The transcription of Kpc fimbrial genes is impeded in the kpcS-ON

pneumoniae cells

To investigate whether the production of KpcA could only be detected in the E.

coli system (Fig. 3.11) but not in the phase ON K. pneumoniae cells, which may due

to no transcription of mRNA, or the instability of kpcA mRNA or KpcA protein, plasmid pKPC-36 was introduced into K. pneumoniae NTUH-K2044. KpcA production was readily observed upon IPTG induction (Fig. 3.12), which implied that transcription of kpcA was low in the ON phase of K. pneumoniae cells.

The kpcSON carried on pSY003 exerting with a remarkably high promoter activity (Fig. 3.9) contains the region between the two inverted repeats. It is possible that other factors participate in the transcriptional control of kpcA through binding to the 83-bp DNA between the IRR and kpcA start codon. To investigate this possibility, the DNA fragment encompassing kpcSON, IRR, and the 83-bp region was PCR amplified and then cloned in front of the promoterless lacZ gene in pLacZ15 by transcriptional fusion, and the resulting plasmid named pAW126 (Fig. 3.13). After the

introduction of pSY003 or pAW126 into the K. pneumoniae NTUH-K2044 ∆lacZ strain, the β-galactosidase activity of the transformants was measured. As shown in Fig. 3.13, the ΔlacZ strain harboring pAW126 exhibited a significantly lower level of β-galactosidase activity than that observed for ΔlacZ [pSY003]. This result suggested that in the ON phase, the transcription of kpcA is impeded by the DNA region between kpcS and kpcA.

3.4. Discussion

The expression of Kpc fimbriae in K. pneumoniae NTUH-K2044 could not be observed with various environmental stimuli, including temperature, starvation, and aeration. Furthermore, no Kpc fimbriae expression was found in the 35 K.

pneumoniae clinical isolates which possessed kpc genes (Table 2.1) grown statically

overnight in LB broth or M9 medium at 25 or 37°C. The heterologous expression system was hence used for functional characterization of the Kpc fimbriae. The displayed Kpc fimbriae were shown to confer the recombinant E. coli a higher biofilm-forming activity. Bacterial biofilm formation on indwelling devices, such as catheters or endotracheal tubes, is a significant medical problem. The Kpc fimbriae may play a role in the development of infections in catheterized patients, and the possibility awaits further investigation. Besides kpcABCD genes, the putative fimbrial genes kpaABCDE and kpbABCD genes have also been heterogeneous expressed on the afimbriate E. coli surface and the fimbriation assessed by transmission electromicroscopy (138).

Since a close association between liver abscess and K1 serotype has been reported, whether Kpc fimbriae could mediate a tissue-tropism in K. pneumoniae liver abscess is worth to study. However, the recombinant Kpc fimbriae on E. coli surface

did not increase the bacterial adherence to the human hepatocellular liver carcinoma cell lines, HepG2 and SK-HEP-1 (data not shown). The kpcC mutation had no apparent effect on K. pneumoniae NTUH-K2044 virulence as assessed using intragastrical inoculation to BALB/c mice (193). No anti-KpcA response could be identified in the sera of the K. pneumoniae liver abscess infection patients (193). How to assess the role of Kpc fimbriae in K. pneumoniae liver abscess pathogenesis remains challenging.

In Pseudomonas aeruginosa, the four types of fimbriae belonged to the chaperone-usher assembly class (CupA, CupB, CupC, and CupD fimbriae) do not express under laboratory growth conditions (167, 213, 222, 254, 304, 305).

Transposon mutagenesis was thus employed to select for the mutants expressing either of the Cup fimbriae for further studies. A transposon-insertion mutant library derived from K. pneumoniae NTUH-K2044 CCW01 strain carrying PkpcA-lacZ was also generated, and the mutants with color changes on the X-gal plate isolated.

However, no blue colony was found in approximate 20,000 individual colonies of the mutant pool grown on X-gal plates.

