Porcine Gastric Mucin Research Articles

Scalable Extraction of Airway Mucins from Porcine Trachea.

Mucins are a major component of the innate defense system in the airways and their biological functions are important to consider in pulmonary disease research. However, the available mucus models for basic research relevant to the lung can be difficult to acquire in sufficient quantity to conduct such studies. Here, we present a new strategy to isolate airway mucins from pig trachea at the milligram to gram scale for use in pulmonary disease research. Using this protocol, we were able to isolate mucins with minimal DNA contamination consisting of ∼70% by weight protein. Compared to porcine gastric mucins extracted with the same procedure, the porcine tracheal extract possessed significantly greater O-linked glycoprotein (mucin) content. Particle tracking microrheology was used to evaluate the biophysical properties of porcine trachea mucins. We found porcine tracheal mucins formed a much tighter mesh network and possessed a significantly greater microviscosity compared to lab extracted porcine gastric mucins. In comparison to mucus harvested from human airway tissue cultures, we found porcine tracheal mucins also possessed a greater microviscosity suggesting these mucins can form into a gel-like material at physiological total solids concentrations. These studies establish an accessible means to isolate airway mucins from porcine trachea at large scale for use in pulmonary disease research.

In Vitro Mucoadhesive Features of Gliadin Nanoparticles Containing Thiamine Hydrochloride.

Gliadins have aroused significant interest in the last decade as suitable biomaterials for food and pharmaceutical applications. In particular, the oral route is the preferred method of administration for gliadin-based formulations, due to the affinity of this biomaterial for the gut mucosa. However, up to now, this has been demonstrated only by means of in vivo or ex vivo studies. This is why, in this study, various in vitro techniques were employed in order to evaluate the ability of polymeric nanoparticles, made up of a commercial grade of the protein and an etheric surfactant, to interact with porcine gastric mucin. The nanosystems were also used for the encapsulation of thiamine hydrochloride, used as a model of a micronutrient. The resulting systems were characterized by a mean diameter of ~160-170 nm, a narrow size distribution when 0.2-0.6 mg/mL of thiamine was used, and an encapsulation efficiency between 30 and 45% of the drug initially employed. The incubation of the gliadin nanosystems with various concentrations of porcine gastric mucin evidenced the ability of the carriers to interact with the mucus glycoprotein, showing a decreased Zeta potential after a 4 h incubation (from ~-30 to -40 mV), while demonstrating that the encapsulation of the drug did not affect its bioadhesive features. Altogether, these data support the conceivable application of gliadin nanoparticles as formulations for the oral administration of bioactive compounds.

Transglutaminase–mucin binding dynamics in gastrointestinal mucus: Interfacial behaviour, thermodynamics and gelation mechanism

Transglutaminase, an enzyme present in epithelial cells and as a residual component in processed foods, is hypothesised to interact with gastrointestinal mucus, impacting its structure and function. To test the physical validity of this phenomenon, this study investigates the binding kinetics and thermodynamics between porcine gastric mucin (PGM) and microbial transglumatinse (TGM) in a model gastrointestinal mucus, followed by the rheological analysis of the resulting systems. At pH 7, TGM exhibited pronounced binding with the PGM interface, characterised by a high surface density (55.34 ± 1.86 µg/m−2) and a low dissociation constant (KD ∼ 4.03 ± 0.10 μM) as determined by surface plasma resonance (SPR). Conversely, at pH 3, TGM showed weak adhesion onto PGM, resulting in a less stable binding, reflected by a lower surface density (10.94 ± 0.67 µg/m−2) and a higher dissociation constant (KD ∼ 6.24 ± 0.22 μM). Regardless of the nature of interaction, the Hill coefficients (nH ≥ 1) indicated that the binding sites were markedly denser at pH 7 (1383.38 ± 46.47 pmol/m−2), than at pH 3 (273.68 ± 16.84 pmol/m−2). Fluorimetry analysis suggested temperature-dependent dynamic binding between PGM and TGM. The resulting Benesi – Hildebrand plots illustrated a linear correlation between PGM and increasing TGM concentration, suggesting a single-step interaction mechanism. The calculated thermodynamic parameters indicated spontaneous interaction between PGM and TGM (ΔG < 0) via endothermic (entropic) interactions (ΔH > 0). Notably, hydrophobic forces played a significant role in the network stabilisation of PGM−TGM complex (ΔS > 0). Rheometry analysis elucidates that the interaction maxima within TGM−PGM systems substantially elevate both shear viscosity (η), and the melting point (Tm), shifting from 6.75 ± 0.28 to 70.94 ± 2.21 (×10−3) Pa s (at 1 s−1), and 36.80 ± 0.80 to 48.20 ± 0.11 °C, respectively. Furthermore, the coexistence of these two macromolecular species results in a 3- to 4-fold dramatic increase in the viscoelastic moduli of the binary complex. These findings build a strong physicochemical basis for the interaction between TGM and PGM, with profound effects on the rhological behaviour of the latter; it also highlights the need to examine the biochemical/enzymatic aspects of said interactions, and their potential effects on mucosa and human physiology.

