MrkI Inversely Regulates the Expression of Type 1 and Type 3 Fimbriae in Klebsiella pneumoniae CG43

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MrkI Inversely Regulates the Expression of Type 1 and Type 3 Fimbriae in Klebsiella pneumoniae CG43

Chien-Chen Wu

1 ([email protected])

, Ching-Ting Lin

2 , Wei-Yun Cheng

1 , and Hwei-Ling Peng

1 *

1 _{Department of Biological Science and Technology, National Chiao Tung University, Hsin Chu, Taiwan, Republic of China}

2 _{School of Chinese Medicine, China Medical University, Taichung, Taiwan. Republic of China}

Type 3 fimbriae play a crucial role in Klebsiella pneumoniae biofilm formation; nevertheless,

how the type 3 fimbrial operon is regulated is largely unknown. We have found that deletion

of mrkI, a LuxR-type regulatory gene, from K. pneumoniae CG43 not only abolished the

production of MrkA, the major pilin of type 3 fimbriae, but also reduced the biofilm-forming

activity. The following quantitative RT-PCR and promoter-reporter assays of mrkA verified

that MrkI regulated type 3 fimbriae expression at the transcriptional level. Electrophoretic

mobility shift assay analysis revealed that MrkI bound the mrkA promoter in the presence of

the phosphodonor acetyl-phosphate. Furthermore, MrkI-mediated type 3 fimbriae expression

was obviously impaired when the aspartate residue (D56), a putative phosphorylation

residue of MrkI, was substituted by alanine. On the other hand, the

D

mrkI strain exhibited an

activation of type 1 fimbriae expression resulting from the OFF-to-ON inversion of the

switch region fimS. This inversion is likely due to MrkI regulation on the expressional level

of the recombinases FimB and FimE. MrkI may also exert an additional regulation because

an MrkI-fimS binding complex was demonstrated in the presence of acetyl-phosphate. Thus,

our results show that a signaling phosphorelay is probably involved in the MrkI-dependent

regulation of type 1 and type 3 fimbriae expressions.

Fig. 1. Deletion of mrkI decreases the expression of type 3 fimbriae and biofilm formation. (A) K. pneumoniae

CG43S3 (WT, wild-type), DmrkI, the mrkI-complement strain CCW41, and the control strain CCW40 were grown

overnight at 37°C with agitation in LB broth. Bacterial total protein, approximately 5 µg per lane, was separated by SDS-PAGE and then subjected to western blot analysis against MrkA antiserum. The MrkA protein is indicated by an arrow. GAPDH (arrowhead) was probed as a protein loading control. (B) Quantification of the K. pneumoniae biofilm forming activity. The results are shown as averages of triplicate samples. Error bars indicate standard deviations. *, P < 0.001 compared with the WT strain. #, P < 0.001 compared with the CCW40 strain.

OD 595 0.0 0.1 0.2 0.3 0.4 0.5 WT _D_mrk A Dmrk I CCW 40 CCW 41 * * #

Fig. 2. Deletion of mrkI decreases transcription of mrkA. (A) The b-galactosidase activities of K. pneumoniae CG43S3DlacZ and its isogenic mrkI-deletion mutant (DlacZDmrkI) carrying each of the reporter plasmids

pmrkA-P1, pmrkA-P2, and pmrkA-P3 were determined from log-phased cultures grown in LB broth. (B) qRT-PCR analysis of the effect of mrkI deletion on the expression of mrkA.

placZ 15 pmrkA -P1 pmrkA -P2 pmrkA -P3 placZ 15 pmrkA -P1 pmrkA -P2 pmrkA -P3 b -g a la ctosi d a se acti v it y ( Mil ler u n it s) 0 200 400 600 800 1000 1200 1400 1600 1800 2000

DlacZ DlacZ DmrkI

+1 100 bp ATG

mrkA

lacZ lacZ lacZ pmrkA-P1 pmrkA-P2 * * pmrkA-P3 re la tiv e am ount o f mRNA (f ol d) 0 1 10 15 20 WT _Dmrk I CCW 40 CCW 41 13.9 0.13

Fig. 3. EMSA of the recombinant MrkI and the mrkA promoter. (A) The mrkA region and the position of the

promoter are outlined. (B) Increasing amounts of the MrkI::His₆ were incubated with different lengths of P_mrkA regions (A1, A2, or A3) in the presence or absence of 50 mM acetyl-phosphate (AcP), as indicated in the margin, for the assay. The DNA-protein complex is indicated by an asterisk.

0 12.5 25 62.5 125 MrkI::His (nM) 0 12.5 25 62.5 125 0 12.5 25 62.5 125 0 12.5 25 62.5 125

A1

* * AcP

_

+

mrkA

Fig. 5. Deletion of mrkI activates the expression of type 1 fimbriae. (A) K. pneumoniae strains were grown

overnight at 37°C with agitation in LB broth. Bacterial total protein, approximately 5 µg per lane, was separated by SDS-PAGE and then subjected to western blot analysis against FimA antiserum (upper panel). The FimA protein is indicated by an arrow. GAPDH (arrowhead) was probed as a protein loading control. Yeast agglutinating activities of the tested strains are also shown in the lower panel. (B) qRT-PCR analysis of the effect of mrkI deletion on the expression of fimA.

