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Characterization of the algC gene expression pattern in the multidrug resistant Acinetobacter baumannii AIIMS 7 and correlation with biofilm development on abiotic surface.

Sahu PK, Iyer PS, Barage SH, Sonawane KD, Chopade BA - ScientificWorldJournal (2014)

Bottom Line: Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05).Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h).Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances).

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411 007, India ; Ispat General Hospital, SAIL, Rourkela 769 005, India.

ABSTRACT
Relative quantification of algC gene expression was evaluated in the multidrug resistant strain Acinetobacter baumannii AIIMS 7 biofilm (3 to 96 h, on polystyrene surface) compared to the planktonic counterparts. Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05). Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h). Cloning, heterologous expression, and bioinformatics analyses indicated algC gene product as the bifunctional enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) of ∼ 53 kDa size, which augmented biofilms significantly in algC clones compared to controls (lacking algC gene), further localized by scanning electron microscopy. Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances). Our observation on differential expression pattern of algC having strong correlation with important biofilm stages, scanning electron-microscopic evidence of biofilm augmentation taken together with predictive enzyme functions via molecular dynamic (MD) simulation, proposes a new basis of A. baumannii AIIMS 7 biofilm development on inanimate surfaces.

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(a)–(d) Molecular modeling of PMM/PGM from A. baumannii AIIMS 7. (a) Four sequential domains of enzyme PMM/PGM after MD simulation; (b) central spherical cavity showing solvent accessible surface area (orange). (c) Superimposed structures before MD (cyan) and after MD simulation (magenta); (d) Mg2+ interacting residues of PMM/PGM enzyme.
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fig7: (a)–(d) Molecular modeling of PMM/PGM from A. baumannii AIIMS 7. (a) Four sequential domains of enzyme PMM/PGM after MD simulation; (b) central spherical cavity showing solvent accessible surface area (orange). (c) Superimposed structures before MD (cyan) and after MD simulation (magenta); (d) Mg2+ interacting residues of PMM/PGM enzyme.

Mentions: To predict the functional association between PMM/PGM enzyme (encoded by algC gene) resulting in alginate/LPS core mediated biofilm formation in A. baumannii, molecular dynamics (MD) simulations up to 10 ns were carried out on homology model of PMM/PGM of A. baumannii AIIMS 7 using GROMACS v4.0.4 program. The generated PMM/PGM model of A. baumannii AIIMS 7 contained four domains of equal size arranged in “heart shaped” manner (Figure 7(a)) which form a compact structure, similar to P. aeruginosa [42]. The polypeptide chain proceeds through each domain sequentially (Figure 7(a)) forming heart shaped geometry. Sequence alignments (Supplementary Figure S4) and model building study showed that the active site was located at the centre of the two domains (domains 1 and 2) in a deep cleft formed by Ser104, Asp244, Asp246, and Asp248. PMM/PGM sequence of A. baumannii AIIMS 7 showed conserved sequence motif in domain 3 from residues 327-331 (GEYAGH) which would act as a sugar binding site. A cluster of positively charged conserved residues found in domain 3 (Lys287) and domain 4 (Arg427, Arg438) could be involved in phosphate binding. The cleft showed solvent accessible surface (SA, Richards' surface) and molecular surface (MS, Connolly's surface) areas as calculated by alpha shape method. The enzyme showed a total cavity of 3641.5 Å and spherical central cavity of 1878.13 Å where substrate could bind as shown in Figure 7(b). To compare the 3D structures obtained before and after MD simulations, structural superpositions were made using PDBeFOLD, results of which showed root mean square deviation (RMSD) of two aligned structures within the range of 2.51 Å (Figure 7(c)). Metal ion Mg2+ interacts with Asp244, Asp246, Asp248, and Ser104 residues (Figure 7(d); Supplementary Table S1). The metal ion Mg2+ is required for enzymatic activity of PMM/PGM of A. baumannii AIIMS 7 (Figure 7(d)), rather than Mn2+ and Zn2+. Specific interactions between metal ion (Mg2+) and protein, water-mediated H-bonds (so-called water bridges), and hydrophobic (Lennard-Jones) interactions have been identified and are listed (Supplementary Table S1).


