Genomic Sequencing Genomic Sequencing of of Mycoplasma Mycoplasma hyopneumoniae,hyopneumoniae,

Virulence Factors, Virulence Factors, and Vaccinesand Vaccines

AcknowledgementsAcknowledgementsThe High Throughput Sequencing CenterThe High Throughput Sequencing CenterUniversity of WashingtonUniversity of Washington

Lee Hood , Greg Mahairas, Steve Lee Hood , Greg Mahairas, Steve Swartzell, Jim WallaceSwartzell, Jim Wallace

The University of Alabama in BirninghamThe University of Alabama in BirninghamElliot LefkowitzElliot Lefkowitz

Iowa State UniversityIowa State UniversityTina VanDyk, Barbara Cleary, Tina VanDyk, Barbara Cleary, Melissa LovekampMelissa Lovekamp

NSW Agriculture, AustraliaNSW Agriculture, AustraliaSteve DjordjevicSteve Djordjevic

How Does How Does M. hyopneumoniaeM. hyopneumoniae Cause Disease?Cause Disease?

• How does How does MhyoMhyo colonize? colonize?

• What genes are expressed?What genes are expressed?

• What do these products do for What do these products do for the pathogen?the pathogen?

• What do these products do to the What do these products do to the host?host?

Functional GenomicsFunctional Genomics

• Define function of every gene Define function of every gene • Establish relationship to Establish relationship to

pathogenesis pathogenesis • Identify virulence factorsIdentify virulence factors• Develop new therapeuticsDevelop new therapeutics

Genome StatisticsGenome Statistics

OrganismOrganism SizeSize G+CG+C C DC D

M. hyopneumoniaeM. hyopneumoniae 903,350903,350 28.6 %28.6 % 93%93%M. genitaliumM. genitalium 580,074580,074 32.0 %32.0 % 90%90%M. pneumoniaeM. pneumoniae 816,394816,394 40.0 %40.0 % 89%89%M. pulmonisM. pulmonis 963,879963,879 26.6 %26.6 % 93%93%U. urealyticumU. urealyticum 751,719751,719 25.5 %25.5 % 93%93%

MhyoMhyo Potential Protein Coding Potential Protein Coding ORFsORFs

• Total Protein Coding ORFs:Total Protein Coding ORFs: 766766– Functional identification:Functional identification: 392392 (51%)(51%)

– Hypothetical:Hypothetical: 374374 (49%)(49%)• Conserved (Database match):Conserved (Database match): 6464 (17%)(17%)

• Unique (No database match):Unique (No database match): 311311 (83%)(83%)

Metabolic AssignmentsMetabolic Assignments

• Amino acid BiosynthesisAmino acid Biosynthesis• Biosynthesis of cofactors, prosthetic groups, and carrierBiosynthesis of cofactors, prosthetic groups, and carrier• Cell envelopeCell envelope• Cellular processesCellular processes• Central intermediary metabolismCentral intermediary metabolism• Energy metabolismEnergy metabolism• Fatty acid and phospholipid metabolismFatty acid and phospholipid metabolism• Purines, pyrimidines, nucleosides, and nucleotidesPurines, pyrimidines, nucleosides, and nucleotides• Regulatory functionsRegulatory functions• ReplicationReplication• TranscriptionTranscription• TranslationTranslation• Transport and binding proteinsTransport and binding proteins• Other categories, UnassignedOther categories, Unassigned• HypotheticalHypothetical

