1st microarray izs te

03/05/2007 1

1st Microarray IZS-TE

Tonelli Alfreda

Tonelli Alfreda

03/05/2007 2

1st Microarray IZS-TE Il Design

Differenziare le specie Presenza di geni di virulenza

03/05/2007 3

Number Array RowArray ColumnRow ColumnName X Location Y Location ch1 Intensitych1 Background85 1 1 8 1 cjcdta70 cdtA cytolethal distending toxin A1180 3350 65535 158,119486 1 1 8 2 cjcdta70 1390 3350 64505,8 113,32836287 1 1 8 3 cjcdta70 1590 3350 63302,2 148,50746288 1 1 8 4 cjcadf70 cadF fibronectin binding protein1790 3350 65535 125,50746289 1 1 8 5 cjcadf70 1990 3350 58875,4 132,94029290 1 1 8 6 cjcadf70 2200 3350 52088,5 131,19403191 1 1 8 7 cjdu70 flmA UDP-GlcNAc C6 dehydratase2390 3350 65477,6 121,32836292 1 1 8 8 cjdu70 2590 3350 53785,9 118,43283893 1 1 8 9 cjdu70 2800 3350 44436,1 77,791046

103 1 1 9 7 cjciab70 ciaB invasion antigen B 2400 3550 46992,2 91,104477104 1 1 9 8 cjciab70 2600 3550 31824,5 111,402985105 1 1 9 9 cjciab70 2800 3550 28335,4 65,895523106 1 1 9 10 cjcdtc70 cdtC cytolethal distending toxin C2990 3550 64726,5 90,492538107 1 1 9 11 cjcdtc70 3200 3550 45088,9 85,671638108 1 1 9 12 cjcdtc70 3400 3550 42539,3 100,731346112 1 1 10 4 cj1431 Cj1431c hypothetical protein Cj1431c1790 3750 12021,6 109,388062113 1 1 10 5 cj1431 1990 3740 11299,6 114,791046114 1 1 10 6 cj1431 2200 3750 11510,2 84,447762115 1 1 10 7 cj1429 Cj1429c hypothetical protein Cj1429c2380 3750 33254,4 82,925377116 1 1 10 8 cj1429 2590 3750 29635,3 109,462685117 1 1 10 9 cj1429 2790 3750 27074,3 94,029854121 1 1 11 1 cjflge70 flgE flagellar hook protein flgE1190 3950 65535 164,253738122 1 1 11 2 cjflge70 1390 3950 61364 156,029846123 1 1 11 3 cjflge70 1590 3950 46825,6 217,522385124 1 1 11 4 cjflgc70 flgC/Cj0527c flagellar basal-body rod protein 1790 3950 65535 175,582092125 1 1 11 5 cjflgc70 1990 3950 65529,4 90,970146126 1 1 11 6 cjflgc70 2190 3950 65421,9 117,014923133 1 1 12 1 cjflha270 flhA flagellar biosynthesisubspprotein1190 4160 65535 176,492538134 1 1 12 2 cjflha270 1400 4160 64946,9 240,253738135 1 1 12 3 cjflha270 1590 4150 53333,3 260,4776136 1 1 12 4 cjflgl70 flgL/Cj0887c flagellar P-ring protein1790 4150 65535 170,059708137 1 1 12 5 cjflgl70 1990 4150 64253,8 129,447754138 1 1 12 6 cjflgl70 2200 4150 62031,1 103,791046139 1 1 12 7 cjflgh70 flgH putative flagellar L-ring protein precursor 2390 4150 65535 98,597015140 1 1 12 8 cjflgh70 2590 4150 54799,4 131,194031141 1 1 12 9 cjflgh70 2800 4150 46258,4 130,2388142 1 1 12 10 cjflgg70 flgG flagellar basal-body rod protein2990 4150 65535 193,940292143 1 1 12 11 cjflgg70 3190 4170 65535 139,104477144 1 1 12 12 cjflgg70 3390 4150 65535 173,447754508 1 3 7 4 ccorp70 10790 3070 65535 166,343277509 1 3 7 5 ccorp70 10990 3080 62996,5 163,641785510 1 3 7 6 ccorp70 11200 3070 58859,7 133,552246517 1 3 8 1 cjj192 cysC/Cj1415cHS:19 putative sulfate adenylyltransferase subunit10190 3280 19588,4 173,865677518 1 3 8 2 cjj192 10390 3280 19488 137,164185519 1 3 8 3 cjj192 10600 3280 19737,9 113,835823520 1 3 8 4 cjdl170 10790 3280 43907,7 85,253731521 1 3 8 5 cjdl170 11000 3280 28233,7 136,985077522 1 3 8 6 cjdl170 11200 3280 25504,1 145,059708535 1 3 9 7 cjj31 HS:31 lipooligosaccharide biosynthesis locus11400 3480 31714,6 91,208954536 1 3 9 8 cjj31 11600 3470 26198,9 81,507462537 1 3 9 9 cjj31 11810 3470 21365,6 87,611938538 1 3 9 10 cjj191 HS:19 lipooligosaccharide biosynthesisubsplocus12000 3470 32255,6 88,8806539 1 3 9 11 cjj191 12200 3470 21422,9 68,910446540 1 3 9 12 cjj191 12410 3470 19974,9 78,850746541 1 3 10 1 cj1415 Cj1415c possible adenylylsulfate kinase10190 3680 44503,8 152,462692542 1 3 10 2 cj1415 10390 3680 31753,7 220,298508543 1 3 10 3 cj1415 10600 3680 28160 122,358208544 1 3 10 4 cj1313a Cj1313 possible flagellar protein10800 3670 11710,9 78,805969545 1 3 10 5 cj1313a 10990 3670 11282,2 76,417908546 1 3 10 6 cj1313a 11200 3680 10164 57,641792547 1 3 10 7 cj1144 Cj1144c & Cj1445c hypothetical protein Cj1144c &Cj1145c11390 3670 40516,8 89,417908548 1 3 10 8 cj1144 11600 3670 38819,4 60,208954549 1 3 10 9 cj1144 11800 3680 37234,2 81,402985553 1 3 11 1 cjp5470 Cjp54 VirB7 (virulence plasmid pV)10200 3880 13858,9 133,522385554 1 3 11 2 cjp5470 10400 3880 13133,7 151,671646555 1 3 11 3 cjp5470 10600 3880 14259,9 66,253731565 1 3 12 1 cjflag70 flaG/Cj0697c possible flagellar protein10190 4090 65535 207,164185566 1 3 12 2 cjflag70 10390 4080 47188,5 122,298508567 1 3 12 3 cjflag70 10600 4080 48334,3 145,7612568 1 3 12 4 cjflab70 fla B/ Cj1339 flagellin B 10800 4090 63685 98,835823569 1 3 12 5 cjflab70 10990 4070 49483,6 95,9403

