Poly- β -(1,6)- N -acetylglucosamine (PNAG), the chief mediator of intercellular adhesion in many bacteria, plays an important role in biofilm formation. The pga ABCD locus was recognized from the whole genome sequence of A. junii SH205. The enzyme glycosyltransferase, PgaC, catalyzes the production of PNAG with N-acetyl-D-glucosamine monomer. In this study, the possibility of PNAG biosynthesis in A. junii SH205 with its own PgaC was explored with the aid of bioinformatics. Multiple alignments of PgaC sequences of different bacteria were used to identify conserved amino acid residues that might be critical for the functioning of the protein. Three-dimensional model of A. junii SH205 PgaC was generated for spatial visualization of amino acid residues. The analyses have shown that the protein PgaC has five conserved amino acids, Asp 140 , Asp 233 , Gln 269 , Arg 272 and Trp 273 , critical for the activity of enzyme. Interaction of UDP- N -acetylglucosamine within the conserved pocket of glycosyltransferase was explored from molecular docking studies.
The genus Acinetobacter belongs to subclass γ-Proteobacteria, family Moraxellaceae, and comprises Gram- staining-negative, strictly aerobic, catalase-positive, non-motile, oxidase-negative, glucose non-fermenting bacteria with a guanine plus cytosine content of 39% - 47%. They are ubiquitous in nature, found in soil and water [
The whole genome sequence of A. junii SH20534 [
The sequence of the translated product of genetic loci similar to pgaC of 12 virulent species from different taxa like Acinetobacter, Escherichia, Staphylococcus, Yersinia, Klebsiella and Chromobacterium have been compared with that of A. junii SH205 using the EMBOSS-Needle program which performs global pairwise sequence alignment based of N-W dynamic programming algorithm (www.ebi.ac.uk/Tools/psa/emboss_needle/). All the thirteen protein sequences of the organisms (
The location and conformation of the conserved amino acids in the enzyme glycosyl transferase (product of pgaC) is important to understand the role of these amino acid residues in the synthesis of PNAG important for the formation the biofilm. This necessitates the prediction of its structure in the absence of any experimental model for the enzyme from A. junii. I-TASSER server [
Molecular docking of glycosyl transferase (homologous to PgaC protein) from A. junii SH205, was performed with the ligand; UDP-GlcNAc (UDP-N-acetylglucosamine). The GUI program of Auto Dock 4 suit [
Species for which the relation between pgaC and virulence is experimentally established | UniProt-KB accession number | Identity and Similarity with A. junii SH205 (UniProt-KB Accn No. D0SLZ4) | |
---|---|---|---|
Identity (%) | Similarity (%) | ||
Yersinia pestis bv. Antiqua (strain Angola) | A9R8A3 | 50.9 | 68.8 |
Acinetobacter calcoaceticus (strain PHEA-2) | F0KG89 | 50.7 | 68.4 |
Escherichia coli (strain K12) | P75905 | 50.7 | 68.2 |
Acinetobacter baumannii | C8YYH7 | 65.3 | 79.7 |
Acinetobacter baumannii NCGM 237 | U3TAR9 | 50.7 | 68.6 |
Acinetobacter baumannii (strain AYE) | B0V7F5 | 68.4 | 83.7 |
Klebsiella pneumoniaesubsp. pneumoniae 1084 | K4H7Y8 | 51.9 | 70.7 |
Acinetobacter baumannii D1279779 | M4R7L7 | 50.7 | 68.6 |
Staphylococcus epidermidis (strain ATCC 35984/RP62A) | Q5HKQ0 | 38.7 | 57.3 |
Acinetobacter gyllenbergii MTCC 11365 | S3Z9V2 | 66.3 | 81.1 |
Acinetobacter junii MTCC 11364 | S7YDM3 | 50 | 68.8 |
Chromobacterium violaceum | Q7NTW2 | 52.6 | 69.1 |
To strengthen the accuracy of binding site prediction for UDP-GlcNAc in the active site of GT, following steps were followed (1) all possible amino acid substitution that can happen on the existing codon for Asp140, Asp233, Gln269, Arg272, and Trp273 in A. junii SH205 translated pgaC sequence by point mutation at each of the base positions were done (2) similar (D140E; D233E; Q269N; R272H; W273L) and dissimilar (D140H; D233H; Q269L; R272L; W273C) amino acids substitutions from step 1 for each of the five target codon were selected. (3) finally 3D structure of the mutated proteins were generated and predicted using the same methodology as was followed for the wild protein and redocked using Autodock 4.0. The same grid and docking parameters were used for the docking analysis and the effect of mutagenesis on binding affinity was analysed.
