NTUH-K2044

Functional characterization of the novel fimbriae in

Klebsiella pneumoniae NTUH-K2044

StudentTeng-Kuei Huang

AdvisorHwei-Ling Peng,Ph.D

June, 2007

.3

.4

.5

.6

.11

.19

.28

.31

.37

.38

JM109[pmrkABCkpfD]...39

.40

DNA ..41

.42

.43

.44

.45

.46

.47

.49

.50

.51

.52

.53

FimH KpfD .55

.56

.57

.58

.59

2

APS

BCIP

CFU

DTT

EDTA

GCAA

HA

IPTG

LB

NBT

PBS

PCR

PVDF

RPM

SDS-PAGE

TEMED

Ammonium persulfate

5-bromo-4-chloro-3-indolyl phosphate

Colony forming unit

1,4-dithio-threitol

NNNN-ethylenediaminetetraacetate

Minimal medium containing 1% glycerol and 0.3% casaminoacid

Hemagglutination

Isopropyl--D-thio-galactopyranoside

Luria-Bertani broth

Nitro blue tetrazolium

Phosphate-buffered saline

Polymerase chain reaction

Polyvinylidene fluoride

Revolution per minute

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

NNNN-tetramethylethyl-endiamide

3

NTUH-K2044 HMMER

fim mrk

kpakpbkpckpdkpekpf kpg

NTUH-K2044

BL21(DE3) SDS-PAGE

kpakpb kpe pilin kpa

MrkD

kpakpbkpckpdkpekpf kpg

KpfD

KpaEKpbDKpdD KpgD

KpfD 0.5 mM kpf

4

Abstract

Fimbrial adhesin is an important bacterial virulence factor which affects the adherence

specificity to host tissues. In the genome of Klebsiella pneumoniae NTUH-K2044, we

found nine fimbrial gene cluters including fim, mrk, and seven novel fimbrial operons,

namely kpa, kpb, kpc, kpd, kpe, kpf, and kpg. These fimbrial gene clusters were cloned

into the expression plasmid pET30b and then transformed to an afimbriated E. coli.

Expression of the major pilin of the recombinant kpa, kpb or kpe fimbriae could be

detected by SDS-PAGE analysis. Moreover, the kpa fimbriae appeared to conferr the

bacteria an adherence ability to HCT-8 cell and also biofilm formation capability. The

adhesin protein at the tip of fimbriae is a major determinant of the adhesion specificity.

To investigate the presence of specific adhesion activity, the mrkD adhesin gene of the

established type 3 fimbriae expression system was replaced respectively by each of

the fimbrial adhesins. Transmission electron microscopy and western blot analysis

revealed that most of the recombinant type 3 fimbriae expressed and assembled

properly. The hemagglutination (HA) activity assay indicated that the recombinant

KpfD could bind to erythrocytes of guinea pig and rabbit. Different binding activity

levels of the recombinant adhesins KpaE, KpbD, KpdD and KpgD to rabbit

erythrocytes were also demonstrated. Moreover, the HA activity of KpfD to guinea

pig erythrocytes was found to be inhibited by 0.5 mM mannose.

5

adhesin

tissue tropism

(15)

K. pneumoniae

(32)K. pneumoniae

(32)

K. pneumoniae K. pneumoniae

(21, 38)

K. pneumoniae (36)

(31)

(36)

(33)

KP K1 K2 K. pneumoniae

6

(14, 42)

Escherichia coli

PDr(28)Salmonella

(6)Pseudomonas aeruginosa

Legionella pneumophilaNeisseria gonorrhoeae

N. meningitidisVibrio cholerae

(7)Actinomyces naeslundii

Corynebacterium diphtheriaeStreptococcus parasanguis

sortase(37)K. pneumoniae

(30)K. pneumoniae

(19)

K. pneumoniae(35)KPF28

(10)CF29K

Caco-2(8)

K. pneumoniae

7

Chaperone/Usher pathway Chaperone

periplasmpilin

Usher

(17)

