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Genomes of bacterial pathogens and their diversity

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Genomes of bacterial pathogens and their diversity. Philippe Glaser - [email protected]. Introduction: general concepts on pathogenic bacteria and their genomes How to sequence a bacterial genome Two examples: the genus Listeria and Streptococcus agalactiae. - PowerPoint PPT Presentation
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Genomes of bacterial pathogens and their diversity Philippe Glaser - [email protected]
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Page 1: Genomes of bacterial pathogens and their diversity

Genomes of bacterial pathogens and their diversity

Philippe Glaser - [email protected]

Page 2: Genomes of bacterial pathogens and their diversity

1. Introduction: general concepts on pathogenic bacteria and their genomes

2. How to sequence a bacterial genome

3. Two examples: the genus Listeria and Streptococcus agalactiae

Page 3: Genomes of bacterial pathogens and their diversity

Examples of bacterial species and diseases

Tuberculosis Mycobacterium tuberculosis Leper Mycobacterium lepraeCholera Vibrio choleraWhooping cough (coqueluche) Bordetella pertussisSoar throat Streptococcus pyogenes and virusesMeningitis Neisseria meningitidis and other bacteria

Gonococci Neisseria gonorrhoeae Plague (la peste) Yersinia pestisDysentery Shigella flexneriGastric cancer, ulcer, gastritis Helicobacter pyloriMultiple diseases Escherichia coli, Staphylococcus aureus….

Page 4: Genomes of bacterial pathogens and their diversity

Published genome sequence of bacterial pathogens

Shigella 2 Chlamydiae 4+1Escherichia coli 4+2 Neisseria 2Salmonella 3 Branhamella 0 2Helicobacter 3 Bordetella 3Pseudomonas 1+2 Pasteurella 1Yersinia 3 Actinobacillus 0 4Stenotrophomonas 0 1 Haemophilus 2Burkholderia 0 12 Bartonella 3 Flavobacterium 0 1 Legionella 3 Acinetobacter 0 3 Leptospira 2Vibrio 4 Borrelia 1Campylobacter 1 Treponema 2Staphylococcus 4 Mycobacterium 5Enterococcus 1 Rickettsia 3Streptococcus 9 Anaplasma 0 2Listeria 4+1 Coxiella 1Nocardia 0 2 Ehrlichia 0 1Corynebacterium 1+3 Clostridium 2+1Mycoplasma 6 Total: > 80 published genomes

Page 5: Genomes of bacterial pathogens and their diversity

Biodiversity of the microbial worldBiodiversity of the microbial world4 000 000 000 000 000 000 000 000 000 000 bacteria on hearth3,5 billion years of evolution5000 culturable species - 500 000 (?) species

Page 6: Genomes of bacterial pathogens and their diversity

Bacterial diversity in a Yellowstone hot spring

Principle of the experiment:

Sample PCR amplification of16 S RNA

Cloning 300 clones

First analysis by restriction

DNA Sequencingof 122 clones

54 bacterialgroups

84 sequences14 phyla

38 sequences

12 new phyla

(Hugenholtz et al., J. Bacteriol 1998, 180 366-376)

Page 7: Genomes of bacterial pathogens and their diversity

Diversity of the non-culturable bacterial worldDiversity of the non-culturable bacterial world

Page 8: Genomes of bacterial pathogens and their diversity

How to define a bacterial species

• For eukaryotes the species definition is based on sexual reproduction.

Not possible for bacteria1. Phenotypic definition2. Molecular definition:

70% of “similarity” by genomic DNA hybridizationMore than 97% of identities between the 16S RNA genes

=>A convenient definition but not fully satisfactory

Page 9: Genomes of bacterial pathogens and their diversity

Interactions between humans (the host) and bacteria

• The human body constitutes multiple ecosystems for bacterial communities:– The digestive tract– The throat– The skin– Other places are normally sterile (urine, milk, blood)

› Symbiotic bacteria› Commensal bacteria› Pathogenic bacteria• Opportunistic pathogens and obligatory pathogens

Page 10: Genomes of bacterial pathogens and their diversity

Bacteria and their environments

ReservoirAnimalsWaterSoilFood

Humanhost

Vectors

The ecology of the pathogenic bacteria or understanding its adaptation to these environments (growth conditions)

Page 11: Genomes of bacterial pathogens and their diversity

Some questions in the study of human bacterial pathogens

• What are the virulence factors and the host - pathogens interaction factors?

