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Characterisation of pellicles formed by Acinetobacter baumannii at the air-liquid interface.

Nait Chabane Y, Marti S, Rihouey C, Alexandre S, Hardouin J, Lesouhaitier O, Vila J, Kaplan JB, Jouenne T, Dé E - PLoS ONE (2014)

Bottom Line: Dispersin B, an enzyme that hydrolyzes poly-N-acetylglucosamine (PNAG) polysaccharide, inhibited A. baumannii pellicle formation, suggesting that this exopolysaccharide contributes to pellicle formation.Also associated with the pellicle matrix were three subunits of pili assembled by chaperon-usher systems: the major CsuA/B, A1S_1510 (presented 45% of identity with the main pilin F17-A from enterotoxigenic Escherichia coli pili) and A1S_2091.The presence of both PNAG polysaccharide and pili systems in matrix of pellicles might contribute to the virulence of this emerging pathogen.

View Article: PubMed Central - PubMed

Affiliation: Unité Mixte de Recherche 6270 CNRS - Laboratory "Polymères, Biopolymères, Surfaces", University of Rouen, Mont-Saint-Aignan, France.

ABSTRACT
The clinical importance of Acinetobacter baumannii is partly due to its natural ability to survive in the hospital environment. This persistence may be explained by its capacity to form biofilms and, interestingly, A. baumannii can form pellicles at the air-liquid interface more readily than other less pathogenic Acinetobacter species. Pellicles from twenty-six strains were morphologically classified into three groups: I) egg-shaped (27%); II) ball-shaped (50%); and III) irregular pellicles (23%). One strain representative of each group was further analysed by Brewster's Angle Microscopy to follow pellicle development, demonstrating that their formation did not require anchoring to a solid surface. Total carbohydrate analysis of the matrix showed three main components: Glucose, GlcNAc and Kdo. Dispersin B, an enzyme that hydrolyzes poly-N-acetylglucosamine (PNAG) polysaccharide, inhibited A. baumannii pellicle formation, suggesting that this exopolysaccharide contributes to pellicle formation. Also associated with the pellicle matrix were three subunits of pili assembled by chaperon-usher systems: the major CsuA/B, A1S_1510 (presented 45% of identity with the main pilin F17-A from enterotoxigenic Escherichia coli pili) and A1S_2091. The presence of both PNAG polysaccharide and pili systems in matrix of pellicles might contribute to the virulence of this emerging pathogen.

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Matrix associated proteins of the pellicles formed by each representative strain of the three morphotypes.A) separated and visualized by SDS-PAGE and silver nitrate staining respectively, after treatment with 0.2% cellulase. B) Histogram giving relative abundances of proteins calculated according to the minimum-maximum normalization method from 3 biological replicates and 2 technical replicates. Error bars represent standard error with a 95% confidence interval.
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pone-0111660-g006: Matrix associated proteins of the pellicles formed by each representative strain of the three morphotypes.A) separated and visualized by SDS-PAGE and silver nitrate staining respectively, after treatment with 0.2% cellulase. B) Histogram giving relative abundances of proteins calculated according to the minimum-maximum normalization method from 3 biological replicates and 2 technical replicates. Error bars represent standard error with a 95% confidence interval.

Mentions: Proteins embedded within the pellicle matrix were difficult to analyse due to the high amount of polysaccharides interfering with migration. Consequently, purified matrices were treated with cellulase previous to SDS-PAGE separation. No high molecular weight proteins were detected associated to the pellicle matrix but different low molecular weight proteins were identified in the samples from each morphogroup (Figure 6 & Table S2). Three proteic bands (located at about 15 kDa) were cut then digested and analysed by LC-MS/MS. The most abundant proteins were identified as pilins. As showed in the Figure 6 (and emphasized by psm values, see Table S2), CsuA/B (A1S_2218) was the most abundant protein in all matrices. This protein as well as CsuA (A1S_2217) and CsuB (A1S_2216), are secreted subunits of the CsuA/BABCDE chaperon-usher pili assembly system, which has been described to be involved in the attachment and biofilm formation of A. baumannii on abiotic surfaces [16]. Another identified pilin was the protein A1S_2091, which could be the major subunit of a chaperone–usher pili assembly (CU) system consisting in 4 proteins (A1S_2088-2091, reversed ORFs) in which A1S_2090 would be the assembly chaperone and A1S_2089 the fimbrial usher. After a sequence analysis and a BLASTP in the data bank, we found this CU system extremely well conserved in A. baumannii (>90% identity except in ACICU strain in which it was absent), and in the other Acinetobacter species, i.e. A. pittii, A. nosocomialis and A. calcoaceticus (>80% identity, see Table S3). Finally, the subunit coded by A1S_1507 gene was also identified as part of an operon coding for a last A1S_1510-1507 CU system, (again reversed ORFs in ATCC 17978). This CU system is constituted by the pilin FimA (A1S_1510 sometimes annotated F17-A protein in A. baumannii genomes), then a PapD chaperone (A1S_1509), a PapC porin (A1S_1508) and the tip adhesion pilin could be the A1S_1507 protein. This operon has already been shown to be involved in biofilm formation on solid support [34]. As shown by the Table S4, this operon is also well conserved in A. baumannii (except in AYE strain where the operon showed the insertion of an ISAbaI in A1S_1508 usher protein), and was found in ACB complex as well as in A. junii.


