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Infection and Immunity, January 2004, p. 54-61, Vol. 72, No. 1
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.1.54-61.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Gene, uge, Is Essential for Klebsiella pneumoniae Virulence

Miguel Regué,1 Beatriz Hita,1 Nuria Piqué,1 Luis Izquierdo,2 Susana Merino,2 Sandra Fresno,2 Vicente Javier Benedí,1,{dagger} and Juan M. Tomás2*

Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, 08071 Barcelona,2 Departamento de Microbiología y Parasitología Sanitarias. División de Ciencias de la Salud, Facultad de Farmacia, Universidad de Barcelona, 08028 Barcelona, Spain1

Received 28 May 2003/ Returned for modification 30 June 2003/ Accepted 15 September 2003


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ABSTRACT
 
Klebsiella pneumoniae strains typically express both smooth lipopolysaccharide (LPS) with O antigen molecules and capsule polysaccharide (K antigen) on the surface. A single mutation in a gene that codes for a UDP galacturonate 4-epimerase (uge) renders a strain with the O-:K- phenotype (lack of capsule and LPS without O antigen molecules and outer core oligosaccharide). The uge gene was present in all the K. pneumoniae strains tested. The K. pneumoniae uge mutants were unable to produce experimental urinary tract infections in rats and were completely avirulent in two different animal models (septicemia and pneumonia). Reintroduction of the single uge wild-type gene in the corresponding mutants completely restored the wild-type phenotype (presence of capsule and smooth LPS) independently of the O or K serotype of the wild type. Furthermore, complemented uge mutants recovered the ability to produce experimental urinary tract infections in rats and virulence in the septicemia and pneumonia animal models.


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INTRODUCTION
 
Klebsiella spp., particularly Klebsiella pneumoniae, are important causes of nosocomial infections (15). K. pneumoniae infections may occur at almost all body sites, but the highest incidence is found in the urinary and respiratory tracts. The main populations at risk are neonates, immunocompromised hosts, and patients predisposed by surgery, diabetes, malignancy, etc (15, 21, 24). The existence of multiply antibiotic-resistant K. pneumoniae strains is notorious and has complicated therapy. Mortality rates of up to 50% have been found in respiratory tract infections. As an alternative to antibiotic treatment, prevention and/or treatment of K. pneumoniae infections by immunotherapy has received increased attention in recent years.

K. pneumoniae typically expresses both smooth lipopolysaccharide (LPS with O antigen) and capsule polysaccharide (K antigen) on its surface, and both LPS and capsule contribute to the pathogenesis of this species. The O antigen is the most external component of LPS, and it consists of a polymer of oligosaccharide repeating units. An interesting feature is the high chemical variability shown by K. pneumoniae O antigens, which is reflected by the genetic variation in the genes involved in O antigen biosynthesis, located in the so-called wb cluster (27, 28, 31).

The genes involved in K. pneumoniae core LPS biosynthesis are known and, as in other Enterobacteriaceae, are found in the waa (rfa) gene cluster (39). Comparison of the known core LPS structures from Enterobacteriaceae reveals that the first outer core residue could be either glucose (Gluc) or a galacturonic acid (GalA) residue. In the five known Escherichia coli core types and in Salmonella enterica, a substitution of the L-glycero-D-manno-heptopyranose II at the O-3 position by a Glcp residue was found (23). In K. pneumoniae, Proteus mirabilis, and Yersinia enterocolitica, a substitution of the L-glycero-D-manno-heptopyranose II at the O-3 position by an {alpha}-D-galacturonic acid residue residue has been described (37, 49, 50). Furthermore, a characteristic of the K. pneumoniae core LPS is the absence of phosphoryl group modification at the L-glycero-D-manno-heptopyranose I and II residues (49) (Fig. 1). This fact suggests that the outer core GalAp residue could play a crucial role in core stability by conferring a negative charge, while in other systems, for instance E. coli, such a role is supposed to be accomplished by inner core phosphoryl modifications.



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  		FIG. 1. (A) Conserved region in the core LPS structure of K. pneumoniae (49) and effect of the uge mutation on the core LPS; 3-deoxy-D-manno-octulopyranosonic acid (Kdop), L-glycero-D-manno-heptopyranose (L,DHepp), D-glucopyranose (Glcp), glucosamine (GlcNp), and galacturonic acid (GalAp). (B) Genetic organization of the uge gene region and the role of the encoded product.