• What is the physiology (the metabolism) of the bacteria in interaction with the host?

• What is the evolution of the bacteria which lead to its adaptation to its host, and the relation with the non-pathogenic related species?

• The identification of diagnostic and typing molecular tools

• The identification on a rational basis of antigens for a-cellular vaccines

• The identification of drug targets

How to use genomics (and post-genomics) to solve these questions

Page 12: Genomes of bacterial pathogens and their diversity

Evolution & Biodiversity

Genome variability

DNA repairBarriers to DNA transfer

Point mutationGenome rearrangementGene duplicationHorizontal gene transfer

Selection Biodiversity

=> virulence and pathogenicity=> virulence and pathogenicity

Page 13: Genomes of bacterial pathogens and their diversity

Size of bacterial genomesSize of bacterial genomes

Nanoarchaeum equitans <500 kbMycoplasma genitalium : 0.580 Mb 481 genesMinimal genome 300-400 genesEscherichia coli 4.6-5.6 4289-5648 genesMesorhizobium loti 7.036 Mb 6752 genesStreptomyces coelicolor : 8.667 Mb 7825 genesHuman 3,000.000 Mb 30000 genes

Page 14: Genomes of bacterial pathogens and their diversity

Adaptation : Transcription regulators - vs genome size

(http://www.regx.de/m_project_bioinformatics.php)

Page 15: Genomes of bacterial pathogens and their diversity

Gene transfers in bacteria

Bacteriophages

PlasmidsTransposons

Competence

Transduction

Conjugation

Transformation

Page 16: Genomes of bacterial pathogens and their diversity

Mobile elements and gene gain

• IS elements => no associated function, gene integration by IS mediated homologous recombination, gene inactivation.

• Transposon => carry functional genes• Integron => a platform to incorporate new functions, multi-antibiotics

resistance.• Phages => may carry virulence genes (cholera toxin)• Pathogenicity (functional) islands• Plasmids => may also carry transposons or integrons• + gene duplication

Identification of such elements in genome sequences

Page 17: Genomes of bacterial pathogens and their diversity

Gene lost

• By homologous recombination

• By insertion of IS elements

• By mutation : gene => pseudogene

Evolutionary impact Reductive evolution (M. leprae, Y. pestis, B. pertussis) Role in virulence: lysine decarboxylase in Shigella (cadA+

derivative are less virulent)

Page 18: Genomes of bacterial pathogens and their diversity

Antigenic variation

• By recombination: a gene cassette is inserted in front of an active promoter or remove from this position. (Brucella, Mycoplamsa galisepticum)

• By mutation: variation of a micro satellite sequence length (homo polymer tract) lead to frameshift deletion or reversion (Helicobacter pylori, Neisseria meningitidis)

Page 19: Genomes of bacterial pathogens and their diversity

Protein families and gene duplications

• May arise by gene duplication or horizontal gene acquisition

• Metabolic functions, surface proteins (antigens)

• Correspond to a specificity of a species

• Frequently discovered after whole genome sequencing

Page 20: Genomes of bacterial pathogens and their diversity

Analysis of the genome of a bacterial pathogen

• Annotation of the genome

• Analysis of regulatory genes

• Analysis of inactivated genes (pseudogenes)

• Identification of protein families and mechanisms of phase variation

• Identification of mobile elements

• Identification of atypical regions (recently acquired)

Information obtained from comparative genomics

Page 21: Genomes of bacterial pathogens and their diversity

DNA sequencingDNA sequencing

Two strategies : directed or random

DNA automated sequencing machines produce 800 bases long sequences with an accuracy of 99 %. => How to sequence a 4 Mb bacterial genome with an accuracy higher than 99.99%?

