In Silico Functional Elucidation of Uncharacterized Proteins of Chlamydia Abortus Strain LLG

Figures & data

Figure 1.  Gene ontology of 51 hypothetical proteins in Chlamydia abortus.

The proteins are classified based on biological, cellular and molecular functions.

Figure 2.  Structural elucidation of two proteins from Chlamydia abortus genome sequence.

(A) WP_006344020.1 (phosphorylase) and (B) WP_006344325.1 (CPAF). The amino acid residues involved in enzymatic activity are shown in red.

Figure 3.  Molecular dynamic analysis of two homology-modeled structures showed stability in the root-mean-square deviations and radius of gyration.

Also the structural flexibility of proteins were analyzed by RMSF of the amino acid residues. Steadiness of proteins were analyzed by SASA during the MD simulations that revealed that the proteins were stable. (A) RMSD of two homology-modeled proteins were plotted, initially both the proteins showed an increase in RMSD and after 2.5 ns, proteins attained stability and consistency in structural behavior, indicating the stability of proteins during MD simulation. (B.I) WP_006344020.1 and (B.II) WP_006344325.1. The proteins had initially less compactness in structures, but at later stages, the proteins showed more compactness and less variation in the radius of gyrations along all spatial axes. (C) Protein WP_006344020.1 (blue) and WP_006344325.1 (red) with amino acid residues, 198 and 565, respectively, showed evenness in the RMSF of amino acid residues of both the proteins with the progression of MD. The uniformity indicates that the proteins are stable. (D) The SASA of the two modeled proteins was constant throughout the MD simulations.

MD: Molecular dynamics; RMSD: Root-mean-square deviations; RMSF: Root-mean-square fluctuations; SASA: Solvent surface accessible area.

Table 1.  List of bioinformatics programs used for functional analysis of hypothetical proteins in Chlamydia abortus.

Sl. No.	Programs	Prediction	Ref.
1	NCBI-CDD	Protein-conserved domain	[18]
2	INTERPROSCAN	Protein domain	[19]
3	SVM-Prot	Protein functional family	[20]
4	Pfam	Protein family	[21]
5	SCOP-Superfamily	SCOP domains in proteins	[22]
6	EMBOOS PepStat	Statistics for protein	[25]
7	TMHMM	Transmembrane α-helices	[26]
8	HMMTOP	Transmembrane α-helices	[27]
9	SOSUI-GramN	Subcellular localization	[28]
10	CELLO	Subcellular localization	[29,30]
11	SignalP 4.1	Signal peptide cleavage site	[31]
12	CELLO2GO	Gene ontology	[32]
13	KEGG	Pathway analysis of proteins	[33]
14	Phyre2	Protein homology modeling	[40]
15	SAVES	Checking protein structure for molecular stereochemical quality, residues parameters, nonbonded interactions, model compatibility, macromolecular volume and the Ramachandran plot	[41–45]
16	GROMACS 5.0	Energy minimization and molecular dynamics of protein	[46]
17	PYMOL	Visualization of tertiary structure of proteins	[47]
18	BLAST	Similarity search between biological sequences	[34]

Table 2.  List of sequence-based analysis of 51 hypothetical proteins, gene IDs and some of their specific functions in Chlamydia abortus.

