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Sequence analysis of the large virulence plasmid pLVPK in Klebsiella pneumoniae CG43 revealed the presence of another mucoid factor encoding gene rmpA besides rmpA2. Promoter activity measurement indicated that the deletion of rmpA reduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carrying rmpA restored CPS production in the rmpA or rmpA2 mutant but not in the rcsB mutant. Transformation of the rmpA deletion mutant with an rcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration of in vivo interaction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5′ noncoding region of rmpA. The promoter activity analysis indicated that the deletion of fur increased the rmpA promoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, the fur deletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis in K. pneumoniae CG43 via an RcsB-dependent manner. The expression of rmpA is regulated by the availability of iron and is negatively controlled by Fur.
Klebsiella pneumoniae, an important nosocomial pathogen, causes a wide range of infections, including pneumonia, bacteremia, urinary tract infection, and life-threatening septic shock (35). Clinically isolated K. pneumoniae strains usually produce a large amount of capsular polysaccharide (CPS), which confers not only a mucoid phenotype to the bacteria but also resistance to engulfment by professional phagocytes or to serum bactericidal factors (27, 37). CPS also plays a role in hindering fimbrial binding (39) and bactericidal effects resulting from antimicrobial peptides (5). The degree of mucoidy has been positively correlated with successful establishment of infection (31, 32). Most recently, the hypermucoviscosity of K. pneumoniae isolates has also been associated with the development of invasive syndrome (51). Among the identified serotypes, K. pneumoniae strains of K1 or K2 CPS are highly virulent in the mouse peritonitis model (30).
Klebsiella CPS resembles the E. coli group I CPS in primary structure and the mechanisms of biosynthesis (48). The chemical composition of Klebsiella K2 CPS, which contains uronic acid as the major component, has been determined as [→4-Glc-(1→3)-α-Glc-(1→4)-β-Man-(3←1)-α-GlcA)-(1→n)] (45). Sequencing of the region responsible for K2 CPS biosynthesis in the K. pneumoniae Chedid strain revealed a total of 17 open reading frames organized into three transcriptional units (1). The two-component system (2CS) RcsBCD, which is the key regulatory system for Escherichia coli colonic acid synthesis, often serves as a model for group I CPS biosynthesis (19, 28). Upon receiving environmental stimuli, the transmembrane sensor kinase RcsC undergoes autophosphorylation, the signal is subsequently relayed to the inner membrane Hpt (for histidine-containing phosphotransfer) module RcsD and eventually to the cytoplasmic response regulator RcsB. The phosphorylated RcsB then interacts with RcsA, an auxiliary transcriptional regulator, and the heterodimer binds to the cps promoters, which in turn activates the biosynthesis of colanic acid capsule. RcsA is highly susceptible to degradation by the Lon protease; hence, lon mutation often leads to the accumulation of colanic acid (16). However, the introduction of multicopy rcsB has been shown to suppress the rcsA-negative phenotype (3).
K. pneumoniae CG43, with a 50% lethal dose (LD50) of 10 CFU for laboratory mice, is a highly mucoid clinical isolate of K2 serotype (7). Its mucoid phenotype has been correlated with the presence of the large virulence plasmid pLVPK; curing of this plasmid has rendered an ~1,000-fold decrease in mouse virulence (12). We have also shown that rmpA2 on pLVPK encodes a transcriptional activator for the cps expression by binding directly to the putative promoters, Porf1-2 and Porf3-15 (23). Interestingly, sequencing of the large virulence plasmid pLVPK revealed an rmpA gene 29 kb away from rmpA2 (8). Excluding the extended 15 amino acids at the N terminus of RmpA2, the deduced RmpA sequence shares an overall 71.4% identity and a conserved C-terminal DNA binding motif with the RmpA2 protein.
An rmpA gene on the 180-kb virulence plasmid pKP100 of K. pneumoniae 52145 was reported 20 years ago (29). The encoding protein RmpA was subsequently demonstrated to be able to enhance colonic acid biosynthesis in E. coli HB101 (31). Later, the rmpA2 gene, carrying an extended 5′ sequences of the rmpA, was isolated and shown to be able to activate K2 capsule production in the recombinant E. coli K-12 harboring Klebsiella K2 cps genes (45). We report here the characterization of RmpA in K2 CPS biosynthesis and comparative analysis of the expression of the two mucoid factor-encoding genes. We have demonstrated the interaction between RmpA and RcsB on the regulation of the CPS biosynthesis, and the involvement of Fur on the expression of rmpA has also been studied.
The bacterial strains and plasmids used in the present study is listed in Table Table1.1. E. coli and K. pneumoniae CG43 (7, 34) and its derivatives were propagated at 37°C in Luria-Bertani (LB) broth or M9 minimal medium as described previously (38). The antibiotics used include ampicillin (100 μg/ml), chloramphenicol (35 μg/ml), kanamycin (25 μg/ml), streptomycin (500 μg/ml), and tetracycline (12.5 μg/ml). The primers used in the present study are listed in Table Table22.