Page 22: Genomes of bacterial pathogens and their diversity

Chromosome Chromosome

Ordering clones of a large-insert library(cosmids, lambda or BAC)

Sequencing clone by cloneof the minimum tiling path

Complete sequence

Random sequencingof a large number of clones

Sequenceassembly

Complete sequence

Directed strategyDirected strategy Random strategyRandom strategy

Page 23: Genomes of bacterial pathogens and their diversity

‘‘Whole genome shotgun’ Whole genome shotgun’

CompleteCompleteGenome sequenceGenome sequence

Chromosome

Large-insert library

(pSYX34 and BAC)

Small-insert library(pcDNA2.1)

closureAnnotation

End-sequencing (small-insert fragments)

End-sequencing (large-insert fragments)

Assembly of sequences in contigs

Page 24: Genomes of bacterial pathogens and their diversity

Organization of a project

Choice of the strategy

Library construction DNA preparation of plasmid clones High throughput sequencing of both ends of inserts

Assembly

Finishing: gap closure and resequencing of low quality regions

Annotation

Page 25: Genomes of bacterial pathogens and their diversity

LibrariesLibraries

Libraries of insufficient quality => No sequence

Important features : coverage of the chromosome, absence of co-ligation, absence of clones without an insert, size of the inserts.

Different types of libraries: * size of the inserts* copy number of the vector

High-copy number vector : 1 to 3 kb inserts 1 to 3 kb inserts Low-copy number vector : 8 to 12 kb inserts 8 to 12 kb inserts Bacterial artificial chromosome : 50 to 100 kb inserts50 to 100 kb inserts

Page 26: Genomes of bacterial pathogens and their diversity

Chromosomal DNA

Nebulization End repair by T4 polymerase

pcDNA: high copynumber vector

Ligation, transformation

Construction of a 1 - 3 kb long inserts library

Recombinant plasmid

TGTGACAC

Two repeated BstXI sites

5’CCAG TGTG ATGG…CCAG CACA CTGG3’3’GGTC ACAC TACC…GGTC GTGT GACC5’

CACAGTGT

Ligation of BstX I adaptors,

Size selection of the inserts

5’pCTTTCCAGCACA3’ 3’GAAAGGTCp 5’

Purification of thedigested vector

(two 5’ protruding ends)

Page 27: Genomes of bacterial pathogens and their diversity

Cloning of DNA fragments of 100- to 300-kb (average, 150 kb) in E. coli

Bacterial artificial chromosome (BAC)

Vector based on naturally occurring F-factor plasmid found in E. coli

» strict copy number control

»stably maintained at 1-2 copies per cell

»lacZ-based color selection of BAC clones with inserts

Page 28: Genomes of bacterial pathogens and their diversity

BAC library construction

Preparation of chromosomal DNA in agarose plugs

Partial digestion with HindIII or BamHI

50 kb100 kb150 kb200 kb

Ligation vector + DNA purified from agarose plugs

Electroporation into E. coli DH10B

Verification of insert size on PFGE gels after NotI digestion

Linearized BAC vector (7kb)

50 kb100 kb150 kb200 kb

Inserts of 70 - 150 kb

Page 29: Genomes of bacterial pathogens and their diversity

AutomationAutomation

Page 30: Genomes of bacterial pathogens and their diversity

High throughput sequencing

DNA Sequencing 15 years ago!DNA Sequencing 15 years ago!