P

fimABCDEFGHI fimA

fimH FimH

mannose-sensitive(30)

mannose (12)

K. pneumoniae

(11)

P pyelonephritis-associated pilus pap

PapG

8

-Gal(1-4)- -Gal (22)P

P

(16)

K. pneumoniae (35)

mrk mrkA mrkB mrkC

UshermrkD mrkF (1)K. pneumoniae

mannose-resistant(34)

(34) K. pneumoniae

CT18

12 Chaperone/Usher pathway (25)

9

(18)

K. pneumoniae NTUH-K2044

(23)

http://genome.nhri.org.tw/kp/index.phpK.

pneumoniae NTUH-K2044chaperone/usher pathway

kpakpbkpckpdkpekpfkpg

LT2sth(25)kpa

28%-51sth(26,

39)K. pneumoniae

kpbkpcK. pneumoniae K1p

DNA

kpakpbkpe

pET30bPCRDNATA

yT&AAkpadk001(NdeI)dk002

yT&Apkpa-1pkpa-1BamHINdeIpET30b

pkpa-4 kpadk004dk005yT&Apkpa-5

pkpa-5KpnIpkpa-4kpaAkpnIpkpa-4

pkpa-6Bkpbpcc070(NdeI)pcc071

yT&Apkpb-1pkpb-1SalINdeIpET30b

pkpb-5kpbpcc072pcc073yT&Apkpb-3 pkpb-3

SalIpkpb-5pkpb-6Ckpedk006(NdeI

)dk007yT&Apkpe-1pkpe-1NdeIHindIII

pET30bpkpe-2

11

K. pneumoniae NTUH-K2044kpaE

kpbDkpcDkpdDkpeDkpfDkpgDfimH

PCRkpaEdk023(StuI

)dk024(ApaI)kpbDdk019(StuI)dk020(ApaI

)kpcDdk013(EcoRV)dk014(ApaI)kpdD

dk025(StuI)dk026(ApaI)kpeDdk021(EcoRV)

dk022(ApaI)kpfDdk017(EcoRV)dk018(ApaI)

kpgDdk027(StuI)dk028(ApaI)fimHdk015(EcoRV

)dk016(ApaI)

pmrkABCpmrkABCEcoRVApaIEcoRV

StuIApaIpmrkABCpmrkABCkpaE

pmrkABCkpbDpmrkABCkpcDpmrkABCkpdD pmrkABCkpeDpmrkABCkpfD

pmrkABCkpgDpmrkABCfimH

PCRpET30

NovaBlue(DE3)

LB0.5 mM IPTG (isopropyl--D-thiogalactopyranoside)

12

His-tag

(nickel resin) (NovagenMadisonWI)

4BALB/c1

KpeA 5-50 g(Freunds

complete adjuvant) 10

20

SDS-PAGE

running gel12.5% geldeionized water 3.7 mlrunning

buffer 1 ml 3 ml Tris-HCl, pH 8.010% SDS 80 l40%W/Vacrylamide

solutionacrylamidebis-acrylamide= 27.513 mlTEMED

N,N,N,N-tetramethylethyl-enediamide9 l10% APS 100 lstacking

gel5% geldeionized water 1.9mlstacking buffer 0.83 ml 1 ml

Tris-HCl, pH 8.010% SDS 33 l40%W/Vacrylamide solutionacrylamide

bis-acrylamide= 27.510.4 mlTEMEDN,N,N,N-tetramethylethyl-enediamide

13

5 l10% APS 60 l 1/5protein lysis

buffer 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 100 mM NaCl

5X loading dye10010

1402090100

Coomassie blue0.25 g Coomassie blue, 45 ml deionized water, 45 ml

methanol, 10 ml glacial acetic acid

109 CFU/mlPBS20 l

(collodion-coated copper grids300 mesh)12%

phosphotungstic acid (PBSpH 7.2)

JEM 1230

A 30 150 l

5 ml PBS 150 l 5 ml 0.01% tannic acid PBS 37C 15

PBS 109 CFU/ml

PBS 3% (vol/vol)A 3% (vol/vol) tannic acid

14

APBS 11 30

PBS

423%1:1

30(27)

