MATERIALS AND METHODS

Study area: The current work was carried out at the pathogen biology Department, Harbin Medical University, from November, 2020 to April, 2021.

Sequence assembly: The hypothetical protein (PA2373) sequence information was obtained through the NCBI database. In a FASTA format, the sequence was taken and then presented for in silico characterization to several prediction servers (Table 1). Similarity searches were carried out with the NCBI Protein Database and Swiss Prot to identify proteins that may have structural similarities to uncharacterized proteins using the “BLAST p tool of the BLAST software.

Alignment of sequences and study of phylogeny: Multiple sequence alignments were achieved using the EBI MUSCLE server¹¹. Phylogenetic analysis was performed using Phylogeny.fr¹².

Analysis of physicochemical properties: Physical and chemical features such as amino acid composition, predictable half-life, the total number of negatively charged residues (Asp +Glu), the total number of positively charged residues (Arg+Lys), aliphatic index, instability index and the grand average of hydropathicity (GRAVY) predictions were determined using the ProtParam tool of ExPASy¹³.

Subcellular localization: The CELLO was used to predict the subcellular localization¹⁴. Upshots were also verified through subcellular localization predictions gained from SOSUIGramN¹⁵ and PSLpred¹⁶ TMHMM¹⁷ and HMMTOP¹⁸.

Conserved domain and family identification: The conserved domains on the Conserved Domains Database (CDD), available on the NCBI webpage has been used for research¹⁹. Two domain search methods, InterProScan²⁰ and Pfam²¹ were also used to confirm the outcomes. The protein motif search was performed using the MOTIF search tool (Genome Net, the Institute for Chemical Research, Kyoto University, Japan)²² and SUPERFAMILY²³. The COILS server was used to classify the coiled-coil conformation into the protein²⁴. The PFP-FunDSeqE server was used to recognize protein folding patterns²⁵. The virulence activity of HPs was predicted using VICMpred²⁶.

Prediction of secondary structure: A PSI blast-based secondary structure prediction (PSIPRED) was implemented to investigate the structure of the protein. The Self-Optimized Prediction Method with Alignment (SOPMA), an online tool was also applied to predict the secondary protein structure²⁷.

Prediction of the three-dimensional structure: The HHpred server predicted the three-dimensional structure²⁸. At The Max Planck Institute for Developmental Biology, Tubingen. The process used the pairwise comparison profile of Hidden Markov Models (HMMs). For advanced precision, the 3D structure was predicted based on the best score template. The homology-modelled protein validated by the expected three-dimensional model evaluated using PROCHECK, Ramachandran plots²⁹, ERRAT³⁰ and ProSA web³¹. The ProB is the server that used the homology-modelled hypothetical protein to classify structurally related protein-binding sites³². The homology-modelled protein was superimposed with the UCSF Chimera 1.10³³.

RESULTS AND DISCUSSION

Sequence similarity data: Homology with the other VgrG proteins were identified in the BLAST (BLASTp search) against non-redundant protein sequence which showed homology with another type VI secretion system tip protein VgrG proteins in the same genus: Pseudomonas, Pseudomonas aeruginosa, Pseudomonas fluorescens with sequence identity (100 and 99.8%) respectively through score 1367 and E-value 0.0. Table 2. Furthermore, Table 3 showed the homology with Type VI secretion system in the different genus of bacteria, Aeromonas hydrophila, Dickeya dadantii, Vibrio cholerae 01and Vibrio cholerae 0395, with sequence identity (34.56, 34.40, 37.11, 36.93, 33.99 and 35.34%) respectively through scores ( 348,342, 341, 316 and 345) and E value Ie-109, 61-109, 8e-108, Ie-107, Ie-93 and 8e-90) in the Swiss Prot databases.

Fig. 1:

Phylogenetic trees of different VgrG proteins

Fig. 2:

Coil depicts the heptads corresponding to the residue windows 14 (green), 21(blue) and 28 (red)

Hypothetical protein (PA2373) FASTA sequences and annotated homologous proteins were systematized. To validate the homology between the proteins, a phylogenetic analysis was also performed. Based on the alignment and BLAST results, a phylogenetic tree was constructed, which provided a similar concept of protein, as shown in Fig. 1.

Physicochemical features: The protein contained 668 amino acids and the molecular weight measured was 7877.95 Da and the theoretical pI was 5.207, representing that the protein was negatively charged. Among the most abundant charged residues, the whole number of positively charged residues (Arg+Lys) was 12 and the whole number of negatively charged residues was 13. The determined index of instability classifies the protein as stable. The measured protein half-life was 7.2 “hrs” in mammalian reticulocytes (in vitro), >20 hrs in yeast (in vivo) and >10 hrs in Escherichia coli (in vivo). The aliphatic index was 71.39, which indicates the stability of the protein in a high-temperature range. The GRAVY value was -0.335. A negative GRAVY value shows that the protein is nonpolar. The molecular protein formula was defined as C₃₅₆H₅₃₉N₉₅O₁₀₂S₃.

Subcellular localization: The hypothetical protein (2373) subcellular localization was expected to be in the extracellular space. The absence of transmembrane helices predicted by THMM and HMMTOP besides enhances the final result of extracellular protein^15-18.