Page 31: Genomes of bacterial pathogens and their diversity

Automated DNA sequencingAutomated DNA sequencing

Page 32: Genomes of bacterial pathogens and their diversity

Automated sequencingAutomated sequencing

Page 33: Genomes of bacterial pathogens and their diversity

Sequence Sequence assemblyassemblyPhred, Phrap, Phred, Phrap, ConsedConsedhttp://www.phrap.org

Page 34: Genomes of bacterial pathogens and their diversity

Statistics and progress of the project

Page 35: Genomes of bacterial pathogens and their diversity

FinishingFinishingRe-sequencing of regions containing low ‘quality’ sequences

Sequencing of ‘missing’ regions

Sequence gapsContig AContig A Contig BContig B

Contig AContig A

Contig BContig B

Contig CContig C

Contig DContig D

Contig EContig E

Contig FContig F

Cloning gaps

Page 36: Genomes of bacterial pathogens and their diversity

Timing of a bacterial genome projectTiming of a bacterial genome project

Library construction and verification (one month)

Plasmid preparation 5000 minipreps per Mb (7 days)

Sequencing : 10000 sequences per Mb (20 days, ABI 3700)

PCR : highly variable (250 reactions per Mb)

Consumable costs : 10 000 Euro per Mb

Page 37: Genomes of bacterial pathogens and their diversity

Listeria monocytogenes

meningitis, encephalitis, septicemia, abortions, neonatal infections, gastroenteritis

foodborne pathogen

dairy products, meat, vegetables, fishTransmission:

Disease:

Population at risk: elderly, newborns, immuno-comprimised,pregnant women

Mortality rate: 30%

Concern for public health Problem for food industry

Page 38: Genomes of bacterial pathogens and their diversity

Ecology of L. monocytogenes

• Ability to survive and to grow in extreme conditions: low temperature, low water activity, broad ranges of pH…

• Ubiquitous in the environment but at very low count

• Variable count depending on the microenvironment and the season at a single location

• Interaction with the vegetal world (silage) and the animal world (waste)

Page 39: Genomes of bacterial pathogens and their diversity

Interaction of Listeria with its hosts

• Carriage is frequent but transient• Low concentration of Listeria in feces• Intracelullar parasite• Ability to cross three barriers: intestinal, hemato-encephalic

and placental barrier • Provokes a broad range of diseases : gastroenteritis,

septicemia, meningitis, encephalitis, abortions• At risk population : immuno-compromised, elderly, pregnant

women and new-born

What are the relations between the two facets of this bacterium?

Page 40: Genomes of bacterial pathogens and their diversity

L. seeligeri

L. innocua

L. monocytogenes

L. welshimeri

L. grayi

B. subtilis

Phylogenetic tree of the genus Listeria

(Pathogenic species)

L. ivanovii

Vaneechoutte et al. Int J Syst Bact. (1998) 48, 127-139

Page 41: Genomes of bacterial pathogens and their diversity

L. monocytogenes

EGDe

L. monocytogenes

4b

L. innocua L. ivanovii

Genome size 2944 kb 2943 kb 3011 kb 2929 kb

rRNA operons 6 6 6 6

CDS 2848 2795 2968 2782

Phages 1 0 5 0

IS 1 (3 copies)

1 transposon

0 0 5

Plasmide -- -- 81.9 kb --

Genome comparison

Page 42: Genomes of bacterial pathogens and their diversity

Listeria monocytogenes

Bac

illu

s su

btil

isL. monocytogenes/B. subtilis synteny

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

0 500000 1000000 1500000 2000000 2500000 3000000

Page 43: Genomes of bacterial pathogens and their diversity

L. monocytogenes EGDe

L. i

nnoc

ua

L. i

van

ovii

L. monocytogenes EGDe

Synteny between Listeria genomes

Absence of rearrangement between genomes Rare translocations : probably deletion + insertion

Page 44: Genomes of bacterial pathogens and their diversity

L. monocytogenesL. monocytogeneschromosome mapchromosome map

L. monocytogenes 270 ‘specific’ genes

L. innocua149 ‘specific’ genes

G+Ccontenthttp://genolist.pasteur.fr/listilist

Page 45: Genomes of bacterial pathogens and their diversity

G+C content of the 270 CDSs specific for L. monocytogenes

0

2

4

6

8

10

12

14

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 52

G+C%

Nb

of C

DS

s (%

)

