International Journal of Genomics
Volume 2016 (2016), Article ID 4512493, 14 pages
http://dx.doi.org/10.1155/2016/4512493

Research Article

Comparative Genomics Analysis of Two Different Virulent Bovine Pasteurella multocida Isolates

Huihui Du,1,2 Rendong Fang,² Tingting Pan,² Tian Li,¹ Nengzhang Li,² Qiang He,¹ Rui Wu,² Yuanyi Peng,² and Zeyang Zhou¹

¹The State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400716, China
²College of Animal Science and Technology, Southwest University, Beibei, Chongqing 400716, China

Received 28 July 2016; Accepted 2 November 2016

Academic Editor: Sylvia Hagemann

Copyright © 2016 Huihui Du et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The Pasteurella multocida capsular type A isolates can cause pneumonia and bovine respiratory disease (BRD). In this study, comparative genomics analysis was carried out to identify the virulence genes in two different virulent P. multocida capsular type A isolates (high virulent PmCQ2 and low virulent PmCQ6). The draft genome sequence of PmCQ2 is 2.32 Mbp and contains 2,002 protein-coding genes, 9 insertion sequence (IS) elements, and 1 prophage region. The draft genome sequence of PmCQ6 is 2.29 Mbp and contains 1,970 protein-coding genes, 2 IS elements, and 3 prophage regions. The genome alignment analysis revealed that the genome similarity between PmCQ2 and PmCQ6 is 99% with high colinearity. To identify the candidate genes responsible for virulence, the PmCQ2 and PmCQ6 were compared together with that of the published genomes of high virulent Pm36950 and PmHN06 and avirulent Pm3480 and Pm70 (capsular type F). Five genes and two insertion sequences are identified in high virulent strains but not in low virulent or avirulent strains. These results indicated that these genes or insertion sequences might be responsible for the virulence of P. multocida, providing prospective candidates for further studies on the pathogenesis and the host-pathogen interactions of P. multocida.

1. Introduction

Pasteurella multocida (P. multocida) is the etiologic agent of bovine pneumonia and hemorrhagic septicemia in cattle which has been estimated to cause huge economic losses. Five capsule types are routinely identified in P. multocida (A, B, D, E, and F) and each is generally associated with, but not completely restricted to, a specific host [1]. P. multocida has the typical characteristics of an opportunistic pathogen that is affected by various host and pathogen specific determinants and can survive in the oral cavity and upper respiratory tract of wild and domestic animals. In both, animals and humans, P. multocida is often associated with chronic as well as acute infections that can lead to significant morbidity (manifested as pasteurellosis, pneumonia, atrophic rhinitis, hemorrhagic septicemia and/or cellulitis, abscesses, and meningitis) and mortality, particularly in animals [2, 3]. Nevertheless, pasteurellosis is still a relatively uncommon cause of mortality in human, even though deaths due to pasteurellosis have increased in recent years in the United States [4, 5], and pasteurellosis in human is often due to bites or scratches by cats or dogs [6, 7].

The first complete genome sequence of P. multocida was Pm70, isolated from avian species in 2001 [8]. Since then, the complete or incomplete genomes of 57 P. multocida isolates have been sequenced, including at least ten complete genomes from the species in the NCBI database. All of the currently available P. multocida genomes are between 1.43 Mbp and 2.44 Mbp in length and comprise a single circular genome with a G+C content between 36.9% and 41%. The available data were used to identify a number of important similarities and differences between these strains and determine their virulence [9].

Several species-specific putative virulence factors, including the capsular and virulence-associated genes, have been proposed to play a key role in the interactions with the host [10, 11]. P. multocida possesses a number of virulence factors which include polysaccharide capsule, endotoxins or lipopolysaccharide (LPS), outer membrane proteins (OMPs), fimbriae, exotoxins, multocidins or siderophores, extracellular enzymes, plasmids, and the virulence-associated genes (tbpA, pfhA, toxA, hgbB, hgbA, Fur, tonB, exbB, hgbB, nanH, nanB, sodA, sodC, ompA, ompH, oma87, PlpB, fimA, hsf-1, hsf-2, tadD, and ptfA) [1, 12, 13]. It is speculated that the virulence factors expressed by P. multocida are likely to play key roles in pathogenesis. Comparative genomics provides an effective source for better understanding the virulence of different isolated strains. In this study, genome sequencing and comparative genomics analysis were carried out to investigate the underlying virulence factors of the high virulent and low virulent bovine P. multocida capsular type A strains, PmCQ2 and PmCQ6, respectively.

