Date post: | 01-Jan-2016 |
Category: |
Documents |
Upload: | martin-thornton |
View: | 219 times |
Download: | 1 times |
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