Total RNA was extracted from log-phase K. pneumoniae cells (optical density at 600 nm [OD600] of 0.6 to 0.8) with the TriReagent (Molecular Research Center, Cincinnati, OH). RQ1 RNase-free DNase (Promega, Madison, WI) was used to eliminate contaminating DNA. Reverse transcription (RT) was carried out with 1 to 2 μg of DNA-free RNA samples using Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI). The resulting cDNA was then used as the template for PCR amplification using Taq polymerase with the primers rmpA06 and rmpA07 (Table (Table2).2). The PCR products were resolved on agarose gels by electrophoresis, and the DNA fragments were transferred onto a nitrocellulose membrane and fixed by using a UV cross-linker (SpectroLinker XL-1000). The membrane was then subjected to hybridization with the fluorescein-labeled rmpA gene probe, and the signal was detected according to the manufacturer's instruction (CDP-Star DNA labeling and detection kit; Roche Molecular Biochemicals, Indianapolis, IN).
Specific gene deletion was introduced into K. pneumoniae CG43 by using an allelic-exchange strategy as previously described (23). In brief, two ~1,000-bp DNA fragments flanking both sides of the deleted region were cloned into pKAS46 (40), a suicide vector containing rpsL, which allows positive selection with streptomycin for vector loss. The resulting plasmids were mobilized from E. coli S17-1λpir (25) to K. pneumoniae CG43S3 or CG43S3ΔlacZ, by conjugation, respectively. The transconjugants, with the plasmid integrated into the chromosome via homologous recombination, were selected with ampicillin and kanamycin on M9 agar plates. Several of the colonies were grown overnight in LB broth at 37°C and then spread onto an LB agar plate containing 500 μg of streptomycin/ml. Streptomycin-resistant and kanamycin-sensitive colonies were selected, and the deletion was verified by PCR and Southern analysis using a gene-specific probe. The resulting mutant strains are listed in Table Table11.
To obtain the complementation plasmids, DNA fragments containing the rmpA, rmpA2, rcsB, and fur loci were PCR amplified by using the primer pairs Yu05/Yu06, rmpA2p06/rmpA2p07, rcsBc01/rcsBc02, and CY007/CY008 (Table (Table2),2), and the PCR products were cloned into pRK415 (20) to generate pRK415-RmpA, pRK415-RmpA2, pRK415-RcsB, and pRK415-Fur, respectively. Plasmid pRK415-RmpAN carries a truncated form of RmpA (RmpAN) of residues 1 to 84 (Table (Table11).
The bacterial CPS was extracted by using a method described earlier (9). Briefly, 500 μl of overnight grown bacteria was mixed with 100 μl of 1% Zwittergent 3-14 (Sigma-Aldrich, Milwaukee, WI) in 100 mM citric acid (pH 2.0) and then incubated at 50°C for 20 min. After centrifugation, 250 μl of the supernatant was transferred to a new tube, and the CPS was precipitated with 1 ml of absolute ethanol. The pellet was dried and dissolved in 200 μl of distilled water, and then 1,200 μl of 12.5 mM borax in H2SO4 was added. The mixture was vigorously mixed, boiled for 5 min, and cooled, and then 20 μl of 0.15% 3-hydroxydiphenol (Sigma-Aldrich, Milwaukee, WI) was added. The absorbance at 520 nm was measured, and the uronic acid content was determined from a standard curve of glucuronic acid and expressed as micrograms per 109 or 1010 CFU.
The virulence in mice was determined as previously described (23). Female BALB/c mice (4 to 5 weeks old) were obtained from the National Laboratory Animal Center and acclimatized in an animal house for 7 days. The tested bacterial strains were cultured in LB medium at 37°C overnight. Four mice of a group were injected intraperitoneally with 0.2 ml of bacterial suspension in saline in 10-fold graded doses. Based on the number of survivors after 14 days, the LD50 values were calculated by using the Reed and Muench method (36) and are expressed as CFU.
Bacterial survival in serum was determined with minor modifications (23). First, 100 μl of bacterial suspension in saline was mixed with 100 μl of pooled serum from healthy volunteers, and the mixture was incubated at 37°C for 30 min. The number of viable bacteria was then determined by plate counting. The survival rate was expressed as the number of viable bacteria treated with human serum compared to the number of those incubated with phosphate-buffered saline. The assay was performed twice, each with triplicate samples. The data from one of the representative experiments are shown and expressed as the mean and standard deviation from the three samples. A 0% survival of K. pneumoniae CG43S3ΔgalU (23) served as a negative control.
The promoter reporter plasmids were individually mobilized into K. pneumoniae strains by conjugation from E. coli S17-1λpir. The β-galactosidase activity was measured as described previously (25). In brief, the bacteria was grown to the log phase in LB medium (OD600 of 0.7) or M9-glucose medium (OD600 of 0.5), and 100 μl of the culture was mixed with 900 μl of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol), 17 μl of 0.1% sodium dodecyl sulfate (SDS), and 35 μl of chloroform, followed by vigorous shaking. After incubation at 30°C for 10 min, 200 μl of a 4-mg/ml concentration of ο-nitrophenyl-β-d-galactopyranoside (ONPG; Sigma-Aldrich, Milwaukee, WI) was added to the mixture to initiate the reaction. When yellow coloration was apparent, the reaction was stopped by adding 500 μl of 1 M Na2CO3 to the mixture, and the absorbance at OD420 was recorded; the activity was expressed as Miller units (29). Each sample was assayed in triplicate, and at least three independent experiments were carried out. The data shown were calculated from one representative experiment and are shown as the means and standard deviation from triplicate samples.