2. Materials and Methods

2.1. Bacterial Strains and Culture Conditions

Two P. multocida isolates (PmCQ2 and PmCQ6) have been previously isolated from the fatal pneumonia lungs of feedlot calves at Gaojiazhen farms in Fengdu (Chongqing, China, longitude/latitude 107.70/29.89) from 2011 to 2012. Based on morphological characteristics, biochemical properties, and 16SrRNA gene sequence analysis, the bacteria were identified as P. multocida. Further analysis with PCR amplification of P. multocida species-specific gene Kmt-1 and serotype-specific genes hyaD-hyaC, bcbD, dcbF, ecbJ, and fcbD [16] indicated that the isolates were P. multocida capsular type A, named as PmCQ2 and PmCQ6, and the virulence of the two strains determined by LD₅₀ in Kunming mice showed that PmCQ2 is a high virulent strain and PmCQ6 is a low virulent strain with 2.2 × 10⁵ CFU and 1.14 × 10⁸ CFU, respectively [17]. Isolated strains were maintained at −80°C in Martin Broth (MB) plus 10% glycerol. PmCQ2 and PmCQ6 were inoculated in 5 mL MB at 37°C overnight with shaking. The concentration was determined by viable cell counting on Martin agar plates at 37°C for 24 h.

2.2. Genome Sequencing and Annotation

Genomic DNAs of the two strains were isolated using the Qiagen DNA extraction kits. Genome sequencing was performed using an Illumina MiSeq platform. A total of 6,394,560 and 525,022,200 paired-end 100 bp reads of each genome were assembled into 7 and 32 contigs for strains PmCQ2 and PmCQ6, respectively. The sequences of PmCQ2 and PmCQ6 were assembled by SOAPdenovo [18]. Assemblies were submitted to NCBI for analysis. Open reading frames (ORFs) were annotated by searching against the Nr, Swiss-Prot, and COG databases with manually curation using BLASTP (-value < ) (Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/4512493). The rRNA and tRNA genes were identified using RNAmmer [19] and tRNAscan [20], respectively. A comprehensive genome map containing coding and noncoding genes, COG annotations, and overall G+C content was plotted using Perl-SVG [21].

2.3. Global Alignment Analysis

MUMmer is ideally suited for aligning genomes when the genome sequences are very similar and provides genome-wide sequence comparisons to determine the maximum unique matches between two sequences [22]. Here, MUMmer and BLASTN (-value of ) were applied for a detailed collinearity analysis of the three bovine Pm genomes, PmCQ2, PmCQ6, and Pm36950 at nucleotide sequence levels. Pm36950 is also bovine P. multocida capsular type A strain and was obtained from the NCBI Genebank and was used as the reference genome sequence.

2.4. BLAST Score Ratio Analysis

Genes that were unique to each strain were also identified using BLASTN. The BLAST score ratio (BSR) method was used to compare peptide identities within three genomes (PmCQ2, PmCQ6, and Pm36950) using a measure of similarity based on the ratio of BLAST scores. The output of the BSR analysis enables global visualization of the degree of proteome similarity among genomes and enables the genomic synteny (conserved gene order) between each genome pair to be assessed [23]. Pm36950 was used as a reference genome sequence. The BSR was calculated by dividing the query score by the reference score for each reference peptide. Following calculation of the BSRs, the four quadrants were derived from a BSR threshold value of 0.4, which was empirically determined to represent approximately 30% amino acid identity over approximately 30% of the peptide length and is a commonly used threshold for peptide similarity [24]. The four quadrants were determined for each of the query genomes and colored accordingly: yellow, unique to the reference, PmCQ2 < 0.4, and PmCQ6 < 0.4; red, common to all three, PmCQ2 ≥ 0.4, and PmCQ6 ≥ 0.4; Green, common between PmCQ2 and Pm36950, but absent in PmCQ6, PmCQ2 < 0.4, and PmCQ6 ≥ 0.4; Blue, common between PmCQ6 and Pm36950, but absent in PmCQ2, PmCQ2 ≥ 0.4, and PmCQ6 < 0.4.

