A genomic insight into evolution and virulence of Corynebacterium

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A genomic insight into evolution and virulence of Corynebacterium diphtheriae Vartul Sangal, Ph.D. Northumbria University, Newcastle [email protected] @VartulSangal Newcastle University 8th January 2016

Corynebacterium diphtheriae • Features: Gram-positive, actinobacterium

aerobic,

non-motile

• Causes diphtheria, a toxin mediated disease of upper respiratory tract • Biochemically divided into four biovars: gravis, intermedius, mitis and belfanti

C. diphtheriae

• Largely been eradicated through the use of diphtheria vaccine • Mortality: ~5000 people per year globally • A recent increase in the number of infections by non-toxigenic C. diphtheriae strains with varying degree of pathogenesis

Biochemical variations between C. diphtheriae biovars

Biovar

Lipophilism Haemolysisa

Ability to utilizeb Starch Glycogen + +

gravis

-

±

Nitrate Reduction +

intermedius

+

-

+

±

±

mitis belfanti

-

± ?

+ -

± -

-

a Biovar

gravis is haemolytic but some strains may be weakly haemolytic. Biovar mitis is weakly haemolytic. We could not find haemolytic properties of biovar belfanti in the literature.

bStrains

of biovar intermedius may utilize glycogen and starch while mitis strains can rarely use starch but not glycogen.

DIPNET Quality Assurance Test

Neal et al., 2009, J Clin. Microbiol.

Sharing of genes between different biovars Core genome = 1717 genes Accessory genome = 1413 genes mitis (NCTC03529) (Sangal et al., 2012a, J. Bacteriology) intermedius (NCTC05011) (Sangal et al., 2012b, J. Bacteriology) gravis (NCTC13129) (Cerdeno-Tarraga et al., 2003, NAR) belfanti INCA402 (Trost et al., 2012, J. Bacteriology)

Function Restriction-modification system mobile genetic elements Phage-associated Hypothetical proteins Others

gravis intermedius mitis belfanti 4 28 4 185 42

6 30 3 69 65

2 25 3 129 67

7 40 3 83 43

Potential biovar specific genes: genes encoding transcription regulators, membrane proteins, fimbrial proteins or other metabolic enzymes were grouped as others.

Biovar specific genes: carbohydrate, lipid, iron and nitrogen metabolism Category

Function

Biovars

DIP1639 C

Carbohydrates/Energy production

dihydrolipoamide acetyltransferase

belfanti, gravis, mitis

DIP0660 I

Carbohydrate/lipid

propionyl-CoA carboxylase complex B subunit

belfanti, gravis, mitis

DIP1011 G

Carbohydrates

putative aldose 1-epimerase

belfanti, gravis, mitis

DIP1302 G DIP1611 E

Carbohydrates Amino acid/ Lipid/carbohydrate

transketolase 5,10-methylenetetrahydrofolate reductase

belfanti, gravis, mitis gravis, intermedius, mitis

Locus COG

Biovar specific nonsense single nucleotide polymorphisms Gene

Biovar

Function

Metabolism

DIP0794

intermedius

Na+/H+ antiporter-like protein

Inorganic ion

DIP2352

intermedius

anthranilate synthase component I

Amino acid/coenzyme

DIP0842

intermedius

putative secreted protein

-

metF (DIP1611)

intermedius

5,10-methylenetetrahydrofolate reductase

Amino acid

Sangal et al., 2014, Infection, Genetics and Evolution

Phylogenetic analyses

Core genome phylogeny

A ML tree from the binary data from the accessory genome

Sangal et al., 2015, BMC Genomics

Genome-wide CDS blast map

Tox phage

D’Afonseca et al., 2012

CRISPR-Cas systems in C. diphtheriae A. Type II-C

B. Type I-E-a

C. Type I-E-b

cas genes genes ending hypothetical proteins mobile genetic elements conserved genes putative septation inhibitor CRISPR arrays: direct repeats shown in colour and spacers are shown in white. Sangal et al., Microbiology, 2013

