Open Access

The genome anatomy of Corynebacterium pseudotuberculosis VD57 a highly virulent strain causing Caseous lymphadenitis

  • Sintia Almeida1Email author,
  • Sandeep Tiwari1,
  • Diego Mariano1,
  • Flávia Souza1,
  • Syed Babar Jamal1,
  • Nilson Coimbra3,
  • Roberto Tadeu Raittz3,
  • Fernanda Alves Dorella2,
  • Alex Fiorine de Carvalho2,
  • Felipe Luiz Pereira2,
  • Siomar de Castro Soares2,
  • Carlos Augusto Gomes Leal2,
  • Debmalya Barh6,
  • Preetam Ghosh7,
  • Henrique Figueiredo2,
  • Lília Ferreira Moura-Costa4,
  • Ricardo Wagner Portela4,
  • Roberto Meyer4,
  • Artur Silva5 and
  • Vasco Azevedo1Email author
Standards in Genomic Sciences201611:29

https://doi.org/10.1186/s40793-016-0149-7

Received: 22 June 2015

Accepted: 30 March 2016

Published: 8 April 2016

Abstract

Corynebacterium pseudotuberculosis strain VD57 (Cp_VD57), a highly virulent, nonmotile, non-sporulating, and a mesophilic bacterium, was isolated from a goat’s granulomatous lesion in the municipality of Juazeiro, Bahia State, Brazil. Here, we describe a set of features of the strain, together with the details of its complete genome sequence and annotation. The genome comprises of a 2.5 Mbp long, single circular genome with 2,101 protein-coding genes, 12 rRNA, 49 tRNA and 47 pseudogenes and a G + C content of 52.85 %. Genetic variation was detected in Cp_VD57 using C. pseudotuberculosis strain 1002 as reference, wherein small genomic insertions and deletions were identified. The comparative analysis of the genome sequence provides means to better understand the host pathogen interactions of this strain and can also help us to understand the molecular and genetic basis of virulence of this bacterium.

Keywords

Biovar ovis Gram-positive pathogen Caseous lymphadenitis Corynebacterium pseudotuberculosis Goat Genome sequencing Ion Torrent PGM

Introduction

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis in sheep and goats, the organism has also been associated with mastitis [13] and can cause ulcerative lymphangitis in horses and cattle [4]. CL is a chronic disease that is characterized by the formation of granulomas in lymph nodes and internal organs, as a response of the host’s immune system against this bacterium that resists to the bactericidal action of phagocytic cells [3].

CL is considered as one of the economically important diseases of small ruminants with losses attributed to reduced wool and hide yields, carcass condemnation, morbidity and rarely mortality [5, 6]. The prevalence of CL has been observed worldwide, including South Africa, Brazil, the USA, Canada, Australia, New Zealand, United Kingdom and Egypt [7].

The pangenome analysis of 15 strains of the pathogen was completed recently [8]. However, as C. pseudotuberculosis is a relatively clonal organism [913], the identification of the virulence mechanisms or nucleotide modifications responsible for making a strain more virulent than another, have not yet been identified.

Sequencing of new genomes coupled with a deeper comparative analysis between the genomes and associating such analyses with the host pathogen interactions can help us understand and identify the differences between genomes and virulence factors. In this context, the present study reports the sequence the genome of the highly virulent strain VD 57 and to understand its virulence factors.

Organism information

Classification and features

C. pseudotuberculosis is a Gram-positive bacteria and belong to a CMNR ( Corynebacterium , Mycobacterium , Nocardia and Rhodococcus ) group that shares characteristics including an outer lipid layer, mycolic acids in the cell wall along with its derivatives including phospholipids and lipomannans [7]. C. pseudotuberculosis is a facultative intracellular pathogen showing pleomorphic forms like coccoids and filamentous rods, non-motile, non-sporulating and possessing fimbriae, with sizes ranging between 0.5–0.6 μm and 1.0–3.0 μm [7].

