The Assembly of Bacteriophage Functional Enzymatic Models in Association with E. coli Proteins' Profiles

Introduction: The invasion of bacteriophage on the associated host bacterium depends on their receptors' orientation that adsorb them to cell surface. During phage replication a valuable number of proteins acts as lytic enzymes for host puncher at the beginning of the infection and other for burst after lytic cycle compilation. Accordingly, the proteomic relationship among phage and bacterium proteins could easily be studied by their protein profiles analysis.

Objective: To detect bacteriophages functional enzymes during lytic cycle.

Methods: The isolation and identification of Escherichia coli and their parasitic T7 phage group was done using bacterial culture and common plaque assay techniques. The investigations and protein-protein interactions' assays were inveterate by proteins profile of phage and bacterium using Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) to find out their molecular weights, where the scaled location of each mobile band was compared to the standards of identified proteins weights in the molecular ladder. Thereafter, Protein model's assembly and bands migration was done by computer analytical software.

Results: Mobilization of the phage' proteins inside the Two Dimensions (2D) gel ranged between 60 and 12 kDa where a model of 4 main bands with molecular weights of (46, 35, 24 and 14 kDa) is corresponded to the host ones, where pure 9 bands with molecular weight ranged between 96-24 kDa. The computational model analysis showed common shared molecular masses of 47, 34 and 16 kDa on plot area of the phage and the bacterium. Model interpretation confirmed that proteins ranged from 47.7 to 34.3 kDa resembles 43.3% of whole phage's proteins that assembled the capsid head and the coil, while the molecular weight mass of 22.5 formed the tail's proteins. The lytic enzymes' molecular weight was ranged between 18-14 kDa according to the function of the enzyme. The study revealed that the 34 kDa band has the common shared peak between T7 phage group and associated Escherichia coli host.

Conclusion: Functional models of analysed proteins during phage assembly, ensures lytic enzymes are built in the capsid head and the lysozyme in the tail, they facilitate the enzymatic decay for bacterial host. This enzymatic function is related to the lytic cycle of the bacteriophages and their phenomenon in employing the bacterial DNA in proteins manufacturing during their replication inside host.

Bioinformatics has provided valuable information in a variety of biological fields that enabled identification of novel components of biological macromolecules via the application of computational techniques to understand and organize the information associated with genomics and its translation into molecules of life-proteins [1-3]. Modern proteomics needs immense powers of resolution, sensitivity, and speed of identification, which so far can be reached through One and/or Two Dimension (1D, 2D) gels in combination with advanced analysis of complex protein distributions in automated fashions considering the large number of different protein interaction domains to provide important data sets [4,5]. Usually, the common technique used to investigate cell protein profiles is Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), which contains separating proteins by isoelectric charge or molecular weight [6-9]. For a deep understanding of the relevant bacteriophages' proteins biology, this information must be integrated with analytical data from various ‘ bioinformatics levels' including DNA sequence, mRNA profiles, protein expression and metabolite concentrations as well as information about dynamic spatio-temporal changes in these molecules [10]. The generation of protein profiles to get the maximum possible distant gave biological information from related sequences by using alignment analytical and computational programs for the preparation of structural protein profiles of bacteriophages [11]. A protein profile of the phages provides additional information regarding the differences among the bacteriophages isolated and characterized [12,13]. Phages breaks down the bacterial DNA to recycle bacterial host genome for their own DNA synthesis during lytic cycle and hamper coexistence between phage and host. Such phages usually encode enzymes involved in nucleotide metabolism and the corresponding genes can be identified through sequence analysis [14]. The phage's tail allows its genome to be translocated into the host cell through internal head proteins that are ejected at the initiation of infection with transport of the leading end of the genome functioning lytic enzymes used by phages (Endolysins) at the end of the replication cycle to degrade bacterial peptidoglycan resulting in a rapid host lysis and the release of phage progeny [15-17]. Phage-associated lytic enzymes have been confirmed in many other bacterial phage-host systems and has been employed to control pathogenic bacteria [18-20]. The P5 protein from bacteriophage phi-6 has been revealed to be a lytic enzyme that cleaves the bacterial peptidoglycan (Murein) layer to facilitate membrane fusion during infection and is also responsible for cell lysis in late infection and was shown to be active against cell walls of various Gram-negative species [21-23]. Depending on the molecular marker which was used for the protein separation process, the molecular weight of lytic enzymes was varied; 14-13 kDa for lysozyme, 16 kDa for the E. coli phage and the estimation of the molecular weight of the lysin was in excess of 100 kDa [24,25]. Two structural proteins were detected in phages, ranging from 41 to 44 kDa and from 48 to 51 kDa, as well as several major and minor proteins were also detected (66 and 14 kDa) and Av-05 (73, 60, 53, 34, 25, and 14 kDa). Bacteriophages of the Myoviridae family commonly show two major structural proteins ranging from 50 to 55 kDa, which are related to the major tail sheath proteins, whereas proteins with a molecular mass around 23-25 kDa are related to the capsid protein [26]. Proteins with a minor molecular mass (6-15 kDa) in phages from the Myoviridae family have been previously reported; however, no specific function has been determined [27-29]. Enzymatic functional reactions usually. Depend on the enzyme targeted substrate. Moreover, reactions are rapid and sensitive. Therefore, the opportunity of detecting and computing bacteria species through specific enzymatic functional model activities has been under study for many years now [30-32].