The Porf1-2::lacZ cassette from pOrf12 (25) was subcloned into pKAS46; the resulting plasmid was mobilized into K. pneumoniae CG43S3ΔlacZ, CG43S3ΔrcsBΔlacZ, or CG43S3ΔrmpAΔlacZ via conjugation from E. coli S17-1λpir. The transconjugants were screened by counterselection on M9 agar plate supplemented with kanamycin and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). After overnight incubation at 37°C, blue colonies, in which the reporter cassette was integrated into the chromosomal cps region via the Porf1-2 by homologous recombination, were isolated. The integration of the reporter cassette in the resulting strains was further confirmed by PCR with the primers CH009/CH010 (Table (Table22).
The DNA fragments encoding full-length RcsA, RmpA, RmpAN, or RmpA2 were PCR amplified with the primer sets rcsAe04/rcsAe07, rmpAe04/rmpAe05, or rmpA2p08/rmpA2p14 (Table (Table2)2) and cloned to the 3′ end of the gene encoding the λ-cI repressor protein domain on the NotI/XhoI site in pTRG to generate pTRG-RcsA, pTRG-RmpA, pTRG-RmpAN, or pTRG-RmpA2, respectively. The DNA fragments encoding the full-length RcsB were PCR amplified with primers rcsBe02/rcsBe04 (Table (Table2)2) and cloned to the 3′ end of the gene encoding the α subunit of RNA polymerase (RNAPα) domain on the NotI/XhoI site in pBT to generate pBT-RcsB. The resulting plasmids were confirmed by DNA sequencing. The pBT and pTRG derived plasmids were cotransformed into E. coli XL1-Blue MRF′ Kan cells, and the transformants were selected on LB agar plates containing 12.5 μg of tetracycline/ml, 25 μg of chloramphenicol/ml, and 50 μg of kanamycin/ml, essentially as described previously (18, 22). To investigate the protein-protein interaction in vivo, the bacteria were grown in LB medium at 30°C until the OD600 reached 0.4 and then diluted serially (10−2, 10−3, 10−4, and 10−5). Two microliters of the bacterial culture was spotted onto the indicator plate (LB agar plate supplemented with 350 μg of carbenicillin/ml, 25 μg of chloramphenicol/ml, 50 μg of kanamycin/ml, 12.5 μg of tetracycline/ml, 50 μg of X-Gal/ml, and 20 μM IPTG (isopropyl-β-d-thiogalactopyranoside). After the plates were incubated at 30°C for 48 h, the growth of the bacterial cells was observed.
For the measurement of β-galactosidase activities, E. coli strains carrying different combinations of the recombinant plasmids were grown for 48 h at 30°C. After washing with LB, the bacteria were grown at 30°C to an OD600 of 0.5. IPTG was then added to a final concentration of 20 μM, and the cultures were incubated at 30°C for another 16 h. Finally, the β-galactosidase activity was determined and calculated as described above.
The coding region of rcsB or fur was PCR amplified with the primer sets rcsBe01/rcsBe02 or CY0010/CY0011 (Table (Table2)2) and cloned into the NdeI/XhoI site in pET30b (Novagen, Madison, WI) or the BamHI/HindIII site in pET30c, respectively. This generates pET30b-RcsB with a carboxyl-terminus His tag (RcsB-His6) or pET30c-Fur with an amino-terminus His tag (His6-Fur). The resulting plasmid pET30b-RcsB or pET30c-Fur was then transformed into E. coli BL21(DE3)/pLysS (Invitrogen), and overproduction of the recombinant protein was induced by the addition of 0.5 mM IPTG for 4 h at 37°C. The recombinant proteins were then purified from the soluble fraction of the total cell lysate by affinity chromatography using His-Bind resin (Novagen). Finally, the purified proteins were dialyzed against 1× TBS (Tris-buffered saline; pH 7.4) containing 10% glycerol at 4°C overnight, followed by condensation with PEG 20000, and the purity was determined by SDS-PAGE.
The RcsB-His6 antiserum was prepared by immunizing 5-week-old female BALB/c mice purchased from the animal center of National Taiwan University intraperitoneally with 10 mg of purified RcsB-His6. Ten days later, the mice were immunized again with 10 mg of the same protein, and the antiserum was obtained via intracardiac puncture.