109 CFU/mlPBS21%

(Saccharomyces cerevisiae AH109)PBS

10

HCT-85% CO224(TPP industries

France)241X105

PBS1X107 CFU/100

l1001X107 CFU5% CO2

1PBS0.1% Triton X-100

15

1/100100 lpolystyrene96

37C1%150

l301% SDS 150 l

10595 nm

600 nm

0.011/10037600 nm

S17-1 pir[pUT mini-Tn5 luxCDABE Km2](41)K.

pneumoniae CG43[pPkpd]LB1:130

(Saline)LB

1 ml:0.5 ml0.1 mlLB

nitrocellulose10

LBX-galLB

ChloramphenicolkanamycinK. pneumoniaeX-gal

16

DNA

K. pneumoniae0.1 mM EDTA LB16

800 l lysis solution (200 mM NaCl, 20 mM EDTA, 40 mM Tris-HCl,

0.2% Triton X-100, 5 mM DTT, 80 g lysosome)3730

Proteinase K0.1 mg/ml50

10250 l

,1013,200 rpm10400 l

1000 l,3

2013,200 rpm15,75%13,200 rpm

1037100 l

4

pUT mini-Tn5 luxCDABE Km2

DNAPstI(ligase)

pUC18(transform)JM109

kanamycinKanamycin

17

SDS-PAGE39 mM glycine, 48 mM Tris-Base, 0.037%

SDS140110(PVDF, ImmobilonTM-P)

0.5% Tween-20PBS1025%

PBS24

1PBS2

5000anti-mouse IgG AP conjugatePBS

NBT 66 lBCIP 33 l

(100 mM NaCl, 5 mM MgCl2, 100 mM Tris-HCl, pH 9.5)

18

K. pneumoniae NTUH-K2044

HMMER K.peumoniae NTUH K2044

kpf kpg NCBI BLAST

chaperon usher C

pilin kpaEkpbDkpcDkpdDkpeDkpfD kpgD

adhesin kpa kpaA

kpaE

kpakpb kpc

kpakpb kpe pET30b

A DNA

B pkpa-6 pkpb-6 pkpe-2 DNA

DNA DNA

kpakpb kpe

DNA

19

kpakpb kpc

kpakpb kpe pkpa-6pkpb-6 pkpe-2

BL21(DE3) IPTG

SDS-PAGE pkpa-6pkpb-6 pkpe-2 1 mM

IPTG Lanes 357

signal peptide KpaA

KpbA pkpe-2 Lanes 7 KpeA

20 kDa

17.5% SDS-PAGE

kpa kpb

KpaA KpbA 20 kDa A kpe

A kpe

Ppellet kpa kpb

usher-chaperone pathway

(single peptide)cytoplasmperiplasm

usher-chaperone pathway

(http://www.cbs.dtu.dk/services/SignalP/)

1 kDa AB

S2 S3

20

kpa kpb

BL21(DE3)[pET30b] A

BL21(DE3)[pkpa-6] BL21(DE3)[pkpb-6] BC

kpa kpb

kpa kpb

polystyrene 96 1 mM IPTG LB 72

BL21(DE3)[pkpa-6] kpa

BL21(DE3)

kpa kpb

BL21(DE3)[pET30b]BL21(DE3)[pkpa-6] HCT-8

kpa K. pneumoniae

21

pmrkABCmrkAmrkBmrkC

chaperoneushermrkABC

mrkDNTUHkpaEkpbDkpcD

kpdDkpeDkpfDkpgDfimH

AC

DNABD

DNA

GCAA LB

LB

JM109[pmrkABCkpdD]

GCAA

GCAA

22

GCAA 20

mrkmrkD

JM109[pmrkABCDNTUH]

MrkA

JM109 rabbit anti-MrkA

MrkA

MrkA 7.5% SDS-PAGE

MrkA

MrkA JM109[pmrkABCkpcD]

