scholarly journals A Novel Outer Membrane Protein, Wzi, Is Involved in Surface Assembly of the Escherichia coli K30 Group 1 Capsule

2003 ◽  
Vol 185 (19) ◽  
pp. 5882-5890 ◽  
Author(s):  
Andrea Rahn ◽  
Kostantinos Beis ◽  
James H. Naismith ◽  
Chris Whitfield
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Outer Membrane ◽  
Capsular Polysaccharide ◽  
Signal Sequence ◽  
Surface Group ◽  
Gene Clusters ◽  
Capsule Assembly ◽  
Free Material ◽  
Group 1

ABSTRACT Escherichia coli group 1 K antigens form a tightly associated capsule structure on the cell surface. Although the general features of the early steps in capsular polysaccharide biosynthesis have been described, little is known about the later stages that culminate in assembly of a capsular structure on the cell surface. Group 1 capsule biosynthesis gene clusters (cps) in E. coli and Klebsiella pneumoniae include a conserved open reading frame, wzi. The wzi gene is the first of a block of four conserved genes (wzi-wza-wzb-wzc) found in all group 1 K-antigen serotypes. Unlike wza, wzb, and wzc homologs that are found in gene clusters responsible for production of exopolysaccharides (i.e., predominantly cell-free polymer) in a range of bacteria, wzi is found only in systems that assemble capsular polysaccharides. The predicted Wzi protein shows no similarity to any other known proteins in the databases, but computer analysis of Wzi predicted a cleavable signal sequence. Wzi was expressed with a C-terminal hexahistidine tag, purified, and used for the production of specific antibodies that facilitated localization of Wzi to the outer membrane. Circular dichroism spectroscopy indicates that Wzi consists primarily of a β-barrel structure, and dynamic light scattering studies established that the protein behaves as a monomer in solution. A nonpolar wzi chromosomal mutant retained a mucoid phenotype and remained sensitive to lysis by a K30-specific bacteriophage. However, the mutant showed a significant reduction in cell-bound polymer, with a corresponding increase in cell-free material. Furthermore, examination of the mutant by electron microscopy showed that it lacked a coherent capsule structure. It is proposed that the Wzi protein plays a late role in capsule assembly, perhaps in the process that links high-molecular-weight capsule to the cell surface.

scholarly journals Impact of Phosphorylation of Specific Residues in the Tyrosine Autokinase, Wzc, on Its Activity in Assembly of Group 1 Capsules in Escherichia coli

2002 ◽  
Vol 184 (23) ◽  
pp. 6437-6447 ◽  
Author(s):  
Anne Paiment ◽  
Jennifer Hocking ◽  
Chris Whitfield
Keyword(s):  
Escherichia Coli ◽  
Capsular Polysaccharide ◽  
Site Directed Mutagenesis ◽  
Detectable Effect ◽  
Tyrosine Residue ◽  
Tyrosine Residues ◽  
Capsule Assembly ◽  
K 12 ◽  
Group 1

ABSTRACT WzcCPS is a tyrosine autokinase essential for the assembly of a high-molecular-weight (HMW) group 1 capsular polysaccharide (CPS) in Escherichia coli. Homologues of Wzc participate in the formation of CPS and exopolysaccharides in a variety of gram-positive and gram-negative bacteria. Phosphorylation of tyrosine residues in the WzcCPS C terminus is essential for HMW CPS assembly. Overexpression of WzbCPS (phosphatase) in a wild-type background caused a 3.7-fold decrease in the amount of cell-associated K30 CPS produced, confirming the importance of WzcCPS phosphorylation for capsule assembly. In this study, the tyrosine-rich region was dissected in an attempt to identify residues critical for WzcCPS phosphorylation and/or capsule expression. Site-directed mutagenesis demonstrated that no single tyrosine residue in this region is sufficient for detectable phosphorylation of WzcCPS in vivo or for HMW CPS expression. Furthermore, no single tyrosine residue is essential for phosphorylation or capsule assembly, since removal of any one tyrosine residue has no detectable effect. Altering combinations of tyrosine residues (from two to five) led to WzcCPS derivatives that were still competent for phosphorylation but that could not support assembly of HMW CPS, showing that phosphorylation of Wzc per se is not an accurate measure of its ability to function in capsule assembly. One interpretation of these data is that the overall level of phosphorylation in this region, rather than the precise combination of residues accessible to phosphorylation, is important for the activity of WzcCPS. Tyrosine 569, a residue shown to modulate the in vitro phosphorylation of WzcCA from E. coli K-12, was also mutated. The derivative with this mutation still functioned in capsule assembly. Quantitation of K30CPS from this mutant revealed no difference in the amount of polymer produced. Finally, dithiobis(succinimidylpropionate) cross-linking was used to confirm that WzcCPS forms complexes in vivo, independent of the phosphorylation state of the protein.