A head-to-head comparison of polymer interaction with mucin from porcine stomach and bovine submaxillary glands

Native mucus is heterogeneous, displays high inter-individual variation and is prone to changes during harvesting and storage. To overcome the lack of reproducibility and availability of native mucus, commercially available purified mucins, porcine gastric mucin (PGM) and mucin from bovine submaxillary gland (BSM), have been widely used. However, the question is to which extent the choice of mucin matters in studies of their interaction with polymers as their composition, structure and hence physicochemical properties differ. Accordingly, the interactions between PGM or BSM with two widely used polymers in drug delivery, polyethylene oxide and chitosan, was studied with orthogonal methods: turbidity, dynamic light scattering, and quartz crystal microbalance with dissipation monitoring. Polymer binding and adsorption to the two commercially available and purified mucins, PGM and BSM, is different depending on the mucin type. PEO, known to interact weakly with mucin, only displayed limited interaction with both mucins as confirmed by all employed methods. In contrast, chitosan was able to bind to both PGM and BSM. Interestingly, the results suggest that chitosan interacts with BSM to a greater extent than with PGM indicating that the choice of mucin, PGM or BSM, can affect the outcome of studies of mucin interactions with polymers.

Iron content of commercial mucin contributes to compositional stability of a cystic fibrosis airway synthetic microbiota community.

In vitro culture models of mucosal environments are used to elucidate the mechanistic roles of the microbiota in human health. These models often include commercial mucins to reflect the in-situ role of mucins as an attachment site and nutrient source for the microbiota. Two types of mucins are commercially available: porcine gastric mucin (PGM) and bovine submaxillary mucin (BSM). These commercial mucins have been shown to contain iron, an essential element required by the microbiota as a co-factor for a variety of metabolic functions. In these mucin preparations, the concentration of available iron can exceed physiological concentrations present in the native environment. This unexpected source of iron influences experimental outcomes, including shaping the interactions between co-existing microbes in synthetic microbial communities used to elucidate the multispecies interactions within native microbiota. In this work, we leveraged the well-characterized iron-dependent production of secondary metabolites by the opportunistic pathogen Pseudomonas aeruginosa to aid in the development of a simple, low-cost, reproducible workflow to remove iron from commercial mucins. Using the mucosal environment of the cystic fibrosis (CF) airway as a model system, we show that P. aeruginosa is canonically responsive to iron concentration in the chemically defined synthetic CF medium complemented with semi-purified PGM, and community composition of a clinically relevant, synthetic CF airway microbial community is modulated, in part, by iron concentration in PGM.

Structural and tribological studies on the interaction of porcine gastric mucin with non- and cationic-modified β-lactoglobulins

β-lactoglobulin (BLG) is the major whey protein with negative charges at neutral pH in aqueous media. Thus, the interaction with mucins, the major polyanionic component of mucus, is very weak due to the electrostatic repulsion between them. The present study postulates that cationization of BLG molecules may reverse the interaction characteristics between BLG and mucin from repulsive to associative. To this end, cationic-modified BLGs were prepared by grafting positively charged ethylenediamine (EDA) moieties into the negatively charged carboxyl groups on the aspartic and glutamic acid residues and compared with non-modified BLG upon mixing with porcine gastric mucin (PGM). To characterize the structural and conformational features of PGM, non/cationized BLGs, and their mixtures, various spectroscopic approaches, including zeta potential, dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy were employed. Importantly, we have taken surface adsorption with optical waveguide lightmode spectroscopy (OWLS), and tribological properties with pin-on-disk tribometry at the sliding interface as the key approaches to determine the interaction nature between them as mixing PGM with polycations can lead to synergistic lubrication at the nonpolar substrate in neutral aqueous media as a result of an electrostatic association. All the spectroscopic studies and a substantial improvement in lubricity collectively supported a tenacious and associative interaction between PGM and cationized BLGs, but not between PGM and non-modified BLG. This study demonstrates a unique and successful approach to intensify the interaction between BLG and mucins, which is meaningful for a broad range of disciplines, including food science, macromolecular interactions, and biolubrication etc.