K. pneumoniae strains constitutively express a polysaccharide capsule that is critical for the organism's ability to resist complement-mediated opsonophagocytic killing (51). More than 90 different types of capsule (K antigens) have been described in Klebsiella. However, only the gene cluster for K. pneumoniae K2 antigen (wcaK2) has been genetically studied (3). The initial aim of this study was to obtain mutants with altered expression of the constitutive K2 capsular polysaccharide in genes outside wcaK2. In order to perform this study we used mini-Tn5 mutagenesis on strain 52145 (O1:K2) and mutant selection by resistance to bacteriophage {phi}2 (a bacteriophage specific for capsular polysaccharide K2).


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions. K. pneumoniae and Escherichia coli strains were grown in LB Miller broth and LB Miller agar (32). LB medium was supplemented with kanamycin (50 µg ml-1), ampicillin (100 µg ml-1), or chloramphenicol (20 µg ml-1) when needed. The plasmids used in this study and their characteristics are shown in Table 1.


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  		TABLE 1. Bacterial strains and plasmids used in this study

Mini-Tn5 Km-1 mutagenesis. Conjugal transfer of transposition element mini-Tn5 Km-1 from E. coli S17-1{lambda}pirKm-1 to rifampin resistant K. pneumonaie 52145 was carried out in a conjugal drop incubated for 6 h at 30°C with the relation 1:5:1 corresponding to S17-1{lambda}pirKm-1, 52145, and HB101/pRK2073 (helper plasmid), respectively. Insertional mutants were selected for rifampin and kanamycin resistance.

General DNA methods. General DNA manipulations were done essentially as described (41). DNA restriction endonucleases, T4 DNA ligase, E. coli DNA polymerase (Klenow fragment), and alkaline phosphatase were used as recommended by the suppliers.

DNA sequencing and computer analysis of sequence data. Double-stranded DNA sequencing was performed by the dideoxy chain termination method (42) with the ABI Prism dye terminator cycle sequencing kit (Perkin Elmer). Oligonucleotides used for genomic DNA amplification experiments and for DNA sequencing were purchased from Pharmacia LKB Biotechnology. The DNA sequence was translated in all six frames, and all open reading frames (ORFs) were inspected. Deduced amino acid sequences were compared with those of DNA translated in all six frames from nonredundant GenBank and EMBL databases by with the BLAST(1;2) network service at the National Center for Biotechnology information and the European Biotechnology Information, respectively. Clustal W (43) was used for multiple sequence alignments.

DNA amplification and plasmid construction. Genomic DNAs from K. pneumoniae strains with different O and K serovars were isolated and used as template in PCR experiments with primers (5'-AGCCAGTGTAAAATCGGCACTTA-3' and 5'-CTTTCTCTCCCCC GTTATATCCCT-3') designed to amplify the K. pneumoniae uge gene. These oligonucleotides were also used to amplify and to subclone in vector pGEMT the uge gene (pGEMT-UGE). An inner 697-bp DNA fragment of ORF1 was obtained from plasmid pGEMT-UGE by PvuII digestion and subcloned in the pir replication-dependent plasmid pSF100 (40). This plasmid construction (pSF-UGE) was used to obtain ORF1-deficient mutants from several K. pneumoniae strains by a single recombination event leading to the generation of two incomplete copies of ORF1 in the chromosome of these mutants, as previously described (40).

Complementation studies. Complementation analysis of the different uge mutants was performed by transformation of the wild-type uge gene cloned in vector pGEMT. Transformants were selected on LB agar containing ampicillin (2 mg ml-1), and LPS was isolated and analyzed in tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

LPS isolation and electrophoresis. Cultures for analysis of LPS were grown in TSB at 37°C. LPS was purified by the method of Galanos (17) resulting in a 2.3% yield. For screening purposes LPS was obtained after proteinase K digestion of whole cells (25), 1983). LPS samples were separated by SDS-PAGE or SDS-tricine-PAGE and visualized by silver staining as previously described (36, 47).