As shown in Fig. 3.10, the recombinant KpcI possessed activity to flip kpcS in both directions; therefore, the induced expression of KpcI in K. pneumoniae may lead

to both ON-to-OFF and OFF-to-ON inversions that occur simultaneously in different cells in the bacterial population. However, whether KpcI could invert the kpcS from ON-to-OFF direction in K. pneumoniae remains to be investigated. In E. coli type 1 fimbriae, the fimS inversion which resulted in changes of the 3-untranslated region of fimE altered the mRNA stability and hence the FimE-mediated phase variation (284).

Although the recombinant KpcI196 and KpcI210 possessed similar activities on the kpcS switch (Fig. 3.10), the possibility that different stability of the two kpcI

transcripts in K. pneumoniae cells differentially control the kpcS inversion remains investigated.

Several reports have shown that DNA recombinases other than FimB/E cause the fimS switch (38, 327). The BLAST search revealed no fimbrial recombinase gene other than fimB, fimE, and kpcI in the K. pneumoniae NTUH-K2044 genome.

Whether a cross-regulation occurs between type 1 and Kpc fimbriae by the recombinases was also analyzed. The expression plasmid carrying fimB, fimE or kpcI was individually introduced into K. pneumoniae NTUH-K2044, the orientation of fimS and kpcS were then determined under the induced-condition. However, inversion of the fimS or kpcS could only be observed by the expression of their cognate recombinase (data not shown).

Fig. 3.1. Transmission electron micrographs of recombinant Kpc fimbriae. Left panel, E. coli Novablue (DE3) [pET30a]; right panel, E. coli Novablue (DE3) [pKPC-7]. Bars, 0.5 μm.

Fig. 3.2. Specificity of the KpcA antiserum. Proteins from total cell lysates of the recombinant bacteria were resolved in 15% (w/v) SDS-polyacrylamide gel and stained with Coomassie brilliant blue (left panel). The gel was subjected to Western blot analysis using KpcA antiserum (right panel). The recombinant protein His6::KpcA (asterisk) and KpcA (arrow) are marked. M, protein marker; Lanes 1, E.

coli NovaBlue(DE3) [pET30a]; 2, E. coli NovaBlue(DE3) [pKPCA]; 3: E. coli NovaBlue(DE3) [pKPC-7].

(A) (B)

Fig. 3.3. Expression of Kpc fimbriae on recombinant E. coli. (A) Anti-KpcA Western blot analysis of E. coli HB101 harboring pETQ (lane 1) or pKPC-36 (lane 2).

The expressed KpcA is indicated by an arrow. (B) Bright-field (left panel) and anti-KpcA immunofluorescence (right panel) microscopic analysis of E. coli HB101 harboring pETQ or pKPC-36 (magnification x630). Bar, 10 μm.

OD595

Fig. 3.4. Biofilm forming activity of E. coli expressing the Kpc fimbriae. The development of biofilms of E. coli HB101 harboring pETQ, pKPC-36, pAW67, and pAW69 was observed (the lower panel) and quantified (the upper panel) as described in Methods. A higher biofilm-forming activity could be observed for E. coli HB101 [pKPC-36]. The results are shown as the average of the triplicate samples. Error bars indicate standard deviations. *, P < 0.001 compared with HB101 [pETQ].

(A) (B)

Fig. 3.5. IPTG-induced expression of the fimbrial genes in E. coli HB101. Plasmid pETQ (lane 1), pKPC-36 (lane 2), pAW67 (lane 3), or pAW69 (lane 4) was introduced into E. coli HB101, respectively. Log-phase grown bacteria were induced with 0.5 mM IPTG for 3 h. (A) The expression of KpcA, indicated by an arrow, was analyzed by SDS-PAGE and anti-KpcA Western blot analysis. The expression of the major pilin FimA (approximately 18.3 kDa) of type 1 fimbriae is marked by an asterisk. M, protein marker. (B) Yeast agglutination. Ten microliter bacterial suspension (10⁷ CFU/ml) was mixed with ten microliter yeast suspension (10 mg/ml) on a glass slide. After gentle shaking on orbital shaker for 5 min, a strong yeast agglutinating activity of E. coli HB101 harboring pAW69 could be observed. PBS, negative control.