General objective

To detect bacteriophages functional enzymes during lytic cycle.

Specific objectives

Isolation and identifications of bacteriophages and E. coli (host) enzymes and proteins.

Build computational model of phage functional enzymes using analytical software.

Phages and bacteria isolation and purification

Bacteriophages and their allied bacteria samples were harvested by using common plaque assay and bacterial loan cultures purification techniques for Escherichia coli.

Samples processed by SDS-PAGE for protein separation

Bacteriophage T7 and corresponding Escherichia coli, cells were. Centrifugated for 3-5 minutes at 15,000 rpm, supernatants were removed and the precipitations were filtered through Millipore filter with a pore size of 0.45 µm (HiMedia Laboratories) and stored at -50°C. Afterward, microorganisms' proteins were crushed by glass rod and homogenized in sterilized distilled water in volume 15 times less than their original volume. To find out the presence of the proteins in selected samples, same volumes of the resulted precipitation was dry freeze to absorb all moister and avoid contamination, kill all microorganisms and keep samples in room temperature in with silica gel granules. The final samples' weight resolved by electrophoresis through SDS-polyacrylamide (12 ± 5% protein assay by HiMedia Laboratories), using methods modified from Laemmli [33]. In this protocol, proteins were denatured and covered with detergent by boiling in the presence of SDS and a reducing agent. Finally, samples were stained with Coomassie blue for visualization. Molecular weight markers (ladders) were included in all gels for interpretation and quality control.

In silico analysis for proteins molecular weight and functions

For the estimation of the molecular weights of the proteins of the bacteriophages T7 and corresponding Escherichia hosts in the samples, the molecular weight mass of the resulted gels were analyzed In silico by the bioinformatics programs [(Lab-Image Platform v. LD300 by Kapelan Bio-Imaging GmbH), (UN – SCAN – IT v6 by Silk Scientific, Inc.) and (ImageJ 136b by Wayne Rasband, USA, National Institute of Health)] for image digitizing. For structural proteins formation, the data mining web page of BLAST – NCBI (National Center for Bioinformatics site) was used for Open Reading Frames (ORFs) identification, peptides and nucleotide sequences and compared with those of other genes.

Functional enzymatic profiles

Detection of functional enzymic activities was done by the molecular analysis of conserved domains of bacteriophage's assembly during their lytic cycle and within the predicted models directly downstream of the proteins' profiles encoded for the lytic enzymes according to their molecular weight for detecting Open Reading Frame (ORF) of the functional enzymes' profiles using the CDD search system at NCBI [34].

SDS-PAGE protein separation

The capability to detect proteins by the Two Dimensions (2D) gel electrophoresis is crucial to its application in proteomics for separating and visualizing protein mixtures. Merging this analysis with computational programs (in silico) for protein identification makes it a very controlling implement technique for characterization of protein mixtures (Table 1).

The protein marker consists of unstained natural protein standards that allow accurate MW determination with uniform band intensities on SDS-PAGE gels stained with Coomassie. The marker was applied for all SDS-PAGE to determine the phage and bacteria proteins molecular weight mass, bands distance migration on gel and plotting graphs (Figure 1).

Figure 1: Detection of bacteriophage proteins' molecular weight on (SDS-PAGE).

Detection of proteins' molecular weight

Sodium Dodecyl Sulphate-Polyacryamide Gel Electrophoresis (SDS-PAGE) profiles indicated that bacteriophage contains four structural proteins. It is necessary to determine how many kilo-Daltons (kDa) make up the protein. This can be done by comparing the results of SDS-PAGE to standard molecular ladder (marker), proteins revealed that four major proteins were observed on the gel, with molecular weight ranging from 46 to 14 kDa (Figure 2).