JM109[pmrkABCfimH] JM109[pmrkABC] kpcD fimH

MrkA

A

23

JM109[pmrkABCkpaE]

JM109[pmrkABCkpbD] JM109[pmrkABCkpdD]JM109[pmrkABCkpeD]

JM109[pmrkABCkpfD]JM109[pmrkABCkpgD]

1/2 1/4 JM109 [pmrkABCkpfD]

1X109 CFU/ml 1/2

1/8

ACDE

JM109 [pmrkABCfimH]FimH

D-mannose

4

D-arabinoseD-fructoseD-galactoseD-glucoseD-mannoseMaltoseLactose

SucroseN-acetyl-glucosamine JM109[pmrkABCkpaE]

24

JM109[pmrkABCkpbD] JM109[pmrkABCkpdD]JM109[pmrkABCkpeD]

JM109[pmrkABCkpgD]

500 mM D-mannose MSHA

(mannose-resistant hemagglutination) JM109[pmrkABCkpfD]

D-mannose D-fructoseD-arabinose

MaltoseSucrose D-mannose

D-fructose 0.5 mM

D-arabinose 50 mMSucrose 75 mM; Maltose 150 mM

mannose 4-nitrophenyl--D-mannoside

Methyl--D-mannoside

mannose 4-nitrophenyl--D-mannoside

0.005 mM; Methyl--D-mannoside 0.001 mM

D-mannose KpfD

D-mannose

FimHK. pneumoniae FimH KpfD

FimH mannose (5)

KpfD mannose

25

JM109[pmrkABC]

AJM109[pmrkABCDNTUH]B

MrkA

JM109[pmrkABCkpfD]

CJM109[pmrkABC]

DKpfD

MrkA

kpd

kpa kpg

kpd

kpd A lacZ

kpd 9000 12

X-gal 10 l

X-gal LB B1B2 B11

26

12 B1 B2 B11

B3B6B7 B12 B4

B5B8B9B10 B8

B1B2B4B8

B1 hns B2 kvhR orfY

B4 orf7 B8 gnd kvhR

orf7 gnd

B1

hns DNA

Hns(29)

27

K. pneumoniae (3, 38)

K. pneumoniae (35)

K. pneumoniae (9, 24) K. pneumoniae

NTUH-K2044 usher-chaperone pathway

K. pneumoniae

K. pneumoniae

BLAST

kpa sth sthABCDE

(39) sthC

(26) kpaABCDE sthABCDE

33%51%44%29%28% kpa sth

kpa

kpa kpb

K1

kpb kpb

kpb

28

MrkD

JM109[pmrkABCkpfD]

mannose

mannose

mannose (30) FimH mannose

(5) KpfD FimH K. pneumoniae

FimH KpfD FimH mannose

mannose 4-nitrophenyl--D-mannoside

methyl--D-mannoside 500-1000 (12) KpfD

4-nitrophenyl--D-mannoside D-mannose

methyl--D-mannoside

methyl--D-mannoside 4-nitrophenyl--D-mannoside

(13) mannose mannose

KpfD D-fructose

(2)

KpfD

29

kpd

hns hns

(29,

40) hns kpd hns

B2 kvhR orfY

4 kDa

30

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43. CG43

44. CG43

MrkDMrkF

45.

36

K. pneumoniae CG43S3lacZ- CG43S3 lacZ (20) pLacZ15 lacZCmr (20) pPkpdA kpdAlacZCmr pkpa-1 dk001 dk002 TAyT&AApr pkpa-4 kpaA NdeI pET30b pkpa-5 dk004 dk005 TAyT&AApr pkpa-6 kpa NdeI pET30b pkpb-1 pcc070 pcc071 TAyT&AApr pkpb-3 pcc072 pcc073TAyT&AApr pkpb-5 kpbAB NdeI pET30b pkpb-6 kpb NdeI pET30b pkpe-1 dk006 dk007TAyT&AApr pkpe-2 kpe NdeI pET30b pmrkABC mrkABCpGEMTApr (17) pmrkABCkpaE dk023 dk024 kpaEEcoRVStuI