Fig. 3.6. Alignment of the amino acid sequences of the fimbrial recombinases.

Identical residues are shaded, the predicted critical residues involved in the DNA recombination are marked by asterisks, and the tyrosine residue which is predicted to be directly involved in the phosphoryl transfer reaction is indicated by an arrow. The difference in the C-terminal fifteen residues between KpcI196 and KpcI210 is boxed.

Fig. 3.7. Sequence analysis of the putative promoter region of the kpc gene cluster.

The 500 bp upstream and 50 bp downstream regions of the kpcA translation start codon ATG (underlined) in the kpcSON phase are shown. The 11 bp inverted repeats are outlined by square boxes. The predicted -10 and -35 promoter regions are shaded.

The kpcI210 translation stop codon TAA is also underlined.

pcc081 pcc082

pcc081 pcc082

AflII

AflII

PCR amplification and digested with AflII

PCR amplification and digested with AflII IRL

PCR amplification and digested with AflII

PCR amplification and digested with AflII IRL

Fig. 3.8. KpcI196-mediated inversion of kpcS. (A) Map of the invertible region, kpcS, in both OFF and ON orientations. The positions of the primers used in the PCR amplification, pcc081 and pcc082, and the sizes of the DNA fragments resulting from AflII digestion are as indicated. IRL, inverted repeat left; IRR, inverted repeat right.

(B) Expression of recombinant KpcI196 resulted in a switch from the OFF to the ON phase. K. pneumoniae NTUH-K2044 transformed with pBAD33 or pKPCI196 was grown in M9 broth supplemented with 0.4% glucose (lanes 1 and 3) or L-arabinose (lanes 2 and 4) for 16 h with agitation at 37^oC. The grown bacterial cultures were collected and then subjected to the switch orientation assay of kpcS. Lanes: M, DNA molecular size markers; 1 and 2, pBAD33 vector as a control; 3 and 4, pKPCI196.

Time (h)

Fig. 3.9. Determination of the promoter activities of kpcSON and kpcSOFF. The β-galactosidase activities (Miller units) of kpcSON::lacZ and kpcSOFF::lacZ in the K.

pneumoniae NTUH-K2044 ΔlacZ strain CCW01 (ΔlacZ) carrying each of the reporter plasmids pSY003 (kpcSON), pSY004 (kpcSOFF), or pLacZ15 (vector only as a negative control) were determined from log-phased cultures grown in LB broth. The results are shown as an average of triplicate samples. Error bars indicate standard deviations. *, P < 0.0001 compared with ΔlacZ [placZ15] in the same growth phase. The growth curve (OD600) of the bacteria is also shown.

(A)

pBAD pBAD pKPCI₁₉₆ pKPCI₂₁₀

pKPC-OFF pKPC-ON

Fig. 3.10. The recombinant KpcI mediated the kpcS inversions in both directions.

(A) Expression of recombinant KpcI196 or KpcI210 resulted in a switch from the OFF to the ON phase. K. pneumoniae NTUH-K2044 carrying pBAD33, pKPCI196, or pKPCI210 was grown in LB broth, supplemented with 0.4% L-arabinose, for 16 h with agitation at 37^oC. Lanes: M, DNA molecular size markers. (B) E. coli JM109 transformed with two plasmids was grown in LB broth, supplemented with 0.4%

L-arabinose, for 16 h with agitation at 37°C. The grown bacterial cultures were collected and then subjected to the switch orientation assay of kpcS. The fragment sizes corresponding to the position of the switches are shown to the right of the panel.

