Identification of the positively selected genes governing host-pathogen arm race in Vibrio sp. through comparative genomics approach

Abstract

Bacterial evolution is due to the adaptive nature of the core bacterial genomes that plays critical role in diversification, fitness and adaptation of the species to different environment. Host-pathogen interactions and environmental settings are such forces that guide the adaptation of bacterial genomes. Since Vibrio cholerae represents an appropriate model organism for studying the interplay of environment and host driven factors shaping the microbial genome structure and function, the current study aims to identify genes that are under these strong forces in V. cholerae. Here, we employed a comparative genomics approach to identify genes that are under positive selection in ten strains of Vibrio sp. including four pathogenic V. cholerae strains. From the available genome sequence data, a total of 421 orthologous genes were identified by reciprocal BLAST best-hit method, recombination breakpoint frequency analysis and tree comparison method. These 421 genes, representing the core genome of Vibrio sp., constituted the dataset to be analyzed for evolutionary selections. The analysis of natural selection, based on Maximum Likelihood method on synonymous and non-synonymous substitution rate, confirms the hypothesis that the bacterial core genomes are mostly under purifying selection with a few positively selected regions. However, our finding also reveals that positively selected sites in the Vibrio genome occur in a wide range of different genes encompassing diverse functional pathways including cell surface proteins (e.g. outer membrane-specific lipoprotein transporter/assembly proteins etc.), cell motility proteins (e.g. flagellar motor switch proteins, flagellar hook and assembly proteins), nutrient acquisition (e.g. amino acid, carbohydrate and phosphate ABC transporters), DNA repair and transcription related proteins. Interestingly, these positively selected gene products are directly involved with host-pathogen interactions and fitness in gastrointestinal environment. Therefore, the collective evidences of these positively selected genes spanning several pathways raise the possibility of their involvement in evolutionary arms races with other bacteria, phages, and/or the host immune system. This finding points to the natural selections which is the responsible factor for the diversification of Vibrio genus.

Full text

Identification of the Positively Selected Genes Governing Identification of the Positively Selected Genes Governing
HostHost--Pathogen Arm Race in Pathogen Arm Race in VibrioVibrio sp. through Comparative sp. through Comparative
Genomics Approach Genomics Approach
Authors:
Atai Rabby1, Sajib Chakraborty1*, Atiqur Rahman1, Shahjalal Soad1,Shamma Shakila
Rahman1,Kaniz Fatima Chanda1, Rajib Chakravorty2
1Department of Biochemistry and Molecular Biology, Faculty of Biological Sciences, University
of Dhaka, Dhaka-1000, Bangladesh.
2Department of EEE, University of Melbourne, National ICT Australia, Victoria 3010, Australia

Background
Vibrio cholerae is a Gram-negative, comma-shaped
bacterium. Some strains of V. cholerae cause the
disease cholera and reside in human Gut or
Intestine.
V. cholerae pathogenicity genes code for proteins
directly or indirectly involved in the virulence of the
bacteria.
V. cholerae has two circular chromosomes , together
totalling 4 million base pairs of DNA sequence and
3,885 predicted genes.
The main reservoirs of V. cholerae are people and aquatic sources such as brackish
water and estuaries.
Two serogroups of V. cholerae, O1 and O139, cause outbreaks of cholera.

The bacterial evolution is due to the adaptive nature of the core bacterial genomes that
plays critical role in diversification, fitness and adaptation of the species to different
environment.
Host-pathogen interaction and environmental setting are such forces that guide the
adaptation of bacterial genomes.
Introduction
Since Vibrio cholerae represents an appropriate model organism for studying the interplay
of environment and host driven factors shaping the microbial genome structure and
function, the current study aims to identify genes that are under these strong forces in
Vibrio cholerae.
Here we have employed a comparative genomics approach to identify genes that are
under positive selection in ten strains of Vibrio sp. including four pathogenic strains of
Vibrio cholerae.

