The KP1_4563 gene is regulated by the cAMP receptor protein and controls type 3 fimbrial function in Klebsiella pneumoniae NTUH-K2044

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen that can adhere to host cells or extracellular matrix via type 1 and type 3 fimbriae. KP1_4563 is a gene encoding a hypothetical protein in K. pneumoniae NTUH-K2044. KP1_4563 is located between the type 1 and type 3 fimbrial gene clusters and is likely associated with fimbrial function given its putative conserved domains of unknown function (DUF1471). Cyclic AMP receptor protein (CRP) regulates virulence-related gene expression and is a crucial transcriptional regulator in many bacteria. The predicted DNA recognition motif of CRP is present in the KP1_4563 promoter region. This study aimed to investigate the function of KP1_4563 in fimbriae and its transcriptional regulation mechanism by CRP. We generated Kp-Δ4563 mutant and complementation strains. We utilized phenotype and adhesion assays to evaluate the role of KP1_4563 in fimbriae. We conducted quantitative RT-PCR (qRT-PCR), LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays to study the transcriptional regulation of KP1_4563 gene by CRP. We found that KP1_4563 negatively regulates the function of type 3 fimbriae. Compared with NTUH-K2044, the absence of KP1_4563 enhanced the ability of Kp-Δ4563 to adhere to A549 cells. CRP negatively regulates KP1_4563 by directly binding to its promoter region. KP1_4563 plays an important role in type 3 fimbrial function. This novel insight will assist in the development of strategies for preventing K. pneumoniae infection.


Bacterial strains, plasmids, primers, and growth conditions
The bacterial strains and plasmids used in this study are listed in Table 1. All the primers used in the present work are listed in Table 2. K. pneumoniae and E. coli were grown in Luria-Bertani (LB) medium or LB medium supplemented with antibiotics at the following concentrations: ampicillin (Ap, 100 μg/ml), kanamycin (Km, 50 μg/ml), and chloramphenicol (Cm, 35 μg/ml). To fully express fimbriae, K. pneumoniae strains were statically cultivated in modified Minka medium for 48 h at 37˚C. Continuous cultivation was conducted for three generations at 1:1000 dilution in the same medium [45].

Construction of gene deletion and complementation strains
Mutant Kp-Δ4563 was constructed via a previously described unmarked deletion method [46,47]. In brief, the upstream and downstream flanking DNA fragments of KP1_4563 were amplified. The two flanking fragments were fused by PCR and then cloned into the temperaturesensitive suicide vector pKO3-Km. The recombinant plasmid was introduced into K2044 by electroporation. Integration (at 30˚C) and excision (at 43˚C) of the plasmid generated Kp-Δ4563, as confirmed by PCR and DNA sequencing.
To construct the complementation strain, the DNA region that contained the intact KP1_4563 gene was amplified via PCR. The DNA fragment was then cloned into the pBAD33 plasmid. Then, the recombinant plasmid was introduced into Kp-Δ4563 via electroporation. The Kpc-Δ4563 complementation strain was selected on LB agar plates supplemented with chloramphenicol and verified via PCR.

Hemagglutination assays
The expression of type 3 fimbriae was examined via mannose-resistant hemagglutination (MRHA) assays as previously described [17,48]. Briefly, strains were statically cultivated in modified Minka medium. The third generations of the strains were harvested, washed once with phosphate-buffered saline (PBS), and resuspended at a concentration 10 10 CFU/ml. Then, 2.5% of fresh human erythrocytes were treated with an equal volume of a 0.003% (wt/vol) tannic acid (Sigma) solution in saline for 10 min at 37˚C and were washed twice with PBS. Tanned erythrocytes were mixed with equal volumes of a series of 2-fold dilutions of the bacterial suspension with or without 0.25% mannose (Sigma) in 96-well, U-bottomed microtiter plates. The plates were gently agitated at room temperature for 1 min. Then, the minimum bacterial density (CFU/ml) required to agglutinate erythrocytes was measured. The expression of type 1 fimbriae was specifically detected via the mannose-sensitive agglutination of guinea pig red blood cells (RBCs) assays [49]. As described above, 2.5% guinea pig RBCs were mixed with a series of 2-fold dilutions of bacterial suspension with or without 0.25% mannose in 96-well, U-bottomed microtiter plates. Two controls were included in this experiment: E. coli DH5α (type 1 fimbriae expression) and PBS (negative control). The plates were gently agitated at room temperature for 1 min. Then, the minimum bacterial density (CFU/ml) required to agglutinate erythrocytes was measured. The experiment was repeated at least thrice.