The operon pgaABCD is present in diverse bacterial species and found to be responsible for the synthesis of the polysachharide PNAG. Gram negative bacteria produced this polysaccharide, based on the gene expression of pgaABCD homologous loci in their genome. The Pga proteins (PgaA, PgaB, PgaC, and PgaD) from diverse bacterial genera have been studied with respect to their role in the biofilm formation and regulation. Although the locus pgaABCD is explored in A. baumanii, but too little is known about the phenotypic expression in A.junii. Since few studies have reported that the risk factors for A. junii infection were similar to the most clinically important Acinetobacter spp, A. baumannii, we were induced to look for the presence of similar proteins or genes for the biosynthesis of PNAG, polysachharide responsible for the biofilm formation, integrity and pathogenicity, in A. junii. BLAST (blastp suite) analysis at NCBI website, enabled the identification of four-gene locus in A. junii SH205 (the strain whose protein database is available). Sequence analysis of the pgalocus in A. Junii SH205 revealed that the predicted proteins encoded by this locus shared 41%, 23.7%, 68.4%, and 42.9% identity with the A. baumannii AYE PgaA, PgaB, PgaC, and PgaD proteins (
PgaC is predicted to encode a 424-amino-acid N-glycosyltransferase (PgaC) that belongs to the glycosyltransferase 2 family. It is a cytoplasmic protein that is required for the synthesis for PNAG. This family includes PgaC, BpsC, HmsR, and IcaA from E. coli, B. pertussis, Y. pestis, and S. aureus, respectively. A BLASTP search homologous loci of other gram-negative and Staphylococuus epidermis (gram positive) bacteria with the NCBI A. junii SH205 nonredundant protein database sequences enabled us to identify pgaC that shares a high degree of similarly with pgaC encoding PNAG. Similarities between PgaC of A. junii SH205 and PgaC of E. coli and some strains of Acinetobacter baumannii, HmsR of Y. pestis and C. violaceum, K. pneumonia, and IcaA of S. epidermidis are shown in
A. baumannii AYE | % Identity | % Similarity | A. junii SH205 |
---|---|---|---|
Biofilm PGA synthesis protein pgaA precursor (YP_001713306.1) | 41 | 61.1 | Biofilm synthesis protein (EEY93049.1) |
Polysaccharide deacetylase PgaB (YP_001712551.1) | 23.7 | 42.0 | Polysaccharide deacetylase (EEY93096.1) |
Biofilm PGA synthesis N-glycosyltransferase PgaC (YP_001714657.1) | 68.4 | 83.7 | Glycosyl transferase (EEY93051.1) |
Biofilm PGA synthesis protein pgaD (YP_001713309.1) | 42.9 | 68.7 | Biofilm PGA synthesis protein pgaD (EEY93052.1) |
PgaC of A. junii SH205 and other similar proteins from certain known virulent pathogenic bacteria we hypothesized that the pgaC loci in A. junii SH205 coding for PNAG might be associated with its virulence too. The ability of several pathogens to adhere to human tissues and medical devices by dint of producing biofilms is a major virulence factor that bears logical correspondence with blanket protection against several antibiotics, phagocytosis, and nutrient-stress. Of the different molecules identified as biofilm component in diverse species of eubacteria, PNAG remains as an important molecule that is widely conserved [
