scholarly journals Differential Expression of Vibrio vulnificus Capsular Polysaccharide

1999 ◽  
Vol 67 (5) ◽  
pp. 2250-2257 ◽  
Author(s):  
Anita C. Wright ◽  
Jan L. Powell ◽  
Mike K. Tanner ◽  
Lynne A. Ensor ◽  
Arthur B. Karpas ◽  
...  
Keyword(s):  
Capsular Polysaccharide ◽  
Vibrio Vulnificus ◽  
Phase Variation ◽  
Surface Expression ◽  
Ruthenium Red ◽  
Cell Surface Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Transposon Mutants ◽  
Phase Variants

ABSTRACT Vibrio vulnificus is a human pathogen whose virulence has been associated with the expression of capsular polysaccharide (CPS). Multiple CPS types have been described; however, virulence does not appear to correlate with a particular CPS composition. Reversible-phase variation for opaque and translucent colony morphologies is characterized by changes in CPS expression, as suggested by electron microscopy of cells stained nonspecifically with ruthenium red. Isolates with opaque colony morphologies are virulent and appear to be more thickly encapsulated than naturally occurring translucent-phase variants, which have reduced, patchy, or absent CPS. Previously, we have shown that the virulence of translucent-phase variants was intermediate between opaque-phase variants and acapsular transposon mutants, suggesting a correlation between virulence and the amount of CPS expressed. In the present study, CPS expression of phase variants and genetically defined mutants of V. vulnificusM06-24/O was examined by using a CPS-specific monoclonal antibody with an enzyme-linked immunosorbent assay, flow cytometry, and immunoelectron microscopy. Semiquantitative analyses of CPS expression correlated well among these assays, confirming that the translucent-phase variant was intermediate in CPS expression and retained type I CPS-specific epitopes. Cell surface expression of CPS varied with the growth phase, increasing during logarithmic growth and declining in stationary culture. Significantly greater CPS expression (P = 0.026) was observed for cells grown at 30°C than for those at 37°C. These studies confirm that phase variation and virulence in V. vulnificus correlate with the amount of CPS expressed and demonstrate the fluidity of bacterial polysaccharide expression in response to environmental conditions.

scholarly journals Identification of a Group 1-Like Capsular Polysaccharide Operon for Vibrio vulnificus

2001 ◽  
Vol 69 (11) ◽  
pp. 6893-6901 ◽  
Author(s):  
Anita C. Wright ◽  
Jan L. Powell ◽  
James B. Kaper ◽  
J. Glenn Morris
Keyword(s):  
Capsular Polysaccharide ◽  
Vibrio Vulnificus ◽  
Phase Variation ◽  
Surface Expression ◽  
Open Reading Frames ◽  
Biosynthetic Genes ◽  
Virulent Phenotype ◽  
Phase Variants ◽  
Group 1

ABSTRACT Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using TnphoA mutagenesis, we identified a V.vulnificus CPS locus, which included an upstreamops element, a wza gene (wza Vv), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wzagene product is required for transport of CPS to the cell surface inEscherichia coli. Polar transposon mutations inwza Vv eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wza Vv was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans. Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions inwza Vv and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V.vulnificus.

scholarly journals Structure, Function, and Regulation of the Essential Virulence Factor Capsular Polysaccharide of Vibrio vulnificus

2020 ◽  
Vol 21 (9) ◽  
pp. 3259 ◽  
Author(s):  
Gregg S. Pettis ◽  
Aheli S. Mukerji
Keyword(s):  
Coastal Waters ◽  
Capsular Polysaccharide ◽  
Vibrio Vulnificus ◽  
Critical Role ◽  
Phase Variation ◽  
Innate Immune ◽  
Repeat Sequences ◽  
Life Threatening ◽  
Inflammatory Cytokine Response ◽  
Group 1

Vibrio vulnificus populates coastal waters around the world, where it exists freely or becomes concentrated in filter feeding mollusks. It also causes rapid and life-threatening sepsis and wound infections in humans. Of its many virulence factors, it is the V. vulnificus capsule, composed of capsular polysaccharide (CPS), that plays a critical role in evasion of the host innate immune system by conferring antiphagocytic ability and resistance to complement-mediated killing. CPS may also provoke a portion of the host inflammatory cytokine response to this bacterium. CPS production is biochemically and genetically diverse among strains of V. vulnificus, and the carbohydrate diversity of CPS is likely affected by horizontal gene transfer events that result in new combinations of biosynthetic genes. Phase variation between virulent encapsulated opaque colonial variants and attenuated translucent colonial variants, which have little or no CPS, is a common phenotype among strains of this species. One mechanism for generating acapsular variants likely involves homologous recombination between repeat sequences flanking the wzb phosphatase gene within the Group 1 CPS biosynthetic and transport operon. A considerable number of environmental, genetic, and regulatory factors have now been identified that affect CPS gene expression and CPS production in this pathogen.