Mannan-binding assay
The quantity of fimbriae from the K2044, Kp-Δ4563, and Kpc-Δ4563 strains was examined by mannan-binding assay as previously described [50]. Mannan (Sigma) derived from Saccharomyces cerevisiae was dissolved in 0.02 M bicarbonate buffer. Then, 100 μl of 20 μg/ml mannan Table 2. Oligonucleotide primers used in this study.

Primers Sequence (5'-3')
Gene deletions Amplification of the KP1_4563 coding region together with AGGAGG, which is a ribosome binding site (underlined) consensus sequence, and AATTCACC (italic), a spacer. Bold letters indicate the respective restriction enzyme site in the primer.

Bacterial adhesion assays
The adhesion ability of the K2044, Kp-Δ4563, and Kpc-Δ4563 strains was examined by adhesion assays as previously described [51]. Briefly, monolayers of A549 human lung epithelial cell lines (8×10 5 ) were infected at a multiplicity of infection (MOI) of 100 in 24-well, flat-bottomed cell culture plates, followed by incubation at 37˚C for 4 h. After incubation, the wells were washed thrice with PBS to remove unattached bacteria. Adherent bacteria were released by the addition of 1 ml of 0.1% Triton per well and were then quantified by plating appropriate dilutions on LB agar plates. Adhesion was expressed as the number of CFU that adhered to the A549 cells. The results were presented as the mean of at least three technical repeat wells in each experiment, and at least three biological replicates were performed.

Quantitative RT-PCR (qRT-PCR)
K2044 and Kp-Δcrp strains were statically cultivated in modified Minka medium at 37˚C for 48 h. The third generations of the strains were diluted 1:1000 in 15 ml of fresh medium and grown until OD 600 = 1.0. The bacteria were pelleted for RNA extraction. RNA was extracted, and the residual DNA was eliminated with an RNAprep Pure Cell/Bacteria Kit (TIANGEN) in accordance with the manufacturer's instructions. RNA quality was determined via 1.2% agarose gel electrophoresis, and RNA quantity was determined with a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific). Equal quantities of RNA were converted to cDNA using the random hexamer primer from a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Real-time PCR was performed with a SYBR Premix Ex Taq II Kit (Takara) and LightCycler System. Data were normalized with 16S rRNA as the endogenous reference. The relative expression ratio of a target gene was calculated using a previously described method [52]. Every sample was tested in triplicate in each experiment. Each experiment was repeated at least thrice.

LacZ fusion and β-galactosidase assay
The promoter region of KP1_4563 was amplified from K2044 using the primers listed in Table 2

Electrophoresis mobility shift assay (EMSA)
The His-CRP protein was purified as previously described [41,55]. For the EMSA [56, 57], the putative promoter region fragments of KP1_4563 were labeled at the 5 0 end with [γ-32 P] ATP and T4 polynucleotide kinase. In the final 10 μl of the reaction volume, the labeled DNA fragment (1000 to 2000 cpm/μl) was incubated with increasing amounts of purified His-CRP protein and 20 mM cAMP at room temperature for 20 min in 5× binding buffer. Three controls were included in the EMSA experiment: 1) cold probe (the same promoter-proximal DNA region unlabeled) as a specific DNA competitor, 2) negative probe (the unlabeled coding region of the 16S rRNA gene) as a non-specific DNA competitor, and 3) unrelated protein as a non-specific protein competitor. After incubation, the samples were analyzed by electrophoresis on 4% native polyacrylamide gels in 0.5× TBE buffer at 80-120 voltage and 4˚C. Radioactivity was detected via autoradiography after exposure to Kodak film at -70˚C.