Total

Specific

Page 46: Genomes of bacterial pathogens and their diversity

Competence operons in L. monocytogenes

comGA B C D E F G

comEA B C

comC

comFA C

27 - 24

37-34 32-21 34-37 30-18 33-18 32-23 31-17

34-3239-6934-44

38-43 35-35

37 : GC%34 : % identities Bs ortholog

2695.1 and 2014.1 two comEC paralogs (DNA binding protein)

Page 47: Genomes of bacterial pathogens and their diversity

0

500

1000

1500

2000

2500

Amino acids

41 surface proteins with an LPXTG motif

InlA-like *= absent from the L. innocua genome

** ** ** ** ** **

****

****

**

**

**

** **** ****

**

**

Page 48: Genomes of bacterial pathogens and their diversity

Sugar PTSHexose phosphate permeaseBile acid hydrolaseArginine deimidaseGlutamate decarboxylase

Metabolic pathways missing in Metabolic pathways missing in L. innocuaL. innocua

Known virulence factors missing in L. innocua

Surface proteins missing in L. innocua

L. monocytogenes / L. inocua comparison

Page 49: Genomes of bacterial pathogens and their diversity

L. monocytogenes - L. ivanovii

L. monocytogenes 345 ‘specific’ genes

L. ivanovii350 ‘specific’ genes

2944 / 0Virulence gene cluster

inlA inlB

inlC

hpt

bsh

Page 50: Genomes of bacterial pathogens and their diversity

The virulence gene cluster

L. monocytogenes

B. subtilisgcaD

prs

ctc

spoVC mfd

yabK

L. innocua

prs ctcprfA plcA hly actA orfAorfBorfZorfXmpl plcB ldh

orfAorfBorfZ ldh ctcprs

orfAorfBorfTorfS ldhprfA plcA hly i-actAmpl plcBprs ctc

L. ivanovii

prs orfB 5,5 kb ldh L. grayi

orfAorfBorfT ldhprs ctcL. welshimeri

prfAprs plcA hly

orfAorfBorfT ldh ctcL. seeligeri

Complex history with several events of insertion and deletions

L. seeligeri

L. innocua

L. monocytogenes

L. welshimeri

L. grayi

B. subtilis

L. ivanovii

Page 51: Genomes of bacterial pathogens and their diversity

L. monocytogenes EGDe

The inlA - inlB region

L. ivanoviilmo0415 amidase inl-like inlA inlB-like inl-like inlB lmo0439

L. innocua

inlA inlB lmo0435 (LPXTG) lmo0439lmo0432lmo0415

17 gènes

wapA-like

lmo0432

lmo0439

Lmo0435 (LPXTG)

lin439

inlA inlBLin439*

lmo0439lmo0432L. monocytogenes 4b

wapA-like

Entrée InlA, InlB

Mouvement intracellulaire ActA

Passage de cellule à celluleActA

Lyse de la double membrane LLO, PlcBLyse de la vacuole

LLO, PlcA

Page 52: Genomes of bacterial pathogens and their diversity

Other virulence genes

L. ivanovii

L. monocytogenes

L. innocua

: pseudogene

hpt, hexose phosphate transport

bsh, bile salt shydrolasebsh2066 groEL

LPXTG groEL

groEL

837 hpt 839295nt 107nt

31nt

L. ivanovii

L. monocytogenes

L. innocua

hpt457nt 153nt

bsh

PrfA box is not conserved

The PrfA box is conserved

Page 53: Genomes of bacterial pathogens and their diversity

Listeria ivanovii - closer to a real pathogen?Some specific functions related to virulence

lmo1240 1241 1242L. monocytogenes and L. innocua

i-inlBi-inlL i-inlKi-inlB2 i-inlGi-inlH

sphingomyelinase-c

lmo1240

i-inlJ i-inlItRNA

L. ivanovii

i-inlF i-inlE

lmo1242

: soluble internalin

Lmo2699 2700L. monocytogenes and L. innocua

L. ivanovii

Lmo2699 2700LPXTG

Capsule biosynthesis ?