Fig. 6. Deletion of mrkI affects the phase variation of type 1 fimbriae. (A) PCR-based detection for assessing the

inversion of the fim promoter. Location of the primers (pcc247 or pcc248 along with pcc249) and the size of the PCR amplicons in the ON or OFF phase are indicated. (B) PCR detection of the ON and OFF orientations of fimS. Bacterial chromosomal DNA (10 ng) purified from the tested strains was used as template for PCR detection of the ON phase (the upper panel) and the OFF phase (the lower panel). M, DNA molecular size markers; C, a nontemplate PCR control; lane 1, K. pneumoniae CG43S3; 2, DmrkI; 3, CCW40; 4, CCW41; 5, DmrkA.

Fig. 7. Regulation of the expression of fimB and fimE by MrkI. qRT-PCR analysis of the effect of mrkI deletion

on the expressions of fimB (A) and fimE (B). (C) MrkI binding to the fim regulatory DNA regions. The upper panel outlines the fimB, fimE, and fimA regions. Increasing amounts of MrkI::His₆, as indicated in the upper panel, were incubated with P_fimB, P_fimE, or fimS in the presence of 50 mM acetyl-phosphate for the assay. The DNA-protein complex is indicated by an asterisk.

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Fig. 4. MrkI is probably a response regulator activated by phosphorylation. (A) Sequences of MrkI and

LuxR-type transcriptional regulators NarL, BvgA, and RcsB were aligned using Vector NTI software. The conserved aspartate (D56) residue of MrkI as a putative target site for phosphorylation is indicated by an arrow. (B) D56 is important for MrkI functionality. K. pneumoniae CG43S3 (WT, wild-type), the DmrkI strain, and the mutant strains

expressing MrkI_D56E (D56E) or MrkI_D56A (D56A) were grown overnight at 37°C with agitation in LB broth. Bacterial total protein, approximately 5 µg per lane, was separated by SDS-PAGE and then subjected to western blot analysis using MrkA antiserum. The MrkA protein is indicated by an arrow. GAPDH (arrowhead) was probed as a protein loading control.

1. K. pneumoniae NTUH-K2044 MrkI

2. E. coli MG1655 NarL

3. B. pertussis Tohama I BvgA

4. E. coli MG1655 RcsB 1. 2. 3. 4. 1 10 20 30 40 50 69 (1)

----LDSILLYTNDNLIGHSIYHYLIDSHENATRLSYADVIHEKHLPLAQTIIFNLINKD ISAIRI---MrkI_K. pneumoniae NTUH-K2044 (1)

MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASN-GEQGIELAESLDPDLILLD LNMPGMNG-NarL_E coli MG1655 (1)

----MYNKVLIIDDHPVLRFAVRVLMEK-EGFEVIGETDN-GIDGLKIAREKIPNLVVLD IGIPKLDG-BvgA_B pertussis Tohama I (1)

---MNNMNVIIADDHPIVLFGIRKSLEQIEWVNVVGEFED-STALINNLPKLDAHVLITDLSMPGDKYG RcsB_E coli MG1655 (1)

71 80 90 100 110 120 130 140 150 160

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-VDLLNALRLSLLRCQQPVLMVKSDIVGLCRELINFDNAMIISEKSPLTLFSSIVQRAKGVSELPPRGL---RKQL

MrkI_K. pneumoniae NTUH-K2044 (63)

-LETLDKLREKSLSGRIVVFSVSNHEEDVVTALKRGADGYLLKDMEPEDLLKALHQAAAGEMVLSEALTPVLAASLRANRATTERDVNQL

NarL_E coli MG1655 (68)

-LEVIARLQSLGLPLRVLVLTGQPPSLFARRCLNSGAAGFVCKHENLHEVINAAKAVMAGYTYFPSTTLSEMRMG--DNAKSDSTLISVL

BvgA_B pertussis Tohama I (63)

GITLIKYIKRHFPSLSIIVLTMNNNPAILSAVLDLDIEGIVLKQGAPTDLPKALAALQKGKKFTPESVSRLLEKISAGGYG---DKRL

RcsB_E coli MG1655 (67)

149 160 170 180 190 200 210 225

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---RKQLSPRECQILELLIANNNNKRIAALLGIAHKTVHSHRIHIMQK LGIDNSRTMNQRIAALHQC---MrkI_K. pneumoniae NTUH-K2044(131)

NRATTERDVNQLTPRERDILKLIAQGLPNKMIARRLDITESTVKVHVKHMLKK MKLKSRVEAAVWVHQERIF---NarL_E coli MG1655(145)

NAKSDSTLISVLSNRELTVLQLLAQGMSNKDIADSMFLSNKTVSTYKTRLLQK LNATSLVELIDLAKRNNLA---BvgA_B pertussis Tohama I(138)

GYG---DKRLSPKESEVLRLFAEGFLVTEIAKKLNRSIKTISSQKKSAMMKLGVENDIALLNYLSSVTLSPADKD RcsB_E coli MG1655(145)

1. 2. 3. 4.

