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D-galactan II is an immunodominant antigen in O1 lipopolysaccharide and affects virulence in Klebsiella pneumoniae: implication in vaccine design.

Hsieh PF, Wu MC, Yang FL, Chen CT, Lou TC, Chen YY, Wu SH, Sheu JC, Wang JT - Front Microbiol (2014)

Bottom Line: Although the composition of the O1 antigen of Klebsiella was resolved more than two decades, the genetic locus involved in the biosynthesis of D-galactan II and the role of D-galactan II in bacterial pathogenesis remain unclear.Serologic analysis of K. pneumoniae clinical isolates demonstrated that D-galactan II was more prevalent in community-acquired pyogenic liver abscess (PLA)-causing strains than in non-tissue-invasive strains.Our findings indicate that both WbbY and WbbZ homologs are sufficient for the synthesis of D-galactan II.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, National Taiwan University College of Medicine Taipei, Taiwan.

ABSTRACT
In the O1 strain of Klebsiella, the lipopolysaccharide (LPS) O-antigen is composed of D-galactan I and D-galactan II. Although the composition of the O1 antigen of Klebsiella was resolved more than two decades, the genetic locus involved in the biosynthesis of D-galactan II and the role of D-galactan II in bacterial pathogenesis remain unclear. Here, we report the identification of the D-galactan II-synthesizing genes by screening a transposon mutant library of an acapsulated Klebsiella pneumoniae O1 strain with bacteriophage. K. pneumoniae strain deleted for wbbY exhibited abrogated D-galactan II production; altered serum resistance and attenuation of virulence. Serologic analysis of K. pneumoniae clinical isolates demonstrated that D-galactan II was more prevalent in community-acquired pyogenic liver abscess (PLA)-causing strains than in non-tissue-invasive strains. WbbY homologs, WbbZ homologs, and lipopolysaccharide structures based on D-galactan II also were present in several Gram-negative bacteria. Immunization of mice with the magA-mutant (K(-) 1 O1) (that is, with a LPS D-galactan II-producing strain) provided protection against infection with an O1:K2 PLA strain. Our findings indicate that both WbbY and WbbZ homologs are sufficient for the synthesis of D-galactan II. D-galactan II represents an immunodominant antigen; is conserved among multiple species of Gram-negative bacteria and could be a useful vaccine candidate.

No MeSH data available.


Related in: MedlinePlus

Analysis of WbbY and WbbZ homologs in K. pneumoniae and other Gram-negative bacteria. (A) PCR detection of wbbY (830 bp) and wbbZ (583 bp)—like genes. lane M, 1 kb DNA ladder; NC, no-template control. (B) EPS specimens were prepared from K. pneumoniae NTUH-K2044; isogenic mutants of K2044; strain A5054; the Klebsiella K72 wild type; K72 wza wzb double mutant; and E. coli O19 ab K−:H7 F8188-41. Extracts from normalized bacterial suspensions (108 CFU) were separated by SDS-PAGE and visualized by immunoblotting with anti-D-Gal II antiserum. (C) EPS extracted from the wbbY and wbbZ single-mutant and Klebsiella K72:O2 strains harboring the indicated plasmid was separated by SDS-PAGE and the LPS patterns were analyzed by immunoblotting with anti-D-Gal II antiserum. (D) Phylogenetic analysis of WbbY and WbbZ homologs from different Gram-negative bacteria using the SMART (Simple Modular Architecture Research Tool) database.
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Figure 4: Analysis of WbbY and WbbZ homologs in K. pneumoniae and other Gram-negative bacteria. (A) PCR detection of wbbY (830 bp) and wbbZ (583 bp)—like genes. lane M, 1 kb DNA ladder; NC, no-template control. (B) EPS specimens were prepared from K. pneumoniae NTUH-K2044; isogenic mutants of K2044; strain A5054; the Klebsiella K72 wild type; K72 wza wzb double mutant; and E. coli O19 ab K−:H7 F8188-41. Extracts from normalized bacterial suspensions (108 CFU) were separated by SDS-PAGE and visualized by immunoblotting with anti-D-Gal II antiserum. (C) EPS extracted from the wbbY and wbbZ single-mutant and Klebsiella K72:O2 strains harboring the indicated plasmid was separated by SDS-PAGE and the LPS patterns were analyzed by immunoblotting with anti-D-Gal II antiserum. (D) Phylogenetic analysis of WbbY and WbbZ homologs from different Gram-negative bacteria using the SMART (Simple Modular Architecture Research Tool) database.