03/05/2007 4


http://euler.bri.nrc.ca/bridna5/

User and Password

Layout View

http://euler.bri.nrc.ca/bridna5/

03/05/2007 5

Cosa chiede il microarray? L’organismo in questione

Arcobacter ? Campylobacter ? Helicobacter ?

Epsilonproteobacteria; Campylobacterales; Campylobacteraceae


03/05/2007 6

Quale specie di Campylobacter? Quali sono i fattori di virulenza presenti nell’organismo?

Aderenza Invasione Motilità Tossine

L’organismo e’virulento?


03/05/2007 7

1st Microarray IZS-TEPer l’identificazione di specie vengono

interrogati 2 “Housekeeping genes” Classificazione Primers universali.

Housekeeping genes Universali In condizioni di crescita specifica il gene non

deve mutare per adattarsi (not show any adaptive mutation to specific growth conditions)

non deve trasferirsi in modo orizzontale

03/05/2007 8


16SrRNA (presenza in abbondanza)

Regioni variabili vengono usate per la distinzione

http://rdp.cme.msu.edu/

http://rdp.cme.msu.edu/

03/05/2007 9

Fig. 1: http://www.molecularassembler.com/KSRM/Figures/4.11.JPG

Fig. 3: http://www.biochem.umd.edu/biochem/kahn/bcbchm465-01/ribosome/16SrRNA.gif

Fig. 2: http://tigger.uic.edu/classes/phys/phys461/phys450/ANJUM04/

http://www.molecularassembler.com/KSRM/Figures/4.11.JPG




http://www.biochem.umd.edu/biochem/kahn/bcbchm465-01/ribosome/16SrRNA.gif




http://tigger.uic.edu/classes/phys/phys461/phys450/ANJUM04/



03/05/2007 10


hsp60 ( groEL or hsp60 ( groEL or chaperonins)chaperonins)Chaperonins sono Chaperonins sono presenti in eukaryotes presenti in eukaryotes e prokaryotes. e prokaryotes.