The C. pseudotuberculosis strain VD57 (Cp_VD57) was isolated from a goat’s granulomatous lesion in the municipality of Juazeiro, Bahia State, Brazil. The bacterial identification was made through Gram’s staining, colonies’ morphology analysis, synergic hemolysis with Rhodococcus equi in Brain Heart Infusion, Blood Agar Medium, and biochemical assays using the API Coryne system (BioMérieux). The strain is maintained in BHI broth at the Microbiology Laboratory of the Federal University of Bahia [14, 15].

C. pseudotuberculosis strain VD57 has been shown to be highly pathogenic to goats and mice [14]. This Cp_VD57 strain was able to induce IFN-gamma production in goats on day 5 after infection. Additionally, it induced a positive antibody titer between 6 and 11 days after infection [16]. Using a murine experimental model, it was observed that, the strain was able to induce a high mortality, when compared to the T1 attenuated strain, confirming its virulent profile [15]. Moura-Costa et al. used Cp_VD57 strain to challenge goats that were immunized with the attenuated T1 strain, obtaining a protection of 33.3 % and a strong humoral response, but the immunization was not able to prevent the spread of this virulent bacteria in the majority of the vaccinated animals [14].

One of the most important fields in the C. pseudotuberculosis study is the definition of genes that are differentially expressed in bacterial cultures and inside the granulomatous lesions. In this regard, VD57 strain was used in a study with the objective to determine reference genes to be used in quantitative real time PCR. It was found that eight of these genes (atpA, dnaG, efp, fusA, gyrA, gyrB, rpoB, and rpoC), mostly participating in DNA replication and transcription, can be useful as candidate reference genes, while DNA gyrase subunit A (gyrA) and elongation factor P (fusA) presented the most suitable profiles to be used in qPCR studies [17]. Figure 1 shows a phylogenetic tree of Corynebacterium pseudotuberculosis strain VD57 based on rpoB gene (β subunit of RNA polymerase). All the classification and general features of C. pseudotuberculosis strain VD57 are summarized in Table 1.
Fig. 1

Phylogenetic tree of C. pseudotuberculosis strain VD57 representing its position relative to type strains in Corynebacteriaceae along with some other type strains of CMNR group. The tree was inferred from 3,537 aligned characters of the rpoB gene sequence using maximum likelihood method and then checked for its agreement with the current classification in Table 1. The branch lengths represent the expected number of substitutions per site. Numbers adjacent to the branches are support values from 1,000 bootstrap replicates, indicated when larger than 60 %. Calculations to determine the phylogenetic distances were done by the software MEGA v6 [40]. The GenBank accession numbers are shown in parentheses

Table 1

Classification and general features of Corynebacterium pseudotuberculosis strain VD57 according to the MIGS recommendations [19]

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [30]

  

Phylum Actinobacteria

TAS [31]

  

Class Actinobacteria

TAS [32]

  

Order Actinomycetales Suborder Corynebacterineae

TAS [32, 33]

  

Family Corynebacteriaceae

TAS [3235]

  

Genus Corynebacterium

TAS [3638]

  

Species Corynebacterium pseudotuberculosis

TAS [37, 39]

 

Gram stain

Positive

TAS [14]

 

Cell shape

Bacilli

TAS [14]

 

Motility

Non-motile

TAS [14]

 

Sporulation

Non-sporulating

TAS [14]

 

Temperature range

Mesophilic

NAS

 

Optimum temperature

37 °C

TAS [14, 18]

 

pH range; Optimum

7.0–7.2

TAS [7]

 

Carbon source

Glucose

TAS [14]

MIGS-6

Habitat

Host

TAS [32]

MIGS-6.3

Salinity

Not reported

 

MIGS-22

Oxygen requirement

Aerobic and Obligate Aerobic

TAS [14, 18]

MIGS-15

Biotic relationship

Intracellular facultative pathogen

TAS [7, 14, 15]

MIGS-14

Pathogenicity

Goat

TAS [14]

MIGS-4

Geographic location

Bahia State, Brazil

TAS [14]

