic strains and species used in the study. Genus Species Strain Origin Bifidobacterium animalis subsp. lactis UABla-12 Human Lactobacillus acidophilus DDS-1 Human Lactobacillus plantarum UALp-05 Plant Streptococcus thermophilus UASt-09 Dairy 2.2. Cell Culture and Reagents Human colonic epithelial cells LS174T were purchased from the American