To obtain the glutathione S-transferase (GST) fusion plasmids, the DNA fragments encoding full-length RcsA, RmpA, RmpAN, or RmpA2 were PCR amplified with the primer sets rcsAe04/rcsAe08, rmpAe04/rmpAe07, or rmpA2p08/rmpA2p17 (Table (Table2)2) and cloned into the BamHI/XhoI site in pGEX-5X-1 (GE Healthcare) to generate pGEX-RcsA, pGEX-RmpA, pGEX-RmpAN, or pGEX-RmpA2 (Table (Table1),1), respectively. To construct an RcsB-His6 expression plasmid compatible with pGEX-5X-1, the DNA fragment containing the entire RcsB-His6 coding sequence and the upstream T7 promoter region was PCR amplified with the primers pET30f2 and pET30r2 (Table (Table2)2) and cloned into an EcoRI site on pACYC184 to generate pACYC184-RcsB. To perform coimmunoprecipitation, different combinations of the expression plasmids were cotransformed into E. coli BL21(DE3). The transformants were grown overnight in LB medium at 37°C and grown in refreshed LB medium at 37°C to an OD600 of 0.5. IPTG was then added to a final concentration of 1 mM, and the cultures were incubated at 25°C for another 16 h. The cells were recovered by centrifugation, resuspended in 500 μl of IP buffer (1× TBS [pH 7.4], 0.1% [wt/vol] bovine serum albumin [BSA], 1× protease inhibitor cocktail set VII [Calbiochem, La Jolla, CA]), and disrupted by sonication on ice. After centrifugation at 4°C, the supernatant was collected as preimmunoprecipitation (Pre-IP) samples. Ten micrograms of each Pre-IP sample was subjected to SDS-PAGE and immunoblotted with anti-GST or anti-His6 monoclonal antibody to ensure the expression of the recombinant proteins. For IP analysis, 500 μg of total protein for each sample was incubated with ~50 μl of glutathione-Sepharose 4 Fast Flow (GE Healthcare) in a final volume of 500 μl. After incubation at 4°C for 3 h with end-over-end mixing, the beads were collected by centrifugation, washed three times with 500 μl of ice-cold IP buffer containing 1% (vol/vol) Triton X-100, and washed once with 500 μl of ice-cold IP buffer containing 1% Triton X-100 and 1 M NaCl. Finally, the beads were washed with 500 μl of ice-cold IP buffer containing 1% Triton X-100 to remove the residual salts and mixed with 50 μl of 1× SDS-PAGE sample buffer. The samples were boiled at 95°C for 10 min and subjected to SDS-PAGE and immunoblot analysis with anti-His6 monoclonal antibody or anti-RcsB-His6 polyclonal antiserum. The presence of proteins was visualized by an alkaline phosphatase-mediated chromogenic substrate reaction.
The DNA fragments P1 to P6 were PCR amplified with the primer sets Piucp01/Piucp02, Pirop01/Pirop02, rmpAp04/rmpAp12, rmpAp04/rmpAc01, rmpAp04/rmpAp13, and rmpAp04/rmpAp11 (Table (Table2),2), respectively, and end labeled with [γ-32P]ATP using T4 polynucleotide kinase as described previously (23). The purified His6-Fur protein was mixed with labeled probes (~0.1 ng) in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 10 mM dithiothreitol, and 0.5 μg of BSA/μl. The mixture was incubated at room temperature for 30 min, mixed with a 0.1× volume of DNA loading dye, loaded onto a 5% nondenaturing polyacrylamide gel containing 5% glycerol, and then run in a 0.5× TB buffer (45 mM Tris-HCl [pH 8.0], 45 mM boric acid) containing 100 μM MnCl2. After electrophoresis at a constant current of 20 mA at 4°C for 2 h, the results were detected by autoradiography.
For the determination of rmpA 5′-mRNA ends, a 5′-RACE (5′ rapid amplification of cDNA ends) PCR was performed by using a 5′-RACE kit (Clontech, Mountain View, CA) according to the manufacturer's instruction. In brief, total RNA was isolated from log-phase K. pneumoniae CG43S3 and Δfur mutant grown in LB medium by using a High-Pure RNA isolation kit (Roche, Mannheim, Germany). One microgram of total RNA was treated with RNase-free DNase I (Roche) and recovered by phenol-chloroform extraction. For the first-strand cDNA synthesis, a 3.75-μl reaction mixture containing 1 μg of DNase-treated RNA and random primer mix (N-15) was incubated at 72°C for 3 min and then reduced to room temperature for 5 min before the addition of 5× first-strand buffer, deionized water, dithiothreitol, deoxynucleoside triphosphate mix, RNase inhibitor, SMARTer II A oligonucleotide, and SMARTScribe reverse transcriptase to a final volume of 10 μl. The reaction mixture was incubated at room temperature for 10 min, 42°C for 90 min, and 72°C for 10 min to terminate the reaction; diluted with 20 μl of Tricine-EDTA buffer; and then stored at −20°C. For the primary PCR, a 50-μl reaction mixture containing diluted cDNA templates, gene-specific primer, universal primer mix, deoxynucleoside triphosphate mix, PCR buffer, deionized water, and DNA polymerase was prepared. The reaction mixture without reverse transcriptase served as a negative control template. The PCR program consisted of 5 cycles of 30 s at 94°C and 3 min at 72°C; 5 cycles of 30 s at 94°C, 30 s at 70°C, and 3 min at 72°C; and 25 cycles of 30 s at 94°C, 30 s at 68°C, and 3 min at 72°C. For the nested PCR, the reaction mixture was essentially the same as the primary PCR mixture except that 100-fold-diluted primary PCR product as the template, gene-specific primer, and nested universal primer were used. The PCR program consisted of 25 cycles of 30 s at 94°C, 30 s at 60°C, and 3 min at 72°C. The PCR products were resolved on an agarose gel by electrophoresis, and the DNA fragments were recovered and cloned into the PCR cloning vector yT&A (Yeastern Biotech, Taiwan, Republic of China). A total of 31 clones were subjected to sequence analysis, and the transcriptional start site of rmpA was determined from the longest DNA fragments.