DNase I footprinting
For the DNase I footprinting [56, 57], the sense or antisense primer of the putative promoter region of the KP1_4563 gene was labeled with [γ-32 P] ATP and T4 polynucleotide kinase. The putative promoter region of KP1_4563 was amplified by PCR with 32 P-labeled primers. In the final 10 μl of the reaction volume, the purified PCR products (15,000 to 20,000 cpm/μl) were incubated with increasing amounts of purified His-CRP protein with 20 mM cAMP in 5× binding EMSA buffer. The reaction volume was incubated at room temperature for 30 min. Optimized DNase I (Promega) was then added to the reaction mixture, and it was incubated at room temperature for 30-70s. The reaction was quenched by adding 9 μl of stop solution (200 mM NaCl, 30 mM EDTA, and 1% SDS). Then, the mixture was incubated at room temperature for 1 min. The digested DNA was extracted with phenol/chloroform and analyzed on 6% polyacrylamide gels with 8 M urea. Radioactivity was detected as above. Footprints were identified by comparison with sequence ladders.

Nucleotide sequence accession number
All sequences obtained in this study were deposited in GenBank under the accession number NC_012731.

Statistical analyses
Statistical analyses were conducted with SPSS 22.0 software. P<0.05 was considered statistically significant.

Results
The KP1_4563 gene negatively regulates the function of type 3 fimbriae We successfully generated Kp-Δ4563 and Kpc-Δ4563 strains. We then utilized phenotype and adhesion assays to investigate the role of KP1_4563 in fimbriae.
To address the influence of KP1_4563 on type 3 fimbrial function, we performed MRHA, a sensitive functional assay for type 3 fimbriae. The minimum bacterial density (CFU/ml) that was required to agglutinate erythrocytes is showed in Fig 1A. Deleting KP1_4563 from K2044 increased the MRHA activity. The minimum bacterial density of Kp-Δ4563 required to agglutinate erythrocytes was 6.67×10 7 CFU/ml, which was approximately 10-fold lower than that of K2044 (6.67×10 8 CFU/ml, P<0.05). The MRHA activity weakened when Kp-Δ4563 was complemented with pBAD33-p4563 plasmid, and the minimum bacterial density of Kpc-Δ4563 required to agglutinate erythrocytes was 3.67×10 8 CFU/ml. Two controls were included in this experiment, E. coli DH5α (type 1 fimbriae expression) and PBS (negative control), which both failed to mediate visible agglutination.
The effect of KP1_4563 on the function of type 1 fimbriae was determined via the mannose-sensitive agglutination assays of guinea pig RBCs. The highest bacterial densities (>1×10 10 CFU/ml, data not showed) of K2044, Kp-Δ4563, and Kpc-Δ4563 examined all failed to induce the visible mannose-sensitive agglutination of guinea pig RBCs. Two controls were included in this experiment, E. coli DH5α, which mediates visible mannose-sensitive agglutination, and PBS, which does not induce agglutination. These results indicated that KP1_4563 is irrelevant to the function of type 1 fimbriae. Type 1 and type 3 fimbriae both bind to yeast surfaces [49]. A binding assay with mannan derived from Saccharomyces cerevisiae (the receptor compound) was performed to investigate the function of fimbriae. As showed in Fig 1B, compared with K2044, Kp-Δ4563 bound to mannan more strongly. Binding was reduced when Kp-Δ4563 was complemented with the expression-complementary pBAD33-p4563 plasmid. These results indicated that KP1_4563 likely negatively regulates the function of type 3 fimbriae in an unknown manner.