Isolation of oligosaccharides. LPS (20 mg) was hydrolyzed with 1% acetate (100°C for 1 h). The resulting precipitate (8 mg) was removed by centrifugation, and the supernatant (10 mg) was analyzed by mass spectrometry. Another sample of LPS (40 mg) was deacylated and purified as described (10), obtaining 6 mg of alditol-oligosaccharides mixture.

LPS chemical analysis. For chemical analysis, either purified LPS or core-LPS oligosaccharide samples were hydrolyzed with 1 N trifluoroacetic acid for 4 h at 100°C. Alditol acetates and methyl glycoside acetates were analyzed on an Agilent Technologies 5973N MS instrument equipped with a 6850A gas chromatograph and an RTX-5 capillary column (Restek, 30 m by 0.25 inner diameter, flow rate 1 ml min-1, He as carrier gas). Acetylated methyl glycoside analysis was performed with the following temperature program: 150° for 5 min, 150° to 250° at 3° min-1, and 250° for 10 min. Acetylated methyl ester lipid analysis was performed as follows: 150° for 3 min, 150° to 280° at 10° min-1, and 280° for 15 min. The alditol acetate mixture was analyzed with the following temperature program: 150°C for 5 min, 150°C to 300°C at 3°C min-1. For partially methylated alditol acetates, the temperature program was 90°C for 1 min, 90°C to 140°C at 25°C min-1, 140°C to 200°C at 5°C min-1, 200°C to 280°C at 10°C min-1, and 280°C for 10 min.

Mass spectrometry studies. Electrospray mass spectra were performed on a Micromass ZQ instrument (Waters). The sample (100 pmol) was deionized on Dowex H+ resin (Fluka) and dissolved in 2% triethylamine in 50% acetonitrile and injected into the ion source at a flow rate of 5 µl min-1. The spectrum was acquired in negative mode. Positive-ion reflection matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra were acquired on a Voyager DE-PRO instrument (Applied Biosystems) equipped with a delayed extraction ion source. Ion acceleration voltage was 20 kV, grid voltage was 14 kV, mirror voltage ratio 1.12, and delay time 100 ns. Samples were irradiated at a frequency of 5 Hz by 337-nm photons from a pulsed nitrogen laser. Postsource decay (PSD) was performed with an acceleration voltage of 20 kV. The reflectron voltage was decreased in 10 successive 25% steps. Mass calibration was obtained with a maltooligosaccharide mixture from corn syrup (Sigma). A solution of 2,5-dihydroxybenoic acid in 20% CH3CN in water at a concentration of 25 mg/ml was used as the MALDI matrix; 1 µl of matrix solution and 1 µl of the sample were mixed and than deposited on the target. The droplet was allowed to dry at ambient temperature. Spectra were calibrated and processed under computer control with the Applied Biosystems Data Explorer software.

Methylation analysis. The alditol oligosaccharide mixture was N-acetylated by dissolving a sample (2 mg) in dry methanol and treating it with 50 µl of acetic anhydride for 16 h. After evaporation of the solvents the sample was methylated as reported (9). Linkage analysis was performed as follows: the methylated sample was carboxymethyl reduced with lithium triethylborohydride (Aldrich), mildly hydrolyzed to cleave the ketosidic linkage, reduced by means of deuterated sodium tetrahydridoborate (NaBD4), totally hydrolyzed, reduced with NaBD4, and finally acetylated as reported (16).

Cell extract production and enzymatic activity measurements. K. pneumoniae cells grown up to late logarithmic phase were centrifuged and washed with 50 mM Tris-HCl, pH 7.5. The bacteria, resuspended in the same buffer, were lysed in a French press at 16,000 lb/in2, and the unbroken cells were removed by centrifugation. The lysate was centrifuged for 60 min at 100,000 x g at 4°C, the pellet was discarded, and the supernatant (cell extract) was kept at -20°C with 50% glycerol until used.