tRNA

A second pathogenicity island

And 96 inactivated genes (pseudogenes)

Page 54: Genomes of bacterial pathogens and their diversity

Conclusions Contrary to the rest of the genome, virulence genes have a complex

history. Possible cycle of virulence genes gain and lost. These cycle may play a

role in the evolution of the genus and in the emergence of species. Functions required for intracellular multiplication are conserved between

the two pathogenic species. Interactions with the host and physiopathology are probably different and

involve different factors. The specialization of L. ivanovii is linked to the presence of specific

genes and to the lost of a large number of functions.

What is the diversity within the species L. monocytogenes

Page 55: Genomes of bacterial pathogens and their diversity

Listeria monocytogenes

Epidemiological dataSerovars

• The great majority of human listeriosis cases is caused by 1/2a, 1/2b and 4b strains

• Serovar 4b strains are responsible for almost all major epidemics of human listeriosis as well as for most of the sporadic cases

1/2a1/2b1/2c

3a

3b3c

4a4ab4b4c

4e4d

7

Page 56: Genomes of bacterial pathogens and their diversity

AscI profiles of L. monocytogenes strains -WHO-multi center study

AscI profiles of L. monocytogenes strains -WHO-multi center study

Page 57: Genomes of bacterial pathogens and their diversity

1/2a, 3a

485

388

242

291

145

194

97

48

23

582

kb

Genomic Division I Genomic Division II

1/2b, 3b 4b , 4d, 4e 1/2c,3c

AscI genomic fingerprints of 62 representative Listeria monocytogenes strains

Brosch et al., 1994, AEM 60:2584-92,

Page 58: Genomes of bacterial pathogens and their diversity

• clinical (epidemic) isolates• food isolates• environmental isolates

hybridisation with chromosomal DNA of

Correlation of genomic and epidemiological data

High density membranes for Listeria

New tools for genomic typingNew accurate methods for diagnostics

Development of:

Identification of genes consistently absent or present in e.g. epidemic and clinical isolates

Should allow the:

Page 59: Genomes of bacterial pathogens and their diversity

L. monocytogenes EGDe 1/2a

L. innocua 6a

gene A gene B gene C

L. monocytogenes 4b

control

Page 60: Genomes of bacterial pathogens and their diversity

Hybridization patterns of L. monocytogenes

L.m.sv. 1/2b

L.m.sv. 1/2b

L.m.sv. 4bL.m.

sv. 4b

Hybridized with genomic DNA of:

Hybridized with genomic DNA of:

L.m.sv. 1/2c

L.m.sv. 1/2a

Page 61: Genomes of bacterial pathogens and their diversity

In total 110 strains belonging to all species of the genus Listeria

Listeria ivanovii 5 strains

Listeria innocua 7 strains

Listeria welshimeri 2 strains

L isteria seeligeri 2 strains

L. monocytogenes 94 strains

Serovar: 1/2a, 1/2c, 1/2b, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e, 7

Origin: Environment, food, animals, production environnementhuman (sporadic and epidemic cases)

Hybridisation with different Listeria strains

Page 62: Genomes of bacterial pathogens and their diversity

Grouping 460 genes for 112 strains of Listeria

Sérovar: 1/2a, 3a

Sérovar: 4b, 4e, 4d

Sérovar: 1/2b, 3b, 7

Sérovar: 1/2c, 3c

Sérovar: 4a, 4c

Listeria sp.