Role of KP1_4563 gene in bacterial adhesion
The ability of K2044, Kp-Δ4563, and Kpc-Δ4563 to adhere to A549 human lung cancer cells were analyzed (Fig 1C). The absence of KP1_4563 dramatically enhanced the adhesion of Kp-Δ4563 to A549 cells compared with that of K2044. Adhesion was weakened when Kp-Δ4563 was complemented with the expression-complementary pBAD33-p4563 plasmid.

CRP negative regulates KP1_4563
To investigate the regulation of KP1_4563 by CRP, qRT-PCR, LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays were performed. We monitored the expression of KP1_4563 in K2044 and the Kp-Δcrp mutant via qRT-PCR (Fig 2A). Compared with K2044, the expression levels of KP1_4563 increased 3.7-fold (P<0.05) in the Kp-Δcrp mutant. The results of the LacZ fusion assays showed that the activity of β-galactosidase in CCW01Δcrp/placZ15-p4563 (6.8×10 4 Miller units) increased approximately 6-fold relative to that of CCW01/placZ15-p4563 (1.14×10 4 Miller units, P<0.05, Fig 2B). The results of qRT-PCR and the LacZ fusion assays revealed that CRP negatively regulates KP1_4563. EMSA was performed to determine the  The results are expressed as the percentage of WT expression. Data are presented as the mean of at least three technical replicates (mean ± standard deviation). Statistical significance was analyzed by independent samples t-test. Significant difference is indicated by * for P<0.05. (B) LacZ fusion assay. The putative promoter region of KP1_4563 was cloned into the lacZ transcriptional fusion placZ15 plasmid and then introduced into CCW01 or CCW01Δcrp to determine promoter activity. The results are expressed as β-galactosidase activity (Miller units) in the cellular extracts. Statistical significance was analyzed by independent samples t-test. Significant difference is indicated by * for P<0.05. (C) EMSA. The radioactively labeled putative promoter region of KP1_4563 was incubated with increasing amounts of purified His-CRP protein with cAMP and was then subjected to 4% (w/v) native polyacrylamide gels electrophoresis. The interaction between His-CRP and the promoter region of KP1_4563 formed a DNA-CRP complex, which produced a retarded DNA band with decreased mobility. (D) DNase I footprinting. A labeled coding or non-coding DNA fragment was incubated with increasing amounts of His-CRP (lanes 1, 2, 3, and 4 specificity of CRP binding to the upstream region of the translation start site of KP1_4563. As showed in Fig 2C, purified His-CRP protein bound to the upstream region of KP1_4563 DNA fragments in a dose-dependent manner. Positive EMSA results were observed for KP1_4563, which demonstrated that the CRP-cAMP complex directly bound to the KP1_4563 promoter. The DNase I footprinting showed that His-CRP protected a single DNA region upstream of the KP1_4563 gene in a dose-dependent manner (Fig 2D). The binding site ranged from 49 bp to 85 bp upstream of the KP1_4563 start codon ATG, where the A in the ATG start codon refers to position 1. Therefore, CRP likely negatively regulates the transcription of KP1_4563 by directly bindng to the promoter region.