Characterization of the algC gene expression pattern in the multidrug resistant Acinetobacter baumannii AIIMS 7 and correlation with biofilm development on abiotic surface.

Sahu PK, Iyer PS, Barage SH, Sonawane KD, Chopade BA - ScientificWorldJournal (2014)

(a)–(d) Molecular modeling of PMM/PGM from A. baumannii AIIMS 7. (a) Four sequential domains of enzyme PMM/PGM after MD simulation; (b) central spherical cavity showing solvent accessible surface area (orange). (c) Superimposed structures before MD (cyan) and after MD simulation (magenta); (d) Mg2+ interacting residues of PMM/PGM enzyme.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4269089&req=5

fig7: (a)–(d) Molecular modeling of PMM/PGM from A. baumannii AIIMS 7. (a) Four sequential domains of enzyme PMM/PGM after MD simulation; (b) central spherical cavity showing solvent accessible surface area (orange). (c) Superimposed structures before MD (cyan) and after MD simulation (magenta); (d) Mg2+ interacting residues of PMM/PGM enzyme.
Mentions: To predict the functional association between PMM/PGM enzyme (encoded by algC gene) resulting in alginate/LPS core mediated biofilm formation in A. baumannii, molecular dynamics (MD) simulations up to 10 ns were carried out on homology model of PMM/PGM of A. baumannii AIIMS 7 using GROMACS v4.0.4 program. The generated PMM/PGM model of A. baumannii AIIMS 7 contained four domains of equal size arranged in “heart shaped” manner (Figure 7(a)) which form a compact structure, similar to P. aeruginosa [42]. The polypeptide chain proceeds through each domain sequentially (Figure 7(a)) forming heart shaped geometry. Sequence alignments (Supplementary Figure S4) and model building study showed that the active site was located at the centre of the two domains (domains 1 and 2) in a deep cleft formed by Ser104, Asp244, Asp246, and Asp248. PMM/PGM sequence of A. baumannii AIIMS 7 showed conserved sequence motif in domain 3 from residues 327-331 (GEYAGH) which would act as a sugar binding site. A cluster of positively charged conserved residues found in domain 3 (Lys287) and domain 4 (Arg427, Arg438) could be involved in phosphate binding. The cleft showed solvent accessible surface (SA, Richards' surface) and molecular surface (MS, Connolly's surface) areas as calculated by alpha shape method. The enzyme showed a total cavity of 3641.5 Å and spherical central cavity of 1878.13 Å where substrate could bind as shown in Figure 7(b). To compare the 3D structures obtained before and after MD simulations, structural superpositions were made using PDBeFOLD, results of which showed root mean square deviation (RMSD) of two aligned structures within the range of 2.51 Å (Figure 7(c)). Metal ion Mg2+ interacts with Asp244, Asp246, Asp248, and Ser104 residues (Figure 7(d); Supplementary Table S1). The metal ion Mg2+ is required for enzymatic activity of PMM/PGM of A. baumannii AIIMS 7 (Figure 7(d)), rather than Mn2+ and Zn2+. Specific interactions between metal ion (Mg2+) and protein, water-mediated H-bonds (so-called water bridges), and hydrophobic (Lennard-Jones) interactions have been identified and are listed (Supplementary Table S1).

Bottom Line: Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05).Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h).Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances).

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411 007, India ; Ispat General Hospital, SAIL, Rourkela 769 005, India.

ABSTRACT
Relative quantification of algC gene expression was evaluated in the multidrug resistant strain Acinetobacter baumannii AIIMS 7 biofilm (3 to 96 h, on polystyrene surface) compared to the planktonic counterparts. Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05). Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h). Cloning, heterologous expression, and bioinformatics analyses indicated algC gene product as the bifunctional enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) of ∼ 53 kDa size, which augmented biofilms significantly in algC clones compared to controls (lacking algC gene), further localized by scanning electron microscopy. Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances). Our observation on differential expression pattern of algC having strong correlation with important biofilm stages, scanning electron-microscopic evidence of biofilm augmentation taken together with predictive enzyme functions via molecular dynamic (MD) simulation, proposes a new basis of A. baumannii AIIMS 7 biofilm development on inanimate surfaces.

Show MeSH
Related in: MedlinePlus