2.5. Virulence Factors

Prophage-associated gene clusters were identified by PhiSpy [25]. Genomic islands (GIs) are clusters of genes in prokaryotic genomes of probable horizontal origin. GIs of P. multocida were predicted with IslandPick [26]. Insertion sequences (ISs) of P. multocida were identified by searching sequences against the IS Database (Table S1) that collects all ISs of bacteria and archaea. ISFinder [27] was implemented to launch BLAST with the -value to search the database. Membrane proteins generally include transmembrane domains and were predicted by TMHMM Server 2.0 [28]. Signal peptide, transmembrane domain, GPI-anchor, and general subcellular localization were predicted with SignalP v3.0 [29], TMHMM Server 2.0, GPI-SOM [30], and PSORTb [31] to screen potential secretory proteins that contain signal peptide and no membrane localization signals. The virulence factor database (VFDB) is an integrated and comprehensive online resource for curating information about virulence factors of bacterial pathogens (Table S1). Based on homologous analysis, some virulent factors (ISs, GIs, VF, secretory proteins, and membrane proteins) were obtained in the sequenced strains. In combination with the potential virulent genes of P. multocida and gene annotation information, putative virulence genes for each strain were presented.

3. Results

3.1. Overview of the P. multocida PmCQ2 and PmCQ6 Genomes

The genome sequences of bothPmCQ2 and PmCQ6 strains were successively sequenced by Illumina MiSeq platform. Using Pm36950 as a reference strain, PmCQ2 genome is 2.32 Mbp in size with 39.12% G+C content, containing 2,000 predicted coding regions, 4 rRNAs operons, and 49 tRNAs. PmCQ6 genome is 2.29 Mbp in size with 40.09% G+C content, containing 1,969 predicted coding regions, 1 rRNA operon, and 43 tRNAs. The single circular genome maps of the two P. multocida genomes were shown in Figure 1. There are no obvious species-specific features of the coding density, and the G+C content is highly conserved. Compared with some other P. multocida strains carrying multiple plasmids that may either be cryptic or carry antibiotic resistance genes, both PmCQ2 and PmCQ6 genomes do not contain any plasmids. Taken together, there are only slightly differences in genome sizes, predicted gene numbers, and G+C contents between PmCQ2 and PmCQ6.

(a)

(b)

Figure 1: Circular genome maps of PmCQ2 (a) and PmCQ6 (b) from inside to outside indicate the following: Circle 1, G+C skew; yellow green, G+C skew > 0; purple, G+C skew < 0; Circle 2, G+C content (median represents the above average content, the outer circle is greater than the average content, and the inner circle is less than the average content); Circle 3, rRNA genes distribution represented in scaffold sequence; Circle 4, tRNA gene distribution represented in scaffold sequence; Circle 5, open reading frame (ORF) distribution, plus strand; and Circle 6, multiple scaffold exhibition.

3.2. COG Classification

The predicted protein sequences were annotated to various COG categories. Some differences in protein numbers among COG categories of PmCQ2 and PmCQ6 were identified (including those listed as protein numbers for PmCQ2 and PmCQ6, resp.): “energy production and conversion” (109 and 111), “amino acid transport and metabolism” (158 and 156), “nucleotide transport and metabolism” (60 and 57), “carbohydrate transport and metabolism” (165 and 166), “coenzyme transport and metabolism” (89 and 86), “translation, ribosomal structure, and biogenesis” (132 and 129), “transcription” (81 and 79), “replication, recombination, and repair” (111 and 100), “cell wall/membrane/envelope biogenesis” (145 and 158), “inorganic ion transport and metabolism” (121 and 120), “general function prediction only” (183 and 181), “function unknown” (158 and 157), “signal transduction mechanisms” (42 and 44), and “intracellular trafficking, secretion, and vesicular transport” (38 and 40) (Figure 2).

Figure 2: Clusters of Orthologous Group annotations for the genomes of PmCQ2 and PmCQ6. Arabic colon-separated numbers in brackets indicate matched proteins in PmCQ2 and PmCQ6.