Probiotic capacity of commensal lactic acid bacteria from the intestine of Guinea pigs (Cavia porcellus)

The intricate balance of intestinal microbiota is significantly influenced by the pivotal role of indigenous lactic acid bacteria (LAB). These LAB not only contribute to antimicrobial activity and enhance animal health and productivity but also serve as defense against intestinal infections. In the present study, the probiotic potential of LAB strains isolated from various intestinal sections of adult and young guinea pigs (Cavia porcellus) was comprehensively assessed. Strains belonging to the genera Ligilactobacillus, Weissella, Enterococcus, and Limosilactobacillus were also identified. The antibacterial activities of the LAB strains against Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus were quantified. Exopolysaccharide production, adherence capacity, antibiotic resistance, and bile salt tolerance (0.15 %, 0.30 %, and 0.45 %) of LAB were quantified. Further analyses focused on the effects of pH (2.9, 5.0, 6.4, and 7.4), temperature (40, 50, and 60 °C) and NaCl concentrations (3.5 % and 6.5 % w/v) on LAB growth. Strains GCI9 and GDE10 (Ligilactobacillus salivarius), isolated from the cecum and intestine of guinea pigs, exhibited significant antimicrobial activity against S. typhimurium, E. coli and S. aureus. Remarkable adherence capacity to porcine gastric mucin was demonstrated by L. salivarius strains, specifically ACI1, GCI9, and GDE10, with the highest exopolysaccharide levels produced by ACI1 and GCI9 (1.71 and 1.76 mg/mL, respectively). The probiotic potential was further underscored by remarkable bile salt tolerance, especially in strain GDE10, and substantial exopolysaccharide production. These strains displayed notable adaptability to varying environmental conditions, including NaCl concentrations at 3.5 % and 6.5 %, temperatures ranging from 40 to 60 °C, and pH levels of 2.9, 5.0, 6.4, and 7.4. This comprehensive assessment of the probiotic properties of L. salivarius strains, particularly ACI1, GCI9, and GDE10, shows promise for the development of probiotic formulations aimed at enhancing the intestinal health of guinea pigs.

Labeling of Mucin-Type O-Glycans for Quantification Using Liquid Chromatography and Fluorescence Detection.

O-glycosylation is a common post-translational modification that is essential for the defensive properties of mucus barriers. Incomplete and altered O-glycosylation is often linked to severe diseases, such as cancer, cystic fibrosis, and chronic obstructive pulmonary disease. Originating from a nontemplate-driven biosynthesis, mucin-type O-glycan structures are very complex. They are often present as heterogeneous mixtures containing multiple isomers. Therefore, the analysis of complex O-glycan mixtures usually requires hyphenation of orthogonal techniques such as liquid chromatography (LC), ion mobility spectrometry, and mass spectrometry (MS). However, MS-based techniques are mainly qualitative. Moreover, LC separation of O-glycans often lacks reproducibility and requires sophisticated data treatment and analysis. Here we present a mucin-type O-glycomics analysis workflow that utilizes hydrophilic interaction liquid chromatography for separation and fluorescence labeling for detection and quantification. In combination with mass spectrometry, a detailed analysis on the relative abundance of specific mucin-type O-glycan compositions and features, such as fucose, sialic acids, and sulfates, is performed. Furthermore, the average number of monosaccharide units of O-glycans in different samples was determined. To demonstrate universal applicability, the method was tested on mucins from different tissue types and mammals, such as bovine submaxillary mucins, porcine gastric mucins, and human milk mucins. To account for day-to-day retention time shifts in O-glycan separations and increase the comparability between different instruments and laboratories, we included fluorescently labeled dextran ladders in our workflow. In addition, we set up a library of glucose unit values for all identified O-glycans, which can be used to simplify the identification process of glycans in future analyses.

