scholarly journals Mutation in a Gene Required for Lipopolysaccharide and Enterobacterial Common Antigen Biosynthesis Affects Virulence in the Plant Pathogen Erwinia carotovora subsp. atroseptica

1999 ◽  
Vol 12 (6) ◽  
pp. 499-507 ◽  
Author(s):  
I. K. Toth ◽  
C. J. Thorpe ◽  
S. D. Bentley ◽  
V. Mulholland ◽  
L. J. Hyman ◽  
...  
Keyword(s):  
Surface Defects ◽  
Single Gene ◽  
Outer Membrane Proteins ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Enteric Bacteria ◽  
Common Antigen ◽  
In Planta ◽  
Enterobacterial Common Antigen ◽  
Multiple Cell

Spontaneous bacteriophage-resistant mutants of the phytopathogen Erwinia carotovora subsp. atroseptica (Eca) SCRI1043 were isolated and, out of 40, two were found to exhibit reduced virulence in planta. One of these mutants, A5/22, showed multiple cell surface defects including alterations in synthesis of outer membrane proteins, lipopolysaccharide (LPS), enterobacterial common antigen (ECA), and flagella. Mutant A5/22 also showed reduced synthesis of the exoenzymes pectate lyase (Pel) and cellulase (Cel), major virulence factors for this pathogen. Genetic analysis revealed the pronounced pleiotropic mutant phenotype to be due to a defect in a single gene (rffG) that, in Escherichia coli, is involved in the production of ECA. We also show that while other enteric bacteria possess duplicate homologues of this gene dedicated separately to synthesis of LPS and ECA, Eca has a single gene.

scholarly journals Crystal Structure of TDP-Fucosamine Acetyltransferase (WecD) from Escherichia coli, an Enzyme Required for Enterobacterial Common Antigen Synthesis

2006 ◽  
Vol 188 (15) ◽  
pp. 5606-5617 ◽  
Author(s):  
Ming-Ni Hung ◽  
Erumbi Rangarajan ◽  
Christine Munger ◽  
Guy Nadeau ◽  
Traian Sulea ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Coenzyme A ◽  
Structural Data ◽  
Enteric Bacteria ◽  
Common Antigen ◽  
Acetyl Coa ◽  
Enterobacterial Common Antigen ◽  
Thiolate Anion ◽  
Acetyl Transfer

ABSTRACT Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose, organized into trisaccharide repeating units having the sequence →3)-α-d-Fuc4NAc-(1→4)-β-d-ManNAcA-(1→4)-α-d-GlcNAc-(1→. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-d-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Å resolution and bound to acetyl-CoA at a 1.66-Å resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.

scholarly journals Identification and Biosynthesis of Cyclic Enterobacterial Common Antigen in Escherichia coli

2003 ◽  
Vol 185 (6) ◽  
pp. 1995-2004 ◽  
Author(s):  
Paul J. A. Erbel ◽  
Kathleen Barr ◽  
Ninguo Gao ◽  
Gerrit J. Gerwig ◽  
Paul D. Rick ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Repeat Unit ◽  
Enteric Bacteria ◽  
Water Soluble ◽  
Ionization Mass ◽  
Common Antigen ◽  
Esi Ms ◽  
Enterobacterial Common Antigen ◽  
Repeat Units ◽  
K 12

ABSTRACT Phosphoglyceride-linked enterobacterial common antigen (ECAPG) is a cell surface glycolipid that is synthesized by all gram-negative enteric bacteria. The carbohydrate portion of ECAPG consists of linear heteropolysaccharide chains comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-d-galactose, ManNAcA is N-acetyl-d-mannosaminuronic acid, and GlcNAc is N-acetyl-d-glucosamine. The potential reducing terminal GlcNAc residue of each polysaccharide chain is linked via phosphodiester linkage to a phosphoglyceride aglycone. We demonstrate here the occurrence of a water-soluble cyclic form of enterobacterial common antigen, ECACYC, purified from Escherichia coli strains B and K-12 with solution nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and additional biochemical methods. The ECACYC molecules lacked an aglycone and contained four trisaccharide repeat units that were nonstoichiometrically substituted with up to four O-acetyl groups. ECACYC was not detected in mutant strains that possessed null mutations in the wecA, wecF, and wecG genes of the wec gene cluster. These observations corroborate the structural data obtained by NMR and ESI-MS analyses and show for the first time that the trisaccharide repeat units of ECACYC and ECAPG are assembled by a common biosynthetic pathway.

scholarly journals Enterobacterial Common Antigen Mutants of Salmonella enterica Serovar Typhimurium Establish a Persistent Infection and Provide Protection against Subsequent Lethal Challenge

2011 ◽  
Vol 80 (1) ◽  
pp. 441-450 ◽  
Author(s):  
Jeremy J. Gilbreath ◽  
Jennifer Colvocoresses Dodds ◽  
Paul D. Rick ◽  
Mark J. Soloski ◽  
D. Scott Merrell ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Enteric Bacteria ◽  
Rank Test ◽  
Common Antigen ◽  
Lethal Challenge ◽  
Heterologous Antigen ◽  
Content Type ◽  
Enterobacterial Common Antigen ◽  
Mutant Strains ◽  
Serovar Typhimurium