Functional annotation and virulence prediction: The conserved domain search tool showed two domains in this hypothetical protein sequence, VI_Rhs_Vgr (accession no TIGR03361) and 5 superfamilies (accession no cl33691). The Pfam server predicted the Phage_GPD at 29–353 amino acid residues with an e-value of 4.9e-82, phage base V at 357-583 with an e-value of 3.1e-13 and Gp5 C at 560-646 amino acid residues with an e-value of 0.0031. The Inter-ProScan server predicted the type VI secretion system, RhsGE-related Vgr protein family subset. Phage GPD family subset, Phage_GPDPhage_base_V, Gp5_C, and DUF3540 domains were also predicted using the MOTIF server. Fold pattern predicted by the PFP-FunDSeqE tool shown the presence of conA-like lectin/glucanases; fold inside the protein sequence. The x-axis of the diagram shows the position in the protein of amino acid number (starting at the N-terminus) and the y-axis represents the coiled-coil whereas ‘Window’ refers to the width of the amino acid in Fig. 2.

Superfamily analyses identified the presence of phage tail proteins and the phage fibre protein superfamily. To predict the bacterial virulence factor of PA2373, we used the VICMpred bioinformatics tool. PA2373 includes a virulent element that may offer clues for future drug design as a treatment objective. The characteristics of HP PA2373 from Pseudomonas aeruginosa PA01 were investigated and studied for the first time. The recent finding was agreed with Wiehlmann L et al.³⁴ who made the report that HPs are attractive therapeutic targets since they are involved in important pathways such as (cell wall integrity e and maintaining genome).

Analysis of the secondary structure: The study using the secondary structure prediction SOPMA tool showed the alpha helix, beta-turn, extended strand and random protein coil proportions as 20.96, 6.8, 28.1 and 44.01%, respectively.

Analysis of the three-dimensional structure: 3D structure prediction was accomplished using the HHpred server with the highest scoring template for 100 percent identification (PDB ID: 4UHV), Fig. 3 and 4. The UHV is a trimetric fold crystal structure that separates two major fragments assembly of the protein, the head and the spike bound by a neck centred on a cord^29-31. This unique molecular structure is identical to the T4 bacteriophage (gp27)3-(gp5)3 complex puncturing-device machinery used by the virus to inject its DNA into the host cell³⁴. The energy minimization server Modrefiner was used for the refined prediction of homology models. For both the energy aspects and geometries, the ProSA web helped evaluate the energy-minimized structures by accessing the Z-score protein structure, representing the overall quality of the model, Fig. 4. It was helpful to verify whether the input structure was within the range of scores typically seen for similar-sized native proteins. The overall model quality Z-score was -6.57, suggesting a similarity between the template and query structure. The Ramachandran plot revealed that 71.9% of the residues were located in the most favourable regions, where the distribution of φ and ψ angle in the model within the limits are shown as illustrated in Fig. 5. The ERRAT server predicted an overall model quality factor of 58.0813, which indicates a good model. The homology-modelled protein was superimposed with the UCSF Chimera 1.10 is presented in Fig. 6.

Analysis of protein-protein interaction: The STRING 10.0 tool was used to establish potential protein functional interaction networks^35,36.

Fig. 3:

Predicted three-dimensional hypothetical protein structure

Fig. 4:

Predicted functional regions of selected hypothetical protein by using ProBis server Highly conserved residues are shown in red and the least conserved regions are shown in blue

The partners of functional listed with scores were; PA2369 (0.938), PA2370 (0.934), PA2367 (0.928), PA2366 (0.915), ClpV1 (0.910), PA2365 (0.909), PA2371 (0.908), PA0088 (0.900), IcmF1 (0.900), PA1660 (0.899). PA2369, PA2370, PA2367, PA2365, PA0088 and PA1660 are hypothetical proteins.

Fig. 5:

Ramachandran plot of modelled structure validated by PROCHECK program

Fig. 6:

Homology modelled proteins are superimposed with the template of (PDB ID: 4uhv) by using UCSF chimera-1.10

Fig. 7:

A string network analysis of the hypothetical protein is identified as PA2373

PA2366 is an uricase, ClpV1 is a protein, which is required for secretion of hcp1 probably by providing an energy source for its translocation, PA2371 is a ClpA/B-type protease and IcmF1 as shown in Fig. 7.

CONCLUSION

The detection of protein functions is crucial to the understanding of biological processes. Therefore, this study aimed to define the biological and structural role of PA2373, a hypothetical protein from P. aeruginosa, via in silico analysis. The hypothetical protein that was identified revealed some characteristics, including extracellular protein, Vgr protein and type VI secretion system, containing two domains. These hypothetical protein characteristics provide basic information about P. aeruginosa. The findings of this study will help determine mechanisms of virulence, drug resistance and pathogenesis of P. aeruginosa infections through extended in vitro experiments to innovate treatment strategies for these infections subsequently.

SIGNIFICANCE STATEMENT

The current research is aimed at describing the structure and function annotation of HPs which have an antibiotic-resistant activity that can help find new targets to improve P. aeruginosa treatment and investigation. Hypothetical proteins in P. aeruginosa have been predicted using several structural and functional annotation informatics servers.

ACKNOWLEDGMENT

We thank our professors and support from the Department of Microbiology, Harbin Medical University, Harbin, China. Taif University Researchers Supporting Project Number (TURSP-2020/140), Taif University, Taif, Saudi Arabia.