Figure 2: Plotting bacteriophage proteins' molecular weight using Lab-Image Platform v. LD300.

A standard curve can was constructed from the distances migrated by each marker protein. Then the distance migrated by the sample proteins was plotted and their molecular weights calculated by interpolation. The molecular weight of the protein of interest were plotted to estimate their values from the curve by linear regression analysis and also determine the band migration and position on the gel by interpolation. The data are fit with a linear regression by the line y = 0.255x + 106.980 with an R2 value of 0.83121. Note that to use the equation to return protein concentration (Figure 3).

Figure 3: Detection of bacteria proteins' molecular weight on (SDS-PAGE).

SDS-PAGE separation of whole cell extracts from E. coli, resolved on 10% acrylamide, the molecular mass of each protein marker is shown on the left. The migrant bands were lied between 97-18 kDa for each sample (Figure 4).

Figure 4: Plotting bacteria proteins' molecular weight using Lab-Image Platform v. LD300.

The molecular weight of the protein was calculated by measuring its relative mobility (R2) and matching that to a calibration curve created from the standards. The relative mobility is calculated by measuring the amount of movement of the protein from the origin and dividing that by the distance of the tracking dye from the origin. A linear plot (y = -0.209x + 112.728) is then constructed from the molecular weights of the protein standards graphed as a function of their R2 values = 0.98017).

Comparing protein profiles of E. coli bacteriophages

In silico analysis was done to determine expressed proteins in E. coli based on the MW of detected peaks in the SDS-PAGE plate. The analysis of the SDS-PAGE image by (UN – SCAN – IT and ImageJ) revealed the bands position on histogram according to their molecular weight (Figure 5).

Figure 5: Histogram of the bacteriophage gel plot area using (UN – SCAN – IT-V6).

The reference markers used were ranged from 97 to 14 kDa, corresponded to standard migration distances of 5, 10, 15,20, 25, 30, 35 cm on the 2D histograms for the bands 46, 34, 24 and 14 kDa, (Figure 7). The 3D plot showed the density of mobilized proteins of the E. coli phage were density and mass of the bands observed (Figure 6). The peaks were compared in all samples and found to be similar.

Figure 6: 3D graph of the phage plot area migration using (UN – SCAN – IT v6).

Figure 7: Bacteriophage protein bands distance using Image J136b.

Protein profiles of the bacteria analysis

The protein profile of E. coli bacteria showed clear nine bands with molecular weight ranged between 96 and 14 figures. The obtained bands of the E. coli phage and the E. coli bacteria were compared, one band of 34 KDa showed typical similarity in both E. coli page and E. coli bacteria (Figure 8).

Figure 8: Histogram of the bacteria gel plots area using (UN – SCAN – IT v6).

Multimodal frequency distribution of protein sizes for E. coli molecular masses were estimated from migration distance in 2D by bacteriophage reference markers, since migration distance is related to the protein molecular mass (Figures 7,10). The bands were plotted on 3D for density comparison (Figures 6,9) the bacterial 3D bands look thicker and higher than the phage's ones.

Figure 9: 3D graph of the bacteria plot area migration using (UN – SCAN – IT v6).

Figure 10: Bacteria protein bands distance ImageJ 136b.

Assembly of structural proteins of E. coli bacteriophages

The structural proteins of the bacteriophages were identified by using BLAST online proteins' data mining tools and found to be related to the capsid and tail proteins, as well as several major and minor proteins related to the lytic enzymes group (Figure 11).

Figure 11: Proteins' assembly of bacteriophage on SDS PAGE.

Structural proteins building up the bacteriophage produced by N-terminal sequences are resulted in size from 47.7 to 34.3 kDa formed the capsid head and the coil, 27 to 22.5 kDa formed the tail proteins and genomic proteins were: Lytic enzymes: Endolysins = 40 -25 kDa, Lysozyme =14,3 kDa (Figure 12).

Figure 12: Detection of Shared Proteins of E. coli on SDS PAGE.

The SDS-PAGE revealed a 34- kDa band corresponding to this peak, which matched the predicted size of nuclease enzyme.