MIGS-5

Sample collection time

2005

[NAS]

MIGS-4.1

Latitude

9°24’S

[IDA]

MIGS-4.2

Longitude

40°30’W

[IDA]

aEvidence codes - IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature), NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence)

De Souza et al. employed VD57 strain to verify the intracellular signaling cascade activation during the infection of splenocytes with the bacterium, and the importance of signaling pathways in the production of different cytokines. The results showed that VD57 strain was able to induce the production of TNF-alpha through the MAPK p38, and IL-10 induction via ERK-1 and −2 pathways. The complete genome sequencing and analysis will help in identifying the genetic background and the genes that may be involved in the infections [18].

Genome sequencing information

Genome project history

In the present study, we determined the nucleotide sequence of the C. pseudotuberculosis strain VD57 (Cp_VD57) genome, isolated from a goat granulomatous lesion. Sequencing, assembly, and annotation were performed at Laboratory of Cellular and Molecular Genetics (LGCM), Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil and Aquacen - National Reference Laboratory for Aquatic Animal Diseases, Federal University of Minas Gerais, Brazil. The Cp_VD57 complete genome sequence and annotation data were deposited in the GenBank under the accession number CP009927. Table 2 presents the project information in accordance with the Minimum Information about a Genome Sequence (MIGS) [19].
Table 2

Genome sequencing project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

Finished

MIGS-28

Libraries used

Fragments

MIGS 29

Sequencing platforms

Semiconductor Ion Torrent PGM

MIGS 31.2

Fold coverage

78.22-fold

MIGS 30

Assemblers

MIRA .4.0CLC Genome Workbench 4.7.2

MIGS 32

Gene calling method

Glimmer v3.02

 

Locus Tag

CpVD57

 

Genbank ID

CP009927 (chromosome)

 

GenBank Date of Release

January 06, 2015

 

BIOPROJECT

PRJNA267107

MIGS 13

Source Material Identifier

BHI broth, VD57

 

Project relevance

Animal Pathogen, Medical

Growth conditions and genomic DNA preparation

Cp_VD57 strain was grown in brain-heart-infusion media (BHI-HiMedia Laboratories Pvt. Ltd, India) under rotation at room temperature (37 °C). Extraction of chromosomal DNA was performed using 30 mL of 48–72 h culture of bacteria, centrifuged at 4 °C and 4000 rpm for 15 min. Re-suspension of cell pellets was done in 600 μL Tris/EDTA/NaCl [10 mM Tris/HCl (pH7.0), 10 mM EDTA (pH 8.0), and 300 mM NaCl], and transferred to tubes with beads for cell lysis using Precellys®24-Dual (2 cycles of 15 s at 6500 rpm with 30 s between them). Thereafter, purification of DNA with phenol/chloroform/isoamyl alcohol (25:24:1) was followed by precipitation with ethanol/NaCl/glycogen (2.5v, 10 % NaCl and 1 % glycogen). The DNA was re-suspended in 30 μL MilliQ®. The concentration was determined by spectrophotometer, and the DNA was visualized in ethidium bromide-stained 0.7 % agarose gel.

Genome sequencing and assembly

The Ion Personal Genome Machine® System (Life Technologies) platform was used for sequencing, using fragment library. The reads with good quality was assembled using de novo strategy through Mira 4.0 software [20]. The assembly produced a total of 15 contigs, coverage of 78.22x with a N50 contig length of 405.436. Additionally, a scaffold was created using the CONTIGuator 2 software [21], taking the genome sequence of C. pseudotuberculosis strain 1002 (NC_017300.1) as reference. The gaps were closed manually using CLC Genomics Workbench 7 software [22].

Genome annotation

The annotation of genes was transferred by our in-house scripts using C. pseudotuberculosis strains 1002, 258 (NC_017945.2) and FRC41 (NC_014329.1) as reference. Manual annotation was performed using Artemis software [23]. Other elements such as rRNA, tRNA, and repetitive regions were predicted using RNAmmer [24], tRNAscan-SE [25], and Tandem Repeat Finder [26], respectively. Enzyme Commission Numbers (EC number) prediction were performed using RAST tool [27].