The multiple alignments of PgaC sequences of different bacteria have revealed that the polypeptide PgaC of A. junii SH205 retains the amino acids that are crucial to the function of the enzyme responsible for building up of the extracellular matrix in biofilms. On the basis of ClustalW and WebLogo results for sequences of PgaC of A. junii SH205, E. coli and some strains of Acinetobacter baumannii, HmsR of Y. pestis and C. violaceum, K. pneumonia, and IcaA of S. epidermidis, certain amino acids were found to be evolutionarily conserved. The result showed conservation of 18 amino acids e.g., Gly, Asn, Glu, Thr, Val, Ile, Asp, Ser, Lys, Ala, Pro, Arg, Gln, Phe, Trp, Cys, Tyr and Leu at 73 positions spread throughout the sequence in different frequencies is shown in
To investigate the role of these amino acid residues, their conformation and location in the structure of the enzyme was explored. The predicted 3D structure generated by I-TASSER with C-score-0.17, when checked for the quality showed ERRAT Overall Quality Score as 74.760. ProCheck result revealed 98.2% residues in the allowed region in Ramachandran plot [
The largest cavity having the surface area of 3329.2 Å2 was calculated using CastP [
PgaC, a cytosolic glycosyltransferase (GT), uses UDP N-acetylglucosamine (GlcNAc) to synthesize the polymer, PNAG. A docking study was carried out by using A.junii SH205 PgaC as receptor and UDP N-acetylgl- ucosamine as ligand. The best model having the binding energy of −4.9 Kcal/mole was selected for analysing the result of the docking experiment. This analysis suggested the involvement of Pro170, Ile190, Lys195, Thr206, Ser208, Ile234, Gln269, Arg270, Arg272 and Trp273 in the interaction with the substrate UDP-GlcNAc (
In silico mutagenesis approach was adopted to reassure the role of five critical amino acids that enables binding of UDP-GlcNAc. The mutated 3D protein models (5 models with substitution of similar amino acids and 5 with dissimilar amino acids) were superimposed, their root-mean square deviation (RMSD) values indicated a good overall structural alignment; as RMSD value of the backbone of whole structures ranged from 0.84 - 1.05 Å (
These analyses suggested that PgaC is a polysaccharide polymerase that uses UDP-GlcNAc as a substrate. The above mentioned residues are among the evolutionary conserved residue list which are found to be present in the periplasmic domain and involved in the enzymatic activity. However, definite evidence can be provided from only additional experiments such as substrate-enzyme reaction kinetics.
The homology search revealed the identity of four-gene locus homologous to various genetic loci encoding proteins for poly β-(1-6)-N-acetylglucosamine biosynthesis in A. junii SH205. The possibility of PNAG synthesis in A. junii SH205 with the aid of its own PgaC was ascertained by Bioinformatics. Based on this study, one can test the virulence potential of A. junii SH205 in cell culture and invent means of control by blocking the synthesis of PNAG.
We sincerely acknowledge, University Grant Commission for financial support under UGC major research grant (F. No. 41-558/2012 (SR) Date: 18 July 2012. BKT is provided with the fellowship from the Department of Biotechnology, Government of India (BT/Bio CARe/06/141/2010-11). AK is provided with the independent UGC-Dr. D.S. Kothari Postdoctoral fellowship (Award No: F.4-2/2006(BSR)/13-1071/2013 (BSR) dated Oct 8, 2013).
Bipransh Kumar Tiwary,Arvind Kumar,Ravi Kant Pathak,Nishtha Pandey,Krishna Kant Yadav,Ranadhir Chakraborty, (2016) The Locus PgaABCD of Acinetobacter junii Putatively Responsible for Poly-β-(1,6)-N-Acetylglucosamine Biosynthesis Might Be Related to Biofilm Formation: A Computational Analysis. Advances in Microbiology,06,222-232. doi: 10.4236/aim.2016.63022