Functional characterization of the mucus barrier on the Xenopus tropicalis skin surface

Mucosal surfaces represent critical routes for entry and exit of pathogens. As such, animals have evolved strategies to combat infection at these sites, in particular the production of mucus to prevent attachment and to promote subsequent movement of the mucus/microbe away from the underlying epithelial surface. Using biochemical, biophysical, and infection studies, we have investigated the host protective properties of the skin mucus barrier of the Xenopus tropicalis tadpole. Specifically, we have characterized the major structural component of the barrier and shown that it is a mucin glycoprotein (Otogelin-like or Otogl) with similar sequence, domain organization, and structural properties to human gel-forming mucins. This mucin forms the structural basis of a surface barrier (∼6 μm thick), which is depleted through knockdown of Otogl. Crucially, Otogl knockdown leads to susceptibility to infection by the opportunistic pathogen Aeromonas hydrophila. To more accurately reflect its structure, tissue localization, and function, we have renamed Otogl as Xenopus Skin Mucin, or MucXS. Our findings characterize an accessible and tractable model system to define mucus barrier function and host–microbe interactions.

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Complete Sequence and Annotation of the Mycoplasma phocicerebrale Strain 1049T Genome

Microbiology Resource Announcements ◽

10.1128/mra.00514-19 ◽

2019 ◽

Vol 8 (25) ◽

Author(s):

Salvatore Frasca ◽

Jessy Castellanos Gell ◽

Gerald F. Kutish ◽

Dina L. Michaels ◽

Daniel R. Brown

Keyword(s):

Genomic Dna ◽

Opportunistic Pathogen ◽

Complete Sequence ◽

Content Type ◽

Library Construction ◽

Susceptibility To Infection

The Mycoplasma phocicerebrale genome was analyzed to better understand this opportunistic pathogen. Amplification with ϕ29 polymerase was used to generate enough genomic DNA for large-insert library construction. Like other mycoplasmas from seals, M. phocicerebrale encodes an immunosuppressor that may predispose susceptibility to infection or influence intercurrent diseases of affected hosts.

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The glycoconjugate-degrading enzymes of Clostridium perfringens: Tailored catalysts for breaching the intestinal mucus barrier

Glycobiology ◽

10.1093/glycob/cwaa050 ◽

2020 ◽

Cited By ~ 3

Author(s):

Kristin E Low ◽

Steven P Smith ◽

D Wade Abbott ◽

Alisdair B Boraston

Keyword(s):

Clostridium Perfringens ◽

Bacterial Species ◽

Opportunistic Pathogen ◽

Glycoside Hydrolases ◽

Necrotic Enteritis ◽

Carbohydrate Active Enzymes ◽

Intestinal Mucus ◽

Mucosal Layer ◽

Potential Component ◽

Mucus Barrier

Abstract The gastrointestinal (GI) tract of humans and animals is lined with mucus that serves as a barrier between the gut microbiota and the epithelial layer of the intestine. As the proteins present in mucus are typically heavily glycosylated, such as the mucins, several enteric commensal and pathogenic bacterial species are well-adapted to this rich carbon source and their genomes are replete with carbohydrate-active enzymes targeted toward dismantling the glycans and proteins present in mucus. One such species is Clostridium perfringens, a Gram-positive opportunistic pathogen indigenous to the gut of humans and animals. The genome of C. perfringens encodes numerous carbohydrate-active enzymes that are predicted or known to target glycosidic linkages within or on the termini of mucus glycans. Through this enzymatic activity, the degradation of the mucosal layer by C. perfringens has been implicated in a number of GI diseases, the most severe of which is necrotic enteritis. In this review, we describe the wide array of extracellular glycoside hydrolases, and their accessory modules, that is possessed by C. perfringens, and examine the unique multimodularity of these proteins in the context of degrading the glycoconjugates in mucus as a potential component of disease.