http://cpndb.cbr.nrc.ca/

Fig.5: http://yalemedicine.yale.edu/ym_fw04/fold.html

Fig. 4:http://yalemedicine.yale.edu/ym_fw04/fold.html

http://cpndb.cbr.nrc.ca/

http://yalemedicine.yale.edu/ym_fw04/fold.html
























03/05/2007 11

1st Microarray IZS-TEgyrB: DNA gyrase negatively supercoils closed circular double-stranded DNA in an ATP-dependent manner and also catalyzes the interconversion of other topological isomers of double-stranded DNA rings, including catenanes and knotted rings

http://seasquirt.mbio.co.jp/icb/

Fig. 4: Corbett et al. The Structural Basis for Substrate Specificity in DNATopoisomerase IV, J. Mol. Biol. (2005) 351, 545–561

http://seasquirt.mbio.co.jp/icb/

03/05/2007 12

1st Microarray IZS-TErpoB: DNA-directed RNA polymerase beta chain protein catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates http://www.bact.wisc.edu/landick/images/geszvain_landick.pdf)

http://www.bact.wisc.edu/landclick/images/gesvain_landkick.pdf

http://www.bact.wisc.edu/landclick/images/gesvain_landkick.pdf

03/05/2007 13

1st Microarray IZS-TENegativo per

A. cryaerophilus C. coli C. concisus C. curvus C. fetus subsp.

fetus C. fetus subsp.

venerealis

C. gracilis C. helveticus C. hominis C. hyointestinalis

ssp. hyointestinalis C. hyointestinalis C. insulaenigrae C. jejuni ssp.

doylei C. lanienae

03/05/2007 14

1st Microarray IZS-TENegativo per

C. lari C. mucosalis C. rectus C. showae C. sputorum C. upsaliensis H. canadensis H. canis H. cinaedi

H. felis H. fennelliae H. muridarum H. mustelae H. nemestrinae H. pullorum H. pylori

03/05/2007 15

1st Microarray IZS-TEPositivo per Campylobacter jejuni ssp jejuni EUB 338

http://www.microbial-ecology.de/probebase/search.aspNegativo per Escherichia coli beta-glucuronidase Citrobacter freundii lactose permease Shigella flexneri conserved hypothetical protein Shigella flexneri hypothetical protein Arabidopsis thaliana chlorophyll synthetase green fluorescent protein mut3.1

http://www.microbial-ecology.de/probebase/search.asp








03/05/2007 16

1st Microarray IZS-TE flgC/Cj0527c

flagellar basal-body rod protein flhAflagellar biosynthesisubspproteinflgL/Cj0887cflagellar P-ring proteinflgHputative flagellar L-ring protein precursor flgGflagellar basal-body rod proteincysC/Cj1415c

HS:19 putative sulfate adenylyltransferase subunit

Cj1415cpossible adenylylsulfate kinaseCj1313 possible flagellar protein

Positivo per i seguenti geni di virulenza

cdtAcytolethal distending toxin AcdtBcytolethal distending toxin B cdtCcytolethal distending toxin CcadFfibronectin binding proteinflmAUDP-GlcNAc C6 dehydrataseciaBinvasion antigen BCj1431chypothetical protein Cj1431cCj1429chypothetical protein Cj1429c

flgE flagellar hook protein flgE

03/05/2007 17

1st Microarray IZS-TEPositivo per i seguenti geni di virulenza

Cj1144c & Cj1445c hypothetical protein Cj1144c &Cj1145cCjp54VirB7 (virulence plasmid pV)flaG/Cj0697cpossible flagellar proteinfla B/ Cj1339flagellin Bfclputative fucose synthetasedocBmethyl accepting chemotaxisubspproteinhtrB/Cj1134putative lipid A biosynthesisubsplauroyl acyltransferasefliYputative flagellar motor switch protein fliR/Cj1179c(2)flagellar biosynthetic protein

fliPflagellar biosynthesi proteinporAmajor outer membrane protein peb1Aamino acid ABC transporter, periplasmic amino-acid binding proteinkpsD/Cj1444c putative capsule polysaccharide export system periplasmicfliG/Cj0059flagellar motor switch proteinfliEputative flagellar hook-basal body complex proteinfliA/Cj0061cputative RNA polymerase sigma factor for flagellar operon

03/05/2007 18


flhBflagellar biosynthetic proteinkpsM/Cj1448cputative capsule polysaccharide export system inner membranekpsF/Cj1443cKpsF protein fliMflagellar motor switch proteinfliIflagellum-specific ATP synthase motAputative flagellar motor proton channel kpsS/Cj1413possible polysaccharide modification protein

waaF/Cj1148ADP-heptose-LPS pheptosyltransferasewaaEputative ADP-heptose synthase

waaF/Cj1148ADP-heptose-LPS pheptosyltransferasewaaEputative ADP-heptose synthase waaC/Cj1133putative lipopolysaccharide heptosyltransferasecdtBcytolethal distending toxin B cadFfibronectin binding proteinwaaMlipooligosaccharide biosynthesis locusCj0371flagellar motility proteincgtBputative beta-1,3-galactosyltransferase Cj1430cputative nucleotide-sugar epimerase/dehydratase flgE2hook protein