Material
Starin RefSeq Accession
Vibrio cholerae O395 NC_009456, NC_009457
Vibrio cholerae O1 NC_002505, NC_002506
Vibrio cholerae MJ NC_012667, NC_012668
Vibrio
cholerae
M
66
NC_
012578
,
NC_
012580
Table : Genome Sequences
Vibrio
cholerae
M
66
NC_
012578
,
NC_
012580
Vibrio splendidus LGP32 NC_011744, NC_011753
Vibrio vulnificus CMCP6 NC_004459, NC_004460
Vibrio vulnificus YJ016 NC_005128, NC_005139, NC_005140
Vibrio fischeri ES114 NC_006840, NC_006841, NC_006842
Vibrio harveyi ATCC NC_009777, NC_009783, NC_009784
Vibrio parahaemolyticus RIMD NC_004603, NC_004605
* Whole Genome Sequence With Genes and Prtein Sequence Was Retrived From NCBI Genome
Database Via FTP.

Method
Starting With All O395 V. Cholerae Genes
Removing Hypothetical and Putative genes
Building Mirror Database
Ugene
422 Orthologous Gens
Building Mirror Database
Reciprocal Blast Best Hit
886 Homologous Gene Sets
Bioedit,Corilog ViewerE-value : 10e-6, Query Coverage : 80%
Gencov, GARD, Chimera
RDP
Remove Paralogous Genes
444 genes
Detection of
Horizontal Gene
Transfer
T-Rex

Method
Homologous
Orthologous
Paralogous
Xenologous
Orthologous
Paralogous
Xenologous
Reciprocal Blast Recombination Detection
Program
(Gencov, GARD, Chimera)
Comparison with rooted tree
(based on 16s rRNA)

Method
422 othologous Genes
422 orthologous Proteins
BlastX
Codon Alignment
Selection with Codon wise dN/dS
RevTrans 1.4
MEGA 5.03

Selection of Species
Figure : Tree of 16s rRNA for Closely
Related Vibrio Species. ( Clade that is
chosen for further Analysis in
indicated with RED box )

COG Categorie of Orthologous Genes
Translation, ribosomal
structure and biogenesis
12%
Transcription
6%
Nucleotide
transport and
metabolism
Coenzyme
metabolism
6%
Lipid metabolism
4%
Secondary metabolites
biosynthesis, transport
and catabolism
1%
General function
prediction only
3%
Function unknown
1%
DNA replication,
recombination and repair
8% Cell division and
chromosome
partitioning
1%
Posttranslational
modification, protein
turnover, chaperones
4%
Cell envelope
biogenesis, outer
membrane
4%
Cell motility and
secretion
5%
Inorganic ion transport
and metabolism
4%
Signal transduction
mechanisms
2%
Energy production
and conversion
9%
Carbohydrate
transport and
metabolism
9%
Amino acid transport and
metabolism
13%
metabolism
7%
Figure : Orthologous Genes In
Different Pathways The
orthologous genes of Vibrio
cholerae O395 was classified
according to their pathway
retrieved from NCBI COG
categories Database.

Pathways that are under influence of Positive Selection
50%
33%
17%
Intracellular trafficing and
secretion
42%
32%
26%
Posttranslational modification:
protein turnover: chaperones
High
38%
19%
43%
Cell motility and secretion
38%
25%
38%
Signal transduction mechanisms
High
Medium
Low

Pathways that are under influence of Positive Selection
36%
14%
50%
Inorganic ion transport and
metabolism
33%
17%
50%
Cell division and chromosome
partitioning
High
24%
41%
35%
Cell envelope biogenesis: outer
membrane
24%
33%
43%
DNA replication: recombination
and repair
High
Medium
Low

Pathways that have Less influence of Positive Selection
21%
25%
54%
Coenzyme metabolism
23%
39%
38%
Energy production and
conversion
High
18%
31%
51%
Amino acid transport and
metabolism
15%
41%
44%
Lipid metabolism
High
Medium
Low

Pathways that have Less influence of Positive Selection
7%
41%
52%
Nucleotide transport and
metabolism
8%
46%
46%
Translation: ribosomal
structure and biogenesis
High
Medium
15%
35%
50%
Transcription
Medium
Low