Reaction mixtures for uridine phosphate galacturonate 4-epimerase (UDPGLE) measurements (33) contained UDP-GlcA (250 nmol) and the extract in a total volume of 250 µl. The reactions performed at 37°C were stopped by addition of cold acetone (1 ml), stored for 90 min at -70°C, and centrifuged (10,000 x g for 15 min at 4°C), discarding the pellet. After the acetone was evaporated, the solution was adjusted to 250 µl with distilled water, the samples were passed through Ultraspin centrifuge filters (cutoff value 10,000 Da), and 20-µl samples were analyzed by high-pressure liquid chromatography (HPLC) (ion-pair reversed-phase) with a column packed with 10 µM Spherisorb ODS-2 as described (30). Known amounts of UDP-GlcA and UDP-GalA (Sigma) were used as standards. One unit of enzyme activity is defined as the amount of enzyme that results in the formation of 1 nmol of product in 5 min (33).

Urinary tract infections in rats. The bacterial strains used to establish infection were grown overnight in LB agar supplemented with antibiotics when needed and gently suspended in phosphate-buffered saline to the appropriate concentration. In each experiment, 12 female Wistar rats (200 to 250 g) of strain CFHB (Interfauna UK, Hungtington, England) were used. Ten animals were infected, and two were used as controls. The infections were established and quantified as previously described (7).

LD50. Albino Swiss female mice (5 to 7 weeks old, Harlan Ibérica, S.L.) were injected intraperitoneally with 0.2 ml of the test samples. Mortality was recorded for up to 7 days, and all deaths occurred within 1 to 5 days. The 50% lethal dose (LD50) was calculated (38).

Murine model of pneumonia. The pneumonia experiments were performed as previously described (11). Briefly, ICR-CDI male mice (Harlan Ibérica, S.L.) were anesthetized and intubated intratracheally with a blunt-ended needle. Approximately 107 CFU of exponential K. pneumoniae cells were suspended in 50 µl of phosphate-buffered saline and inoculated through the blunt-ended needle. The mice were observed daily, and bacteremia was assessed at days 2, 4, and 6 by culturing the blood obtaining from the tail vein (approximately 20 µl) on LB agar plates. Lung and spleen tissues from surviving animals and dead animals were aseptically removed, homogenized, and plated for quantitative bacterial cultures. Each experiment was performed with nine animals.

Nucleotide sequence accession number. The nucleotide sequence of the uge gene described here has been assigned GenBank accession number AY150065.


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RESULTS
 
After mini-Tn5 mutagenesis of a rifampin-resistant isolate of the K. pneumoniae wild-type strain 52145 (O1:K2), kanamycin-resistant mutants were screened for resistance to bacteriophage {phi}2 [a specific bacteriophage for the capsular polysaccharide K2 (8)]. Mutant KT3412 was among 1,500 mutants that were initially screened and showed complete resistance to bacteriophage {phi}2 compared to the wild-type strain, which is sensitive to this bacteriophage. Strain KT3412 was resistant to other Klebsiella bacteriophages (FC3-1, FC3-2, and FC3-10) whose receptor is the LPS (6, 44, 46), indicating that the transposon was affecting some gene involved in capsular polysaccharide and LPS production.

Whole cells of mutant KT3412 were unable to react in an enzyme immunoassay with specific antiserum against capsular polysaccharide K2 and was completely unencapsulated when observed by electron microscopy (4). As can be observed in Fig. 2, the LPS of strain KT3412 migrated faster than the LPS obtained from the wild-type strain 52145. In addition, this faster migrating LPS was devoid of O1 antigen. Neither whole KT3412 cells nor its purified LPS were able to react with specific antiserum against O1 antigen LPS in enzyme immunoassays (45). No major differences were found in the growth rate between mutant strain KT3412 (generation time = 40 min) and the wild-type strain 52145 (generation time = 38 min). Comparative SDS-PAGE analysis of the outer membrane protein profile indicated that the mutant and the wild-type strain showed essentially the same protein bands, but some differences could be observed in the relative intensity of some protein bands (a decrease in the major band of 37 to 39 kDa and a small change in mobility of the 14-kDa band). Nevertheless, these changes in outer membrane proteins are rather small in comparison to the capsule and LPS changes.