Page 63: Genomes of bacterial pathogens and their diversity

Lmo0171Lmo0172Lmo0525Lmo0734Lmo0735Lmo0736Lmo0737Lmo0738Lmo0739Lmo1060Lmo1061Lmo1062Lmo1063Lmo1968Lmo1969Lmo1971Lmo1973Lmo1974

ORF2819ORF3840ORF2568ORF1761ORF0029

ORF0799ORF2372ORF2110

L. monocytogenes

I II III

I.1 I.2 II.1II.2

Page 64: Genomes of bacterial pathogens and their diversity

Conclusion

The L. monocytogenes species shows a broad genomic diversity

Genomes are stable and horizontal genetic exchanges are rare.

The species and subspecies are well defined by a set of genes and it seems that there is no continuum between groups.

The notion of species is probably not only an arbitrary one. DNA array is a powerful genome-level typing tool for

epidemiological studies and research.

Page 65: Genomes of bacterial pathogens and their diversity

Streptococcus agalactiaeStreptococcus agalactiae (group B) (group B)

=> Surveillance of pregnant women to avoid mother-infant transmission=> Development of a vaccine ?

Part of the normal flora colonizing the gastrointestinal tract, of an important part of the population, and may colonizethe urogenital tract.

Disease: Rare infections of immuno-compromised adults Leading cause of invasive infections in neonates

septicemia (early onset disease)

pneumonia (early onset disease)

meningites (late onset disease)

Page 66: Genomes of bacterial pathogens and their diversity

Biodiversity within the species S. agalactiae

Characterstics Human Bovine mastitis__________________________________________________

Pigment + -Lactose - +Salicin + +Beta-galactosidase - +Bacitracine sensitivity - +Protein antigens R, Icp X (Finch & Martin, 1984)

Other animal origins: diseases in various mammals and fishes

Human origin: carriage or invasive strains

MLEE, MLST pointed the existence of an hypervirulent lineage.

Two ecovars

Q. What is the genomics basis of this diversity?

Page 67: Genomes of bacterial pathogens and their diversity

S. mutans

S. bovis

S. salivarus

S. pneumoniae

S. mitis

S. sanguis

S. anginosusS. equi

S. pyogenesS. agalactiae

S. uberis

S. suis

S. pleomorphus

Phylogenetic relationship among Streptoccocci

(from Kawamura et al. J. Syst. Bacteriol. 1995)

Page 68: Genomes of bacterial pathogens and their diversity

S. agalactiaeNEM316

S. pneumoniae

2 206 kb

8

2182

2160 kb

4

2236

Size of the genome

Ribosomal operons

CDSs

S. pyogenes

1852 kb

6

1752

Mobile elements

8 IS 12 phage like

integrases2 integrated plasmid

(1 with 3 copies, 42kb)

17 IS4 bacteriophages

105 IS

Genome comparison

Page 69: Genomes of bacterial pathogens and their diversity

SyntenySynteny between S. agalactiae between S. agalactiae and and S. pneumoniaeS. pneumoniae((1141 pairs of orthologous genes)

S. agalactiae

S. p

neum

onia

e

0

500000

1000000

1500000

2000000

0 500000 1000000 1500000 2000000

Page 70: Genomes of bacterial pathogens and their diversity

SyntenySynteny between S. agalactiae between S. agalactiae and and S. pyogenesS. pyogenes ((1170 pairs of orthologous genes)

S. agalactiae

S. p

yoge

nes

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

0 500000 1000000 1500000 2000000

36 recombination breakpoints

Page 71: Genomes of bacterial pathogens and their diversity

14 mobile islands (532 genes)

G+C%

G+C/G-C

Page 72: Genomes of bacterial pathogens and their diversity

Genes related to mobile element within the islands

I

II

III VII VIII

IV

V

VI

IX

X

XI

XII

XIII

XIV

18 kb

16 kb

46 kb

19 kb

11 kb

59 kb

46 kb

46 kb

25 kb

33 kb

86 kb

45 kb

23 kb

int rep tra

int rep rep mob.