3.3. Global Alignment Analysis

The colinearity analysis at the nucleotide level provides information on sequence insertion or deletion [32]. By aligning the genome at the nucleotide level, there was no significant differences among the large segments between high virulent PmCQ2 and low virulent PmCQ6, and the two strains revealed high colinearity with Pm36950 (Figures 3(a)–3(c)). Direct comparison of the complete nucleotide sequences using BLAST revealed the similarity between PmCQ2 and Pm36950, PmCQ6 and Pm36950, and PmCQ2 and PmCQ6 is 90%, 90%, and 99%, respectively. PmCQ2 and PmCQ6 showed higher homology as indicated by matched CDS (Figure 3(d)). By BSR analysis, the protein sequences shared a high degree of synteny among PmCQ2, PmCQ6, and Pm36950, using Pm36950 as a reference strain (Figure 4). However, some unique proteins were identified, PmCQ2 and PmCQ6 (BLAST score ratio is less than 0.4). There are 32 unique proteins in PmCQ2 genome (including transposase IS200, elongation factor Tu-A-1/2, SrfC, lsrR, TolA, and peptidase B) and only two unique proteins found in PmCQ6 genome (Pasteurella filamentous hemagglutinin protein and mercuric transport protein MerT). The relative chromosomal locations of the unique proteins (red thick marks) of PmCQ2 and PmCQ6 were shown in Figure 5.

(a)

(b)

(c)

(d)

Figure 3: The global alignment analysis of three bovine Pm capsular type A genomes. Aligned segments are represented as dots or line. The alignment was generated by the mummerplot script and the Unix program gnuplot. (a) PmCQ2 and PmCQ6 genome sequences are given on the - and -axis, respectively. (b) Pm36950 and PmCQ2 genome sequences are given on the - and -axis, respectively. (c) Pm36950 and PmCQ6 genome sequences are given on the - and -axis, respectively. Dot plot indicted the alignment blocks of two genome alignment sequences; red and blue indicted the forward and the reverse sequence, respectively. (d) Direct comparison of the three nucleotide sequences using BLAST. The vertical coordinates are the number of genes. Percentage of genetic similarity is indicated by color coding.

Figure 4: The distribution diagram of BLAST score ratio (BSR) between PmCQ2, PmCQ6, and Pm36950. Pm36950 was obtained from NCBI and used as a reference genome sequence. The color coding is as follows: yellow: PmCQ2 < 0.4 and PmCQ6 < 0.4; red: PmCQ2 ≥ 0.4 and PmCQ6 ≥ 0.4; green: PmCQ2 < 0.4 and PmCQ6 ≥ 0.4; blue: PmCQ2 ≥ 0.4 and PmCQ6 < 0.4.

Figure 5: Venn diagram illustrating the number of putative proteins associated with each organism and the number shared with the intersecting organism. Red thick marks on each circle represent the location of the unique proteins (BLAST score ratio less than 0.4) on the PmCQ2 and PmCQ6 genome. Chromosomal comparison: jacinth, PmCQ2; blue, PmCQ6; green, Pm36950.

Using a Venn diagram of three bovine P. multocida strains, the majority of homologous gene groups and unique gene groups were identified. The unique gene groups were significantly different among three strains, containing 37, 29, and 245 gene groups in PmCQ2, PmCQ6, and Pm36950, respectively (Figure 5).

3.4. Virulence Factors

The pathogenicity of P. multocida is associated with different virulence factors. The major virulence factors identified in P. multocida are capsule proteins, lipopolysaccharides, membrane proteins, and secreted proteins. Here, together with genome sequences of PmCQ2 and PmCQ6, published genome sequences of high virulent strains (Pm36950 and PmHN06) and avirulent strains (Pm3480 and Pm70) from NCBI were selected for comparative genomics analysis (Table 1). Comparing the PmCQ2 and PmCQ6 genomes with the complete genome sequences of Pm36950 (G+CA_000234745.1), PmHN06 (G+CA_000255915.1), Pm3480 (G+CA_000259545), and Pm70 (G+CA_000006825.1) using BLAST, a number of virulence-associated genes were identified that were absent or present in all of the comparison strains (Table 2).

Table 1: Genome features of sequenced P. multocida strains.

Strains
Capsular type
Host
Virulence
Size (Mbp)
Genes
References

PmCQ2
A
Bovine
Highly virulent
2.33
2,002
This study

PmCQ6
A
Bovine
Lowly virulent
2.29
1,970
This study

Pm36950
A
Bovine
Highly virulent
2.35
2,182
[14]

PmHN06
D
Swine
Highly virulent
2.41
2,292
[15]

Pm3480
A
Swine
Avirulent
2.37
2,242
NCBI database

Pm70
F
Avian
Avirulent
2.26
2,090
[8]

Table 2: The difference of virulence-associated genes in some or all comparison genomes using BLAST.