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  		FIG. 2. Silver-stained polyacrylamide gel of LPS samples from different K. pneumoniae strains prepared according to Darveau and Hancock (12). Lanes: 1, 52145 (O1:K2); 2, KT3412; 3, KT3412 harboring pGEMT-UGE; 4, B5055 (O1:K2); 5, pSF-UGE insertional mutant from B5055; 6, mutant strain in lane 5 harboring pGEMT-UGE; 7, KT769 (O5:K57); 8, pSF-UGE insertional mutant from KT769; 9, mutant strain in lane 8 harboring pGEMT-UGE; 10, C3 (O8:K66); 11, pSF-UGE insertional mutant of C3; and 12, mutant strain in lane 11 harboring pGEMT-UGE.

Southern blot analysis with a specific probe for the transposon demonstrated that mutant KT3412 had a single copy of the minitransposon in its genome (data not shown). In order to identify the gene(s) responsible for the observed phenotype, genomic DNA was isolated from mutant KT2312, partially digested with EcoRV, and DNA fragments of about 4.0 kb were ligated to vector pBCSK. The DNA ligation mixture was transformed into E. coli DH5{alpha}, and colonies growing in kanamycin plates were recovered, and analyzed again by Southern blot with the mini-Tn5-specific probe to identify clones containing the transposon and surrounding chromosomal DNA from mutant KT3412.

The nucleotide sequence of the insert of one such recombinant plasmid was determined with oligonucleotides 5'-AGATCTGATCAAGAGACAG-3' and 5'-ACTTGTG TATAAGAGTCAG-3' (from the mini-Tn5Km1 flanking regions) and oligonucleotides T3 and T7 from the plasmid vector. Analysis of the sequence allowed the identification of an ORF, interrupted by the mini-Tn5, which encoded a protein with high similarity (75% identity) to several nucleotide sugar epimerases from different bacteria, such as E. coli WbnF or Vibrio cholerae WbfW.

Cloning and sequencing of a K. pneumoniae 52145 genomic region encoding a nucleotide sugar epimerase-like gene in E. coli K-12 strains. A cosmid-based genomic library of K. pneumoniae 52145 in E. coli DH5{alpha} has been constructed as previously described (27). This library was screened by colony blotting with a DNA probe to the ORF1 (nucleotide sugar epimerase-like locus) of K. pneumoniae 52145. Several positive recombinant clones were identified, of which clone COS-NSE was chosen for further analysis because it was able to completely complement the KT3412 mutation by rescuing the wild-type strain pattern of bacteriophage sensitivity. The DNA sequence of the nucleotide sugar epimerase-like gene (ORF1), where the mini-Tn5 was inserted, as well as the surrounding region in cosmid COS-NSE indicates the presence of two flanking ORFs (ORF1u and ORF1d) which are transcribed in opposite directions to ORF1 (Fig. 1). The upstream ORF1u was found to be nearly identical to the E. coli ugd gene, which codes for a UDP glucose dehydrogenase. The downstream ORF1d showed some similarities but not complete identity to rmlB genes from different Enterobacteriaceae, including K. pneumoniae.

The K. pneumoniae 52145 putative sugar-epimerase presented the same size (334 amino acid residues) and was practically identical to (with only three different residues) the ORF20 from contig 324 of the K. pneumoniae MGH78578 unfinished genomic sequence project (htpp://pedant.gsf.de/cgi-bin/wwwfly.pl). In addition a similar genetic organization was found in strain MGH 78578, with udg (ORF19) and rmlB-like (ORF20) genes flanking the putative sugar-epimerase-like gene. This genetic organization and the transcriptional direction for these three genes strongly suggest that the mutant KT3412 phenotype is attributable only to the sugar-epimerase insertional mutation, since no polar effects on downstream genes should be expected. This was confirmed since reintroduction of the single gene (plasmid pGEMT-UGE) rescued the complete wild-type phenotype in mutant KT3412, i.e., phage sensitivity, presence of K2 capsular polysaccharide, O1 antigen LPS, and wild-type LPS migration pattern (Fig. 2).

We studied the presence of ORF1 in different Klebsiella strains (n = 50) by PCR fragment DNA amplification with genomic DNAs from these strains and oligonucleotides binding to regions flanking the ORF1 (5'-AGCCAGTGTAAAATCGGCACTTA-3' and 5'-CTTTCTCTCCCCCGTTATATCCCT-3'). In all the strains tested, a single 1,965-bp fragment was amplified. To confirm that ORF1 was indeed present, the nucleotide sequence of the DNA-amplified fragments from several Klebsiella strains was determined. Thus, a gene (ORF1) coding for a putative sugar-epimerase was found in all the Klebsiella strains tested.