M 1 2 3 4 5 6 7 8 [pBAD33] [pKPCI₁₉₆] kDa

25

15

10 11 12 [pKPCI₂₁₀] 9

M 1 2 3 4 5 6 7 8

[pBAD33] [pKPCI₁₉₆] kDa

25

15

10 11 12 [pKPCI₂₁₀] 9

Fig. 3.11. KpcI-mediated expression of KpcA in E. coli. Plasmid pBAD33, pKPCI196, or pKPCI210, as marked above the panels, was introduced into E. coli JM109 [pAW73]. E. coli carrying the two plasmids was grown in LB broth, and when growth reached mid-exponential phase, the expression of KpcI was induced by varying concentrations of L-arabinose: lanes 1, 5, and 9, no induction; lanes 2, 6, and 10, 0.002% ; lanes 3, 7, and 11, 0.02%; lanes 4, 8, and 12, 0.2% L-arabinose induction.

After 3 h induction, the bacteria were analyzed by anti-KpcA Western blot hybridization. The expression of KpcA is marked by an arrow.

Fig. 3.12. The T5lac promoter driven expression of kpcABCD genes in K.

pneumoniae. Plasmid pETQ (lane 1) and pKPC-36 (lane 2) were introduced into K.

pneumoniae NTUH-K2044, respectively. The IPTG-induced expression of kpcA was analyzed by SDS-PAGE (left panel) and anti-KpcA Western blot hybridization (right panel). Expression of KpcA is indicated by an arrow. M, protein marker.

Time (h)

Fig. 3.13. Determination of the promoter activities of kpcSON and kpcSON*. The β-galactosidase activities (Miller units) of kpcSON::lacZ and kpcSON*::lacZ in the K.

pneumoniae NTUH-K2044 ΔlacZ strain CCW01 (ΔlacZ) carrying each of the reporter plasmids pSY003 (kpcSON), pAW126 (kpcSON*), or pLacZ15 (vector only as a negative control) were determined from log-phased cultures grown in LB broth. The results are shown as an average of triplicate samples. Error bars indicate standard deviations. The growth curve (OD600) of the bacteria is also shown. The results are shown as the average of the triplicate samples. Error bars indicate standard deviations.

*, P < 0.0001 compared with ΔlacZ [pSY003] in the same growth phase.

CHAPTER 4

Regulation of the Expression of Type 3 Fimbriae

in Klebsiella pneumoniae CG43

4.1. Abstract

Type 3 fimbriae play an important role in Klebsiella pneumoniae biofilm formation. Nevertheless, how the type 3 fimbrial operon, mrkABCDF, is regulated is largely unknown. Downstream to the mrkF are three putative regulatory genes named mrkH, mrkI, and mrkJ. MrkH is a PilZ domain protein of putative binding activity to

the second messenger c-di-GMP. MrkI is predicted as a LuxR-type transcriptional regulator. MrkJ has been reported as a c-di-GMP phosphodiesterase.

Reverse-transcription PCR analysis showed that mrkH, mrkI, and mrkJ could be transcribed in a polycistronic mRNA. Furthermore, deletion of mrkI from K.

pneumoniae CG43S3 appeared to abolish the production of MrkA, the major pilin of

type 3 fimbriae, as assessed by Western blot analysis. The following promoter-reporter assay of mrkA verified that MrkI regulated the type 3 fimbriae expression at transcriptional level. Moreover, mutation of a conserved aspartate residue (D56), which is predicted as a putative target site for phosphorylation, of MrkI affected the type 3 fimbriae expression. MrkA production was slightly increased by the mrkJ-deletion, whereas no obvious effect was found by the mrkH-deletion.

Nevertheless, an increased expression of type 3 fimbriae could be observed upon the induced expression of MrkH.

Analysis of the putative promoter sequences of mrkA and mrkHIJ operon revealed the ferric uptake regulator Fur binding elements. Western blot analysis showed that the deletion of fur from K. pneumoniae CG43S3 abolished the expression of MrkA. Moreover, the promoter activity of mrkA and mrkH were reduced in the Δfur strain. These suggested that Fur acted as an activator for the type 3 fimbriae expression. Interestingly, the overproduction of YdeH, an Escherichia coli c-di-GMP cyclase, appeared to activate the MrkA expression, whereas this activation was suppressed by deletion of mrkI or fur from K. pneumoniae CG43S3 [pYdeH]. Finally, we also found that the availability of oxygen could affect the expression of type 3 fimbriae. The findings concluded a multi-factorial regulation of the expression of type 3 fimbriae in K. pneumoniae CG43.