Number of ORFs Identified

0 10 20 30 40 50 60 70

Amino acid biosynthesis

Cofactors, prosthetic groups and carriers

Cell envelope

Cellular processes

Central intermediary metabolism

Energy metabolism

Fatty acid & phospholid metabolism

Nucleic acid metabolism

Regulatory functions

Replication

Transcription

Translation

Transport & binding proteins

16SIleSer

Gly5S

Ser

Leu

Thr

TyrGln

Arg

Ser

Phe

Met

Asp

Pro

Met

Met

Ala

Arg Trp

LysLeu

HisLeuTrp

ValThr

Asn

Leu

Glu

Mycoplasma hyopneumoniaeMycoplasma hyopneumoniae

900 kb900 kb

Ribosomal and Transfer RNAs

PTSPTS

Gluc6PGluc6P

Fruc6PFruc6PFruc1,6PFruc1,6P

GA3PGA3P

DHAPDHAP

PyruvatePyruvateAc-CoAAc-CoA

Ac-PAc-P

AcetateAcetate LactateLactate

G3PG3P

ABCABC+ATP+ATP +NAD+NAD++

+ATP + NADH+ATP + NADH

+NAD+NAD++

GlycolysisGlycolysis

Ribose5PRibose5P

5Phosphoribose diphosphate5Phosphoribose diphosphate

AdenineAdenine

ThymidineThymidine

UracilUracil

GuanineGuanine

AdenosineAdenosine

ThymineThymine

UMPUMP

AMPAMP

dTMPdTMP

CMPCMP

GMPGMP

Uridine

Guanosine

ADPADP

dTDPdTDP

CDPCDP

GDPGDP

NDPNDPNTPNTP

dNDPdNDPdNTPdNTP

+ATP+ATP

+5PRPP+5PRPP

dUMPdUMP

+5PRPP+5PRPP

CytidineCytidine

UDPUDP

+5PRPP+5PRPP

??

??

??

Purine and Purine and Pyrimidine Pyrimidine MetabolismMetabolism

Membrane TransportMembrane Transport

FOF1

ATPase

ADPADP

ATPATP

HH++ /Na/Na++

~45~45lipoproteinslipoproteins

P-typeATPas

e

ADPADP

ATPATP

PTSPTS

mglAmglAMgtEMgtEMgMg2+2+//

CoCo2+2+

FeFe3+3+ / /haeminhaemin

phosphatphosphateespermidinespermidineputrescineputrescine

oligopeptideoligopeptidess

cationscations

carbohydratecarbohydrate

amino acids,bases, amino acids,bases, nucleosides ??nucleosides ??

ChaperonesChaperones

• DnaKDnaK

• DnaJDnaJ

• GrpEGrpE

• Trigger FactorsTrigger Factors

• No GroEL-GroESNo GroEL-GroES

SecretionSecretion

• SecASecA

• SecYSecY

• ClpBClpB

Mycoplasma hyopneumoniaeMycoplasma hyopneumoniae

900 kb900 kb

Locations of Putative LipoproteinsLocations of Putative Lipoproteins

Mycoplasma hyopneumoniaeMycoplasma hyopneumoniae

• Attaches only to Attaches only to ciliated epitheliumciliated epithelium

• Attaches exclusively Attaches exclusively to the ciliato the cilia

cilium bindingcilium binding

90009000800080007000700060006000500050004000400030003000200020001000100000

P102P102P97P97

ORF 1ORF 1

ORF 2ORF 2

ORF 5ORF 5

ORF 6ORF 6

F2G5 F2G5 NP97NP97

F2G5F2G5NP97NP97NP97NP97

Immunogold EM Image of M. hyopneumoniae reacted with F2G5 monoclonal antibody

R1 R2

∆NP97 NP97 F1B6

Immunogold labeling of in vitro grown Immunogold labeling of in vitro grown M. hyopneumoniaeM. hyopneumoniae with with -∆NP97 peptide antiserum-∆NP97 peptide antiserum

R1 R2

∆NP97 NP97 F1B6

Immunogold EM Images of M. hyopneumoniae reacted with anti-NP97.2 mouse antiserum

R1 R2

∆NP97 NP97 F1B6

Immunogold EM Image of M. hyopneumoniae reacted with anti-P102 mouse antiserum

66

Mycoplasma hyopneumoniaeMycoplasma hyopneumoniae cilium adhesin-related cilium adhesin-related

operonsoperons

11

2233

44

55

penitol phosphotransferase

uniqueP102*

1

2

3

4

5

P110 P97 paralog

M-6-P isomeraseunique

6

P102*P102* gyrBrnpBrps15

P97 P102 uniqueUnkrps10

unique pheS

P146MsbA MsbA Unique

unique P102*

P102*

P102*

unique

P97 paralog

P97 paralog

435612

200

200

400

400

600

600

800

800

1000

1000

1200 1400 1600 1800

1200 1400 1600 1800

P97 homolog alignmentP97 homolog alignment

P102 homolog alignmentP102 homolog alignment

4 3B 3C 3A 5 2 6

100

100

200

200

300

300

400

400

500

500

600

600

700

700

800

800

900

900

1000 1100 1200

1000 1100 1200

Therapeutic TargetsTherapeutic Targets

• Lipoproteins as vaccine componentsLipoproteins as vaccine components

• Membrane transportersMembrane transporters

• Adherence-related proteinsAdherence-related proteins

• P97 and P102 paralogsP97 and P102 paralogs

• Unique membrane activitiesUnique membrane activities