VARIOUS ADHERENT-INVASIVE E. COLI (AIEC) AND NON-AIEC STRAINS ISOLATED FROM CROHN’S DISEASE PATIENTS GROW DIFFERENTLY WITH RUMINOCOCCUS GNAVUS PRE-DIGESTED MUCUS AND R. GNAVUS-RELEASED PRE-DIGESTED ILEAL AND COLONIC MUCUS MONOSACCHARIDES

Abstract BACKGROUND Mucus-digesting Ruminococcus gnavus (R. gnavus), adherent-invasive E. coli (AIEC), and hydrogen sulfide (H2S) are enriched in active Crohn’s disease (CD) patients compared to healthy subjects. Dual colonization of R. gnavus and E. coli LF82 in ex-germ-free mice enhanced distal colitis and H2S production in cecal contents. R. gnavus pre-digestion of mucus enhanced the ileal CD isolate AIEC LF82 growth and H2S production in vitro. H2S enhanced LF82 adhesion and invasion both in vitro and in vivo along with related LF82 adhesion gene expression and Caco-2 cell expression of CEACAM6, a receptor for AIEC ligand FimH. Interactions between other CD-derived AIEC/non-AIEC and R. gnavus are unknown. HYPOTHESIS Different AIEC and non-AIEC may grow and produce H2S differently using mucus digested by R. gnavus, which releases mucus-derived monosaccharides and sulfur substrates. METHODS We studied CD-derived clinical isolates of AIEC 541-15 (phylotype A), 541-1 (B1), LF82 and NRG857c (B2) and non-AIEC T75 (A) and lab strain K12 (A). M9 minimal media was supplemented with porcine gastric mucin (PGM), ileal (MIM) and colonic (MCoM) mucus isolated from germ-free mice. We measured growth kinetics and H2S production by these AIEC and non-AIEC following culture with sterile supernatants of each mucin/mucus media precultured with or without R. gnavus, monosaccharides obtained by R. gnavus digestion of mucus, and several sulfur substrates in the gut added to M9 minimal media. RESULTS AIEC, especially LF82 (B2), grew better with ileal mucus aerobically and, to a lesser extent, anaerobically than non-AIEC strains, with even greater AIEC growth with R. gnavus pre-digested mucus (Fig.1). AIEC produced more H2S with R. gnavus-precultured mucus than with mucus alone. R. gnavus digestion of PGM released mucus monosaccharides (Fig.2A). AIEC strains grew differently with each monosaccharide constituent of R. gnavus pre-digested PGM. AIEC grew better than non-AIEC with each monosaccharide in aerobic conditions, but AIEC (B2) grew better than others in all monosaccharides in anaerobic conditions (Fig.2B). Amongst mucus-relevant sulfur substrates, L-cysteine was the only substrate to enhance E. coli H2S production by all AIEC and non-AIEC strains. All mucus-relevant sulfur substrates supported R. gnavus survival for 24 hrs, while H2S sustained R. gnavus for 48 hrs in minimum media. CONCLUSIONS AIEC grew better and produced more H2S with R. gnavus pre-digested mucus suggesting that co-existence of AIEC with R. gnavus mutually support each other and may explain why they are often observed together in active CD patients. In addition, enhanced AIEC growth with ileal vs. colonic mucus may explain why AIEC are frequently isolated from ileal CD patients. AIEC phylotype growth differences with mucus substrates may explain their persistence in the ileal mucosa. Figure 1 Figure 2

On the mucoadhesive properties of synthetic and natural polyampholytes

HypothesisThe mucoadhesive characteristics of amphoteric polymers (also known as polyampholytes) can vary and are influenced by factors such as the solution's pH and its relative position against their isoelectric point (pHIEP). Whilst the literature contains numerous reports on mucoadhesive properties of either cationic or anionic polymers, very little is known about these characteristics for polyampholytes ExperimentsHere, two amphoteric polymers were synthesized by reaction of linear polyethylene imine (l-PEI) with succinic or phthalic anhydride and their mucoadhesive properties were compared to bovine serum albumin (BSA), selected as a natural polyampholyte. Interactions between these polymers and porcine gastric mucin were studied using turbidimetric titration and isothermal titration calorimetry across a wide range of pHs. Model tablets were designed, coated with these polymers and tested to evaluate their adhesion to porcine gastric mucosa at different pHs. Moreover, a retention study using fluorescein isothiocyanate (FITC)-labelled polyampholytes deposited onto mucosal surfaces was also conducted FindingsAll these studies indicated the importance of solution pH and its relative position against pHIEP in the mucoadhesive properties of polyampholytes. Both synthetic and natural polyampholytes exhibited strong interactions with mucin and good mucoadhesive properties at pH < pHIEP.