Mentions: A previous study indicated that the E. coli O19 antigen is recognized by an anti-Klebsiella O1 monoclonal antibody (McCallum et al., 1989; Fricke et al., 2008). We therefore investigated the link between the wbbY and wbbZ genes and structures based on D-Gal II synthesis in the E. coli O19-type bacterium. Specifically, we carried out PCR analysis using primer pairs located within the wbbY and wbbZ genes. Positive signals were obtained from the acapsulated E. coli O19ab K−:H7 strain, F8188-41; the amplicons were of the same sizes as those obtained from K. pneumoniae NTUH-K2044 and A5054, which are known O1-type strains (Figure 4A). We then carried out immunoblotting analysis using the anti-D-Gal II antiserum. All wbbY-, wbbZ-positive strains, including F8188-41, showed positive signals. In addition, D-Gal II was immunologically detected in all of the wbbY and wbbZ single-mutant and the K72 harboring the F8188-41 wbbY-wbbZ-expressing plasmid strains (Figures 4B,C). These results suggested that D-Gal II is present in the LPS of the E. coli O19 group. Moreover, we also determined the DNA sequences of the wbbY and wbbZ genes from F8188-41, which were clustered on the chromosome and showed 95% DNA sequence identity compared with those from the NTUH-K2044 and another O19-type E. coli strain, SMS-3-5. The WbbY and WbbZ homologs from K. pneumoniae NTUH-K2044 and E. coli SMS-3-5 showed 99% protein sequence identity and belong to the same branch by phylogenetic tree analysis (Figure 4D); the E. coli F8188-41 homologs showed lower protein sequence similarity and sorted as a separate phylogenetic branch. To investigate the presence of WbbY and WbbZ homologs in other bacteria, we searched the NCBI database of encoded sequences from other bacterial genomes by BLASTp. WbbY and WbbZ homologs were detected in another member of the Klebsiella genus (K. oxytoca) and in four other more distantly related Gram-negative bacteria (E. coli, Erwinia piriflorinigrans, Serratia plymuthica, and Commensalibacter intestine). In each of these species, the wbbY and wbbZ gene were adjacently located on the respective chromosome and also as divergent transcription units (Table 2). These observations suggested that the D-Gal II structure described in the present study also may be present in Gram-negative bacteria of multiple genera.


D-galactan II is an immunodominant antigen in O1 lipopolysaccharide and affects virulence in Klebsiella pneumoniae: implication in vaccine design.

Hsieh PF, Wu MC, Yang FL, Chen CT, Lou TC, Chen YY, Wu SH, Sheu JC, Wang JT - Front Microbiol (2014)