pmrkABCApr

pmrkABCkpbD dk019 dk020 kpbDEcoRVStuIpmrkABCApr

pmrkABCkpcD dk013 dk014 kpcDEcoRVApaIpmrkABCApr

pmrkABCkpdD dk025 dk026 kpdDEcoRVStuIpmrkABCApr

pmrkABCkpeD dk021 dk022 kpeDEcoRVApaIpmrkABCApr

pmrkABCkpfD dk017 dk018 kpfDEcoRVApaIpmrkABCApr

pmrkABCkpgD dk027 dk028 kpgDEcoRVStuIpmrkABCApr

pmrkABCfimH dk015 dk016 fimHEcoRVApaIpmrkABCApr

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(5 3)

dk001 5'-TTGTCGTTTCATATGGATATGGGAA-3' kpaBCDE 5

dk002 5'-GATTCCCCTTTCTCCATTCAACA-3' kpaBCDE 3

dk004 5'-GGGCAATGTGATGAAGAATAACG-3' kpaBCDE 5 dk005 5'-GCCAGGCGCATTAATCGTCTT-3' kpaBCDE 3

dk006 5'-TCATATGAAAGAAAAAGGCACCCTG-3' kpeABCD 5

dk007 5'-TCAGAGCAATTCCTTCATCCG-3' kpeABCD 3 dk011 5'-ATTCATTAAAGAGGAGAAATTAACATATG-3'kpaBCDE 5

dk012 5'-CATATGTTAATTTCTCCTCTTTAATGAAT-3' kpaBCDE 3

dk013 5'-GGATATCaGGAAACTGAACAGGAG-3' kpcD 5 dk014 5'-GGGCCCaTGCTCTTAAGTGTCGGC3' kpcD 3

dk015 5'-GATATCaAAGGCACCATTCAGGCGG-3' fimH 5

dk016 5'-CGAGCAGGGCCCaGAGAGGATA-3' fimH 3 dk017 5'-GCCTGGCGTGAGATATCaCTTA-3' kpfD 5

dk018 5'-CAATGAGGGCCCaAGCAACTT-3' kpfD 3

dk019 5'-TCGCAGGCCTaAAGCATCGTTAG-3' kpbD 5 dk020 5'-GGGCCCaGGTGCAGAACATTAGGT-3' kpbD 3

dk021 5'-GATATCaACCCCGACGCTGG-3' kpeD 5

dk022 5'-TTCGGGCCCaGGCGTGACAAG-3' kpeD 3 dk023 5'-GCACCCAGGCCTaTCAATCTATAAA-3' kpaE 5

dk024 5'-GGGCCCaAGTTTATCCGTTCACA-3' kpaE 3

dk025 5'-GCAGCAGGCCTaTAGCAGCGG-3' kpdD 5 dk026 5'-GGGCCCaGATGACCACGTTTAC-3' kpdD 3

dk027 5'-CGGATAAGGCCTaCACAGCAGAGTC-3' kpgD 5

dk028 5'-TCCAGGGCCCaGAAAGTCACCAT-3' kpgD 3 dk029 5-GTTGCGCTGCCCGGATTACAG-3 a

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JM109[pmrkABCkpfD]

D-arabinose 50 mM

D-fructose 0.5 mM

D-galactose -

D-glucose -

D-mannose 0.5 mM

Maltose 150 mM

Lactose -

Sucrose 75 mM

N-acetyl-glucosamine -

N-acetyl-galactosamine -

4-nitrophenyl--D-mannoside 0.005 mM

Methyl--D-mannoside 0.001 mM

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NCBI BLAST

40

DNA K. pneumoniae NTUH-K2044 DNA PCR DNA ()TA yT&A pET30b() kpaABCDEkpbABCD kpeABCD [pET30b] pkpa-6(A)pkpb-6(B) pkpe-2(C)