scholarly journals Interaction of the Colicin K Bactericidal Toxin with Components of Its Import Machinery in the Periplasm of Escherichia coli

2010 ◽  
Vol 192 (22) ◽  
pp. 5934-5942 ◽  
Author(s):  
Aurélie Barnéoud-Arnoulet ◽  
Marthe Gavioli ◽  
Roland Lloubès ◽  
Eric Cascales
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Outer Membrane ◽  
Signal Sequence ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Outer Membrane Receptor ◽  
Terminal Domain

ABSTRACT Colicins are bacterial antibiotic toxins produced by Escherichia coli cells and are active against E. coli and closely related strains. To penetrate the target cell, colicins bind to an outer membrane receptor at the cell surface and then translocate their N-terminal domain through the outer membrane and the periplasm. Once fully translocated, the N-terminal domain triggers entry of the catalytic C-terminal domain by an unknown process. Colicin K uses the Tsx nucleoside-specific receptor for binding at the cell surface, the OmpA protein for translocation through the outer membrane, and the TolABQR proteins for the transit through the periplasm. Here, we initiated studies to understand how the colicin K N-terminal domain (KT) interacts with the components of its transit machine in the periplasm. We first produced KT fused to a signal sequence for periplasm targeting. Upon production of KT in wild-type strains, cells became partly resistant to Tol-dependent colicins and sensitive to detergent, released periplasmic proteins, and outer membrane vesicles, suggesting that KT interacts with and titrates components of its import machine. Using a combination of in vivo coimmunoprecipitations and in vitro pulldown experiments, we demonstrated that KT interacts with the TolA, TolB, and TolR proteins. For the first time, we also identified an interaction between the TolQ protein and a colicin translocation domain.

scholarly journals Wzi Is an Outer Membrane Lectin that Underpins Group 1 Capsule Assembly in Escherichia coli

Structure ◽  
2013 ◽  
Vol 21 (5) ◽  
pp. 844-853 ◽  
Author(s):  
Simon R. Bushell ◽  
Iain L. Mainprize ◽  
Martin A. Wear ◽  
Hubing Lou ◽  
Chris Whitfield ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Capsule Assembly ◽  
Group 1

scholarly journals Structural and Functional Variation in Outer Membrane Polysaccharide Export (OPX) Proteins from the Two Major Capsule Assembly Pathways Present inEscherichia coli

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Caitlin Sande ◽  
Catrien Bouwman ◽  
Elisabeth Kell ◽  
Nicholas N. Nickerson ◽  
Sharookh B. Kapadia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Outer Membrane ◽  
Abc Transporter ◽  
Capsular Polysaccharides ◽  
Content Type ◽  
E Coli ◽  
Capsule Production ◽  
Group 2 ◽  
Group 1

ABSTRACTCapsular polysaccharides (CPSs) are virulence factors for many important pathogens. InEscherichia coli, CPSs are synthesized via two distinct pathways, but both require proteins from the outer membrane polysaccharide export (OPX) family to complete CPS export from the periplasm to the cell surface. In this study, we compare the properties of the OPX proteins from the prototypical group 1 (Wzy-dependent) and group 2 (ABC transporter-dependent) pathways inE. coliK30 (Wza) andE. coliK2 (KpsD), respectively. In addition, we compare an OPX fromSalmonella entericaserovar Typhi (VexA), which shares structural properties with Wza, while operating in an ABC transporter-dependent pathway. These proteins differ in distribution in the cell envelope and formation of stable multimers, but these properties do not align with acylation or the interfacing biosynthetic pathway. InE. coliK2, murein lipoprotein (Lpp) plays a role in peptidoglycan association of KpsD, and loss of this interaction correlates with impaired group 2 capsule production. VexA also depends on Lpp for peptidoglycan association, but CPS production is unaffected in anlppmutant. In contrast, Wza and group 1 capsule production is unaffected by the absence of Lpp. These results point to complex structure-function relationships between different OPX proteins.IMPORTANCECapsules are protective layers of polysaccharides that surround the cell surface of many bacteria, including that ofEscherichia coliisolates andSalmonella entericaserovar Typhi. Capsular polysaccharides (CPSs) are often essential for virulence because they facilitate evasion of host immune responses. The attenuation of unencapsulated mutants in animal models and the involvement of protein families with conserved features make the CPS export pathway a novel candidate for therapeutic strategies. However, appropriate “antivirulence” strategies require a fundamental understanding of the underpinning cellular processes. Investigating export proteins that are conserved across different biosynthesis strategies will give important insight into how CPS is transported to the cell surface.

