Genetic and biochemical analysis of Shigella dysenteriae 1 O antigen polysaccharide biosynthesis in Escherichia coli K-12: 9 kb plasmid of S. dysenteriae 1 determines addition of a galactose residue to the lipopolysaccharide core

ABSTRACTThe outer membrane of Gram-negative bacteria presents a significant barrier for molecules entering the cell. Nevertheless, colicins, which are antimicrobial proteins secreted byEscherichia coli, can target otherE. colicells by binding to cell surface receptor proteins and activating their import, resulting in cell death. Previous studies have documented high rates of nonspecific resistance (insensitivity) of variousE. colistrains toward colicins that is independent of colicin-specific immunity and is instead associated with lipopolysaccharide (LPS) in the outer membrane. This observation poses a contradiction: why doE. colistrains have colicin-expressing plasmids, which are energetically costly to retain, if cells around them are likely to be naturally insensitive to the colicin they produce? Here, using a combination of transposon sequencing and phenotypic microarrays, we show that colicin insensitivity of uropathogenicE. colisequence type 131 (ST131) is dependent on the production of its O-antigen but that minor changes in growth conditions render the organism sensitive toward colicins. The reintroduction of O-antigen intoE. coliK-12 demonstrated that it is the density of O-antigen that is the dominant factor governing colicin insensitivity. We also show, by microscopy of fluorescently labelled colicins, that growth conditions affect the degree of occlusion by O-antigen of outer membrane receptors but not the clustered organization of receptors. The result of our study demonstrate that environmental conditions play a critical role in sensitizingE. colitoward colicins and that O-antigen in LPS is central to this role.IMPORTANCEEscherichia coliinfections can be a major health burden, especially with the organism becoming increasingly resistant to “last-resort” antibiotics such as carbapenems. Although colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent colicin insensitivity in a clinically relevant uropathogenicE. colistrain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that colicin insensitivity amongE. coliorganisms has been greatly overestimated, and as a consequence, colicins could in fact be effective species-specific antimicrobials targeting pathogenicE. colisuch as uropathogenicE. coli(UPEC).

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The lipopolysaccharide of the mastitis isolate Escherichia coli strain 1303 comprises a novel O-antigen and the rare K-12 core type

Microbiology ◽

10.1099/mic.0.046912-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1750-1760 ◽

Cited By ~ 13

Author(s):

Katarzyna A. Duda ◽

Buko Lindner ◽

Helmut Brade ◽

Andreas Leimbach ◽

Elżbieta Brzuszkiewicz ◽

...

Keyword(s):

Escherichia Coli ◽

Inner Core ◽

Bovine Mastitis ◽

Outer Core ◽

Coli Strain ◽

Core Oligosaccharide ◽

2D Nmr Spectroscopy ◽

E Coli ◽

O Antigen ◽

K 12

Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease are Escherichia coli and Staphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection. Here, we report the O-antigen (OPS, O-specific polysaccharide) structure of the LPS from bovine mastitis isolate E. coli 1303. The structure was determined utilizing chemical analyses, mass spectrometry, and 1D and 2D NMR spectroscopy methods. The O-repeating unit was characterized as -[→4)-β-d-Quip3NAc-(1→3)-α-l-Fucp2OAc-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→]- in which the O-acetyl substitution was non-stoichiometric. The nucleotide sequence of the O-antigen gene cluster of E. coli 1303 was also determined. This cluster, located between the gnd and galF genes, contains 13 putative open reading frames, most of which represent unknown nucleotide sequences that have not been described before. The O-antigen of E. coli 1303 was shown to substitute O-7 of the terminal ld-heptose of the K-12 core oligosaccharide. Interestingly, the non-OPS-substituted core oligosaccharide represented a truncated version of the K-12 outer core – namely terminal ld-heptose and glucose were missing; however, it possessed a third Kdo residue in the inner core. On the basis of structural and genetic data we show that the mastitis isolate E. coli 1303 represents a new serotype and possesses the K-12 core type, which is rather uncommon among human and bovine isolates.

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Sequence Analysis of Four Shigella boydii O-Antigen Loci: Implication for Escherichia coli andShigella Relationships

Infection and Immunity ◽

10.1128/iai.69.11.6923-6930.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 6923-6930 ◽

Cited By ~ 44

Author(s):

Lei Wang ◽

Wenjia Qu ◽

Peter R. Reeves

Keyword(s):

Escherichia Coli ◽

Gene Clusters ◽

Shigella Dysenteriae ◽

E Coli ◽

Shigella Boydii ◽

Antigen Gene ◽

O Antigen ◽

Atypical Features ◽

Serotype 5 ◽

O Antigens

ABSTRACT Shigella strains are in reality clones ofEscherichia coli and are believed to have emerged relatively recently (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567–10572, 2000). There are 33 O-antigen forms in these Shigella clones, of which 12 are identical to O antigens of other E. coli strains. We sequenced O-antigen gene clusters from Shigella boydiiserotypes 4, 5, 6, and 9 and also studied the O53- and O79-antigen gene clusters of E. coli, encoding O antigens identical to those of S. boydii serotype 4 and S. boydii serotype 5, respectively. In both cases the S. boydii and E. coli O-antigen gene clusters have the same genes and organization. The clusters of both S. boydii 6 and S. boydii 9 O antigens have atypical features, with a functional insertion sequence and a wzx gene located in the orientation opposite to that of all other genes in S. boydii serotype 9 and an rmlC gene located away from other rml genes in S. boydii serotype 6. Sequences of O-antigen gene clusters from another threeShigella clones have been published, and two of them also have abnormal structures, with either the entire cluster or one gene being located on a plasmid in Shigella sonnei orShigella dysenteriae, respectively. It appears that a high proportion of clusters coding for O antigens specific toShigella clones have atypical features, perhaps indicating recent formation of these gene clusters.

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