scholarly journals The Level of CD81 Cell Surface Expression Is a Key Determinant for Productive Entry of Hepatitis C Virus into Host Cells

2006 ◽  
Vol 81 (2) ◽  
pp. 588-598 ◽  
Author(s):  
George Koutsoudakis ◽  
Eva Herrmann ◽  
Stephanie Kallis ◽  
Ralf Bartenschlager ◽  
Thomas Pietschmann
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Surface ◽  
Surface Expression ◽  
Hcv Infection ◽  
Host Cells ◽  
Productive Infection ◽  
Cell Surface Expression ◽  
Type I ◽  
Cell Clones

ABSTRACT Recently a cell culture model supporting the complete life cycle of the hepatitis C virus (HCV) was developed. Searching for host cell determinants involved in the HCV replication cycle, we evaluated the efficiency of virus propagation in different Huh-7-derived cell clones. We found that Huh-7.5 cells and Huh7-Lunet cells, two former replicon cell clones that had been generated by removal of an HCV replicon by inhibitor treatment, supported comparable levels of RNA replication and particle production, whereas virus spread was severely impaired in the latter cells. Analysis of cell surface expression of CD81 and scavenger receptor class B type I (SR-BI), two molecules previously implicated in HCV entry, revealed similar expression levels for SR-BI, while CD81 surface expression was much higher on Huh-7.5 cells than on Huh7-Lunet cells. Ectopic expression of CD81 in Huh7-Lunet cells conferred permissiveness for HCV infection to a level comparable to that for Huh-7.5 cells. Modulation of CD81 cell surface density in Huh-7.5 cells by RNA interference indicated that a certain amount of this molecule (∼7 × 104 molecules per cell) is required for productive infection with a low dose of HCV. Consistent with this, we show that susceptibility to HCV infection depends on a critical quantity of CD81 molecules. While infection is restricted in cells expressing very small amounts of CD81, susceptibility rapidly rises within a narrow range of CD81 levels, reaching a plateau where higher expression does not further increase the efficiency of infection. Together these data indicate that a high density of cell surface-exposed CD81 is a key determinant for productive HCV entry into host cells.

scholarly journals Atypical Fusion Peptide of Nelson Bay Virus Fusion-Associated Small Transmembrane Protein

2005 ◽  
Vol 79 (3) ◽  
pp. 1853-1860 ◽  
Author(s):  
LiTing T. Cheng ◽  
Richard K. Plemper ◽  
Richard W. Compans
Keyword(s):  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Transmembrane Protein ◽  
Syncytium Formation ◽  
Surface Expression ◽  
Fusion Peptide ◽  
Cell Surface Expression ◽  
Type I ◽  
Fusion Activity ◽  
Hydrophobic Domain

ABSTRACT A 10-kDa nonstructural transmembrane protein (p10) encoded by a reovirus, Nelson Bay virus, has been shown to induce syncytium formation (34). Sequence analysis and structural studies identified p10 as a type I membrane protein with a central transmembrane domain, a cytoplasmic basic region, and an N-terminal hydrophobic domain (HD) that was hypothesized to function as a fusion peptide. We performed mutational analysis on this slightly hydrophobic motif to identify possible structural requirements for fusion activity. Bulky aliphatic residues were found to be essential for optimal fusion, and an aromatic or highly hydrophobic side chain was found to be required at position 12. The requirement for hydrophilic residues within the HD was also examined: substitution of 10-Ser or 14-Ser with hydrophobic residues was found to reduce cell surface expression of p10 and delayed the onset of syncytium formation. Nonconservative substitutions of charged residues in the HD did not have an effect on fusion activity. Taken together, our results suggest that the HD is involved in both syncytium formation and in determining p10 transport and surface expression.

scholarly journals HFE interacts with the BMP type I receptor ALK3 to regulate hepcidin expression