Measurement of Mucinase Activity.

Mucinase consists of some proteases, glycosidases, sulfatases, and sialidases. It is not practical to measure individual enzyme activities when measuring mucinase activity. In this method, mucinase activity is measured using porcine gastric mucin as a substrate and feces as an enzyme source. This description includes fecal pellet preparation, reaction procedure of mucinase, measurement of reducing sugars liberated during the procedure, and determination of nitrogen content in the fecal preparations.

Adhesion Inhibition Assay for Helicobacter pylori to Mucin by Lactobacillus.

The ability of Lactobacillus to adhere to mucin is a parameter for evaluating the effectiveness of probiotics. In particular, a competitive inhibition assay of pathogenic bacteria using mucin-adherent lactobacilli is useful for identifying Lactobacillus strains capable of preventing mucus from being colonized by pathogens. Here, we describe an adhesion inhibition assay method for Helicobacter pylori to porcine gastric mucin by Limosilactobacillus reuteri.

Examining the efficiency of porcine gastric mucin-coated magnetic beads in extraction of noroviruses from frozen berries

Human norovirus is the leading cause of foodborne gastroenteritis worldwide. Due to the low infectious dose of noroviruses, sensitive methodologies are required to detect and characterize small numbers of viral particles that are found in contaminated foods. The ISO 15216 method, which is internationally recognized for detection of foodborne viruses from high-risk food commodities, is based on viral precipitation, followed by RNA extraction and identification of the viral genome by RT-PCR. Although the ISO 15216 method is efficient, it is time consuming and tedious, does not report on the viral infectivity, and is sensitive to the presence of RT-PCR inhibitors. Norovirus capture by the porcine gastric mucin conjugated magnetic beads (PGM-MB) was developed as an alternative virus recovery method. It relies on the integrity of the viral capsid being able to bind to PGM. PGM contains a variety of histo-blood group antigens (HBGAs) that act as norovirus receptors. Therefore, the PGM-MB method allows for extraction of noroviruses, with potentially intact viral capsids, from complex food matrices. The viral genome can then be released through heat-shock of the captured virus. For this reason, we performed a parallel comparison between the ISO 15216 method and the PGM-MB method in isolation and quantification of noroviruses from frozen raspberries. We have demonstrated that the efficiency of the PGM-MB method in extraction of murine norovirus (MNV) and human norovirus GII.4 from raspberries is equal or better than the ISO 15216 method, while the PGM-MB has fewer steps and shorter turnaround time. Moreover, the PGM-MB method is more efficient in removing the inhibitors prior to RT-PCR analysis.

Development of recombinant oyster heat shock protein 70 mediated in situ capture RT-qPCR to detect human norovirus and Tulane virus

Human noroviruses (HuNoVs) are the major foodborne pathogens that cause non-bacterial gastroenteritis globally. Conventional RT-qPCR is prone to counting free RNA in the sample, resulting in an inflated infectious virus titer. Porcine gastric mucin (PGM), broadly used as the capture unit of in situ capture RT-qPCR (ISC-RT-qPCR), can precisely capture intact norovirus without adsorbing free RNA. However, PGM is a complex mixture that is difficult to obtain, and the concentration of histo-blood group antigens in it is unclear, hampering its widespread use. This study introduced a newly discovered capture unit, recombinant oyster heat shock protein 70 (roHSP70), to replace PGM. The roHSP70-mediated ISC-RT-qPCR (roHSP-ISC-RT-qPCR) were developed and compared with the PGM-mediated ISC-RT-qPCR (PGM-ISC-RT-qPCR) and conventional column-based RNA extraction RT-qPCR (CBE-RT-qPCR). The results showed that roHSP70 can capture various genotypes of HuNoVs. The smaller concentration of roHSP70 (20.0 μg/mL) captured HuNoVs to a similar extent compared to PGM (1.0 mg/mL). In addition, roHSP-ISC-RT-qPCR can accurately detect the amount of authentic intact virus particles in heat-treated Tulane virus (TV) samples with an accuracy comparable to that of TCID50. Interestingly, roHSP-ISC-RT-qPCR gave lower titers than CBE-RT-qPCR when measuring cell-cultured TV with high titers. However, when measuring lower-titer TV in artificially contaminated oysters, roHSP-ISC-RT-qPCR instead gave significantly higher titers than CBE-RT-qPCR. Overall, roHSP-ISC-RT-qPCR had a similar excellent performance as the PGM-ISC-RT-qPCR in detecting intact norovirus in samples. Moreover, roHSP70 has broader application scenarios than PGM due to its single-identified component and easier accessibility.