Genome properties

The genome is 2,337,177 bp long and comprises one main circular chromosome with a 52.19 % GC content. A total of 2,148 genes were predicted, among which 2,101 were protein coding genes, and 61 RNAs. Forty seven pseudogenes were also identified. The properties and statistics of the Cp_VD57 strain genome are listed in Table 3. The distributions of genes according to the COGs functional categories is presented in Table 4, followed by a cellular overview diagram in Fig. 2 and a summary of metabolic network statistics shown in Table 5.
Table 3

Genome Statistics

Attribute

Value

% of Total

Genome size (bp)

2,337,177

100.0

DNA coding (bp)

1,998,286.

85.5

DNA G + C (bp)

1,235,198

52.9

DNA scaffolds

1

 

Total genesa

2,148

100.0

Protein coding genesa

2,101

97.8

RNA genes

61

2.83

Pseudo genes

47

2.2

Genes in internal clusters

NA

NA

Genes with function prediction

1,578

73.5

Genes assigned to COGs

1,629

75.8

Genes with Pfam domains

1,682

80,1

Genes with signal peptides

158

7.36

Genes with transmembrane helices

605

28.8

CRISPR repeats

NA

NA

aThe total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome

Table 4

Number of genes associated with the general COG functional categories

Code

Value

% agea

Description

J

148

7.04

Translation, ribosomal structure and biogenesis

A

2

0.09

RNA processing and modification

K

113

5.37

Transcription

L

104

4.95

Replication, recombination and repair

B

0

0.00

Chromatin structure and dynamics

D

20

0.95

Cell cycle control, cell division, chromosome partitioning

Y

0

0.00

Nuclear structure

V

31

1.47

Defense mechanisms

T

51

2.42

Signal transduction mechanisms

M

93

4.42

Cell wall/membrane biogenesis

N

5

0.23

Cell motility

Z

1

0.04

Cytoskeleton

W

0

0.0

Extracellular structures

U

33

1.57

Intracellular trafficking and secretion

O

82

3.90

Posttranslational modification, protein turnover, chaperones

C

100

4.75

Energy production and conversion

G

115

5.47

Carbohydrate transport and metabolism

E

191

9.09

Amino acid transport and metabolism

F

69

3.28

Nucleotide transport and metabolism

H

103

4.90

Coenzyme transport and metabolism

I

62

2.95

Lipid transport and metabolism

P

128

6.09

Inorganic ion transport and metabolism

Q

31

1.47

Secondary metabolites biosynthesis, transport and catabolism

R

193

9.18

General function prediction only

S

141

6.71

Function unknown

-

472

22.46

Not in COGs

Totalb

2288

104.42

 

aThe percentage is based on the total number of protein coding genes in the annotated genome

bThe total does not correspond to 1,537 CDSs, because some genes are associated with more than one COG functional categories

Fig. 2

Graphical circular map of the genome [41]. From center to the outside: In wine Ovis strains, in blue Equi strains, RNA genes (tRNAs green, rRNAs orange, tRNAs red), GC content in black, GC skew

Table 5

Metabolic Network Statistics

Attribute

Value

Total genes

2145

Enzymes

599

Enzymatic reactions

1197

Metabolic pathways

232

Compounds

912

Insights from the genome sequence

Genetic variation seems to be limited in C. pseudotuberculosis , which has been shown previously as genetically homogenous [913]. The MLST findings of the 64 biovar ovis strains show seven STs and all were clonally derived by eBURST analysis when a complex was deemed to share 7/8 loci; the strain Cp_VD57 was included in this analysis [28]. Although it is evident that there is very little genetic variation, we analyzed the fully sequenced Cp_VD57 genome to detect the presence of SNPs. The detected SNPs are listed in Table 6.
Table 6

Total number of SNP’s in C. pseudotuberculosis VD57 in comparison to other strains