The reaction mixtures include primer sets RTrmpA01/RTrmpA02 or 23SR/23SF (Table (Table2)2) and the diluted cDNA templates from the first-strand synthesis in 5′-RACE PCR. The PCR program consisted of 30 cycles of 30 s at 94°C, 30 s at 50°C, and 20 s at 72°C. For a quantitative comparison of the rmpA expression levels, the intensity of the band resolved on agarose gel was analyzed and normalized with 23S rRNA gene by using ImageJ software (National Institutes of Health). The expression level of rmpA from the undiluted wild-type cDNA was set as 100.
A Student t test was used to determine the significance of the differences between the CPS amounts and the levels of β-galactosidase activity. The P values of <0.05 were considered statistically significant.
On the basis of the sequence annotation, the DNA fragment containing the rmpA2 gene with upstream vagC and vagD and downstream iucABCDiutA genes was labeled as PAI-1 (for pathogenicity island 1), while the rmpA-, fecIRA-, and iroBCD-containing region was named PAI-2 (Fig. (Fig.11 A). The positive regulatory role of RmpA2 in the expression of the major virulence factor, CPS, has been demonstrated (20). However, whether RmpA plays a functional role remains unknown. PCR with a primer pair that could differentiate rmpA from rmpA2 and Southern hybridization analysis were performed. As shown in Fig. Fig.1B,1B, the PCR products representing the rmpA-specific transcript could be detected using the reverse transcribed cDNA templates from K. pneumoniae CG43S3 or the ΔrmpA2 mutant strain. This indicates that the rmpA on pLVPK is a functional gene and that the expression of rmpA is independent of RmpA2.
To assess the functional role of RmpA, the rmpA deletion mutant was generated by using the allelic-exchange strategy. The colony of ΔrmpA mutant on the LB agar plate was found to be smaller than its parental strain, and the degree of mucoidy was reduced significantly, as determined by a string test (23), which refers to the ability to form a string when the bacterial colony was picked with toothpick. As shown in the sedimentation test in Fig. Fig.2A,2A, the ΔrmpA mutant, as well as the ΔrcsB mutant, could be rapidly precipitated by low-speed centrifugation. The loss of mucoid phenotype in the ΔrmpA mutant could be complemented with the transformation of pRK415-RmpA, or pRK415-RmpA2. Interestingly, the mucoid phenotype could not be restored by introducing the rcsB expression plasmid pRK415-RcsB. The sedimentation analysis also revealed that the introduction of pRK415-RmpA or pRK415-RmpA2 was able to increase the mucoviscosity of the rmpA rmpA2 double mutant (Fig. (Fig.2A),2A), indicating the independent regulatory activity of RmpA and RmpA2. The effect of rcsB deletion could only be complemented by transformation of the ΔrcsB mutant with pRK415-RcsB but not with pRK415-RmpA. As assessed by measuring the glucuronic acid content, which served as an indicator for Klebsiella K2 CPS (33), deletion of rmpA or rcsB caused a ca. 25% reduction in the amount of CPS compared to that of CG43S3 (Fig. (Fig.2B).2B). The effect of rmpA or rcsB deletion could only be restored by transformation of ΔrmpA with pRK415-RmpA or transformation of ΔrcsB with pRK415-RcsB (Fig. (Fig.2C),2C), which is consistent with the findings in Fig. Fig.2A2A.
Furthermore, the rmpA deletion appeared to increase LD50 from 1 × 104 CFU to 5 × 105 CFU in the mouse peritonitis model and reduced the resistance to human serum from >95% to >70% (Table (Table3).3). The deficiency in serum resistance could be reverted by the introduction of pRK415-RmpA, suggesting a role of RmpA in bacterial virulence.
To investigate whether the CPS-deficient phenotype of ΔrmpA mutant was a result of altered expression of the cps genes, three reporter plasmids, pOrf12 (Porf1-2::lacZ), pOrf315 (Porf3-15::lacZ), and pOrf1617 (Porf16-17::lacZ), each carrying a lacZ transcriptional fused to the putative promoter region of the K2 cps gene cluster (25), were used to transform K. pneumoniae strains CG43S3ΔlacZ, CG43S3ΔrmpAΔlacZ, CG43S3ΔrmpA2ΔlacZ, or CG43S3ΔrcsBΔlacZ individually. The promoter activity measurements shown in Fig. Fig.33 A reveal that the deletion of rmpA reduced the activity of Porf1-2::lacZ and Porf16-17::lacZ. A reduction in the activity Porf1-2::lacZ or Porf16-17::lacZ was also observed in the ΔrcsB mutant. Interestingly, the deletion of rmpA2 had less effect on the activity of Porf16-17::lacZ compared to the rmpA deletion. As shown in Fig. Fig.3B,3B, the deleting effect of rmpA, rmpA2, or rcsB on Porf3-15::lacZ activity was only apparent when the strains were grown in M9-glocuse minimal medium. Compared to that of rmpA2, the deletion of rmpA resulted in a more drastic reduction in the activity of Porf1-2::lacZ and Porf16-17::lacZ implying a differential regulation of RmpA and RmpA2 on the cps promoters. Nevertheless, the results suggested that the expression of RmpA, RcsB, and RmpA2 is required for cps expression.