Characterisation of pellicles formed by Acinetobacter baumannii at the air-liquid interface.

Nait Chabane Y, Marti S, Rihouey C, Alexandre S, Hardouin J, Lesouhaitier O, Vila J, Kaplan JB, Jouenne T, Dé E - PLoS ONE (2014)

Matrix associated proteins of the pellicles formed by each representative strain of the three morphotypes.A) separated and visualized by SDS-PAGE and silver nitrate staining respectively, after treatment with 0.2% cellulase. B) Histogram giving relative abundances of proteins calculated according to the minimum-maximum normalization method from 3 biological replicates and 2 technical replicates. Error bars represent standard error with a 95% confidence interval.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216135&req=5

pone-0111660-g006: Matrix associated proteins of the pellicles formed by each representative strain of the three morphotypes.A) separated and visualized by SDS-PAGE and silver nitrate staining respectively, after treatment with 0.2% cellulase. B) Histogram giving relative abundances of proteins calculated according to the minimum-maximum normalization method from 3 biological replicates and 2 technical replicates. Error bars represent standard error with a 95% confidence interval.
Mentions: Proteins embedded within the pellicle matrix were difficult to analyse due to the high amount of polysaccharides interfering with migration. Consequently, purified matrices were treated with cellulase previous to SDS-PAGE separation. No high molecular weight proteins were detected associated to the pellicle matrix but different low molecular weight proteins were identified in the samples from each morphogroup (Figure 6 & Table S2). Three proteic bands (located at about 15 kDa) were cut then digested and analysed by LC-MS/MS. The most abundant proteins were identified as pilins. As showed in the Figure 6 (and emphasized by psm values, see Table S2), CsuA/B (A1S_2218) was the most abundant protein in all matrices. This protein as well as CsuA (A1S_2217) and CsuB (A1S_2216), are secreted subunits of the CsuA/BABCDE chaperon-usher pili assembly system, which has been described to be involved in the attachment and biofilm formation of A. baumannii on abiotic surfaces [16]. Another identified pilin was the protein A1S_2091, which could be the major subunit of a chaperone–usher pili assembly (CU) system consisting in 4 proteins (A1S_2088-2091, reversed ORFs) in which A1S_2090 would be the assembly chaperone and A1S_2089 the fimbrial usher. After a sequence analysis and a BLASTP in the data bank, we found this CU system extremely well conserved in A. baumannii (>90% identity except in ACICU strain in which it was absent), and in the other Acinetobacter species, i.e. A. pittii, A. nosocomialis and A. calcoaceticus (>80% identity, see Table S3). Finally, the subunit coded by A1S_1507 gene was also identified as part of an operon coding for a last A1S_1510-1507 CU system, (again reversed ORFs in ATCC 17978). This CU system is constituted by the pilin FimA (A1S_1510 sometimes annotated F17-A protein in A. baumannii genomes), then a PapD chaperone (A1S_1509), a PapC porin (A1S_1508) and the tip adhesion pilin could be the A1S_1507 protein. This operon has already been shown to be involved in biofilm formation on solid support [34]. As shown by the Table S4, this operon is also well conserved in A. baumannii (except in AYE strain where the operon showed the insertion of an ISAbaI in A1S_1508 usher protein), and was found in ACB complex as well as in A. junii.

Bottom Line: Dispersin B, an enzyme that hydrolyzes poly-N-acetylglucosamine (PNAG) polysaccharide, inhibited A. baumannii pellicle formation, suggesting that this exopolysaccharide contributes to pellicle formation.Also associated with the pellicle matrix were three subunits of pili assembled by chaperon-usher systems: the major CsuA/B, A1S_1510 (presented 45% of identity with the main pilin F17-A from enterotoxigenic Escherichia coli pili) and A1S_2091.The presence of both PNAG polysaccharide and pili systems in matrix of pellicles might contribute to the virulence of this emerging pathogen.

View Article: PubMed Central - PubMed

Affiliation: Unité Mixte de Recherche 6270 CNRS - Laboratory "Polymères, Biopolymères, Surfaces", University of Rouen, Mont-Saint-Aignan, France.

ABSTRACT
The clinical importance of Acinetobacter baumannii is partly due to its natural ability to survive in the hospital environment. This persistence may be explained by its capacity to form biofilms and, interestingly, A. baumannii can form pellicles at the air-liquid interface more readily than other less pathogenic Acinetobacter species. Pellicles from twenty-six strains were morphologically classified into three groups: I) egg-shaped (27%); II) ball-shaped (50%); and III) irregular pellicles (23%). One strain representative of each group was further analysed by Brewster's Angle Microscopy to follow pellicle development, demonstrating that their formation did not require anchoring to a solid surface. Total carbohydrate analysis of the matrix showed three main components: Glucose, GlcNAc and Kdo. Dispersin B, an enzyme that hydrolyzes poly-N-acetylglucosamine (PNAG) polysaccharide, inhibited A. baumannii pellicle formation, suggesting that this exopolysaccharide contributes to pellicle formation. Also associated with the pellicle matrix were three subunits of pili assembled by chaperon-usher systems: the major CsuA/B, A1S_1510 (presented 45% of identity with the main pilin F17-A from enterotoxigenic Escherichia coli pili) and A1S_2091. The presence of both PNAG polysaccharide and pili systems in matrix of pellicles might contribute to the virulence of this emerging pathogen.

Show MeSH
Related in: MedlinePlus