To determine if the putative sugar-epimerase gene (ORF1) is also involved in the production of non-O1 and non-K2 antigens, we constructed ORF1 mutants in several K. pneumoniae strains belonging to different O and K serovars (B5055, KT769, and C3). All the ORF1 mutants obtained with plasmid pSF-UGE were unencapsulated by electron microscopy and lacked the O antigen LPS, and its LPS migrated faster than the LPS of the corresponding wild-type strains (Fig. 2). These results are in agreement with the KT3412 mutant phenotype and are independent of the K and O serotypes of the wild-type strains. All the phenotypic changes observed in the mutants can be complemented by the reintroduction of ORF1 in plasmid pGEMT-UGE (Fig. 2).

Mutant KT3412 LPS characterization. Comparative analysis on SDS-PAGE-tricine of LPS obtained from mutant KT3412 and wild-type strain showed not only an absence of O antigen in the mutant LPS but also an LPS banding pattern similar to that of previously described K. pneumoniae core LPS mutants (Fig. 2) (26). Thus, it was hypothesized that mutant KT3412 LPS would be truncated at the core level. To test this possibility, LPS obtained from mutant KT3412 and the wild-type strain were subjected to mild acid hydrolysis (1% acetic acid) to cleave the acid-labile ketosidic linkage between Kdo and lipid A.

The lipid A fraction was removed by high-speed centrifugation, and the core oligosaccharides were recovered by Sephadex G-50 chromatography. Chemical composition analysis of the core oligosaccharide fractions revealed that GalA and GlcN were absent from mutant KT3412, suggesting that in this mutant the core LPS is truncated at the level of the GalA residue (Fig. 1). To further prove this point, the oligosaccharide fraction from mutant KT3412 was analyzed by electrospray ionization MS. This experiment revealed the presence of major signals at 783.33 and 764.49 m/z (data not shown). These signals could correspond to oligosaccharides containing Kdo, Hep2, Hex (Mr 783.67),or the corresponding anhydro form, as previously described for LPS samples hydrolyzed with acetic acid (35). Other minor signals were detected, such as 1002.70 m/z, that could be assigned to oligosaccharide-containing Kdo2, Hep2, Hex, and 621.59 m/z (Kdo, Hep2).

In addition, the sample was analyzed by the MALDI-PSD technique to determine the oligosaccharide sequence. The positive-ion PSD spectrum of this sample (Fig. 3, see fragmentation scheme) contains fragment ions, most of them attributable to B-type ions (14). In addition to the signals reported in the fragmentation scheme the fragment ion at m/z 745.0 can be attributed to the decarboxylated anhydro core structure (M-18 to -44) (19). The two signals at m/z 376.5 and 358.3 are attributable to internal fragmentation (18) as they might arise from a loss of the terminal heptose residue from the signal at 568.4, leaving a hydroxyl group (m/z 376.5) or a double bond (m/z 358.3).



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  		FIG. 3. PSD spectrum of m/z 807.2 of K. pneumoniae 889{Delta}wabG core oligosaccharide after acidic release of lipid A, in the positive-ion mode. Insert shows the proposed structure and fragmentation pattern.

Thus, compositional and electrospray ionization MS analysis data of the core LPS oligosaccharide fraction obtained from mutant KT3412 agree with a core LPS truncated at the level of the first outer core residue GalA, according to the known K. pneumoniae core LPS structure (Fig. 3) (49). This conclusion was further supported by analysis of the O,N-deacylated LPS (see Materials and Methods) and by the methylation analysis experiments of the N-acetylated oligosaccharide alditol mixture obtained from the K. pneumoniae KT3412 mutant. In this last experiment, the presence of 3,4-linked Hep and terminal Hep and Glc confirmed the proposed structure for mutant KT3412 core LPS (Fig. 3).