Plasm. Phage int

int tnp tnp

tnp

rep parA plasm.tra ssb rep

tra

mob plasm. tra phage rep

tnp tnp pol int rep int rel hel tra rep

Int phage

rep phage int int

int

tRNA-A

tRNA-L

tRNA-TtRNA-R

tRNA-A

tRNA-K

Page 73: Genomes of bacterial pathogens and their diversity

NEM316 - SAG2603 genome comparison

• No chromosomal rearrangement between the two strains

• No integrated plasmid in SAG2603 but three prophages

•1799 orthologs among these two genomes (633 100%

identical)

=> 241 Nem316 genes are missing in SAG2603 (37,

backbone)

=> 258 Sag2603 genes are missing in NEM316 (42,

backbone)

Although highly variable 10 mobile islands are conserved.

Page 74: Genomes of bacterial pathogens and their diversity

NEM316 / SAG2603 - conserved backbone0

gbs0046-47

sga0046

gbs0086-87

sag0086-88

gbs0162-163

gbs0493

cpsJDNMH

gbs1240-1242

gbs1400-1401ABC transporter

sag1330-1331protein R5

gbs1740-1749ABC transporter sag1697-1703

gbs1823His triad prot

sag1780

NEM316

SAG2603

Page 75: Genomes of bacterial pathogens and their diversity

Comparative analysis of island XII

NEM316

98%< <100%95%< <98%90%< <95%80%< <90%70%< <80%60%< <70% <60%

Lactose utilizationLmb scpB

SAG2603 A/B

Mercuric and cadmium resistance

Page 76: Genomes of bacterial pathogens and their diversity

MLST results for S. agalactiae

NEM316

« Hypervirulent »

Sag2603

(Jones et al., 2003 Int. J. Clin. Microbiol.)

adhP : alcohol dehydrogenasepheS : Phenylalanyl tRNA synthetaseatr Amino acid transporterglnA glutamine synthetasesdhA serine dehydrataseglcK glucokinasetkt transketolase

Page 77: Genomes of bacterial pathogens and their diversity

DNA arrays hybridization for genome characterization

Page 78: Genomes of bacterial pathogens and their diversity

• 10 invasive ST-17 strains (MLST study)• BM110, hypervirulent clone defined by

MLEE• 18 invasive strains (Hôpital Necker)• 13 carriage strains (Hôpital Necker)• 14 strains from bovine mastitis• 12 strains of animal origin (horse, dog, cat,

rabbit, guinea pig, fish)

68 strains analyzed by MLST and hybridization

Page 79: Genomes of bacterial pathogens and their diversity

Genome diversity is essentially located within genomic islands

0

50

100

150

200

250

300

inv_b9inv_h11inv_j81inv_j95inv_mk2inv_wc3inv_1000inv_1572inv_1002inv_1560inv_1568inv_318inv_1573port_38bisport_41bisport_37.39port_65.8bisport_60_36bisbov_411.07bov_527.25bov_543.05bov_547.25bov_549.13bov_501_19bov_44poisson_2_22chat_3448_97chat_693chien_928662gui_pig_622lapin_6144_98

islandsbackbone

Page 80: Genomes of bacterial pathogens and their diversity

st17st1st10,6,9

st23st23 st103

Hierarchical clustering of 69 strains and comparison with MLST data

st19

Page 81: Genomes of bacterial pathogens and their diversity

Two loci heterogeneously distributed among isolates

rofA hemagglutinin Glycosyl transferase secY secA

rofA and rogB are mutated in sag2603

fibronectin binding protein srtB srtCLPXTG LPXTGrogB

I

II

I ------------------++++++++++++++++++++++++++++++++--++++++++++II ------------------++++++++++++++++++++++++++++--------+-+-++++

Page 82: Genomes of bacterial pathogens and their diversity

Conclusion

• Strains from different origin do not cluster except invasive ST17 strains.

• ST17 strains constitute a highly homogenous group

• Diversity reside mostly within islands

• Antigenic diversity is highlighted by genome analysis and is

found both within and outside islands

• DNA arrays, a powerful method for molecular epidemiology

Page 83: Genomes of bacterial pathogens and their diversity

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