Database
PmCQ2
PmCQ6
Pm36950
PmHN06
Pm3480
Pm70
Annotation

Phages-associated genes
PmCQ2_1g0197

Pmu_00900
PMCN06_0843

IS200 transposase

PmCQ2_1g0267

Pmu_13570

IS200 transposase

PmCQ2_1g0316

Pmu_13960

IS200 transposase

PmCQ2_1g0378

Pmu_16290

IS200 transposase

PmCQ2_2g0113

Pmu_17660

IS200 transposase

PmCQ2_4g0323

Pmu_18340

IS200 transposase

PmCQ2_4g0359

IS200 transposase

PmCQ2_2g0080

Pmu_00570
PMCN06_0130
NT08PM_0122
PM1098
Glycyl-tRNA synthetase subunit alpha

PmCQ2_2g0148

Pmu_01310
PMCN06_0200
NT08PM_0197
PM1029
Peptidase B

PmCQ2_2g0349

Pmu_04490
PMCN06_0404
NT08PM_0930
PM0385
Electron transport complex protein RnfC

PmCQ2_2g0231

Pmu_15590

NT08PM_1618
PM1778
Hypothetical protein

PmCQ2_6g0020

Hypothetical protein

PmCQ2_2g0066

Transposase IS605

PmCQ2_2g0097

Transposase IS605

PmCQ2_6g0026

Transposase IS605

PmCQ2_2g0228

NT08PM_0285

Hypothetical protein

PmCQ2_2g0229

NT08PM_0286

Hypothetical protein

PmCQ2_2g0230

NT08PM_0288

Hypothetical protein

PmCQ2_2g0233

NT08PM_0294

Phage major capsid protein

PmCQ2_2g0234

NT08PM_0295

Hypothetical protein

PmCQ2_2g0235

NT08PM_0298

Prophage integrase, putative

PmCQ2_2g0067

Transposase IS200

PmCQ2_2g0096

Transposase IS200

PmCQ2_2g0232

Hypothetical protein

PmCQ2_2g0088

ModB, partial

PmCQ6_9g0009

PMCN06_2102

Hypothetical protein PMCN06_2102

PmCQ6_9g0008

PMCN06_2103

Hypothetical protein PMCN06_2103

PmCQ6_9g0006

PMCN06_2105

Hypothetical protein PMCN06_2105

PmCQ6_9g0003

Tail assembly protein I

PmCQ6_9g0001

Host specificity protein, putative, partial

PmCQ6_9g0005

Tail protein

PmCQ6_9g0004

Tail assembly protein

Genomic islands
PmCQ2_1g0197

Pmu_00900
PMCN06_0843

IS200 transposase

PmCQ2_1g0267

Pmu_13570

IS200 transposase

PmCQ2_1g0316

Pmu_13960

IS200 transposase

PmCQ2_1g0378

Pmu_16290

IS200 transposase

PmCQ2_2g0113

Pmu_17660

IS200 transposase

PmCQ2_4g0323

Pmu_18340

IS200 transposase

PmCQ2_4g0359

IS200 transposase

PmCQ2_2g0231

Pmu_15590

NT08PM_1618
PM1778
Hypothetical protein

PmCQ2_6g0025

PMCN06_0111
NT08PM_0103

Hypothetical protein PMCN06_0111

PMCN06_1438

PMCN06_2110

PmCQ2_5g0021

PMCN06_0084
NT08PM_0084

Hypothetical protein PMCN06_0084

PMCN06_2086

PmCQ2_5g0019

PMCN06_0086
NT08PM_0086

Phage terminase, large subunit, pbsx family, putative

PMCN06_2088

PmCQ2_2g0228

NT08PM_0285

Hypothetical protein

PmCQ2_2g0229

NT08PM_0286

Hypothetical protein

PmCQ2_2g0230

NT08PM_0288

Hypothetical protein

PmCQ2_2g0233

NT08PM_0294

Phage major capsid protein

PmCQ2_2g0234

NT08PM_0295

Hypothetical protein

PmCQ2_2g0235

NT08PM_0298

Prophage integrase, putative

PmCQ2_7g0006

PMCN06_0066

Transcriptional regulator

PmCQ2_5g0013

PMCN06_0092

Hypothetical protein PMCN06_0092

PmCQ2_5g0025

PMCN06_2080

Hypothetical protein

PmCQ6_17g0009

PMCN06_0073
NT08PM_0074

Site-specific DNA-methyltransferase (adenine-specific)