Analysis of WbbY and WbbZ homologs in K. pneumoniae and other Gram-negative bacteria. (A) PCR detection of wbbY (830 bp) and wbbZ (583 bp)—like genes. lane M, 1 kb DNA ladder; NC, no-template control. (B) EPS specimens were prepared from K. pneumoniae NTUH-K2044; isogenic mutants of K2044; strain A5054; the Klebsiella K72 wild type; K72 wza wzb double mutant; and E. coli O19 ab K−:H7 F8188-41. Extracts from normalized bacterial suspensions (108 CFU) were separated by SDS-PAGE and visualized by immunoblotting with anti-D-Gal II antiserum. (C) EPS extracted from the wbbY and wbbZ single-mutant and Klebsiella K72:O2 strains harboring the indicated plasmid was separated by SDS-PAGE and the LPS patterns were analyzed by immunoblotting with anti-D-Gal II antiserum. (D) Phylogenetic analysis of WbbY and WbbZ homologs from different Gram-negative bacteria using the SMART (Simple Modular Architecture Research Tool) database.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Analysis of WbbY and WbbZ homologs in K. pneumoniae and other Gram-negative bacteria. (A) PCR detection of wbbY (830 bp) and wbbZ (583 bp)—like genes. lane M, 1 kb DNA ladder; NC, no-template control. (B) EPS specimens were prepared from K. pneumoniae NTUH-K2044; isogenic mutants of K2044; strain A5054; the Klebsiella K72 wild type; K72 wza wzb double mutant; and E. coli O19 ab K−:H7 F8188-41. Extracts from normalized bacterial suspensions (108 CFU) were separated by SDS-PAGE and visualized by immunoblotting with anti-D-Gal II antiserum. (C) EPS extracted from the wbbY and wbbZ single-mutant and Klebsiella K72:O2 strains harboring the indicated plasmid was separated by SDS-PAGE and the LPS patterns were analyzed by immunoblotting with anti-D-Gal II antiserum. (D) Phylogenetic analysis of WbbY and WbbZ homologs from different Gram-negative bacteria using the SMART (Simple Modular Architecture Research Tool) database.
Mentions: A previous study indicated that the E. coli O19 antigen is recognized by an anti-Klebsiella O1 monoclonal antibody (McCallum et al., 1989; Fricke et al., 2008). We therefore investigated the link between the wbbY and wbbZ genes and structures based on D-Gal II synthesis in the E. coli O19-type bacterium. Specifically, we carried out PCR analysis using primer pairs located within the wbbY and wbbZ genes. Positive signals were obtained from the acapsulated E. coli O19ab K−:H7 strain, F8188-41; the amplicons were of the same sizes as those obtained from K. pneumoniae NTUH-K2044 and A5054, which are known O1-type strains (Figure 4A). We then carried out immunoblotting analysis using the anti-D-Gal II antiserum. All wbbY-, wbbZ-positive strains, including F8188-41, showed positive signals. In addition, D-Gal II was immunologically detected in all of the wbbY and wbbZ single-mutant and the K72 harboring the F8188-41 wbbY-wbbZ-expressing plasmid strains (Figures 4B,C). These results suggested that D-Gal II is present in the LPS of the E. coli O19 group. Moreover, we also determined the DNA sequences of the wbbY and wbbZ genes from F8188-41, which were clustered on the chromosome and showed 95% DNA sequence identity compared with those from the NTUH-K2044 and another O19-type E. coli strain, SMS-3-5. The WbbY and WbbZ homologs from K. pneumoniae NTUH-K2044 and E. coli SMS-3-5 showed 99% protein sequence identity and belong to the same branch by phylogenetic tree analysis (Figure 4D); the E. coli F8188-41 homologs showed lower protein sequence similarity and sorted as a separate phylogenetic branch. To investigate the presence of WbbY and WbbZ homologs in other bacteria, we searched the NCBI database of encoded sequences from other bacterial genomes by BLASTp. WbbY and WbbZ homologs were detected in another member of the Klebsiella genus (K. oxytoca) and in four other more distantly related Gram-negative bacteria (E. coli, Erwinia piriflorinigrans, Serratia plymuthica, and Commensalibacter intestine). In each of these species, the wbbY and wbbZ gene were adjacently located on the respective chromosome and also as divergent transcription units (Table 2). These observations suggested that the D-Gal II structure described in the present study also may be present in Gram-negative bacteria of multiple genera.

Bottom Line: Although the composition of the O1 antigen of Klebsiella was resolved more than two decades, the genetic locus involved in the biosynthesis of D-galactan II and the role of D-galactan II in bacterial pathogenesis remain unclear.Serologic analysis of K. pneumoniae clinical isolates demonstrated that D-galactan II was more prevalent in community-acquired pyogenic liver abscess (PLA)-causing strains than in non-tissue-invasive strains.Our findings indicate that both WbbY and WbbZ homologs are sufficient for the synthesis of D-galactan II.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, National Taiwan University College of Medicine Taipei, Taiwan.

ABSTRACT
In the O1 strain of Klebsiella, the lipopolysaccharide (LPS) O-antigen is composed of D-galactan I and D-galactan II. Although the composition of the O1 antigen of Klebsiella was resolved more than two decades, the genetic locus involved in the biosynthesis of D-galactan II and the role of D-galactan II in bacterial pathogenesis remain unclear. Here, we report the identification of the D-galactan II-synthesizing genes by screening a transposon mutant library of an acapsulated Klebsiella pneumoniae O1 strain with bacteriophage. K. pneumoniae strain deleted for wbbY exhibited abrogated D-galactan II production; altered serum resistance and attenuation of virulence. Serologic analysis of K. pneumoniae clinical isolates demonstrated that D-galactan II was more prevalent in community-acquired pyogenic liver abscess (PLA)-causing strains than in non-tissue-invasive strains. WbbY homologs, WbbZ homologs, and lipopolysaccharide structures based on D-galactan II also were present in several Gram-negative bacteria. Immunization of mice with the magA-mutant (K(-) 1 O1) (that is, with a LPS D-galactan II-producing strain) provided protection against infection with an O1:K2 PLA strain. Our findings indicate that both WbbY and WbbZ homologs are sufficient for the synthesis of D-galactan II. D-galactan II represents an immunodominant antigen; is conserved among multiple species of Gram-negative bacteria and could be a useful vaccine candidate.

No MeSH data available.


Related in: MedlinePlus