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Enzymatically active biomimetic micropropellers for the penetration of mucin gels

Science Advances ◽

10.1126/sciadv.1500501 ◽

2015 ◽

Vol 1 (11) ◽

pp. e1500501 ◽

Cited By ~ 113

Author(s):

Debora Walker ◽

Benjamin T. Käsdorf ◽

Hyeon-Ho Jeong ◽

Oliver Lieleg ◽

Peer Fischer

Keyword(s):

Physical State ◽

Defense Mechanism ◽

Drug Carriers ◽

The Body ◽

Artificial System ◽

Epithelial Surface ◽

Efficient Delivery ◽

Biomimetic Approach ◽

Acidic Conditions ◽

Mucus Barrier

In the body, mucus provides an important defense mechanism by limiting the penetration of pathogens. It is therefore also a major obstacle for the efficient delivery of particle-based drug carriers. The acidic stomach lining in particular is difficult to overcome because mucin glycoproteins form viscoelastic gels under acidic conditions. The bacteriumHelicobacter pylorihas developed a strategy to overcome the mucus barrier by producing the enzyme urease, which locally raises the pH and consequently liquefies the mucus. This allows the bacteria to swim through mucus and to reach the epithelial surface. We present an artificial system of reactive magnetic micropropellers that mimic this strategy to move through gastric mucin gels by making use of surface-immobilized urease. The results demonstrate the validity of this biomimetic approach to penetrate biological gels, and show that externally propelled microstructures can actively and reversibly manipulate the physical state of their surroundings, suggesting that such particles could potentially penetrate native mucus.

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Molecular Design, Functional Characterization and Structural Basis of a Protein Inhibitor Against the HIV-1 Pathogenicity Factor Nef

PLoS ONE ◽

10.1371/journal.pone.0020033 ◽

2011 ◽

Vol 6 (5) ◽

pp. e20033 ◽

Cited By ~ 24

Author(s):

Sebastian Breuer ◽

Simone I. Schievink ◽

Antje Schulte ◽

Wulf Blankenfeldt ◽

Oliver T. Fackler ◽

...

Keyword(s):

Molecular Design ◽

Functional Characterization ◽

Structural Basis ◽

Protein Inhibitor ◽

Pathogenicity Factor ◽

Hiv 1

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MUCI, glycans and the cell-surface barrier to embryo implantation

Biochemical Society Transactions ◽

10.1042/bst0290153 ◽

2001 ◽

Vol 29 (2) ◽

pp. 153-156 ◽

Cited By ~ 57

Author(s):

J. D. Aplin ◽

M. Meseguer ◽

C. Simon ◽

M. E. Ortíz ◽

H. Croxatto ◽

...

Keyword(s):

Cell Adhesion ◽

Epithelial Cells ◽

Cell Surface ◽

Embryo Implantation ◽

Human Embryos ◽

Surface Barrier ◽

Epithelial Surface ◽

Epithelial Monolayers ◽

Maternal Rejection ◽

Normal Expression

As it approaches the maternal surface, the attaching embryo encounters the epithelial glycocalyx, which contains the mucin, MUC1. A high density of MUC1 at the cell surface can inhibit cell adhesion. This raises the possibility of the existence of a uterine barrier to implantation that might allow maternal rejection of poorer quality embryos. To investigate the mechanism of implantation, human embryos were incubated with endometrial epithelial monolayers. Hatched blastocysts were found to attach readily to the epithelial surface. MUC1 was lost from epithelial cells beneath and near to the attached embryo, while normal expression persisted in neighbouring cells.

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Germline Variants of CYBA and TRPM4 Predispose to Familial Colorectal Cancer

10.20944/preprints202112.0354.v1 ◽

2021 ◽

Author(s):

Lizhen Zhu ◽

Beiping Miao ◽

Dagmara Dymerska ◽

Magdalena Kuświk ◽

Elena Bueno-Martínez ◽

...

Keyword(s):