To investigate the possibility of an interaction between RmpA and RcsB for cps expression, the lacZ reporter cassette on pOrf12 was cloned into a suicide vector, and the plasmid was mobilized into K. pneumoniae CG43S3ΔlacZ, CG43S3ΔrmpAΔlacZ, or CG43S3ΔrcsBΔlacZ individually. The resulting strain harboring a chromosomally integrated Porf1-2::lacZ cassette was then transformed with different complementation plasmids, and the β-galactosidase activities were determined. As shown in Fig. Fig.3C,3C, the introduction of pRK415-RmpA or pRK415-RcsB could enhance Porf1-2::lacZ activity in the parental strain, suggesting the functional activity of RmpA or RcsB. Functional RmpA could enhance cps expression in the rmpA deletion strain but not the rcsB deletion strain. Consistent with the phenotype observed in Fig. Fig.2B,2B, the functional RcsB carried by pRK415-RcsB could not restore cps expression in the rmpA deletion strain. The introduction of pRK415-RmpAN, which encoded a truncated RmpA without the carboxyl-terminal DNA binding region, into the rmpA deletion strain also failed to restore cps expression. The results suggest that RmpA activated cps expression in an RcsB-dependent manner and that its DNA-binding motif is required for regulation.
Since the cooperation of RcsA and RcsB for regulation on K2 cps expression has been demonstrated (47), we thought of using EMSA to investigate whether the RmpA exerts RcsA-like activity to interact with RcsB in order to bind to the Porf1-2 region cooperatively. However, the overproduction of RmpA using the pET expression system appeared to impair cell growth significantly; hence, the bacterial two-hybrid assay was used instead. Therefore, plasmids pTRG-RcsA, pTRG-RmpA, pTRG-RmpA2, pTRG-RmpAN, and pBT-RcsB, which harbored the λ-cI-RcsA, λ-cI-RmpA, λ-cI-RmpA2, λ-cI-RmpAN, and α-RNAP-RcsB coding sequences, respectively, were constructed. The interaction between α-RNAP and λ-cI fusion proteins would allow binding of λ-cI to the operator sequence and recruitment of α-RNAP to initiate the transcription of both ampR and lacZ genes in the reporter cassette harbored in the E. coli reporter strain. The interaction between the recombinant proteins could be verified by bacterial growth on the X-Gal indicator plate supplemented with carbenicillin, and the level of interaction could be quantified by measuring the activation of the LacZ reporter.
As shown in Fig. Fig.44 A, the strain carrying pBT-RcsB/pTRG-RcsA or the positive control plasmids grew well on the indicator plate. Substitution of pBT-RcsB and pTRG-RcsA with pBT or pTRG resulted in poor or no growth. The strains carrying pBT-RcsB/pTRG-RmpA, pBT-RcsB/pTRG-RmpA2, or pBT-RcsB/pTRG-RmpAN also grew on the indicator plate (Fig. (Fig.4A).4A). As shown in Fig. Fig.4B,4B, the strain carrying pBT-RcsB/pTRG-RcsA exhibited relatively higher activity than the strains carrying pBT-RcsB/pTRG-RmpA, pTRG-RmpA2, or pTRG-RmpAN. The transformants harboring pBT-RcsB and pTRG-RmpA, pTRG-RmpA2, or pTRG-RmpAN also exhibited higher activity compared to strains wherein which pBT-RcsB was replaced by pBT (Fig. (Fig.4B,4B, right panel). The results suggested an in vivo interaction between RcsB and RmpA, and the N-terminal peptide (residues 1 to 84) of RmpA may play an important role in the interaction.
To confirm the results from the two-hybrid analysis, coimmunoprecipitation was also performed. The full-length RcsA, RmpA, RmpA2, and RmpAN coding regions were cloned into pGEX-5X-1 to generate plasmids pGST-RcsA, pGST-RmpA, pGST-RmpA2, and pGST-RmpAN, respectively. The plasmid pACYC184-RcsB, which is compatible with pGEX-5X-1, was also constructed. Using anti-GST or anti-His monoclonal antibody, the amounts of the recombinant GST fusion proteins and the recombinant RcsB in the Pre-IP samples were determined (Fig. (Fig.55 A). As shown in Fig. Fig.5B,5B, the immunoprecipitates of pGEX-RcsA/pACYC184-RcsB, pGEX-RmpA/pACYC184-RcsB, pGEX-RmpAN/pACYC184-RcsB, or pGEX-RmpA2/pACYC184-RcsB pulled down with the glutathione-Sepharose all contained the RcsB-His6, as determined by using anti-His monoclonal antibody or anti-RcsB polyclonal antibody. The amounts of recombinant RcsB protein precipitated by GST-RcsA and GST-RmpA were much lower than those pulled down by GST-RmpAN and GST-RmpA2 (Fig. (Fig.5B).5B). This may have resulted from instability of the GST-RcsA and GST-RmpA fusion proteins, as reflected by very low amounts of both proteins obtained after IP (data not shown). The results further supported a role of RmpA or RmpA2 as an auxiliary factor, like RcsA, for RcsB in regulating the CPS biosynthesis.