Sl. No.	Protein ID	Gene ID	Protein function
1.	WP_006344227.1	493388305	DNase and RNase activity
2.	WP_006342697.1	493386499	RNA-binding domain
3.	WP_006343813.1	493387621	Involved in binding peptidoglycans
4.	WP_035395401.1	737414395	Plays roles in distinct ADP–ribose pathway
5.	WP_006344461.1	493388269	Regulates protein phosphorylation
6.	WP_006343888.1	493387696	Periplasmic-binding protein
7.	WP_006344021.1	493387829	Periplasmic-binding protein
8.	WP_006344141.1	493387949	Binds and stabilizes newly synthesized polypeptides
9.	WP_006343819.1	493387627	Stress response protein
10.	WP_006344147.1	493387955	Nucleic-acid-binding protein
11.	WP_006344255.1	493388063	Plays role in regulation of enzymes and changes the subcellular localization of signaling pathway components
12.	WP_006343878.1	493387686	Involved in CMP-Kdo biosynthesis pathway
13.	WP_006344558.1	493388367	TIM barrel proteins
14.	WP_006343973.1	493387781	DNA-binding proteins
15.	WP_006344427.1	493388235	Role in cell motility, the Ras pathway, vesicle trafficking and secretion, immune cell activation and apoptosis
16.	WP_006343829.1	493387637	P-53-associated protein
17.	WP_006344570.1	493388379	DNA double-strand break repair
18.	WP_006343995.1	493387803	Co-enzyme metabolism
19.	WP_006344048.1	493387856	DNA polymerase III
20.	WP_006344566.1	493388375	Catalyzes addition of l-glutamate
21.	WP_035395338.1	737414331	Type-1 glutamine amidotransferase (GATase1)-like domain
22.	WP_006344020.1	493387828	Catalytic and methylthioadenosine nucleosidase activity
23.	WP_006344334.1	493388142	Involved in bacterioruberin synthesis
24.	WP_006343689.1	493387496	Rhodanese homology
25.	WP_006343852.1	493387660	Ribokinase activity and carbohydrate phosphorylation
26.	WP_006343738.1	493387545	Involved in coupling of ATP degradation to H^+ translocation
27.	WP_006343847.1	493387655	Involved in structural integrity
28.	WP_006343848.1	493387656	Involved in structural integrity
29.	WP_006343935.1	493387743	ChlamPMP_M superfamily
30.	WP_006343849.1	493387657	Forms disulfide bonds to provide rigidity to the cell wall
31.	WP_006344111.1	493387919	OmpH superfamily (characterizes as a molecular chaperon that interacts with unfolded proteins)
32.	WP_006343706.1	493387513	Tri chaperone protein like
33.	WP_006343695.1	493387502	Type III secretion system
34.	WP_006343696.1	493387503	Type III secretion system
35.	WP_006344485.1	493388293	Type III secretion system
36.	WP_006344486.1	493388294	Type III secretion system
37.	WP_006344325.1	493388133	Promotes the release of all-trans retinol
38.	WP_006344533.1	493388342	Architecture and deduce catalytic mechanism of PP2C phosphatase
39.	WP_006344534.1	493388343	Architecture and deduce catalytic mechanism of PP2C phosphatase
40.	WP_006343947.1	493387755	Function in fertility regulation
41.	WP_006343725.1	493387532	TPR domain (mitochondrial import protein)
42.	WP_006344218.1	493388026	Involved in protein–protein interactions, chaperons, cell cycle and transcription
43.	WP_035395294.1	737414287	Function in the ABC transport of ferric siderophores and metal ions such as Mn^2+, Fe^3+, Cu^2+ and/or Zn^2+
44.	WP_006344406.1	493388214	Alkaline shock protein
45.	WP_006344365.1	493388173	Shows anti-apoptotic effect and role in pathogen resistance
46.	WP_006343744.1	493387552	Globin-like protein (plays various roles in all three kingdoms of life)
47.	WP_006344477.1	493388285	Plays vital roles in the ISC and NIF systems of Fe–S protein maturation
48.	WP_006344274.1	493388082	Required for PSII to be fully operation in vivo
49.	WP_006343831.1	493387639	Metal-dependent hydrolase
50.	WP_006344578.1	493388387	Nucleotide transport and metabolism
51.	WP_006344167.1	493387975	Protein act as a virulence factor

ABC: ATP-binding cassette; CMP-Kdo: Cytidine 5-monophospho-3-deoxy-d-octulosonic acid; ISC: Iron-sulphur cluster; NIF: Nitrogen fixation; TIM: Triosephosphate isomerase; TPR: Tetratricopeptide repeat.

Table 3.  List of hypothetical proteins of Chlamydia abortus that were showing sequence homology with selected six host proteins.

