Capsular Polysaccharides and Lipopolysaccharides from Two Strains of Bacteroides fragilis

ABSTRACT Bacteroides fragilis produces a capsular polysaccharide complex (CPC) that is directly involved in its ability to induce abscesses. Two distinct capsular polysaccharides, polysaccharide A (PS A) and PS B, have been shown to be synthesized by the prototype strain for the study of abscesses, NCTC9343. Both of these polysaccharides in purified form induce abscesses in animal models. In this study, we demonstrate that the CPC of NCTC9343 is composed of at least three distinct capsular polysaccharides: PS A, PS B, and PS C. A previously described locus contains genes whose products are involved in the biosynthesis of PS C rather than PS B as was originally suggested. The actual PS B biosynthesis locus was cloned, sequenced, and found to contain 22 genes in an operon-type structure. A mutant with a large chromosomal deletion of the PS B biosynthesis locus was created so that the contribution of PS B to the formation of abscesses could be assessed in a rodent model. Although purified PS B can induce abscesses, removal of this polysaccharide does not attenuate the organism's ability to induce abscesses.

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Expression of a Uniquely Regulated Extracellular Polysaccharide Confers a Large-Capsule Phenotype to Bacteroides fragilis

Journal of Bacteriology ◽

10.1128/jb.01519-07 ◽

2007 ◽

Vol 190 (3) ◽

pp. 1020-1026 ◽

Cited By ~ 22

Author(s):

Maria Chatzidaki-Livanis ◽

Michael J. Coyne ◽

Hazeline Roche-Hakansson ◽

Laurie E. Comstock

Keyword(s):

Bacteroides Fragilis ◽

Extracellular Polysaccharide ◽

Inverted Repeats ◽

Deletion Mutants ◽

Capsular Polysaccharides ◽

Phase Variable ◽

Dna Inversion ◽

Percoll Density Gradient ◽

Active Processes ◽

Polysaccharide Layer

ABSTRACT Bacteroides fragilis synthesizes eight distinct capsular polysaccharides, more than any described bacterium outside the order Bacteroidales. Here, we show that this organism also produces a high-molecular-weight extracellular polysaccharide (EPS). Expression of the EPS results in the formation of a large polysaccharide layer around the bacteria which prevents them from forming a tight pellet upon centrifugation and from entering a Percoll density gradient. Like expression of the capsular polysaccharides, expression of the EPS is phase variable and dictated by DNA inversion of its promoter. EPS expression is regulated at one level by the DNA invertase Tsr19, which is encoded by a gene immediately upstream of the EPS locus and inverts the EPS promoter, causing an on or off phenotype. Expression of the EPS is also regulated at another level, which dictates the amount of EPS produced. By analyzing a panel of tsr19 deletion mutants, we found that the number of inverted repeats (IRs) flanking the promoter is variable. Transcription into the EPS genes is greater in mutants with a single IR between the promoter and the downstream EPS genes than in mutants with more than one IR in this region, correlating with the synthesis of more EPS. By analyzing the relative orientations of the EPS promoter of bacteria obtained from human fecal samples, we showed that both DNA inversion and variation in the number of IRs are active processes of B. fragilis in the endogenous human intestinal ecosystem.

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Genetic Diversity of the Capsular Polysaccharide C Biosynthesis Region of Bacteroides fragilis

Infection and Immunity ◽

10.1128/iai.68.11.6182-6188.2000 ◽

2000 ◽

Vol 68 (11) ◽

pp. 6182-6188 ◽

Cited By ~ 12

Author(s):

Laurie E. Comstock ◽

Annalisa Pantosti ◽

Dennis L. Kasper

Keyword(s):

Genetic Diversity ◽

Capsular Polysaccharide ◽

Bacteroides Fragilis ◽

Hybridization Analysis ◽

Clinical Isolates ◽

Pcr Analysis ◽

Capsular Polysaccharides ◽

Genetic Approach ◽

Polysaccharide Biosynthesis ◽

Putative Aminotransferase

ABSTRACT A genetic approach was used to assess the heterogeneity of the capsular polysaccharide C (PS C) biosynthesis locus ofBacteroides fragilis and to determine whether distinct loci contain genes whose products are likely to be involved in conferring charged groups that enable the B. fragilis capsular polysaccharides to induce abscesses. A collection of 50 B. fragilis strains was examined. PCR analysis demonstrated that the genes flanking the PS C biosynthesis region are conserved, whereas the genes within the loci are heterogeneous. OnlycfiA + B. fragilis strains, which represent 3% of the clinical isolates of B. fragilis, displayed heterogeneity in the regions flanking the polysaccharide biosynthesis genes. Primers were designed in the conserved regions upstream and downstream of the PS C locus and were used to amplify the region from 45 of the 50 B. fragilis strains studied. Fourteen PS C genetic loci could be differentiated by a combination of PCR and extended PCR. These loci ranged in size from 14 to 26 kb. Hybridization analysis with genes from the PS C loci of strains 9343 and 638R revealed that the majority of strains contain homologs ofwcgC (N-acetylmannosamine dehydrogenase),wcfF (putative dehydrogenase), and wcgP(putative aminotransferase). The data suggest that the synthesis of polysaccharides that have zwitterionic characteristics rendering them able to induce abscesses is common in B. fragilis.