Differential control likely explains the coexistence of the two functional mucoid factors, RmpA and RmpA2, in the cps expression in the bacteria. To verify this, the DNA fragments encompassing the putative promoter region of rmpA or rmpA2 was cloned in front of the promoterless lacZ to generate placZ15-PrmpA and placZ15-PrmpA2. The resulting plasmids were then transformed individually into K. pneumoniae CG43S3ΔlacZ for promoter activity measurements. In contrast to the negative autoregulation for rmpA2 expression (23), the rmpA expression appeared to be independent of autoregulatory control (data not shown).
Since rmpA and rmpA2 are, respectively, located next to the iron acquisition genes iucABCD, iutA, and iroBCDN (Fig. (Fig.1A),1A), we sought to determine whether any iron uptake regulator is involved in their expression. A close inspection of the rmpA and rmpA2 upstream regions revealed a Fur box-like sequence (2, 11) on PrmpA but not on PrmpA2. This element was also identified in front of a set of Fur-regulated genes including iucABCD, iroBCD, feoAB, and fur (data not shown). To investigate whether Fur, the global regulator for iron uptake regulation, participated in the control of the gene expression, the expression levels of PrmpA::lacZ and PrmpA2::lacZ were measured in the wild-type strain and in the fur deletion mutant. As shown in Fig. Fig.66 A, fur deletion resulted in a higher level of activity for the Fur-dependent promoters PiucA and PiroB in LB medium. An increased level of PrmpA::lacZ expression in the Δfur mutant was even more profound when the bacterial culture was switched from LB medium to the M9-glucose medium (Fig. (Fig.6B).6B). In the presence of iron scavenger 2,2-dipyridyl, the activity of PiucA::lacZ, Piro::lacZ, or PrmpA::lacZ was higher in M9-glucose medium but less affected by Fur. On the other hand, the deletion of fur or the addition of iron scavenger had no apparent effect on the expression of PrmpA2::lacZ in either medium (Fig. (Fig.6).6). The results suggest that Fur negatively regulate the expression of PrmpA but does not participate in the regulation of rmpA2 expression.
The Fur box-like sequence could be predicted upstream of iucA, iroB, and rmpA, as shown in Fig. Fig.77 A. To further ascertain the effect of Fur on rmpA expression, an EMSA was performed. The DNA fragments encompassing PiucA (P1), PiroB (P2), and PrmpA (P3) and the truncated forms P4, P5, and P6, as depicted in Fig. Fig.7A,7A, were isolated and isotope labeled for the analysis. As shown in Fig. Fig.7B,7B, the purified recombinant His6-Fur protein was able to bind to the DNA probes P1, P2, and P3. By using different lengths of PrmpA, the binding of His6-Fur could be observed for P3, P4, and P5 but not for P6 (Fig. (Fig.7C).7C). This suggested that the His6-Fur binding site may be located between −226 to −184 relative to the RmpA start codon (Fig. (Fig.7A).7A). As shown in Fig. Fig.7D,7D, the formation of the PrmpA/Fur complex could be observed as the amount of His6-Fur increased, and the binding specificity was demonstrated as the complex diminished in the presence of excess nonlabeled P3 or P5 acting as specific competitor DNA. The binding of His6-Fur on PrmpA remained unaffected by the addition of excess amounts of pT7-7 (42), a plasmid without a Fur box-containing sequence, pUC19 DNA, rmpA gene, or P6 DNA fragment. The results support the conclusion that the recombinant Fur protein could specifically interact with PrmpA DNA.
As shown in Fig. Fig.88 A, a single DNA band has been obtained from the 5′-RACE analysis using either primer pair. Sequence analysis of a total of 31 clones revealed the transcription start site at the G nucleotide at position −80 relative to the translational start site of RmpA. As shown in Fig. 8B, a conserved −10 and −35 promoter sequence of σ70 could be readily identified. Limiting-dilution RT-PCR was subsequently carried out to determine whether the rmpA transcription would be affected by Fur. As shown in Fig. Fig.8C,8C, a stronger signal for rmpA transcript in the fur mutant than in the parental strain also supports a negative regulatory role of Fur on the rmpA expression.
If rmpA expression is negatively regulated by Fur, then the CPS level in the fur deletion mutant should increase. As shown in Fig. Fig.99 A, the Δfur mutant formed more mucoid and glistening colonies compared to the parental strain. In a string test, the Δfur mutant could form a string at least 3-fold longer than its parental strain (data not shown). Introduction of pRK415-Fur carrying a functional fur allele in Δfur mutant resulted in small colonies (Fig. (Fig.9A)9A) and a readily precipitated phenotype (Fig. (Fig.9B).9B). The changes in CPS production were also evident in the glucuronic acid content measurement. As shown in Fig. Fig.9C,9C, the fur mutant exhibited a >2-fold increase in the glucuronic acid production, while transformation with pRK415-Fur resulted in an ca. 50% reduction of the glucuronic acid.
The presence of rmpA and rmpA2 on pLVPK as two independent loci 29 kb apart has been demonstrated (8). As shown in Fig. Fig.1A,1A, the DNA fragments containing rmpA2 and rmpA also harbored an iron acquisition gene cluster and insertion sequence, which are characteristics of a pathogenicity island, and hence named PAI-1 and PAI-2, respectively. Why and how the evolutionary convergence of the similar gene organization occurred remained to be explained. In the present study, we show that rmpA and rmpA2 genes are present in one bacterial strain and that both encode a mucoid factor contributing to K2 CPS biosynthesis. Presumably, differential control on the expression of these two mucoid factors is required for an efficient regulatory function with no redundant activity in the bacteria.
