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Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.

Lubin JB, Lewis WG, Gilbert NM, Weimer CM, Almagro-Moreno S, Boyd EF, Lewis AL - Infect. Immun. (2015)

Bottom Line: Here we demonstrate that sialic acid-like molecules are present on the lipopolysaccharide of V. vulnificus, are required for full motility and biofilm formation, and also contribute to the organism's natural resistance to polymyxin B.Further experiments in a murine model of intravenous V. vulnificus infection demonstrated that expression of nonulosonic acids had a striking benefit for bacterial survival during bloodstream infection and dissemination to other tissues in vivo.In fact, levels of bacterial persistence in the blood corresponded to the overall levels of these molecules expressed by V. vulnificus isolates.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, USA.

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V. vulnificus LPS is modified with NulO, which contributes to polymyxin B resistance. (A) V. vulnificus LPS was analyzed by SDS-PAGE with staining of fluorescent glycans (Pro-Q Emerald 300). (B) Sensitivity to the cationic antimicrobial peptide polymyxin B was determined by measuring zones of clearance following overnight growth on plates with discs containing 100 μg of polymyxin B. ****, P < 0.0001. (C) Resistance of V. vulnificus to other antibiotics.
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Figure 2: V. vulnificus LPS is modified with NulO, which contributes to polymyxin B resistance. (A) V. vulnificus LPS was analyzed by SDS-PAGE with staining of fluorescent glycans (Pro-Q Emerald 300). (B) Sensitivity to the cationic antimicrobial peptide polymyxin B was determined by measuring zones of clearance following overnight growth on plates with discs containing 100 μg of polymyxin B. ****, P < 0.0001. (C) Resistance of V. vulnificus to other antibiotics.

Mentions: The LPS O antigen is a frequent site of NulO modification in Gram-negative organisms (16, 29–31). To investigate whether V. vulnificus LPS contains NulO residues, LPS was isolated from the WT and Δnab2 strains. Polyacrylamide gel electrophoresis followed by glycan visualization using the Pro-Q Emerald stain demonstrated a lower-molecular-weight band in the Δnab2 strains than in the WT strain (Fig. 2A), suggesting a truncation of LPS. The higher-molecular-weight band seen with WT LPS was partially restored in the Δnab2/pnab2 strain (Fig. 2A). This finding is consistent with the intermediate levels of NulO measured in the Δnab2/pnab2 complemented strain (Fig. 1C). The material used for electrophoresis was also subjected to fluorescence derivatization and HPLC analysis, verifying that NulOs were present in the LPS preparations (data not shown). In contrast, HPLC of flagellum subunits from isolated SDS-PAGE bands did not yield evidence of the presence of NulOs, despite the fact that positive controls analyzed in parallel (bacterial species known to produce glycosylated flagella) gave the expected result (data not shown). We note that a more sensitive detection method may be required to completely rule out the possibility of direct glycosylation of flagellar proteins in V. vulnificus.


Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.

Lubin JB, Lewis WG, Gilbert NM, Weimer CM, Almagro-Moreno S, Boyd EF, Lewis AL - Infect. Immun. (2015)

V. vulnificus LPS is modified with NulO, which contributes to polymyxin B resistance. (A) V. vulnificus LPS was analyzed by SDS-PAGE with staining of fluorescent glycans (Pro-Q Emerald 300). (B) Sensitivity to the cationic antimicrobial peptide polymyxin B was determined by measuring zones of clearance following overnight growth on plates with discs containing 100 μg of polymyxin B. ****, P < 0.0001. (C) Resistance of V. vulnificus to other antibiotics.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496609&req=5

Figure 2: V. vulnificus LPS is modified with NulO, which contributes to polymyxin B resistance. (A) V. vulnificus LPS was analyzed by SDS-PAGE with staining of fluorescent glycans (Pro-Q Emerald 300). (B) Sensitivity to the cationic antimicrobial peptide polymyxin B was determined by measuring zones of clearance following overnight growth on plates with discs containing 100 μg of polymyxin B. ****, P < 0.0001. (C) Resistance of V. vulnificus to other antibiotics.
Mentions: The LPS O antigen is a frequent site of NulO modification in Gram-negative organisms (16, 29–31). To investigate whether V. vulnificus LPS contains NulO residues, LPS was isolated from the WT and Δnab2 strains. Polyacrylamide gel electrophoresis followed by glycan visualization using the Pro-Q Emerald stain demonstrated a lower-molecular-weight band in the Δnab2 strains than in the WT strain (Fig. 2A), suggesting a truncation of LPS. The higher-molecular-weight band seen with WT LPS was partially restored in the Δnab2/pnab2 strain (Fig. 2A). This finding is consistent with the intermediate levels of NulO measured in the Δnab2/pnab2 complemented strain (Fig. 1C). The material used for electrophoresis was also subjected to fluorescence derivatization and HPLC analysis, verifying that NulOs were present in the LPS preparations (data not shown). In contrast, HPLC of flagellum subunits from isolated SDS-PAGE bands did not yield evidence of the presence of NulOs, despite the fact that positive controls analyzed in parallel (bacterial species known to produce glycosylated flagella) gave the expected result (data not shown). We note that a more sensitive detection method may be required to completely rule out the possibility of direct glycosylation of flagellar proteins in V. vulnificus.

Bottom Line: Here we demonstrate that sialic acid-like molecules are present on the lipopolysaccharide of V. vulnificus, are required for full motility and biofilm formation, and also contribute to the organism's natural resistance to polymyxin B.Further experiments in a murine model of intravenous V. vulnificus infection demonstrated that expression of nonulosonic acids had a striking benefit for bacterial survival during bloodstream infection and dissemination to other tissues in vivo.In fact, levels of bacterial persistence in the blood corresponded to the overall levels of these molecules expressed by V. vulnificus isolates.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, USA.

Show MeSH
Related in: MedlinePlus