1. K. pneumoniae NTUH-K2044 MrkI

2. E. coli MG1655 NarL

3. B. pertussis Tohama I BvgA

4. E. coli MG1655 RcsB 1. 2. 3. 4. 1 10 20 30 40 50 69 (1)

----LDSILLYTNDNLIGHSIYHYLIDSHENATRLSYADVIHEKHLPLAQTIIFNLINKD ISAIRI---MrkI_K. pneumoniae NTUH-K2044 (1)

MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASN-GEQGIELAESLDPDLILLD LNMPGMNG-NarL_E coli MG1655 (1)

----MYNKVLIIDDHPVLRFAVRVLMEK-EGFEVIGETDN-GIDGLKIAREKIPNLVVLD IGIPKLDG-BvgA_B pertussis Tohama I (1)

---MNNMNVIIADDHPIVLFGIRKSLEQIEWVNVVGEFED-STALINNLPKLDAHVLITDLSMPGDKYG RcsB_E coli MG1655 (1)

71 80 90 100 110 120 130 140 150 160

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-VDLLNALRLSLLRCQQPVLMVKSDIVGLCRELINFDNAMIISEKSPLTLFSSIVQRAKGVSELPPRGL---RKQL

MrkI_K. pneumoniae NTUH-K2044 (63)

-LETLDKLREKSLSGRIVVFSVSNHEEDVVTALKRGADGYLLKDMEPEDLLKALHQAAAGEMVLSEALTPVLAASLRANRATTERDVNQL

NarL_E coli MG1655 (68)

-LEVIARLQSLGLPLRVLVLTGQPPSLFARRCLNSGAAGFVCKHENLHEVINAAKAVMAGYTYFPSTTLSEMRMG--DNAKSDSTLISVL

BvgA_B pertussis Tohama I (63)

GITLIKYIKRHFPSLSIIVLTMNNNPAILSAVLDLDIEGIVLKQGAPTDLPKALAALQKGKKFTPESVSRLLEKISAGGYG---DKRL

RcsB_E coli MG1655 (67)

149 160 170 180 190 200 210 225

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---RKQLSPRECQILELLIANNNNKRIAALLGIAHKTVHSHRIHIMQK LGIDNSRTMNQRIAALHQC---MrkI_K. pneumoniae NTUH-K2044(131)

NRATTERDVNQLTPRERDILKLIAQGLPNKMIARRLDITESTVKVHVKHMLKK MKLKSRVEAAVWVHQERIF---NarL_E coli MG1655(145)

NAKSDSTLISVLSNRELTVLQLLAQGMSNKDIADSMFLSNKTVSTYKTRLLQK LNATSLVELIDLAKRNNLA---BvgA_B pertussis Tohama I(138)

GYG---DKRLSPKESEVLRLFAEGFLVTEIAKKLNRSIKTISSQKKSAMMKLGVENDIALLNYLSSVTLSPADKD RcsB_E coli MG1655(145) 1. 2. 3. 4. MrkA D56E DmrkI D56A S3 GAPDH re lati ve amount of mRNA (f old) 0 15 20 1 WT _Dmrk I CCW4 0 CCW4 1 13.4 0.4 re lati ve amount of mRNA (f old) 0 15 20 1 WT _Dmrk I CCW4 0 CCW4 1 re lati ve amount of mRNA (f old) 0 15 20 1 WT _Dmrk I CCW4 0 CCW4 1 13.4 0.4 P_fim pcc249 fimA fimE pcc247 pcc248 ON OFF P_fim pcc249 fimA fimE pcc248 pcc247 599 bp 478 bp fimS inversion P_fim pcc249 fimA fimE pcc247 pcc248 ON OFF P_fim pcc249 fimA fimE pcc248 pcc247 599 bp 478 bp fimS inversion re lative amount of mRNA (f old) 0.0 0.5 1.0 1.5 2.0 1.53 0.52 WT _Dmrk I CCW 40 CCW 41 re lative amount of mRNA (f old) 0.0 0.5 1.0 1.5 2.0 1.3 0.67 WT _Dmrk I CCW 40 CCW 41 MrkI::His (nM) 0 12.5 25 62.5 125 P_fimE (478 bp) 0 12.5 25 62.5 125 fimS (586 bp)

100 bp _fimB _fimE _fimA

P_fimB (520 bp) P_fimE (478 bp) fimS (586 bp)

IRL IRR * * 0 12.5 25 62.5 125 P_fimB (520 bp) *

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ABSTRACT

SUMMARY

[ mrkA ]

[ fimA ]

[ fimB ]

[ fimE ]

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MrkI controls biofilm formation in K. pneumoniae by activating type 3 fimbriae expression.

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MrkI represses the expression of type 1 fimbriae by modulation of the phase variation.

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MrkI Inversely Regulates the Expression of Type 1 and Type 3 Fimbriae in Klebsiella pneumoniae CG43