41

(A) pkpa-6pkpb-6 pkpe-2 (B)pkpa-6pkpb-6 pkpe-2

42

37 LB 1/20 LB 2 (A) 1 mM IPTG 3 lanes 1357) IPTG 3 lanes 246 13.5% SDS-PAGE Coomassie Blue (B) 1 mM IPTG P1234 S1234 17.5% SDS-PAGE (P234 )(P)()(P234S23)KpaA(P2S2 ) KpbA(P3S3 )

43

37 LB 1/20 LB2 1 mM IPTG 3 phosphotungstic acid 25,000-30,000

44

37 LB 1/100 1 mM IPTG LB polystyrene 96 37 72 1% SDS 595 nm

45

37 LB 1/20 LB 2 1 mM IPTG 3 HCT-8

46

47

pmrkABC ApaI EcoRV StuI pmrkABC AC BD DNA

48

GCAA LB 37 20 1/100 GCAA LB 600 nm

49

37GCAA 1/100GCAA polystyrene 96 72 1% SDS 595 nm 1JM109[pmrkABC]2JM109[pmrkABCDNTUH]3JM109[pmrkABCkpaE]4JM109[pmrkABCkpbD]5JM109[pmrkABCkpcD]6JM109[pmrkABCkpdD]7JM109[pmrkABCkpeD]8JM109[pmrkABCkpfD]9JM109[pmrkABCkpgD]10JM109[pmrkABCfimH]

50

37 GCAA 20 95 10 13.5% SDS-PAGE Coomassie Blue (A) anti-MrkA MrkA (B) M1JM109[pGEMT-easy]2JM109[pmrkABC]3JM109[pmrkABCDNTUH]4JM109[pmrkABCkpaE]5JM109[pmrkABCkpbD]6JM109[pmrkABCkpcD]7JM109[pmrkABCkpdD]8JM109[pmrkABCkpeD]9JM109[pmrkABCkpfD]10JM109[pmrkABCkpgD]11JM109[pmrkABCfimH]

51

37GCAA 20 7.5% SDS-PAGECoomassie Blue(A)anti-MrkAMrkA (B)M 1, JM109[pmrkABC]; 2, JM109[pmrkABCDNTUH]; 3, JM109[pmrkABCkpaE]; 4, JM109[pmrkABCkpbD]; 5, JM109[pmrkABCkpcD]; 6, JM109[pmrkABCkpdD]; 7, JM109[pmrkABCkpeD]; 8,JM109[pmrkABCkpfD]; 9, JM109[pmrkABCkpgD]; 10, JM109[pmrkABCfimH].

52

53

1x109 CFU/ml 1/2 PBS1JM109[pmrkABC]2JM109[pmrkABCDNTUH]3JM109[pmrkABCkpaE]4JM109[pmrkABCkpbD]5JM109[pmrkABCkpcD]6JM109[pmrkABCkpdD]7JM109[pmrkABCkpeD]8JM109[pmrkABCkpfD]9JM109[pmrkABCkpgD]10JM109[pmrkABCfimH] 37 GCAA 20 PBS 1X109 CFU/ml 1/2 3%(A)3%(B)A(C)(D)(E)11

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FimHKpfD FimHK. pneumoniae NTUH-K2044FimHFimH reviewmannoseFimH(5)FimHmannose

55

37 GCAA 20 PBS phosphotungstic acid 30,000 JM109[pmrkABC](A) 1/10 JM109[pmrkABC] JM109[pmrkABCkpfD] 50%(C) 0.5 m (D) 0.5 m JM109[pmrkABC]

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pPkpdkpdK. pneumoniae CG43 S3lacZ- [pPkpd]kpdLB 10 lX-gal 50 lX-gal WT pPkpdN1~N29000 B1~B12 9000

57

pPkpdkpdK. pneumoniae CG43 S3lacZ- [pPkpd]kpd 37LBK. pneumoniae 3000 rpm 5 WT N1~N29000 B1~B12 9000

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() () B1B2B4 B8 B1B2B4 B8hns kvhR orf7 gnd

59