Reference

Total SNPs

SNP coding regions

SNP intergenic regions

C. pseudotuberculosis 31 Equi

25,609

19,811

5,798

C. pseudotuberculosis 258 Equi

25,706

21,303

4,403

C. pseudotuberculosis 106A Equi

24,352

18,085

6,267

C. pseudotuberculosis 5297 Equi

25,866

20,017

5,849

C. pseudotuberculosis 162 Equi

24,274

18,501

5,773

C. pseudotuberculosis 316 Equi

25,905

20,911

4,994

C. pseudotuberculosis 1002 Ovis

35

28

7

C. pseudotuberculosis C231 Ovis

952

741

211

C. pseudotuberculosis P54B56 Ovis

999

754

245

C. pseudotuberculosis I19 Ovis

968

762

206

C. pseudotuberculosis FRC41 Ovis

471

374

97

C. pseudotuberculosis 267 Ovis

2,404

1,869

535

C. pseudotuberculosis PAT10 Ovis

1,060

804

256

C. pseudotuberculosis 4202 Ovis

956

735

221

C. pseudotuberculosis 3/99-5 Ovis

502

411

91

C. pseudotuberculosis 48252

521

394

127

C. pseudotuberculosis CS_10

516

392

124

C. pseudotuberculosis Ft_2193

492

380

112

To run SNP detection programs with MUMmer [29], default parameters were assigned. The results for SNP are in agreement with the literature, despite the fact that these strains were isolated from several hosts in different countries thereby verifying that C. pseudotuberculosis strains show limited genetic differences between worldwide strains.

Small genomic insertions and deletions were identified using the reference strain 1002, which is closer to Cp_VD57. MUMmer [29] identified 425 indels in Cp_VD57, 18 of which were in coding regions. However, three major regions of indel were identified comparing 1002 and VD57 strains: two insertion regions and one deletion. The first insertion region is located at coordinates 966430 to 968875 and comprises 2445 pb; this region has 4 genes and is present in biovar Equi strains. The second insertion region is located at coordinates 1182765 to 1182855 (90 pb), and is located within a hypothetical protein. Finally, the deletion region is located at 1002 strain (1575360–1576000) and comprises 640pb aceF pseudogenes.

Conclusions

Isolates from the C. pseudotuberculosis are genetically homogenous. Multi-locus sequence typing and comparative genomic analysis show that the isolates ovis seem to fall into the same clades. Despite the general similarity between the strains from C. pseudotuberculosis , some are more virulent, as C. pseudotuberculosis strain VD57 presented in this paper. Comparative studies with genome sequences of different C. pseudotuberculosis strains and Cp_VD57 can be performed and these analyses may be useful in identification of genome variations.

Abbreviations

CL: 

caseous lymphadenitis

Cp_VD57: 

Corynebacterium pseudotuberculosis strain VD57

Declarations

Acknowledgements

We would like to acknowledge the help of all the team members & the financing agencies. Scholarship from the CNPq under the “TWAS-CNPq Postgraduate Fellowship Programme” for doctoral studies. This work was partially executed by Rede Paraense de Genômica e Proteômica supported by FAPESPA (Fundação de Amparo à Pesquisa do Estado do Pará), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil), FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brasil) and FAPESB (Fundação de Amparo à Pesquisa do Estado da Bahia)

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Institute of Biologic Sciences, Federal University of Minas Gerais
(2)
Aquacen - National Reference Laboratory for Aquatic Animal Diseases, Federal University of Minas Gerais
(3)
Laboratory of Bioinformatics, Professional and Technological Education Sector, Federal University of Paraná
(4)
Institute of Health Sciences, Federal University of Bahia
(5)
Institute of Biologic Sciences, Federal University of Para
(6)
Centre for Genomics and Applied Gene Technology, Institute of Integrative Omics and Applied Biotechnology (IIOAB)
(7)
Department of Computer Science, Virginia Commonwealth University

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© Almeida et al. 2016