Fineran & Charpentier , 2012, Virology

Spacer diversity in Type II CRISPR-Cas system csn2 cas4

Type II-A (Type II-BII-C)

C. diphtheriae

Total unique spacers: 93 Shared spacers: 18 Duplicated spacers: 2

CRISPR direct repeats that are common between different strains are shown in the same colour and spacers are shown in white. Shared and duplicated spacers are numbered. Sangal et al., Microbiology, 2013

Variants of Type I-E CRISPR-Cas system

Type I-E-a Total unique spacers: 101 Shared spacers: 12 Duplicated spacers: 3

Type I-E-b Total unique spacers: 25 Shared spacers: 4 Duplicated spacers: 5 Sangal et al., Microbiology, 2013

CRISPR-Cas phylogeny A. NJ tree from the repeat sequences

B. NJ tree from the cas1 gene alignment

G+C content = 47.8%

G+C content = 49.5%

G+C content = 46%

Average genomic G+C content = 53% Sangal et al., Microbiology, 2013

Unique evolutionary histories of C. diphtheriae strains

I-E-a (15) II-C (7)

II-C (28) II-C (6)

II-C (7) & I-E-b (26)

II-C (4)

II-C (11)

I-E-a (19)

I-E-a (17)

I-E-a (15)

II-C (15) & I-E-b (4) I-E-a (12)

II-C (15) & I-E-b (4) II-C (5)

II-C (14)

Variation in the degree of pathogenicity of non-toxigenic C. diphtheriae ISS 3319 (mitis), ISS 4060 (gravis), ISS 4746 (gravis) and ISS 4749 (gravis) from patients with severe pharyngitis and tonsilitis in Italy.

Adhesion to D562 cell lines

Hunolstein et al., 2003, J Med. Microbiol. Pulti et al., 2006, J Med. Microbiol. Ott et al., 2012, BMC Microbiology

Variation in potentially virulence associated proteome ST26

ST5

ST32

Protein category ISS 3319 ISS 4060 ISS 4746 ISS 4749 ISS 4746 - ISS4749 Lipoproteins 2 1 2 Secreted proteins 2 5 4 25 6 5 Trans membrane 21 37 4 5 13 non-classical Sec 22 19 Cytoplasmic 72 83 22 11 93 Ambiguous 2 6 3 2 11 Trans membrane proteins include TM-Sec and LPXTG proteins

Predicted proteome (20 genomes)

Variation in the pilus gene clusters

Sangal et al., 2015, BMC Genomics

1. A lack of correlation between gene content and biochemical differentiation.

Conclusions 3. Three novel configurations of CRISPR-Cas systems 4. Horizontally acquired three independent events.

2. Bio-typing is not suitable for epidemiological & evolutionary studies.

in

CRISPR-Cas Systems

Biochemical differentiation Genomics

5. The variation in number and organization of pilus gene clusters is associated with the variation in adhesive properties. 6. Strain specific lipoproteins and secretory proteins are potentially responsible for the variation in the degree of pathogenesis.

Variation in the degree of virulence

Evolutionary insights 7. Distinctive phylogeny and unique spacers suggest that unique evolutionary events in different environments led to current genomic diversity. 8. Recombination and gain/loss of the gene function introduced variation, even within a clonal group.

Acknowledgements Paul Hoskisson University of Strathclyde, Glasgow, UK Peter Fineran University of Otago, New Zealand

Iain C. Sutcliffe Northumbria University, Newcastle, UK Jochen Blom Justus-Liebig-University, Giessen, Germany Andreas Burkovski Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen, Germany

Thank You.

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A genomic insight into evolution and virulence of Corynebacterium

A genomic insight into evolution and virulence of Corynebacterium diphtheriae Vartul Sangal, Ph.D. Northumbria University, Newcastle [email protected]

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