03/05/2007 19


flgDputative flagellar hook assembly protein flhBflagellar biosynthetic protein FlhBflhAflagellar biosynthesis proteinflgK/Cj1466putative flagellar hook-associated proteinflgG2putative flagellar basal-body rod proteinmapAouter membrane lipoproteinCj0397chypothetical protein Cjo397cCj1416cputative sugar nucleotidyl transferaseCj1313 possible flagellar proteinCj1312 possible flagellar protein docA/Cj0020c

docA/Cj0020ccytochrome C551 peroxidaseflgB/Cj0528cflagellar biosynthetic protein FlhB flaD/Cj0887cputative flagellin flaA/ Cj1338flagellin A docCmethyl accepting chemotaxis proteiniamA/Cj1647hypothetical integral membrane protein

http://www.genome.jp/kegg/pathway/eco/eco02040.html











03/05/2007 20


fliSflagellar protein fliQflagellar biosynthetic protein pflA/Cj1565cparalysed flagellum proteinkpsE/Cj1445cputative capsule polysaccharide export system inner membranekpsC/Cj1413possible polysaccharide modification proteinfliHputative flagellar assembly proteinfliFflagellar M-ring proteinfliDputative flagellar hook-associated protein

flhFflagellar biosynthesisubspproteinfliNflagellar motor switch proteinfliLpossible flagellar proteinpeb1Aamino acid ABC transporter, periplasmic amino-acid binding proteinmotBputative flagellar motor protein waaV/Cj1146putative glucosyltransferasewaaF/Cj1148ADP-heptose-LPS pheptosyltransferasewaaDADP-L-glycero-D-manno-heptose-6-epimerase

03/05/2007 21

1st Microarray IZS-TENegativo per i seguenti geni di virulenza

Cj1135 putative two-domain glycosyltransferase

Cj1136putative galactosyltransferase Cj1137/chypothetcal protein Cj1137c Cj1138putative galactosyltransferase wlaN/Cj1139cputative galactosyltransferase cst/Cj1140

hypothetical protein Cj1140 neuA1/Cj1443acylneuraminate cytidylyltransferase neuB1/Cj1141

N-acetylneuraminic acid synthetase neuC1/Cj1442

N-acetylglucosamine-6-phosphate 2-epimerase/N- acetylglucosamine-6-phosphatase

Implicati nella differenziazione del LOS

03/05/2007 22


Cj1421c possible sugar transferase

Cj1422c possible sugar transferase

Cj1423c putative sugar-phosphate nucleotidyltransferase

Cj1425c putative sugar kinase

Cj1426c hypothetical protein Cj1426c

Cj1427c putative sugar-nucleotide epimerase/dehydratease

Cj1432cputative sugar transferase Cj1433chypothetical protein Cj1433c Cj1434cputative sugar transferase Cj1435chypothetical protein Cj1435c

Cj1436cputative aminotransferase Cj1437cputative aminotransferase Cj1438cputative sugar transferase Cj1440c putative sugar transferase Cj1442c putative sugar transferase glfUDP-galactopyranose mutase kfiD/Cj1441c

putative UDP-glucose 6-dehydrogenase kpsT/Cj1447cputative capsule polysaccharide export ATP-binding protein

Implicati nella sintesi capsulare

03/05/2007 23


ptmA putative oxidoreductase (flagellin modification)

ptmB acylneuraminate cytidylyltransferase

VirB10 VirB11 VirB4 VirB8 VirB9 virBII

virulence plasmid VirD4 wlaN

putative galactosyl transferase

Implicati nella modificazione del flagellum,

ptmA ptmB

Implicati nella Type IV secretion system

http://www.genome.jp/dbget-bin/get_pathway?org_name=ko&mapno=03080

VirB10 VirB11 VirB4 VirB8 VirB9 virBII (PLASMID)






















03/05/2007 24

1st Microarray IZS-TEThe infectivity of C. jejuni has been found to

progressively decrease under storage conditions of starvation and aerobic stress (Hald et al., 2001).

There is relationship between the genetically determined outer membrane composition, strain survivability and infectivity and consequently risk potential for human health (On et al.,2006)

C. jejuni has a weak clonal population

structure, and that intra- and interspecies horizontal genetic exchange is a common event (Dingle et al.,2001a).

Campylobacter jejuni ssp jejuni ATCC33291

1st microarray izs te

Science