PMCN06_2074

PmCQ6_17g0011

PMCN06_0074
NT08PM_0075

Putative bacteriophage protein

PMCN06_2076

PmCQ6_5g0001

PMCN06_0082
NT08PM_0082

Lysozyme

PMCN06_2084

PmCQ6_17g0004

PMCN06_1403

Hypothetical protein

PMCN06_2067

PmCQ6_C4143g0001

PMCN06_1416

Glycoside hydrolase

PmCQ6_17g0007

PMCN06_2072

Hypothetical protein PMCN06_2072

PmCQ6_17g0008

PMCN06_2073

Putative replicative DNA helicase

PmCQ6_17g0010

PMCN06_2075

Hypothetical protein PMCN06_2075

PmCQ6_9g0009

PMCN06_2102

Hypothetical protein PMCN06_2102

PmCQ6_9g0008

PMCN06_2103

Hypothetical protein PMCN06_2103

PmCQ6_9g0006

PMCN06_2105

Hypothetical protein PMCN06_2105

ISs
PmCQ2_1g0197

Pmu_00900
PMCN06_0843

IS200 transposase

PmCQ2_1g0267

Pmu_13570
PMCN06_0843

IS200 transposase

PmCQ2_1g0316

Pmu_13960
PMCN06_0843

IS200 transposase

PmCQ2_1g0378

Pmu_16290
PMCN06_0843

IS200 transposase

PmCQ2_2g0113

Pmu_17660
PMCN06_0843

IS200 transposase

PmCQ2_4g0323

Pmu_18340
PMCN06_0843

IS200 transposase

PmCQ2_4g0359

PMCN06_0843

IS200 transposase

PmCQ6_12g0001

Putative transposase for insertion sequence IS1162

VFDB
PmCQ2_4g0316

Pmu_13500
PMCN06_1329
NT08PM_1414
PM1994
UDP-3-O-[3-hydroxymyristoyl]; UDP-3-O-acylglucosamine N-acyltransferase

PmCQ2_1g0631

Pmu_21120
PMCN06_2192
NT08PM_2001
PM1666
Noncanonical purine NTP pyrophosphatase, RdgB/HAM1 family

PmCQ2_4g0241

Pmu_12710
PMCN06_1257
NT08PM_1342
PM0051
Iron-binding protein FbpA

PmCQ2_3g0252

Pmu_08050
PMCN06_0796
NT08PM_0537
PM0734
Periplasmic serine protease do/hhoA-like protein

PmCQ2_2g0162

Pmu_01460
PMCN06_0215
NT08PM_0212
PM1015
Hypothetical protein PM1015

PmCQ2_1g0106

Pmu_15140
PMCN06_1551
NT08PM_1574
PM1820
Putative virulence effector, SrfC

PmCQ2_1g0553

Pmu_15880
PMCN06_1607
NT08PM_1650
PM1357
Elongation factor Tu, partial

PmCQ2_1g0157

Pmu_20230
PMCN06_2025
NT08PM_2100
PM1746

PmCQ2_3g0367

Nonspecific tight adherence protein D, partial

PmCQ6_2g0065
Pmu_09310

NT08PM_0414
PM0846
Nonspecific tight adherence protein D

PmCQ6_6g0048

Nucleoside-diphosphate sugar epimerase/dehydratase

Secreted proteins
PmCQ2_1g0033

Pmu_14370
PMCN06_1474
NT08PM_1498
PM1897
Hypothetical protein, uncharacterized lipoprotein PM1897

PmCQ2_3g0252

Pmu_08050
PMCN06_0796
NT08PM_0537
PM0734
Periplasmic serine protease do/hhoA-like protein