These results clearly show that a mutation in a putative sugar-epimerase produces an LPS core devoid of the outer core and thus explain the O antigen deficiency of this LPS. Since GalA is the first outer core residue in K. pneumoniae, a possible explanation for this core defect would be that the ORF1-encoded sugar-epimerase is involved in the formation of UDP-GalA from UDP-GlcA. Assuming that UDP-GalA is the substrate for GalA addition to core LPS, a mutation in uridine phosphate galacturonate 4-epimerase (UDPGLE) precluding UDP-GalA formation will generate the observed LPS phenotype.

UDPGLE enzymatic activity. The genetic analysis as well as the LPS-core structure of the mutants prompted us to study the enzymatic activity that allows the production of UDP-GalA from UDP-GlcA. K. pneumoniae wild-type strains (independently of the O and K serotypes) showed a high UDPGLE activity, e.g., 96 U (mg of protein)-1 for strain 52145 when measured as described in Materials and Methods. However, mutant strains KT3412 and KT3412 with the plasmid vector (pGEMT) showed a complete lack of UDPGLE activity in this assay, which could be rescued by the reintroduction of the single gene with plasmid pGEMT-UGE. From the results obtained, orf1 was named uge for uridine diphosphate galacturonate 4-epimerase.

Colonization and virulence studies. As a colonization model, we used the experimental urinary tract infections in rats. As can be observed in Table 2, mutant strains KT3412 and KT3412 with the plasmid vector (pGEMT) were completely unable to induce experimental urinary tract infections in rats (completely unable to colonize the rat urinary tract). Transformations of the mutant strains with the plasmid harboring the uge wild-type gene (pGEMT-UGE) rescued the ability to induce experimental urinary tract infections in rats to the wild-type strain level.


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  		TABLE 2. Experimental urinary tract infection studies in rats with different K. pneumoniae mutant strainsa

Virulence was tested in two different models: a septicemia model in mice by intraperitoneal injection and recording the mortality (LD50), and a murine model of pneumonia by intratracheal injection. When we measured the virulence of the strains in the septicemia model, mutant strains KT3412 and KT3412 with the plasmid vector (pGEMT) showed higher LD50 values (107.5) than the wild-type strain (102.1). Thus, the uge mutant showed an approximately 5-log increase in its LD50 value in comparison to the wild-type strain. When the virulence of these strains was assayed in the murine pneumonia model (Table 3), strains KT3412 and KT3412 with the plasmid vector (pGEMT) were completely avirulent. Mutant strain KT3412 transformed with the plasmid harboring the uge wild-type gene (pGEMT-UGE) recovered LD50 values similar to those of the wild-type strain and was as virulent as the wild-type strain in the murine pneumonia model (Table 3).


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  		TABLE 3. Experimental pneumonia induced by different K. pneumoniae strains


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DISCUSSION
 
The initial isolation of a mutant (KT3412) resistant to different Klebsiella bacteriophages with either the capsular polysaccharide or the LPS as receptors led us to the characterization of a gene related to sugar nucleotide epimerases. This gene, named uge (for uridine diphosphate galacturonate 4-epimerase), was clearly located outside of the O1 antigen LPS (wb), LPS-core (waa), and K2 capsular polysaccharide (wacK12) gene clusters from the wild-type strain 52145 or similar O1:K2 strains sequenced previously (3, 28, 39). The uge gene was found to be present in all the Klebsiella strains tested. Several uge insertional mutants showing the same phenotype as the KT3412 mutant strain were obtained. This phenotype was characterized by a complete lack of capsule and by an LPS lacking the O antigen and the outer core (faster migration of core oligosaccharides in SDS-PAGE in comparison to the wild-type strains). Furthermore, this O-:K- phenotype was independent of the wild-type strain's O and K serotypes.

The uge gene was found to be identical to orf20 from contig 324 of the K. pneumoniae MGH78578 unfinished genomic sequence (htpp://pedant.gsf.de/cgi-bin/wwwfly.pl). Furthermore, the same uge upstream and downstream genes were found in strains 52145 and MGH78578. The mutation of the uge gene by either mini-Tn5 or pSF-UGE insertion could not affect by polarity the downstream gene since the genes are transcribed in opposite directions (Fig. 1). Furthermore, the reintroduction of the single uge wild-type gene by transformation with plasmid pGEMT-UGE rescued the wild-type phenotype.