Blood ◽  
2014 ◽  
Vol 124 (8) ◽  
pp. 1335-1343 ◽  
Author(s):  
Xing-gang Wu ◽  
Yang Wang ◽  
Qian Wu ◽  
Wai-Hang Cheng ◽  
Wenjing Liu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Proteasomal Degradation ◽  
Surface Expression ◽  
Bmp Signaling ◽  
Cell Surface Expression ◽  
Type I ◽  
Hepcidin Expression ◽  
Key Points

Key Points HFE increases Smad1/5/8 phosphorylation and hepcidin expression, and inhibition of BMP signaling abolishes HFE-induced hepcidin expression. HFE interacts with ALK3, inhibits ALK3 ubiquitination-proteasomal degradation, and increases ALK3 cell-surface expression.

scholarly journals Analysis of the Role of N-glycosylation in Cell-surface expression, Function and Binding Properties of SARS-CoV-2 receptor ACE2

2021 ◽  
Author(s):  
Alberto Brandariz-Nuñez ◽  
Raymond R Rowland
Keyword(s):  
Cell Surface ◽  
Viral Entry ◽  
Biological Activities ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Type I ◽  
Protein Activity ◽  
Host Cell Membrane ◽  
S Protein

Human angiotensin I-converting enzyme 2 (hACE2) is a type-I transmembrane glycoprotein that serves as the major cell entry receptor for SARS-CoV and SARS-CoV-2. The viral spike (S) protein is required for attachment to ACE2 and subsequent virus-host cell membrane fusion. Previous work has demonstrated the presence of N-linked glycans in ACE2. N-glycosylation is implicated in many biological activities, including protein folding, protein activity, and cell surface expression of biomolecules. However, the contribution of N-glycosylation to ACE2 function is poorly understood. Here, we examined the role of N-glycosylation in the activity and localization of two species with different susceptibility to SARS-CoV-2 infection, porcine ACE2 (pACE2) and hACE2. The elimination of N-glycosylation by tunicamycin (TM) treatment or mutagenesis, showed that N-glycosylation is critical for the proper cell surface expression of ACE2 but not for its carboxiprotease activity. Furthermore, nonglycosylable ACE2 localized predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Our data also revealed that binding of SARS-CoV and SARS-CoV-2 S protein to porcine or human ACE2 was not affected by deglycosylation of ACE2 or S proteins, suggesting that N-glycosylation plays no role in the interaction between SARS coronaviruses and the ACE2 receptor. Impairment of hACE2 N-glycosylation decreased cell to cell fusion mediated by SARS-CoV S protein but not SARS-CoV-2 S protein. Finally, we found that hACE2 N-glycosylation is required for an efficient viral entry of SARS-CoV/SARS-CoV-2 S pseudotyped viruses, which could be the result of low cell surface expression of the deglycosylated ACE2 receptor.

Metalloproteinase-mediated release of the ectodomain of L1 adhesion molecule

1999 ◽  
Vol 112 (16) ◽  
pp. 2667-2675 ◽  
Author(s):  
S. Beer ◽  
M. Oleszewski ◽  
P. Gutwein ◽  
C. Geiger ◽  
P. Altevogt
Keyword(s):  
Cell Surface ◽  
Adhesion Molecule ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Tnf Alpha ◽  
Type I ◽  
Ig Superfamily ◽  
Cell Adhesion And Migration ◽  
Membrane Bound ◽  
And Migration

The L1 adhesion molecule is an approx. 200–220 kDa type I membrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. L1 can bind in a homotypic fashion and was shown to support integrin-mediated binding via RGDs in the 6th Ig-like domain. In addition to its cell-surface expression, L1 can occur in the extracellular matrix (ECM). Here we demonstrate that L1 is constitutively released from the cell surface by membrane-proximal cleavage. L1 shed from B16F10 melanoma cells remains intact and can serve as substrate for integrin-mediated cell adhesion and migration. The release of L1 occurs in mouse and human cells and is blocked by the metalloproteinase inhibitor TAPI (Immunex compound 3). This compound has been shown previously to block release of L-selectin and TNF-alpha which is mediated by the membrane-bound metalloproteinase TNF-alpha converting enzyme (TACE). Using CHO cells that are low in TACE expression and do not release L-selectin we demonstrate that L1 release is distinct from L-selectin shedding. We propose that cell-surface release may be necessary for the conversion of L1 from a membrane into an ECM protein.

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