Integrated analysis of natural glycans using a versatile pyrazolone-type heterobifunctional tag ANPMP

Glycans mediate various biological processes through carbohydrate-protein interactions, and glycan microarrays have become indispensable tools for understanding these mechanisms. However, advances in functional glycomics are hindered by the absence of convenient and universal methods for obtaining natural glycan libraries with diverse structures from glycoconjugates. To address this challenge, we have developed an integrative approach that enables one-pot release and simultaneously capture, separation, structural characterization, and functional analysis of N/O-glycans. Using this approach, glycoconjugates are incubated with a pyrazolone-type heterobifunctional tag-ANPMP to obtain glycan-2ANPMP conjugates, which are then converted to glycan-AEPMP conjugates. We prepared a tagged glycan library from porcine gastric mucin, soy protein, human milk oligosaccharides, etc. Following derivatization by N-acetylation and permethylation, glycans were subjected to detailed structural characterization by ESI-MSn analysis, which revealed >83 highly pure glycan-AEPMPs containing various natural glycan epitopes. A shotgun microarray is constructed to study the fine details of glycan-bindings by proteins and antisera.

Unraveling the Intertwined Effect of pH on Helicobacter pylori Motility and the Microrheology of the Mucin-Based Medium It Swims in.

The gastric pathogen, Helicobacter pylori bacteria have to swim across a pH gradient from 2 to 7 in the mucus layer to colonize the gastric epithelium. Previous studies from our group have shown that porcine gastric mucin (PGM) gels at an acidic pH < 4, and H. pylori bacteria are unable to swim in the gel, although their flagella rotate. Changing pH impacts both the rheological properties of gastric mucin and also influences the proton (H+)-pumped flagellar motors of H. pylori as well as their anti-pH sensing receptors. To unravel these intertwined effects of acidic pH on both the viscoelastic properties of the mucin-based mucus as well as the flagellar motors and chemo-receptors of the bacterium, we compared the motility of H. pylori in PGM with that in Brucella broth (BB10) at different pH values using phase contrast microscopy to track the motion of the bacteria. The results show that the distribution of swimming speeds and other characteristics of the bacteria trajectories exhibit pH-dependent differences in both media. The swimming speed exhibits a peak at pH 4 in BB10, and a less pronounced peak at a higher pH of 5 in PGM. At all pH values, the bacteria swam faster and had a longer net displacement in BB10 compared to PGM. While the bacteria were stuck in PGM gels at pH < 4, they swam at these acidic pH values in BB10, although with reduced speed. Decreasing pH leads to a decreased fraction of motile bacteria, with a decreased contribution of the faster swimmers to the distributions of speeds and net displacement of trajectories. The body rotation rate is weakly dependent on pH in BB10, whereas in PGM bacteria that are immobilized in the low pH gel are capable of mechano-sensing and rotate faster. Bacteria can be stuck in the gel in various ways, including the flagella getting entangled in the fibers of the gel or the cell body being stuck to the gel. Our results show that in BB10, swimming is optimized at pH4, reflecting the combined effects of pH sensing by anti-pH tactic receptors and impact on H+ pumping of flagellar motors, while the increase in viscosity of PGM with decreasing pH and gelation below pH 4 lead to further reduction in swimming speed, with optimal swimming at pH 5 and immobilization of bacteria below pH 4.

Flagellin is essential for initial attachment to mucosal surfaces by Clostridioides difficile.