It seems clear that the uge gene codes for a UDPGLE enzyme responsible for the conversion of UDP-GlcA to UDP-GalA, as can be judged from the results of UDPGLE activity. Furthermore, similar proteins such as E. coli WbnF and V. cholerae Wbfw are found in gene clusters encoding polysaccharides containing GalA residues. Also, the Uge protein showed some similarity (35% identity) to Streptococcus pneumoniae Cap1J, a UDPGLE enzyme codified by one of the 15 genes responsible for the synthesis of the type 1 capsule (33).

From the presence of GalA residues in the K. pneumoniae core LPS structure (49), we hypothesize that one of the waa genes (39) should be responsible for the transfer of UDP-GalA to the LPS core backbone to allow full core LPS extension (Fig. 1). The presence of GalA as the first outer core LPS residue is a common feature for all the Klebsiella strains studied (49). If no UDPGalA is available because no conversion of UDP-GlcA to UDP-GalA is produced, then according to our hypothesis an O-deficient and truncated core LPS should be expected (Fig. 1). Chemical compositional, electrospray ionization MS, PSD fragmentation (Fig. 3), and permethylation analysis of LPS obtained from mutant KT3412 showed that this is indeed the case. All these results are in agreement with the fact that uge gene is found in all Klebsiella isolates studied, and uge mutants showed an LPS profile in gels lacking the O antigen LPS (O-) and faster migration of core oligosaccharides in comparison with the wild-type strain's LPS because of their truncated LPS core (48).

The fact that uge mutants are unencapsulated (K-) may not be explained on the basis of UDP-GalA absence. For instance, neither K2 nor K57 or K66 capsules presented GalA in their chemical composition (29). Furthermore, the lack of capsule was found in all the Klebsiella uge mutants isolated that belong to different K serotypes (unpublished results). One possible explanation could be the fact that deep LPS-core mutants (like uge mutants) are altered in different outer membrane components (5). If the capsular polysaccharide is linked to the LPS outer core directly or to some other outer membrane molecule, the uge mutants may be altered enough in these outer membrane components to prevent the attachment of the different capsular polysaccharides. This hypothesis is in agreement with the fact that mutant KT3412 seems to produce capsular polysaccharide K2 by an enzyme immunoassay with culture supernatants, but is not linked to the bacterial surface according to the results from the same assay performed with pelleted whole cells (data not shown).

The effects of the uge mutation on colonization and virulence experiments were studied in the K. pneumoniae 52145 background because this strain is highly virulent and able to colonize different surfaces. The uge mutation drastically reduced the colonization ability of K. pneumoniae in induced experimental urinary infections (Table 2). In addition, this mutation also resulted in a 5-log increase in LD50 in mice and was completely avirulent in an experimental model of pneumonia (Table 3). From these results we can conclude that the capsule and/or the LPS is essential in K. pneumoniae colonization of the urinary tract and its virulence whether tested as LD50 in mice inoculated intraperitoneally or in the experimental model of pneumonia. However, some differences in outer membrane protein profile could be observed between mutant strains and the corresponding wild-type strains. These small differences could also contribute to the decrease in colonization and virulence of the uge mutants. Finally, all the changes observed in the K. pneumoniae uge mutants in colonization and virulence experiments were rescued by the introduction of the corresponding single wild-type gene, but not by the introduction of the plasmid vector alone. Work is in progress to evaluate the feasibility of targeting this enzyme (Uge) for K. pneumoniae experimental therapy.


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ACKNOWLEDGMENTS
 
This work was supported by grants Plan Nacional de I+D (Ministerio de Ciencia y Tecnología, Spain) and Generalitat de Catalunya. L.I., B.H., and S.F. were supported by FPI fellowships from the Ministerio de Ciencia y Tecnología (Spain) and Universidad de Barcelona.

We thank Maite Polo for technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departamento de Microbiología. Facultad de Biología. Universidad de Barcelona. Diagonal 645, 08071 Barcelona, Spain. Phone: 34-934021486. Fax: 34-93-4110592. E-mail: juant{at}porthos.bio.ub.es. Back

Editor: D. L. Burns

{dagger} In memoriam. Back


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Infection and Immunity, January 2004, p. 54-61, Vol. 72, No. 1
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.1.54-61.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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