Pectin Modification in Seed Coat Mucilage by In Vivo Expression of Rhamnogalacturonan-I- and Homogalacturonan-Degrading Enzymes

2021 ◽  
Author(s):  
Robert McGee ◽  
Gillian H Dean ◽  
Di Wu ◽  
Yuelin Zhang ◽  
Shawn D Mansfield ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Wall ◽  
Seed Coat ◽  
Epidermal Cells ◽  
Wall Structure ◽  
Rhamnogalacturonan I ◽  
Degrading Enzymes ◽  
Developing Seed ◽  
Extracellular Matrix Components

Abstract The cell wall is essential for plant survival. Determining the relationship between cell wall structure and function using mutant analysis or overexpressing cell wall-modifying enzymes has been challenging due to the complexity of the cell wall and the appearance of secondary, compensatory effects when individual polymers are modified. In addition, viability of the plants can be severely impacted by wall modification. A useful model system for studying structure-function relationships among extracellular matrix components are the seed coat epidermal cells of Arabidopsis thaliana. These cells synthesize relatively simple, easily-accessible, pectin-rich mucilage that is not essential for plant viability. In this study, we expressed enzymes predicted to modify polysaccharide components of mucilage in the apoplast of seed coat epidermal cells and explored their impacts on mucilage. The seed coat epidermal-specific promoter TESTA ABUNDANT2 (TBA2) was used to drive expression of these enzymes to avoid adverse effects in other parts of the plant. Mature transgenic seeds expressing Rhamnogalacturonate lyase A (RglA) or Rhamnogalacturonate lyase B (RglB) that degrade the pectin rhamnogalacturonan-I (RG-I), a major component of mucilage, had greatly reduced mucilage capsules surrounding the seeds and concomitant decreases in the monosaccharides that comprise the RG-I backbone. Degradation of the minor mucilage component homogalacturonan (HG) using the HG-degrading enzymes Pectin Lyase A (PLA) or ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2) resulted in developing seed coat epidermal cells with disrupted cell-cell adhesion and signs of early cell death. These results demonstrate the feasibility of manipulating the seed coat epidermal cell extracellular matrix using a targeted genetic engineering approach.

Cell-extracellular matrix interactions under in vivo conditions during interstitial cell migration in Hydra vulgaris

Development ◽  
1994 ◽  
Vol 120 (2) ◽  
pp. 425-432 ◽  
Author(s):  
X. Zhang ◽  
M.P. Sarras
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Synthetic Peptide ◽  
Interstitial Cell ◽  
Extracellular Matrix Components ◽  
Matrix Interactions ◽  
Apical Half ◽  
Matrix Components ◽  
Extracellular Matrix Interactions

Interstitial cell (I-cell) migration in hydra is essential for establishment of the regional cell differentiation pattern in the organism. All previous in vivo studies have indicated that cell migration in hydra is a result of cell-cell interactions and chemotaxic gradients. Recently, in vitro cell adhesion studies indicated that isolated nematocytes could bind to substrata coated with isolated hydra mesoglea, fibronectin and type IV collagen. Under these conditions, nematocytes could be observed to migrate on some of these extracellular matrix components. By modifying previously described hydra grafting techniques, two procedures were developed to test specifically the role of extracellular matrix components during in vivo I-cell migration in hydra. In one approach, the extracellular matrix structure of the apical half of the hydra graft was perturbed using beta-aminopropionitrile and beta-xyloside. In the second approach, grafts were treated with fibronectin, RGDS synthetic peptide and antibody to fibronectin after grafting was performed. In both cases, I-cell migration from the basal half to the apical half of the grafts was quantitatively analyzed. Statistical analysis indicated that beta-aminopropionitrile, fibronectin, RGDS synthetic peptide and antibody to fibronectin all were inhibitory to I-cell migration as compared to their respective controls. beta-xyloside treatment had no effect on interstitial cell migration. These results indicate the potential importance of cell-extracellular matrix interactions during in vivo I-cell migration in hydra.