Sequence analysis revealed that RmpA and RmpA2 belong to the UhpA-LuxR family of transcription factors, which also include RcsA and RcsB (41). The involvement of RcsB in Klebsiella K2 capsule biosynthesis of the recombinant E. coli K-12 has been demonstrated (44). Previous studies have found that RcsB must interact with RcsA to form a heterodimer in order to bind specifically to the cps promoter for transcription initiation (21). In E. coli, the cellular level of RcsA was limited at 37°C due to its degradation by the Lon protease, and thus the colonic acid capsule was overproduced only at lower temperatures (16). In K. pneumoniae CG43, however, a profound production of CPS was observed at 37°C. We assumed that RmpA or RmpA2 could take the place of RcsA to regulate the cps expression at 37°C. This hypothesis was supported by the fact that the deletion of rcsA in K. pneumoniae CG43 did not affect the mucoid phenotype or the CPS amount at 37°C (data not shown). Moreover, the two-hybrid analysis and coimmunoprecipitation assay further demonstrated that RmpA or RmpA2, in addition to RcsA, was able to interact with RcsB. Therefore, in K. pneumoniae, multiple accessory factors may be used in order to increase the CPS biosynthesis in response to different environmental stimuli.
A conserved RcsAB box sequence has been identified in Klebsiella K2 cps Porf1-2 (47), and analysis of the Porf16-17 sequence also revealed a semiconserved RcsAB box. This suggested that the decrease of the CPS production in the ΔrmpA mutant may have been due to a reduction in the expression of cps-orf1-2 and cps-orf16-17. The existence of an rmpA gene has been correlated to the hypermucoviscosity phenotype in K. pneumoniae clinical isolates (51). It is interesting that no rmpA homologue in any other bacterial genomes could be identified. The BLAST (http://www.ncbi.nlm.nih.gov) search in the released genome sequences of K. pneumoniae NTUH-K2044 (49), MGH78578 (http://genome.wustl.edu/), and 342 (14) revealed the presence of rmpA only in NTUH-K2044, which is a heavy encapsulated clinical isolate of K1 serotype (12, 51). We have also observed that rmpA was more prevalent in the strains belonging to K1/K2 serotypes (36 out of 36 isolates [100%]) than in the non-K1/K2 clinical isolates (28 out of 83 isolates [33.73%]) collected in our laboratory. The presence of rmpA in K. pneumoniae K1 or K2 isolates has also been associated with virulence in mice (4). These findings suggest that the profound expression of K. pneumoniae CPS, which is a major virulence factor, could be attributed to the regulation by RmpA or its homologue.
Similar to rmpA2, rmpA harbored a poly(G) tract in the coding sequence. The occurrence of DNA slip-strand synthesis may result in a truncated RmpA of abnormal function. Conceivably, the frequency of the mutation caused by DNA slip-strand synthesis in rmpA or rmpA2 may play a role in the differential expression of the two regulatory genes. A Fur-box-like sequence was identified within −146 and −104 upstream of the transcription start site (Fig. (Fig.8B).8B). This site was not uncommon compared to a genome-wide study in Yersinia pestis (15), in which 17 of 34 predicted Fur binding sites were located at −100 relative to each transcriptional start site. Also, as shown in Fig. Fig.8C,8C, the rmpA transcript was increased upon the deletion of fur, indicating a negative regulation of Fur on rmpA expression in K. pneumoniae.
Fur governs iron uptake by repressing the transcription of the genes involved in the biosynthesis of siderophore, iron storage, iron sparing, and respiration (2, 24, 43). In addition, it is also involved in virulence properties (46, 50), acid stress response (13), osmotic shock (17), and chemotaxis (10). In the present study, a negative role of Fur in CPS biosynthesis that is achieved by the reduction of the RmpA expression is reported for the first time. The findings imply that some K. pneumoniae strains may face iron shortage during infection; accordingly, the genes involved in both the biosynthesis of the iron acquisition system and the production of CPS are upregulated. Therefore, coordination between CPS production and iron uptake may play an important role in the pathogenesis of the bacteria. Nevertheless, cps expression was also subjected to regulation by the 2CS response regulators KvgA, KvhA, and KvhR (26). How the interplay between Fur, RmpA, and the 2CS determines the control of the cps expression remains to be elucidated.
In summary, our results indicate that in K. pneumoniae CG43, the mucoid factor RmpA exerts a regulatory role on cps expression in an RcsB-dependent manner. The fur deletion increased rmpA expression but had no effect on rmpA2 expression. In addition, the recombinant Fur could bind specifically to the putative promoter of rmpA. This suggests that Fur plays a role in the differential expression of RmpA and RmpA2. This is also the first report that investigates the role of Klebsiella Fur in the regulation of CPS biosynthesis.
The study is supported by grants from the Veterans General Hospital/Taiwan University System (VGHUST94-P5-27), the National Research Program for Genome Medicine (NSC96-3112-B009-001), and the National Science Council (97-2320-B-009-001-MY3).
Published ahead of print on 9 April 2010.