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Structure and Function of Bacteroides fragilis Capsular Polysaccharides: Relationship to Induction and Prevention of Abscesses

Clinical Infectious Diseases ◽

10.1093/clinids/20.supplement_2.s132 ◽

1995 ◽

Vol 20 (Supplement_2) ◽

pp. S132-S140 ◽

Cited By ~ 25

Author(s):

Arthur O. Tzianabos ◽

Dennis L. Kasper ◽

Andrew B. Onderdonk

Keyword(s):

Bacteroides Fragilis ◽

Structure And Function ◽

Capsular Polysaccharides ◽

And Function

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Analysis of a Capsular Polysaccharide Biosynthesis Locus of Bacteroides fragilis

Infection and Immunity ◽

10.1128/iai.67.7.3525-3532.1999 ◽

1999 ◽

Vol 67 (7) ◽

pp. 3525-3532 ◽

Cited By ~ 40

Author(s):

Laurie E. Comstock ◽

Michael J. Coyne ◽

Arthur O. Tzianabos ◽

Annalisa Pantosti ◽

Andrew B. Onderdonk ◽

...

Keyword(s):

Capsular Polysaccharide ◽

Bacteroides Fragilis ◽

Open Reading Frames ◽

Capsular Polysaccharides ◽

Nucleotide Sugar ◽

Polysaccharide Biosynthesis ◽

Abdominal Abscesses ◽

Intra Abdominal Infection ◽

Reading Frames ◽

Nucleotide Sugar Biosynthesis

ABSTRACT A major clinical manifestation of infection with Bacteroides fragilis is the formation of intra-abdominal abscesses, which are induced by the capsular polysaccharides of this organism. Transposon mutagenesis was used to locate genes involved in the synthesis of capsular polysaccharides. A 24,454-bp region was sequenced and found to contain a 15,379-bp locus (designated wcf) with 16 open reading frames (ORFs) encoding products similar to those encoded by genes of other bacterial polysaccharide biosynthesis loci. Four genes encode products that are similar to enzymes involved in nucleotide sugar biosynthesis. Seven genes encode products that are similar to sugar transferases. Two gene products are similar toO-acetyltransferases, and two products are probably involved in polysaccharide transport and polymerization. The product of one ORF, WcfH, is similar to a set of deacetylases of the NodB family. Deletion mutants demonstrated that the wcf locus is necessary for the synthesis of polysaccharide B, one of the two capsular polysaccharides of B. fragilis 9343. The virulence of the polysaccharide B-deficient mutant was comparable to that of the wild type in terms of its ability to induce abscesses in a rat model of intra-abdominal infection.

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Structural elucidation of two capsular polysaccharides from one strain of Bacteroides fragilis using high-resolution NMR spectroscopy

Biochemistry ◽

10.1021/bi00131a026 ◽

1992 ◽

Vol 31 (16) ◽

pp. 4081-4089 ◽

Cited By ~ 133

Author(s):

H. Baumann ◽

A. O. Tzianabos ◽

J. R. Brisson ◽

D. L. Kasper ◽

H. J. Jennings

Keyword(s):

Nmr Spectroscopy ◽

High Resolution ◽

Bacteroides Fragilis ◽

Structural Elucidation ◽

Capsular Polysaccharides ◽

High Resolution Nmr

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A Family of Transcriptional Antitermination Factors Necessary for Synthesis of the Capsular Polysaccharides of Bacteroides fragilis

Journal of Bacteriology ◽

10.1128/jb.00500-09 ◽

2009 ◽

Vol 191 (23) ◽

pp. 7288-7295 ◽

Cited By ~ 32

Author(s):

Maria Chatzidaki-Livanis ◽

Michael J. Coyne ◽

Laurie E. Comstock

Keyword(s):

Mutational Analysis ◽

Bacteroides Fragilis ◽

Small Region ◽

Capsular Polysaccharides ◽

Single Strain ◽

Conserved Residues ◽

Polysaccharide Biosynthesis ◽

Transcriptional Termination ◽

Amino Acid Segment ◽

By Products

ABSTRACT A single strain of Bacteroides fragilis synthesizes eight distinct capsular polysaccharides, designated PSA to PSH. These polysaccharides are synthesized by-products encoded by eight separate polysaccharide biosynthesis loci. The genetic architecture of each of these eight loci is similar, including the fact that the first gene of each locus is a paralog of the first gene of each of the other PS loci. These proteins are designated the UpxY family, where x is replaced by a to h, depending upon the polysaccharide locus from which it is produced. Mutational analysis of three separate upxY genes demonstrated that they are necessary and specific for transcription of their respective polysaccharide biosynthesis operon and that they function in trans. Transcriptional reporter constructs, reverse transcriptase PCR, and deletion analysis demonstrated that the UpxYs do not affect initiation of transcription, but rather prevent premature transcriptional termination within the 5′ untranslated region between the promoter and the upxY gene. The UpxYs have conserved motifs that are present in NusG and NusG-like proteins. Mutation of two conserved residues within the conserved KOW motif abrogated UpaY activity, further confirming that these proteins belong to the NusG-like (NusGSP) family. Alignment of highly similar UpxYs led to the identification of a small region of these proteins predicted to confer specificity for their respective loci. Construction of an upaY-upeY hybrid that produced a protein in which a 17-amino-acid segment of UpaY was changed to that of UpeY altered UpaY's specificity, as it was now able to function in transcriptional antitermination of the PSE biosynthesis operon.

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