PmCQ2_2g0088

ModB, partial

Membrane proteins
PmCQ2_1g0666

Pmu_21540
PMCN06_2233
NT08PM_2236
PM1230
Penicillin-binding protein 1A

NT08PM_2237

PmCQ2_4g0293

Pmu_13260
PMCN06_1306
NT08PM_1389
PM0004
Bicyclomycin resistance protein-1

PmCQ6_1g0027
Pmu_21770

NT08PM_2255
PM1212
Mercuric transport protein MerT

PmCQ6_C4143g0001

PMCN06_1416

Glycoside hydrolase

PmCQ6_17g0010

PMCN06_2075

Hypothetical protein PMCN06_2075

PmCQ6_9g0003

Tail assembly protein I

PmCQ6_14g0071

C4-dicarboxylate ABC transporter permease

PmCQ6_23g0011

Hypothetical protein, partial

A number of genes or gene clusters have been implicated as important for virulence of P. multocida [9]. Some of these genes encoding putative virulence factors are universally present in all six P. multocida genomes, including genes encoding prophage, genomic islands, insertion sequences, virulence factor, secretory proteins, and outer membrane proteins.

By comparing the high virulent strains (PmCQ2, Pm36950, and PmHN06) with low virulent strain (PmCQ6) and avirulent strains (Pm3480 and Pm70), unique genes which were correlated with virulence and only presented in high virulent strains were identified. For instance, insertion sequence (transposase IS200) only existed in three high virulent strains, suggesting that IS200 elements are not conserved sequences and do not spread among all P. multocida strains. IS605 and secreted protein PmCQ2_2g0088 (ModB) and nonspecific tight adherence protein D PmCQ2_3g0367 were presented only in PmCQ2 genome (Table 2).

In addition, genomic islands (GIs) are clusters of genes in prokaryotic genomes and are probable horizontal origin. GIs of Pm70, Pm3480, Pm36950, and PmHN06 were predicted with IslandPick. Homology analysis of these GIs with the draft genomes of PmCQ2 and PmCQ6 was carried out using ORTHOMCL1.4 (BLAST value , percent identity cutoff 60%, and percent match cutoff 60%). The result showed that transcriptional regulator PmCQ2_7g0006 and hypothetical proteins PmCQ2_5g0013 and PmCQ2_5g0025 are present in high virulent strains (PmCQ2 and PmHN06) but absent in low virulent strain PmCQ6 and the avirulent strains (Pm70 and Pm3480).

Taken together, comparative genomics analysis supplies essential information for understanding the virulence of different capsular type (A, D, and F) and different host origin (bovine, avian, and swine) strains. Five unique genes and two insertion sequences were identified only in high virulent strains, providing candidate virulence factors for further studies on the pathogenesis of different P. multocida strains (Table 3).

Table 3: The distribution of predicted virulence factors among different P. multocida strains.

Virulence
Strains
IS 200
PmCQ2_5g0025
PmCQ2_7g0006
PmCQ2_5g0013
IS 605
PmCQ2_2g0088
PmCQ2_3g0367
Capsular type
Host

Highly virulent
PmCQ2

A
Bovine

Highly virulent
Pm36950

A
Bovine

Highly virulent
PmHN06

D
Swine

Lowly virulent
PmCQ6

A
Bovine

Avirulent
Pm3480

A
Swine

Avirulent
Pm70

F
Avian

+ stands for the gene present in certain strain; − stands for the gene absent in certain strain.

4. Discussion

Moreover, comparative genomic analysis allows the identification of core genes and/or disease-specific factors. The first complete P. multocida genome was sequenced from strain Pm70 in 2001, from which 104 putative virulence-associated genes were identified [8]; this facilitated new approaches for studying the pathogenesis of P. multocida. Until now, the complete and incomplete genomes of 57 P. multocida have been sequenced in NCBI database. In this study, two bovine P. multocida capsular type A genomes (high virulent PmCQ2 and low virulent PmCQ6) were sequenced. Comparative genomics analysis was performed among PmCQ2, PmCQ6, and four other P. multocida genomes (Pm36950, PmHN06, Pm3480, and Pm70) from NCBI. Some virulence genes were identified among different virulent strains; five genes and two insertion sequences were only identified in high virulent strains, which might be responsible for the virulence differences among high virulent, low virulent, and avirulent strains.