scholarly journals Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1039-1049 ◽  
Author(s):  
Sheila Patrick ◽  
Simon Houston ◽  
Zubin Thacker ◽  
Garry W. Blakely
Keyword(s):  
Capsular Polysaccharide ◽  
Mutational Analysis ◽  
Bacteroides Fragilis ◽  
Gene Clusters ◽  
Opportunistic Pathogen ◽  
Extracellular Polysaccharides ◽  
Phase Variable ◽  
Multiple Gene ◽  
Polysaccharide Biosynthesis ◽  
Group 1

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.

scholarly journals Initial Steps of Colicin E1 Import across the Outer Membrane of Escherichia coli

2007 ◽  
Vol 189 (7) ◽  
pp. 2667-2676 ◽  
Author(s):  
Muriel Masi ◽  
Phu Vuong ◽  
Matthew Humbard ◽  
Karen Malone ◽  
Rajeev Misra
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Outer Membrane ◽  
Vitamin B ◽  
Whole Cells ◽  
E Coli ◽  
Colicin E1 ◽  
Unfolded State ◽  
Translocation Domain

ABSTRACT Data suggest a two-receptor model for colicin E1 (ColE1) translocation across the outer membrane of Escherichia coli. ColE1 initially binds to the vitamin B12 receptor BtuB and then translocates through the TolC channel-tunnel, presumably in a mostly unfolded state. Here, we studied the early events in the import of ColE1. Using in vivo approaches, we show that ColE1 is cleaved when added to whole cells. This cleavage requires the presence of the receptor BtuB and the protease OmpT, but not that of TolC. Strains expressing OmpT cleaved ColE1 at K84 and K95 in the N-terminal translocation domain, leading to the removal of the TolQA box, which is essential for ColE1's cytotoxicity. Supported by additional in vivo data, this suggests that a function of OmpT is to degrade colicin at the cell surface and thus protect sensitive E. coli cells from infection by E colicins. A genetic strategy for isolating tolC mutations that confer resistance to ColE1, without affecting other TolC functions, is also described. We provide further in vivo evidence of the multistep interaction between TolC and ColE1 by using cross-linking followed by copurification via histidine-tagged TolC. First, secondary binding of ColE1 to TolC is dependent on primary binding to BtuB. Second, alterations to a residue in the TolC channel interfere with the translocation of ColE1 across the TolC pore rather than with the binding of ColE1 to TolC. In contrast, a substitution at a residue exposed on the cell surface abolishes both binding and translocation of ColE1.

scholarly journals Partially Assembled K99 Fimbriae Are Required for Protection

2005 ◽  
Vol 73 (11) ◽  
pp. 7274-7280 ◽  
Author(s):  
Miguel A. Ascón ◽  
Javier Ochoa-Repáraz ◽  
Nancy Walters ◽  
David W. Pascual
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Protective Immunity ◽  
Immunoglobulin A ◽  
Gene Clusters ◽  
T Cell Epitopes ◽  
Partial Reduction ◽  
Fimbrial Subunit ◽  
Cytokine Responses ◽  
Antibody Titers

ABSTRACTAntibodies to K99 fimbriae afford protection to F5+bovine enterotoxigenicEscherichia coli(ETEC). Previous studies show that murine dams immunized withSalmonellavaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaptation of the K99 scaffold to display heterologous B- and T-cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished immunoglobulin A (IgA) and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion offanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial reduction in protective immunity. By contrast, deletion offanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion offanGHis sufficient to confer protection.

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