Reconstitution of O-specific lipopolysaccharide expression in Burkholderia cenocepacia strain J2315, which is associated with transmissible infections in patients with cystic fibrosis

Abstract

Burkholderia cenocepacia is an opportunistic bacterium that infects patients with cystic fibrosis. B. cenocepacia strains J2315, K56-2, C5424, and BC7 belong to the ET12 epidemic clone, which is transmissible among patients. We have previously shown that transposon mutants with insertions within the O antigen cluster of strain K56-2 are attenuated for survival in a rat model of lung infection. From the genomic DNA sequence of the O antigen-deficient strain J2315, we have identified an O antigen lipopolysaccharide (LPS) biosynthesis gene cluster that has an IS402 interrupting a predicted glycosyltransferase gene. A comparison with the other clonal isolates revealed that only strain K56-2, which produced O antigen and displayed serum resistance, lacked the insertion element inserted within the putative glycosyltransferase gene. We cloned the uninterrupted gene and additional flanking sequences from K56-2 and conjugated this plasmid into strains J2315, C5424, and BC7. All the exconjugants recovered the ability to form LPS O antigen. We also determined that the structure of the strain K56-2 O antigen repeat, which was absent from the LPS of strain J2315, consisted of a trisaccharide unit made of rhamnose and two N-acetylgalactosamine residues. The complexity of the gene organization of the K56-2 O antigen cluster was also investigated by reverse transcription-PCR, revealing several transcriptional units, one of which also contains genes involved in lipid A-core oligosaccharide biosynthesis.

FIG. 1.

(A) Genetic organization of a ca. 29-kb region of the ET12 genome in B. cenocepacia strains K56-2 and J2315 containing genes for core-lipid A and O antigen biosynthesis. The flanking genes ureG and apaH are indicated in black. The four genes represented with gray shading encode proteins putatively involved in lipid A-core biosynthesis. waaA, 3-deoxy-d-manno-octulosonic acid transferase; wbxY, conserved hypothetical protein; waaC, heptosyltransferase I; manB, phosphomannomutase; wzx, O antigen exporter; wbxA, glycosyltransferase; wbxB, glycosyltransferase; galE, UDP-glucose epimerase; wecA, UDP-N-acetylglucosamine 1-P transferase; wbiI, nucleotide sugar epimerase-dehydratase; wbiH, UDP-UDP-N-acetylglucosamine 1-P transferase; wbiG, nucleotide sugar epimerase-dehydratase; wbiF, glycosyltransferase; wbxC, acetyltransferase; wbxD, glycosyltransferase; wbxE, glycosyltransferase; vioA, nucleotide sugar aminotransferase; wzt, ATPase; wzm, permease; rmlDCAB, dTDP-rhamnose biosynthesis. The locations of the transposon insertions in the K56-2 mutants 33H3, 32D2, 38C2, and 34D8, as well as the location of the IS402 element in strain J2315, are indicated. The boundaries of the DNA inserts from plasmids pXO3, pXO4, and pXO7 are depicted. (B) Transcriptional organization of the LPS cluster of B. cenocepacia K56-2. Dotted lines with arrowheads indicate the transcriptional regions (regions A through F) deduced from the expected PCR products for the RT-PCR analysis (indicated by numbers 1 to 8). Asterisks indicate that no PCR product was obtained.

FIG. 2.

Electrophoretic profiles of LPS extracted from K56-2 mutants 33H3, 32D2, 38C2, and 34D8 in comparison to the profile of the parental K56-2 strain. The loading of the LPS was standardized on the basis of the number of cells used to prepare the samples. Samples were run in 14% polyacrylamide gels in a Tricine-SDS system and developed by silver staining as described in Materials and Methods. Asterisks indicate novel bands probably corresponding to core-lipid A replaced with partial O antigen subunits.

FIG. 3.

The serum sensitivity of wild-type K56-2 and mutants 33H3 and 32D2 and mutants J2315 and J2315(pXO4) was determined by incubating bacterial cultures in 40% fresh and heat-inactivated pooled human serum. The percentages of survival were calculated by comparing the growth (measured in CFU per milliliter) of each strain in heat-inactivated serum (100% survival). Bars represent the means of at least three experiments; standard errors are also indicated.

FIG. 4.

Association of O antigen production with the absence of an insertion element in the O antigen biosynthesis gene cluster of strain K56-2. (A) LPS electrophoretic profiles for B. cenocepacia ET12 strains. Samples were run in 14% polyacrylamide gels in a Tricine-SDS system and developed by silver staining. (B) PCR amplification experiment to investigate the presence of IS402 interrupting the wbxE gene on B. cenocepacia in ET12 strains. Amplified products were run in a 0.7% agarose gel. M, 1-kb DNA ladder.

FIG. 5.

LPS electrophoretic profiles for B. cenocepacia ET12 strains in the presence of the complementing plasmid pXO3 or vector control pSCRhaB3.

FIG. 6.

LPS electrophoretic profiles for B. cenocepacia ET12 strains K56-2 and J2315 in the presence of the vector control pSCRhaB3 and complementing plasmids pXO3, pXO4, and pXO7.

FIG. 7.

The GC-MS profiles of the glycosyl residues present in strain K56-2 (top panel) and strain J2315 (bottom panel). The various TMS methyl glycoside peaks are as labeled. Inositol was added to each sample as an internal control. The y axis denotes ion intensity.

FIG. 8.

The proton spectrum of the O-chain polysaccharide isolated from strain K56-2. The anomeric, N-acetyl methyl, and Rha methyl protons are indicated. The remaining glycosyl residue ring protons resonate between 3.5 and 4.7 ppm. A complete proton assignment was determined as described in the text and as given in Table 3.