4.2. Introduction

Klebsiella pneumoniae type 3 fimbriae, which are encoded by the mrkABCDF

operon, play an important role in biofilm formation on biotic and abiotic surfaces (71, 139, 148). By analyzing the available genome sequences of K. pneumoniae, three ORFs (namely mrkH, mrkI, and mrkJ) were found to locate downstream to the mrkF gene (Fig. 4.1). MrkH is predicted as a PilZ domain protein which is able to bind to c-di-GMP (12, 34, 56, 210, 245, 258), and MrkI is predicted as a LuxR-type transcriptional factor (153). MrkJ, an EAL domain protein, has been reported as a functional c-di-GMP phosphodiesterase (153). Deletion of mrkJ was found to increase the type 3 fimbriae expression and biofilm-forming activity which is speculated to be resulted from the accumulation of intracellular c-di-GMP (153). However, how MrkH and MrkI exert regulation on the expression of type 3 fimbriae awaits investigation.

Iron is essential to most bacteria for growth and reproduction by playing as a cofactor for electron transport chain and various enzymes (221). Under anaerobic conditions, iron is in the ferrous form (Fe²⁺), which can be taken up by bacteria directly using transporter such as EfeUOB, FeoAB, MntH or SitABCD (14, 40, 111).

Under aerobic conditions, Fe²⁺ is oxidized to the ferric (Fe³⁺) state, which forms insoluble ferric-hydroxides at neutral pH resulting in poor iron availability (221).

Thus, bacteria utilized intricate iron transporting systems, which are able to dissolve and transport ferric iron under aerobic conditions. Such systems involve the secretion of high-affinity, low-molecular-weight, Fe³⁺-chelating compounds called siderophores to form ferrisiderophore complexes with Fe³⁺ (166, 221). These complexes are subsequently taken up by bacteria through specific transport systems (166).

Iron-uptake systems are also considered as virulence factors of pathogenic bacteria since iron availability is generally restricted in vivo (266).

Under aerobic conditions, however, excess iron tends to catalyze the generation of damaging free radicals which causes toxicity to bacteria (16). A tight regulation of iron-uptake systems is thus required. Genes responsible for the uptake and metabolism of iron are generally regulated by the ferric uptake regulator (Fur) in many bacteria (6, 41, 84, 120). Under iron-repletion conditions, Fur binds iron and dimerized, and the Fe²⁺-Fur dimers bind to a 19-bp consensus DNA sequence, the Fur box (GATAATGATwATCATTATC; w=A or T), in target promoters (19, 85, 110).

Binding of Fur at the promoters impedes the binding of RNA polymerase, thereby preventing transcription from these genes. Not only is Fur involved in regulating iron homeostasis, it is also participated in bacterial colonization, oxidative stress response, toxin secretion and virulence (41). In some cases, Fur functions as an activator and even to regulate certain genes in the absence of the iron (41). Recently, we have

shown that Fur repressed the expression of the mucoid factor RmpA and then decreased the biosynthesis of capsular polysaccharide (CPS) in K. pneumoniae CG43 (53).

Bis-(3’-5’)-cyclic dimeric guanosine monophosphate (Cyclic-di-GMP or c-di-GMP) is an ubiquitous second messenger which regulates a variety of cellular processes, including biogenesis of fimbriae, flagella, and capsule, in bacteria (151, 239, 250, 256, 293). The elevated level of intracellular c-di-GMP has been reported to activate the expression of type 3 fimbriae and fur in K. pneumoniae and in Escherichia coli, respectively (153, 209). In this study, deletion effects of mrkH, mrkI, mrkJ and fur on the type 3 fimbriae expression were analyzed. The presence of Fur-mediated regulation on the transcription of mrk genes was also investigated.

4.3. Results