Fibronectin enhances Campylobacter fetus interaction with extracellular matrix components and INT 407 cells

2008 ◽  
Vol 54 (1) ◽  
pp. 37-47 ◽  
Author(s):  
L. L. Graham ◽  
T. Friel ◽  
R. L. Woodman
Keyword(s):  
Extracellular Matrix ◽  
Solid Phase ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Bacterial Attachment ◽  
Binding Assays ◽  
Rgd Sequence ◽  
Campylobacter Fetus ◽  
Extracellular Matrix Components

Campylobacter fetus is a recognized pathogen of cattle and sheep that can also infect humans. No adhesins specific for C. fetus have to date been identified; however, bacterial attachment is essential to establish an infecting population. Scanning electron microscopy revealed C. fetus attachment to the serosal surface of human colonic biopsy explants, a location consistent with the presence of the extracellular matrix (ECM). To determine whether the ECM mediated C. fetus adherence, 7 C. fetus strains were assessed in a solid-phase binding assay for their ability to bind to immobilized ECM components. Of the ECM components assayed, adherence to fibronectin was noted for all strains. Attachment to ECM components was neither correlated with S-layer expression nor with cell-surface hydrophobicity. Ligand immunoblots, however, identified the S-layer protein as a major site of fibronectin binding, and modified ECM binding assays revealed that soluble fibronectin significantly enhanced the attachment of S-layer-expressing C. fetus strains to other ECM components. Soluble fibronectin also increased C. fetus adherence to INT 407 cells. This adherence was inhibited when INT 407 cells were incubated with synthetic peptides containing an RGD sequence, indicating that integrin receptors were involved in fibronectin-mediated attachment. Together, this data suggests that C. fetus can bind to immobilized fibronectin and use soluble fibronectin to enhance attachment to other ECM components and intestinal epithelial cells. In vivo, fibronectin would promote bacterial adherence, thereby, contributing to the initial interaction of C. fetus with mucosal and submucosal surfaces.

scholarly journals Rapid in vivo development of resistance to daptomycin in vancomycin-resistant Enterococcus faecium due to genomic rearrangements

2021 ◽  
Author(s):  
Sarah Mollerup ◽  
Christine Elmeskov ◽  
Heidi Gumpert ◽  
Mette Pinholt ◽  
Tobias Steen Sejersen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecium ◽  
Chromosomal Rearrangements ◽  
Resistance Mechanisms ◽  
Resistant Isolate ◽  
Wall Structure ◽  
Whole Genome ◽  
Cyclic Lipopeptide ◽  
Vancomycin Resistant

AbstractBackgroundDaptomycin is a cyclic lipopeptide used in the treatment of vancomycin-resistant Enterococcus faecium (VREfm). However, the development of daptomycin-resistant VREfm challenges the treatment of nosocomial VREfm infections. Resistance mechanisms of daptomycin are not fully understood. Here we analysed the genomic changes leading to a daptomycin-susceptible VREfm isolate becoming resistant after 40 days of daptomycin and linezolid combination therapy.MethodsThe two isogenic VREfm isolates (daptomycin-susceptible and daptomycin-resistant) were analysed using whole genome sequencing with Illumina and Nanopore.ResultsWhole genome comparative analysis identified the loss of a 46.5 kb fragment and duplication of a 29.7 kb fragment in the daptomycin-resistant isolate, with many implicated genes involved in cell wall synthesis. Two plasmids of the daptomycin-susceptible isolate were also found integrated in the chromosome of the resistant isolate. One nonsynonymous SNP in the rpoC gene was identified in the daptomycin-resistant isolate.ConclusionsDaptomycin resistance developed through chromosomal rearrangements leading to altered cell wall structure. Such novel types of resistance mechanisms can only be identified by comparing closed genomes of isogenic isolates.

scholarly journals Agrin-induced reorganization of extracellular matrix components on cultured myotubes: relationship to AChR aggregation.

1990 ◽  
Vol 111 (3) ◽  
pp. 1161-1170 ◽  
Author(s):  
R M Nitkin ◽  
T C Rothschild
Keyword(s):  
Extracellular Matrix ◽  
Type Iv Collagen ◽  
Neuromuscular Junctions ◽  
Synaptic Organization ◽  
Type Iv ◽  
Extracellular Matrix Components ◽  
Cultured Myotubes ◽  
Matrix Components ◽  
Induced Aggregation

Agrin, an extracellular matrix-associated protein extracted from synapse-rich tissues, induces the accumulation of acetylcholine receptors (AChRs) and other synaptic components into discrete patches on cultured myotubes. The appearance of agrin-like molecules at neuromuscular junctions suggests that it may direct synaptic organization in vivo. In the present study we examined the role of extracellular matrix components in agrin-induced differentiation. We used immunohistochemical techniques to visualize the spatial and temporal distribution of laminin, a heparan sulfate proteoglycan (HSPG), fibronectin, and type IV collagen on cultured chick myotubes during agrin-induced aggregation of AChRs. Myotubes displayed significant amounts of laminin and HSPG, lesser amounts of type IV collagen, and little, if any, fibronectin. Agrin treatment caused cell surface laminin and HSPG to patch, while collagen and fibronectin distributions were generally unaffected. Many of the agrin-induced laminin and HSPG patches colocalized with AChR patches, raising the possibility of a causal relationship between matrix patching and AChR accumulations. However, patching of AChRs (complete within a few hours) preceded that of laminin or HSPG (not complete until 15-20 h), making it unlikely that matrix accumulations initiate AChR patching at agrin-induced sites. Conversely, when AChR patching was blocked by treatment with anti-AChR antibody mAb 35, agrin was still able to effect patching of laminin and HSPG. Taken together, these findings suggest that agrin-induced accumulations of AChR and laminin/HSPG are not mechanistically linked.