The detection of bacteriophage lytic enzymes was done after equilibration of the proteins where the resulting focused bands were separated according to size using modified classical methods (SDS-PAGE) and other reducing agents discovered by [33,35,36]. The proteins' bands migration were plotted on 2D and 3D graphs for distance and density properties to predict the size of the proteins and similarity in the phage and bacterium, [37] reported that the calibration curve of SDS-MW size standards plotting the molecular weight towards mobility values estimated from migration distance in gels. The gels' digital images were acquired into computer programs and other device using imaging technology for advance analysis [38]. The in silico simulation of protein profiles analysed data by the ImageJ programme showed the protein migration plot area of the bacteriophage and their bacterial host, while similar peaks of bacteriophage's bands, in the meantime, the bacterial host E. coli protein profile showed more bands and more peaks than the bacteriophages ones, this is related to the lytic cycle of the bacteriophages and their phenomenon in utilising the bacterial proteins during their multiplication inside the host, the revealed agreed [39] who reported that the SDS-PAGE of whole-cell protein extracts of E. coli strains produced patterns containing 26 to 35 discrete bands with molecular weights of 65- 20 kDa. The molecular mass of the protein was estimated by comparing the mobility of a band with protein standards [40] reported that; Natural gel electrophoresis (mobility shift) assays might be applied to obtain quantitative information about the bands'location, distribution, equilibria and kinetics of protein-DNA interactions. The calibration curve of electrophoretic mobility against the molecular weight of known protein size was established and the relative MW of unknown protein was calculated based on the calibration curve for bacteriophage coefficient (R² = 0.83121) and corresponding bacterial host (R² = 0.98017), this was in agreement with [41] and their coefficient of determination (R² = 0.998) was nearly to the findings of this study. However in the present study, the molecular analysis of SDS PAGE purified bacteriophages showed four major bands with molecular masses of 46, 34, 24 and 14 kDa this range nearly to findings of [42] who revealed 15 protein bands of lytic phage SboM-AG3 virions ranging from approximately 12 to 126 kDa based on comparison to size markers ranged from 200-15 kDa. The molecular and computational analysis of enzymes activities indicates that the encoded proteins are associated to different assigned functions according to their molecular weight scaling [43]. During their lytic cycle and assembly structural proteins building up the bacteriophage produced by N-terminal sequences are resulted in size from 47.7 to 34.3 kDa resembles 43.3% of total phage and formed the capsid head and the coil Cps (NH2-DIMSSTNNGA, encoded by ORF6). While the molecular weight mass of 27 to 22.5 kDa formed the tail proteins Tsh (NH2-PEVVNTRRXG, encoded by ORF11) corresponds to 12.7% of the total protein. The amino acid sequence minor structural protein gp16, 2.1% total phage's protein content (NH2- MKYIQTKVVY ORF16) size of 55.1 kDa. The results also agreed [44] who found the major capsid component results in size from 47.7 to 34.3 KDa resembles 43.3% of total phage protein and the tail protein corresponds to 12.7% with an apparent size of 27 kDa. The length of the phage tail is thought to be determined by a ruler mechanism, dependent on the size of the so-called tape measure protein [40]. During phage assembly phage genomic proteins were: Lytic enzymes: endolysins = 40 -25 kDa, Lysozyme =14, 3 kDa, (lysine –amino acid- = 146 kDa) [45-49]. The common shared peak 34 kDa of phage and hosted bacterium was found to be nuclease enzyme of the gp32, which is a typical model assembled for capsid protein genes of T7 and T3 phages, also the calculated molecular masses of polypeptide fragments 35 and 34 kDa, assuming that they extend to the C terminus of Escherichia coli Protein RecE [50,51]. The predicted models of lytic enzymes were assumed to have different sizes ranged between 35-12.5 KDa, [52-54]. Lytic phages manipulate the bacterial synthetic machinery to produce viral rather than bacterial nucleic acids and proteins, leading to phage assembly [55]. These phages developed a complicated lysis mechanism with many lytic enzymes in approaching and attacking bacteria, during their lytic cycle when bursting the host and release new phage progeny. This natural phenomenon makes them the best alternative solution for antibiotics resistant bacteria [56-59].

Protein separation by SDS-PAGE is a valuable system for detecting proteins, their molecular weights, and functions by the assessment of bioinformatics tools and data-mining. During the phage protein assembly, the lytic enzymes are constructed in the capsid head and the lysozyme in the tail, the lytic enzymes mediate the enzymatic cleavage for bacterial host lysing.

We would like to acknowledge with gratitude the assistance received from Dr. Mubarak Karsany (Karary University), Dr. Abdelazim Ali and Dr. Jehan Elhaboub (University of Khartoum) and their cooperative laboratories staff.

Funding: This work was supported by Elbarkal Multi Activities, Dept. Research & Consultancy [Local fund sources No; 4]; Khartoum- Sudan.

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