Mucins are glycoproteins which can be found in host cell membranes and as a gelatinous surface formed from secreted mucins. Mucosal surfaces in mammals form a barrier to invasive microbes, particularly bacteria, but are a point of attachment for others. Clostridioides difficile is an anaerobic bacterium, which colonizes the mammalian gastrointestinal (GI) tract and is a common cause of acute GI inflammation leading to a variety of negative outcomes. Although C. difficile toxicity stems from secreted toxins, colonization is a prerequisite for C. difficile disease. While C. difficile is known to associate with the mucous layer and underlying epithelium, the mechanisms underlying these interactions that facilitate colonization are less well understood. To understand the molecular mechanisms by which C. difficile interacts with mucins, we used ex vivo mucosal surfaces to test the ability of C. difficile to bind to mucins from different mammalian tissues. We found significant differences in C. difficile adhesion based upon the source of mucins, with highest levels of binding observed to mucins purified from the human colonic adenocarcinoma line LS174T and lowest levels of binding to porcine gastric mucin. We also observed defects in adhesion by mutants deficient in flagella but not type IV pili. These results imply that interactions between host mucins and C. difficile flagella facilitate the initial host attachment of C. difficile to host cells and secreted mucus. Importance Clostridioides difficile is one of the leading causes of hospital-acquired infections worldwide and presents challenges in treatment due to recurrent gastrointestinal disease after treatment with antimicrobials. The mechanisms by which C. difficile colonizes the gut represent a key gap in knowledge, including its association with host cells and mucosa. Our results show the importance of flagellin for specific adhesion to mucosal hydrogels and can help to explain prior observations of adhesive defects in flagellin and pilin mutants.

Shear-reversible clusters of HIV-1 in solution: stabilized by antibodies, dispersed by mucin.

The phenomenon of reversible clustering is expected to further nuance HIV immune stealth because virus surfaces can escape interaction with antibodies (Abs) by hiding temporarily within clusters. It is well known that mucin reduces HIV virulence, and the current perspective is that mucin aggregates HIV-1 to reduce infections. Our findings, however, suggest that mucin is dispersing HIV clusters. The study proposes a new paradigm for how HIV-1 may broadly evade Ab recognition with reversible clustering and why mucin effectively neutralizes HIV-1.

Exploring magnetic capture to improve the detection of human norovirus in strawberries

AbstractHuman norovirus is a leading cause of foodborne illness worldwide, and its detection in foods remains a challenge due to the difficulty of eluting and enriching trace amounts of viral particles. In this study, we developed porcine gastric mucin–labeled magnetic capture probes and optimized the conditions for their binding with norovirus, elution of viral particles, and RNA isolation based on the previous research. We evaluated the magnetic capture‐based pretreatment method in artificially contaminated strawberry samples and compared it with the conventional polyethylene glycol precipitation–based method using reverse transcriptional‐quantitative PCR (RT‐qPCR). The results revealed that Tris·HCl (pH 9.5) was the optimal buffer for the binding of magnetic probes with viral particles, and RNA recovery rate obtained by heating‐based release was significantly higher than that obtained by a commercial kit, with only active virus being captured. This buffer was similar to the RT‐qPCR buffer, which facilitated the sensitivity of RT‐qPCR. Furthermore, the buffer was an effective solution for eluting viral particles from foods in previous research and was optimized to ensure maximum elution and no inhibitory effect on viral capture. We explored multiple factors or steps to ensure that the sensitivity of our method was three orders of magnitude higher than that of the conventional method. Our findings suggest that magnetic capture‐based detection has great potential for the sensitive detection of human norovirus.

Host-microbe interaction and pathogen exclusion mediated by an aggregation-prone surface layer protein of Lactobacillus helveticus

Probiotic surface layer proteins (Slps) have multiple functions and bacterial adhesion to host cells is one of them. The precise role of Slps in cellular adhesion is not well understood due to its low native protein yield and self-aggregative nature. Here, we report the recombinant expression and purification of biologically active Slp of Lactobacillus helveticus NCDC 288 (SlpH) in high yield. SlpH is a highly basic protein (pI = 9.4), having a molecular weight of 45 kDa. Circular Dichroism showed a prevalence of beta-strands in SlpH structure and resistance to low pH. SlpH showed binding to human intestinal tissue, enteric Caco-2 cell line, and porcine gastric mucin, but not with fibronectin, collagen type IV and laminin. SlpH inhibited the binding of the enterotoxigenic E. coli by 70 % and 76 % and that of Salmonella Typhimurium SL1344 by 71 % and 75 % to enteric Caco-2 cell line in the exclusion and competition assays, respectively. The pathogen exclusion and competition activity and tolerance to harsh gastrointestinal conditions show the potential for developing SlpH as a prophylactic or therapeutic agent against enteric pathogens.