Cell wall alterations in staphylococci growing in situ in experimental osteomyelitis

1987 ◽  
Vol 33 (2) ◽  
pp. 142-150 ◽  
Author(s):  
J. W. Costerton ◽  
D. W. Lambe Jr. ◽  
K.-J. Mayberry-Carson ◽  
B. Tober-Meyer
Keyword(s):  
Cell Wall ◽  
Cell Size ◽  
Wall Structure ◽  
Specific Cell ◽  
Experimental Osteomyelitis ◽  
Rich Media ◽  
Rabbit Tibia ◽  
Rabbit Bone

When cells of both Staphylococcus aureus and Staphylococcus epidermidis are grown in batch culture in nutrient-rich media, their cell walls are regular in thickness, their cell size is within the normal range for each species, and their septation patterns are orderly. When cells of each of these species are examined directly in infected tissue in the rabbit tibia model infection, their cell wall thickness is often much increased and very irregular around the circumference of the cell, their cell size is often increased, and their septation patterns are often severely deranged. All of these alterations in cell wall structure occur in the absence of antibiotics, and we suggest that they may be an expression of phenotypic plasticity in response to altered environmental conditions such as specific nutrient limitations, the presence of antibacterial factors, and growth of the cells on hard surfaces such as rabbit bone or plastic catheters. Some of these specific cell wall alterations are also seen when staphylococcal cells are exposed, in vitro or in vivo, to antibiotics such as clindamycin, but we emphasize that growth in tissue alone is sufficient for their induction.

scholarly journals Laminin promotes neuritic regeneration from cultured peripheral and central neurons.

1983 ◽  
Vol 97 (6) ◽  
pp. 1882-1890 ◽  
Author(s):  
M Manthorpe ◽  
E Engvall ◽  
E Ruoslahti ◽  
F M Longo ◽  
G E Davis ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Neurite Growth ◽  
Sensory Ganglia ◽  
Culture Methods ◽  
Fetal Rat ◽  
Extracellular Matrix Components ◽  
Ciliary Ganglion Neurons ◽  
Ganglion Neurons

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co-purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.

scholarly journals Streptococcus Adherence and Colonization

2009 ◽  
Vol 73 (3) ◽  
pp. 407-450 ◽  
Author(s):  
Angela H. Nobbs ◽  
Richard J. Lamont ◽  
Howard F. Jenkinson
Keyword(s):  
Cell Wall ◽  
Molecular Techniques ◽  
Host Cells ◽  
In Vivo Studies ◽  
Regulatory Pathways ◽  
Mucosal Tissues ◽  
Extracellular Matrix Components ◽  
Host Tissues

SUMMARY Streptococci readily colonize mucosal tissues in the nasopharynx; the respiratory, gastrointestinal, and genitourinary tracts; and the skin. Each ecological niche presents a series of challenges to successful colonization with which streptococci have to contend. Some species exist in equilibrium with their host, neither stimulating nor submitting to immune defenses mounted against them. Most are either opportunistic or true pathogens responsible for diseases such as pharyngitis, tooth decay, necrotizing fasciitis, infective endocarditis, and meningitis. Part of the success of streptococci as colonizers is attributable to the spectrum of proteins expressed on their surfaces. Adhesins enable interactions with salivary, serum, and extracellular matrix components; host cells; and other microbes. This is the essential first step to colonization, the development of complex communities, and possible invasion of host tissues. The majority of streptococcal adhesins are anchored to the cell wall via a C-terminal LPxTz motif. Other proteins may be surface anchored through N-terminal lipid modifications, while the mechanism of cell wall associations for others remains unclear. Collectively, these surface-bound proteins provide Streptococcus species with a “coat of many colors,” enabling multiple intimate contacts and interplays between the bacterial cell and the host. In vitro and in vivo studies have demonstrated direct roles for many streptococcal adhesins as colonization or virulence factors, making them attractive targets for therapeutic and preventive strategies against streptococcal infections. There is, therefore, much focus on applying increasingly advanced molecular techniques to determine the precise structures and functions of these proteins, and their regulatory pathways, so that more targeted approaches can be developed.

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