Genes with higher rate of Positive selection
Genes Under Influence of Positive Selection
Intracellular trafficing and secretion
MotA----TolQ----ExbB proton channel family protein
preprotein translocase subunit SecF
Signal peptidase I
Posttranslational modification, protein turnover, chaperones
Genes Under Influence of Positive Selection
Cell motility and secretion
methyl-accepting chemotaxis protein
flagellar motor switch protein
flagellar basal body rod protein FlgB
flagellar
motor protein
MotB
Posttranslational modification, protein turnover, chaperones
3-oxoacyl-(acyl carrier protein) synthase II
peptidyl-prolyl cis-trans isomerase, FKBP-type
cytochrome c-type biogenesis protein CcmF
trigger factor
bifunctional glutamine-synthetase adenylyltransferase----
deadenyltransferase
protease IV
SsrA-binding protein
co-chaperone HscB
Peptide Methionine sulfoxide reductase
flagellar
motor protein
MotB
flagellin
flagellar hook protein FlgE
flagellar basal body rod modification protein
flagellar hook-associated protein FlgK
flagellar MS-ring protein
flagellar basal body L-ring protein
Signal transduction mechanisms
regulatory protein CsrD
aerobic respiration control sensor protein ArcB
fused phosphoenolpyruvate-protein phosphotransferase
PtsP...GAF domain

Result

Result : Comparison With House-kepping Genes as control
S

Codon Wise Selection
0
0.5
1
1.5
2
2.5
3
1
26
51
76
101
126
151
176
201
226
251
dN/dS
flagellar basal body L-ring protein
P-Value : ≤ 0.05
1
26
51
76
101
126
151
176
201
226
251
Codons
0
1
2
3
4
5
1
26
51
76
101
126
151
176
201
226
251
276
301
326
351
376
401
426
451
476
501
526
dN/dS
Codons
methyl-accepting chemotaxis protein
P-Value : ≤ 0.05

Codon Wise Selection
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
1
26
51
76
101
126
151
176
201
226
251
276
301
326
351
dN/dS
flagellin
P-Value : ≤ 0.05
1
26
51
76
101
126
151
176
201
226
251
276
301
326
351
Codons
0
0.5
1
1.5
2
2.5
3
3.5
4
1
26
51
76
101
126
151
176
201
226
251
276
301
326
351
376
401
426
451
476
501
526
551
576
601
dN/dS
Codons
flagellar hook-associated protein FlgK
P-Value : ≤ 0.05

Exploring 3-D structure for PS Sites
Methyl
-
accepting chemotaxis protein
Methyl
-
accepting chemotaxis protein
Flagellin
Flagellar basal body rod modification protein Flagellar MS-ring protein

Flagellar Assembly Proteins

Region in between genes
0
0.5
1
1.5
2
2.5
3
3.5
4
Signal peptidase I
0
1
2
3
4
5
6
7
8
preprotein translocase subunit SecF
35 - 62
107 - 297
82 - 149
0
1 26 51 76 101 126 151 176 201 226 251 276
0
0.5
1
1.5
2
2.5
3
3.5
1 26 51 76 101 126 151 176 201 226 251 276 301 326 351 376 401 426 451
MotA/TolQ/ExbB proton channel family
0
1 26 51 76 101 126 151 176 201 226 251 276 301
305 - 432

Conclusion
Positive Selection of Positive Selection of VibrioVibrio choleraecholerae is a Genome wide Phenomena.is a Genome wide Phenomena.
Genes which come direct contact with host interaction are influenced by the Genes which come direct contact with host interaction are influenced by the
selection process .selection process .
FlagellarFlagellar Assembly Proteins are in High Selection influence, which explain the Assembly Proteins are in High Selection influence, which explain the
importance of motility for importance of motility for VibrioVibrio choleraecholerae pathogenic strain.pathogenic strain.
FlagellarFlagellar Assembly Assembly OperonOperon is probably Under Selection Pressure.is probably Under Selection Pressure.
Pathways that are highly influenced by selection process indicating possible Pathways that are highly influenced by selection process indicating possible
immergence of immergence of VibrioVibrio choleraecholerae due to environmental pressure, interaction with host due to environmental pressure, interaction with host
and facility during food intake.and facility during food intake.

Further Investigation
Exploring Every genes for functional site with selection Pressure.
Exploring each genes 3-D structure with high substitution rate.
More Insights With Selection Method
Human Immune System
Micobacterium Tuberculosis