ABSTRACTInfection withSalmonellaspp. is a significant source of disease globally. A substantial proportion of these infections are caused bySalmonella entericaserovar Typhimurium. Here, we characterize the role of the enterobacterial common antigen (ECA), a surface glycolipid ubiquitous among enteric bacteria, inS.Typhimurium pathogenesis. Construction of a defined mutation in the UDP-N-acetylglucosamine-1-phosphate transferase gene,wecA, in two clinically relevant strains ofS.Typhimurium, TML and SL1344, resulted in strains that were unable to produce ECA. Loss of ECA did not affect the gross cell surface ultrastructure, production of lipopolysaccharide (LPS), flagella, or motility. However, thewecAmutant strains were attenuated in both oral and intraperitoneal mouse models of infection (P< 0.001 for both routes of infection; log rank test), and virulence could be restored by complementation of thewecAgene intrans. Despite the avirulence of the ECA-deficient strains, thewecAmutant strains were able to persistently colonize systemic sites (spleen and liver) at moderate levels for up to 70 days postinfection. Moreover, immunization with thewecAmutant strains provided protection against a subsequent lethal oral or intraperitoneal challenge with wild-typeS.Typhimurium. Thus,wecAmutant (ECA-negative) strains ofSalmonellamay be useful as live attenuated vaccine strains or as vehicles for heterologous antigen expression.

scholarly journals A New Look at the Enterobacterial Common Antigen Forms Obtained during Rough Lipopolysaccharides Purification

2021 ◽  
Vol 22 (2) ◽  
pp. 701
Author(s):  
Tomasz K Gozdziewicz ◽  
Anna Maciejewska ◽  
Alona Tsybulska ◽  
Czeslaw Lugowski ◽  
Jolanta Lukasiewicz
Keyword(s):  
Current Knowledge ◽  
Membrane Integrity ◽  
Gel Filtration ◽  
Common Antigen ◽  
Mild Acid Hydrolysis ◽  
Enterobacterial Common Antigen ◽  
Lipopolysaccharide Endotoxin ◽  
Lipid Moiety ◽  
Detailed Composition ◽  
Mild Acid

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide − endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC®HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.

scholarly journals Secretion of Anti-Plasmodium Effector Proteins from a Natural Pantoea agglomerans Isolate by Using PelB and HlyA Secretion Signals

2011 ◽  
Vol 77 (13) ◽  
pp. 4669-4675 ◽  
Author(s):  
Dawn C. Bisi ◽  
David J. Lampe
Keyword(s):  
Control Strategies ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Individual Characteristics ◽  
Pantoea Agglomerans ◽  
Effector Proteins ◽  
World Health ◽  
Content Type ◽  
E Coli ◽  
The Individual

ABSTRACTThe insect-vectored disease malaria is a major world health problem. New control strategies are needed to supplement the current use of insecticides and medications. A genetic approach can be used to inhibit development of malaria parasites (Plasmodiumspp.) in the mosquito host. We hypothesized thatPantoea agglomerans, a bacterial symbiont ofAnophelesmosquitoes, could be engineered to express and secrete anti-Plasmodiumeffector proteins, a strategy termed paratransgenesis. To this end, plasmids that include thepelBorhlyAsecretion signals from the genes of related species (pectate lyase fromErwinia carotovoraand hemolysin A fromEscherichia coli, respectively) were created and tested for their efficacy in secreting known anti-Plasmodiumeffector proteins (SM1, anti-Pbs21, and PLA2) inP. agglomeransandE. coli.P. agglomeranssuccessfully secreted HlyA fusions of anti-Pbs21 and PLA2, and these strains are under evaluation for anti-Plasmodiumactivity in infected mosquitoes. Varied expression and/or secretion of the effector proteins was observed, suggesting that the individual characteristics of a particular effector may require empirical testing of several secretion signals. Importantly, those strains that secreted efficiently grew as well as wild-type strains under laboratory conditions and, thus, may be expected to be competitive with the native microbiota in the environment of the mosquito midgut.

scholarly journals Synthetic Enterobacterial Common Antigen (ECA) for the Development of a Universal Immunotherapy for Drug-ResistantEnterobacteriaceae

2015 ◽  
Vol 54 (37) ◽  
pp. 10953-10957 ◽  
Author(s):  
Lin Liu ◽  
Jingying Zha ◽  
Antonio DiGiandomenico ◽  
Douglas McAllister ◽  
C. Kendall Stover ◽  
...  
Keyword(s):  
Common Antigen ◽  
Enterobacterial Common Antigen

scholarly journals Regulated Delayed Expression of rfaH in an Attenuated Salmonellaenterica Serovar Typhimurium Vaccine Enhances Immunogenicity of Outer Membrane Proteins and a Heterologous Antigen

2009 ◽  
Vol 77 (12) ◽  
pp. 5572-5582 ◽  
Author(s):  
Qingke Kong ◽  
Qing Liu ◽  
Kenneth L. Roland ◽  
Roy Curtiss
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Lethal Dose ◽  
Enteric Bacteria ◽  
Lymphoid Tissues ◽  
Heterologous Antigen ◽  
O Antigen ◽  
Mutant Strains ◽  
Serovar Typhimurium

ABSTRACT RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A ΔrfaH mutant strain is attenuated in mice (50% lethal dose [LD50], >108 CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC PBAD promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the PBAD promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, ΔPrfaH178, was introduced into the attenuated vaccine strain χ9241 (ΔpabA ΔpabB ΔasdA) expressing the pneumococcal surface protein PspA from an Asd+ balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic ΔrfaH derivative or the isogenic RfaH+ parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD50 of virulent S treptococcus pneumoniae WU2. Immunization with ΔPrfaH178 mutant strains led to increased levels of protection compared to that of the parent χ9241 and of a ΔrfaH derivative of χ9241.

scholarly journals Enterobacterial common antigen.

1976 ◽  
Vol 40 (3) ◽  
pp. 591-632 ◽  
Author(s):  
P H Mäkelä ◽  
H Mayer
Keyword(s):  
Common Antigen ◽  
Enterobacterial Common Antigen
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