The genome sequences of high virulent PmCQ2 and the low virulent PmCQ6 have high similarity, but the virulence of two strains is significantly different. It could be speculated that the unique genes may play a key role in virulence. Compared with PmCQ6, the five genes and two insertion sequences are predicted virulence-associated genes in PmCQ2 and other high virulent strains. Further studies to construct mutant strains targeting these genes would be of great importance to prove their contributions to virulence. Besides, PmCQ2 has more than 30 other unique genes that might also orchestrate the virulence differences of PmCQ2 and PmCQ6. These genes include recombinase, phage-related genes, phage N-6-adenine-methyltransferase, phage terminase, and prophage integrase.

Based on homology analysis, prophage-associated genes, GIs, ISs, secretory proteins, and membrane proteins were screened for different virulence-associated genes among different virulent strains. Insertion sequences usually only carry genes of transposon sequences for the transposition in bacteria and can also induce a variety of genomic rearrangements; they also play an important role in bacterial host specificity and virulence [33, 34]. Transposase IS200 was found in three high virulent isolated strains encoding the 7 genes (PmCQ2_1g0197, PmCQ2_1g0267, PmCQ2_1g0316, PmCQ2_1g0378, PmCQ2_2g0113, PmCQ2_4g0323, and PmCQ2_4g0359), but IS200 was not present in the low virulent strains (PmCQ6, Pm70) or the avirulent strain (Pm3480). The IS200 elements may adapt to different hosts in closely related genera but stochastic loss can appear in some low virulent or avirulent strains. According to previous reports, IS200-related transposons may have already existed in remote stages of bacterial evolution, such as Salmonellae, and IS200-based methods have been described for the identification of certain Salmonella serovars [35]. The function and host range of transposase IS200 in P. multocida still need to be further studied.

PmCQ2_2g0088 has been suggested to encode a subfamily of ATP-binding cassette (ABC) transporters that have a possible role in remodeling the cell envelope and entry of the pathogen into nonphagocytic cells [36]. Bacterial ABC transporters are essential for the uptake of nutrients, including rare elements such as molybdenum [37]. ABC transporters are integral membrane proteins that actively transport molecules across cell membranes [38], and these three proteins are coded by modA, modB, and modC genes, respectively. The ModA, ModB, and ModC proteins are very similar in various organisms (Escherichia coli, Haemophilus influenzae, Azotobacter vinelandii, and Rhodobacter capsulatus) [39]. In this study, PmCQ2_2g0088 (ModB) is only present in virulent PmCQ2 but absent in PmCQ6. PmCQ2_2g0088 contains a signal peptide and a SBP_bac_11 structural domain. The SBP-box gene family is specific to plants and encodes a class of zinc finger-containing transcription factors with a broad range of functions [40]. However, the function of the ModB protein family has not been clearly established; PmCQ2_2g0088 might affect the virulence of strain and needs to be further studied as a candidate virulence factor.

The present study revealed that P. multocida strains carry different virulence genes which may indicate variation in the pathogenicity. It could be speculated that the specific genes of different strains play the most important role for the difference of pathogenicity. By extensive genomics and proteomics analysis, the intensive study on virulence genes provides deeper understanding of host specificity and pathogenesis and also provides insights into the host-microbe interactions and the immunologic mechanism, contributing to the development of novel vaccines.

Additional Points

Availability of Data and Materials. The genome sequences of PmCQ2 and PmCQ6 have been deposited at GenBank under the accession numbers of LIUN00000000 and LIUO00000000, respectively.

Competing Interests

The authors declare no conflict of interests.

Authors’ Contributions

Huihui Du and Rendong Fang contributed equally to the present study. Yuanyi Peng and Zeyang Zhou conceived and designed the experiments. Huihui Du and Tingting Pan performed the experiments. Huihui Du and Rendong Fang wrote the paper. Nengzhang Li, Qiang He, Tian Li, and Rui Wu contributed to data analysis. Rendong Fang, Yuanyi Peng, and Zeyang Zhou supervised the project.

Acknowledgments

This work was supported by the earmarked fund for China Agriculture Research System (Beef/Yak Cattle, CARS-38), Chongqing Science & Technology Commission (cstc2015shmszx8022, cstc2015jcyjBX0108, cstc2015shmszx80010, and cstc2015jcyjA80021), the National Natural Science Foundation of China (31400762), and the Fundamental Research Funds for the Central Universities (XDJK2015B002).

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