Sl. No.	Protein ID	Gene ID	Sequence homology with host proteomes						Virulence factor
			Sheep	Goat	Bovine	Yak	Pig	Humans
1.	WP_006344227.1	493388305	×	×	×	×	×	×	Yes
2.	WP_006342697.1	493386499	×	×	×	×	×	×	Yes
3.	WP_006343813.1	493387621	×	×	×	×	×	×	×
4.	WP_035395401.1	737414395	×	×	×	×	×	×	×
5.	WP_006344461.1	493388269	×	×	×	×	×	×	×
6.	WP_006343888.1	493387696	×	×	×	×	×	×	Yes
7.	WP_006344021.1	493387829	×	×	×	×	×	×	×
8.	WP_006344141.1	493387949	×	×	×	×	×	×	×
9.	WP_006343819.1	493387627	×	×	×	×	×	×	×
10.	WP_006344147.1	493387955	×	×	×	×	×	×	Yes
11.	WP_006344255.1	493388063	×	×	×	×	×	×	×
12.	WP_006343878.1	493387686	×	×	×	×	×	×	Yes
13.	WP_006344558.1	493388367	Yes	Yes	Yes	×	×	Yes	Rejected
14.	WP_006343973.1	493387781	×	×	×	×	×	×	×
15.	WP_006344427.1	493388235	×	×	×	×	×	×	Yes
16.	WP_006343829.1	493387637	×	Yes	Yes	×	×	Yes	Rejected
17.	WP_006344570.1	493388379	×	×	×	×	×	×	×
18.	WP_006343995.1	493387803	×	×	×	×	×	×	Yes
19.	WP_006344048.1	493387856	×	×	×	×	×	×	×
20.	WP_006344566.1	493388375	×	×	×	×	×	×	Yes
21.	WP_035395338.1	737414331	×	×	×	×	×	×	Yes
22.	WP_006344020.1	493387828	×	×	×	×	×	×	Yes
23.	WP_006344334.1	493388142	×	×	×	×	×	×	Yes
24.	WP_006343689.1	493387496	Yes	Yes	Yes	×	×	Yes	Rejected
25.	WP_006343852.1	493387660	×	×	×	×	×	×	Yes
26.	WP_006343738.1	493387545	×	×	×	×	×	×	Yes
27.	WP_006343847.1	493387655	×	×	×	×	×	×	Yes
28.	WP_006343848.1	493387656	×	×	×	×	×	×	Yes
29.	WP_006343935.1	493387743	×	×	×	×	×	×	Yes
30.	WP_006343849.1	493387657	×	×	×	×	×	×	Yes
31.	WP_006344111.1	493387919	×	×	×	×	×	×	Yes
32.	WP_006343706.1	493387513	×	×	×	×	×	×	Yes
33.	WP_006343695.1	493387502	×	×	×	×	×	×	Yes
34.	WP_006343696.1	493387503	×	×	×	×	×	×	Yes
35.	WP_006344485.1	493388293	×	×	×	×	×	×	Yes
36.	WP_006344486.1	493388294	×	×	×	×	×	×	×
37.	WP_006344325.1	493388133	×	×	×	×	×	×	Yes
38.	WP_006344533.1	493388342	×	×	×	×	×	×	Yes
39.	WP_006344534.1	493388343	×	×	×	×	×	×	×
40.	WP_006343947.1	493387755	Yes	Yes	Yes	×	×	Yes	Rejected
41.	WP_006343725.1	493387532	Yes	Yes	Yes	×	×	Yes	Rejected
42.	WP_006344218.1	493388026	×	×	×	×	×	×	Yes
43.	WP_035395294.1	737414287	×	×	×	×	×	×	Yes
44.	WP_006344406.1	493388214	×	×	×	×	×	×	Yes
45.	WP_006344365.1	493388173	×	×	×	×	×	×	Yes
46.	WP_006343744.1	493387552	×	×	×	×	×	×	Yes
47.	WP_006344477.1	493388285	×	Yes	×	×	×	×	Rejected
48.	WP_006344274.1	493388082	×	×	×	×	×	×	×
49.	WP_006343831.1	493387639	×	×	×	×	×	×	Yes
50.	WP_006344578.1	493388387	×	×	×	×	×	×	Yes
51.	WP_006344167.1	493387975	×	×	×	×	×	×	×

The virulence factors were checked in the nonhomologous proteins. Six proteins showing homology with proteins present in host were rejected because they could also be targeted.

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