Discussion
The region between the fim and mrk fimbrial gene clusters is highly conserved in different K. pneumoniae isolates. Sequence analysis of the fimbrial region from K. pneumoniae C132-98, C747, and C4712 has revealed the presence of homologues of the five ORFs described in K. pneumoniae C3091 [26]. KP1_4563 is a hypothetical protein-encoding gene in the fimbrial region. The KP1_4563 protein has putative conserved domains of unknown function (DUF1471). This study describes, for the first time, the role of KP1_4563 in type 3 fimbrial function and elucidates the transcriptional regulation of KP1_4563 by CRP. The function of KP1_4563 reported here will facilitate understanding of the functions of other DUF1471 proteins' function. Understanding the effect of the KP1_4563 on type 3 fimbrial function will aid in the development of strategies for preventing K. pneumoniae infection.
We investigated fimbrial types in K2044, Kp-Δ4563, and Kpc-Δ4563. The majority of the K. pneumoniae strains express the type 1 and type 3 fimbriae [58-60]. Type 1 fimbrial expression is specifically detected by the mannose-sensitive agglutination of guinea pig RBCs, whereas type 3 fimbrial expression is detected by the agglutination of tannic acid-treated human erythrocytes in a mannose-resistant manner [17,49]. This study is the first to describe the effects of the KP1_4563 gene on fimbriae. Unexpectedly, K2044, Kp-Δ4563, and Kpc-Δ4563 all failed to mediate the visible mannose-sensitive agglutination of guinea pig RBCs at the highest tested bacterial density (>1×10 10 CFU/ml, data not showed). These results indicated that KP1_4563 does not influence type 1 fimbrial function. The minimum bacterial density of Kp-Δ4563 required to agglutinate tannic acid-treated human erythrocytes was approximately 10-fold lower than that of K2044. The results of the mannan-binding assay further confirmed the negative regulatory role of KP1_4563 in type 3 fimbriae.
The ability of bacteria to adhere to host structures plays a major role in the development of infections. Given that type 3 fimbriae can adhere to epithelial cells in the respiratory tract, A549 human lung epithelial cell lines were selected as target cells to identify the effect of KP1_4563 on adhesion in K. pneumoniae. As expected, the ability of Kp-Δ4563 to adhere to A549 cells was dramatically enhanced. The results showed that the absence of KP1_4563 increased bacterial adhesion to A549 cells.
We studied the transcriptional regulation mechanism of the KP1_4563 gene by CRP. The results indicated that CRP negatively regulates KP1_4563 by directly binding to the promoter region of KP1_4563 and that KP1_4563 negatively regulates the function of type 3 fimbriae in an unknown manner. Overall, CRP may indirectly and positively regulate the function of type 3 fimbriae. These results corroborate the importance of CRP in regulating virulence-related genes in K. pneumoniae [41].
CRP has been reported to be required for fimbrial production, and the deletion of crp lead to a huge attenuation of the ability to agglutinate yeast cells [61]. Our results clarify that CRP may indirectly and positively regulate the function of type 3 fimbriae by directly regulating the KP1_4563 gene at the molecular level, which might aid in understanding the relationship of CRP and fimbriae. Lin reported that CRP down-regulates type 3 fimbriae expression indirectly through the c-di-GMP signaling pathway [62]. c-di-GMP is a second messenger molecule in bacteria, and the synthesis and decomposition of c-di-GMP is accomplished by diguanylate cyclases with conserved GGDEF domains and phosphodiesterases (PDEs) with EAL domains [63]. The mrkJ gene is immediately adjacent to the mrkABCDF operon that encodes the structural and assembly components of type 3 fimbriae. MrkJ has homology to EAL domain-containing phosphodiesterases (PDEs). Overexpression of mrkJ results in a significant decrease in the intracellular concentration of c-di-GMP and down-regulates type 3 fimbriae expression [64]. Bioinformatic analysis showed that the KP1_4563 protein does not contain conserved GGDEF or EAL domains, which indicates that KP1_4563 negatively regulates the function of type 3 fimbriae without affecting the intracellular concentration of c-di-GMP, but did show that KP1_4563 protein has the putative conserved DUF1471 domains. In E. coli K-12, the YcfR protein has DUF1471 domains, and deleting ycfR caused changes in bacterial cell surface structures and properties by affecting cell surface protein gene expression, which further affects cell aggregation. The remarkable changes in the ycfR mutant may be due to regulation by CRP, as EMSA results showed that CRP binds to the upstream region of the ycfR gene [65]. We suspect that KP1_4563 regulates the function of type 3 fimbriae, which may be associated with changes in bacterial cell surface structures and properties. Further study is needed to elucidate the precise role of KP1_4563 in type 3 fimbrial function.
In conclusion, we found that KP1_4563 negatively regulates type 3 fimbrial function, but does not influence type 1 fimbrial function. Adherence to A549 cells is considerably enhanced in the absence of KP1_4563. Moreover, CRP negatively regulates the transcription of KP1_4563 by directly binding to the upstream KP1_4563 promoter region.