Colorectal Cancer ◽

Stop Codon ◽

Bacterial Colonization ◽

Functional Characterization ◽

Premature Stop Codon ◽

Familial Colorectal Cancer ◽

Intestinal Mucus ◽

Healthy Elderly ◽

Predisposing Genes ◽

Mucus Barrier

Familial colorectal cancer (CRC) is only partially explained by known germline predisposing genes. We performed whole genome sequencing in 15 Polish families of many affected individuals, without mutations in known CRC predisposing genes. We focused on loss-of-function variants and functionally characterized them. We identified a frameshift variant in the CYBA gene (c.246delC) in one family and a splice site variant in the TRPM4 gene (c.25-1 G>T) in another family. While both variants were absent or extremely rare in gene variant databases, we identified four additional Polish familial CRC cases and two healthy elderly individuals with the CYBA variant (odds ratio 2.46, 95% confidence interval 0.48-12.69). Both variants led to a premature stop codon and to a truncated protein. Functional characterization of the variants showed that knockdown of CYBA or TRPM4 depressed generation of reactive oxygen species (ROS) in LS174T and HT-29 cell lines. Knockdown of TRPM4 resulted in decreased MUC2 protein production. CYBA encodes a component in the NADPH oxidase system which generates ROS and controls, e.g., bacterial colonization in the gut. Germline CYBA variants are associated with early onset inflammatory bowel disease, supported with experimental evidence on loss of intestinal mucus barrier function due to ROS deficiency. TRPM4 encodes a calcium-activated ion channel, which in a human colonic cancer cell line controls calcium-mediated secretion of MUC2, a major component of intestinal mucus barrier. We suggest that the gene defects in CYBA and TRPM4 mechanistically involve intestinal barrier integrity through ROS and mucus biology, which converges in chronic bowel inflammation.

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Exploring Potential COPD Immunosuppression Pathways Causing Increased Susceptibility for MAC Infections among COPD Patients

Clinics and Practice ◽

10.3390/clinpract11030077 ◽

2021 ◽

Vol 11 (3) ◽

pp. 619-630

Author(s):

Shafaa Munjal ◽

Shalok Munjal ◽

Jingya Gao ◽

Vishwanath Venketaraman

Keyword(s):

Mycobacterium Avium ◽

Opportunistic Pathogen ◽

Developed Countries ◽

The United States ◽

Chronic Obstructive ◽

Obstructive Pulmonary Disease ◽

Susceptibility To Infection ◽

Copd Patients ◽

Respiratory Conditions ◽

Increased Susceptibility

Although there has been a drastic decline in the cases of Tuberculosis in the United States, the prevalence of infections caused by Mycobacterium avium Complex (MAC) has steadily increased in the past decades. Mycobacterium avium (M. avium) is one of the most abundant microorganisms in the MAC species. The mycobacterium genus is divided into two major groups: tuberculosis causing mycobacteria and non-tuberculous mycobacteria. MAC is most prominent among the non-tuberculous mycobacteria. MAC is an opportunistic pathogen that is present in soil, water, and droplets in the air. MAC infections can result in respiratory disease and can disseminate in affected patients. MAC infections are especially prevalent in patients with preexisting respiratory conditions such as Chronic Obstructive Pulmonary Disease (COPD). COPD is one of the most common lung conditions in the world with the primary cause being smoking in developed countries. COPD involves chronic inflammation of lung tissue resulting in increased susceptibility to infection. There is a lack of research regarding the pathophysiology that leads COPD patients to be susceptible to MAC infection. Our review paper therefore aims to investigate how the pathogenicity of MAC bacteria and immune decline seen in COPD patients leads to a greater susceptibility to MAC infection among COPD patients.

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Mechanism of hERG inhibition by gating-modifier toxin, APETx1, deduced by functional characterization

BMC Molecular and Cell Biology ◽

10.1186/s12860-020-00337-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Kazuki Matsumura ◽

Takushi Shimomura ◽

Yoshihiro Kubo ◽

Takayuki Oka ◽

Naohiro Kobayashi ◽

...

Keyword(s):

Potassium Channel ◽

Resting State ◽

Mutational Analysis ◽

Functional Characterization ◽

Structural Basis ◽

Anthopleura Elegantissima ◽

Gating Modifier ◽

Peptide Toxin ◽

Key Residues ◽

Voltage Sensing Domain

Abstract Background Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be conducted as only natural resources have been available until now. Therefore, it remains unclear where and how APETx1 interacts with the VSD in the resting state. Results We established a method for preparing recombinant APETx1 and determined the NMR structure of the recombinant APETx1, which is structurally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, F15, Y32, F33, and L34, in APETx1, and F508 and I521 in hERG, in addition to a previously reported acidic hERG residue, E518, play key roles in the inhibition of hERG by APETx1. Our hypothetical docking models of the APETx1-VSD complex satisfied the results of mutational analysis. Conclusions The present study identified the key residues of APETx1 and hERG that are involved in hERG inhibition by APETx1. These results would help advance understanding of the inhibitory mechanism of APETx1, which could provide a structural basis for designing novel ligands targeting the VSDs of KV channels.

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