Departamento de Microbiología y Parasitología Sanitarias, División de Ciencias de la Salud, Facultad de Farmacia, Universidad de Barcelona, Av. Joan XXIII s/n, Barcelona 08028, Spain1
Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain2
Author for correspondence: Miguel Regué. Tel: +34 3 4024496. Fax: +34 3 4024498. e-mail: regue{at}farmacia.far.ub.es
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ABSTRACT |
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Keywords: LPS, waaE homologues, non-polar mutant characterization, cross-complementation
Abbreviations: -D-Gal(p)A,
-D-galacturonic acid; GlcN(p), glucosamine; ß-D-Glc(p), ß-D-glucopyranose; L,D-Hep(p),
-L-glycero-D-manno-heptopyranose; Kdo(p), 3-deoxy-D-manno-octulopyranosonic acid; BATH, bacterial adherence to hydrocarbons; HIC, hydrophobic interaction chromatography; NHS, non-immune human serum
b The GenBank accession number for the waaE gene sequences of P. mirabilis CECT170, Y. enterocolitica R102 and Ent. aerogenes CECT684 reported in this paper are AY075039, AY075041 and AY075040, respectively.
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INTRODUCTION |
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Comparison of the known structures of core LPS from different Enterobacteriaceae reveals that the inner core structure contains two residues of 3-deoxy-D-manno-octulopyranosonic acid (Kdop) and three residues of L-glycero-D-manno-heptopyranose (L,D-HeppI, L,D-HeppII and L,D-HeppIII) (Radziejewska-Lebrecht et al., 1994 ; Heinrichs et al., 1998
; Vinogradov et al., 2000
; Vinogradov & Perry, 2001
). In addition, some Enterobacteriaceae, like E. coli, contain a phosphoryl group modification at the L,D-HeppI residue (Heinrichs et al., 1998
). In other species, like Klebsiella pneumoniae, a substitution of L,D-HeppI at the O-4 position by a ß-D-glucopyranose residue (ß-D-Glcp) is found (Vinogradov & Perry, 2001
). Furthermore, in K. pneumoniae, Proteus mirabilis and Yersinia enterocolitica a substitution of L,D-HeppII at the O-3 position by an
-D-galacturonic acid residue (
-D-GalpA) residue is found (Radziejewska-Lebrecht et al., 1994
; Vinogradov et al., 2000
; Vinogradov & Perry, 2001
), while a substitution of L,D-HeppII at the O-3 position by a Glcp residue is found in E. coli (Heinrichs et al., 1998
).
The waaE gene is present in K. pneumoniae and Serratia marcescens waa gene clusters, but not in waa gene clusters from E. coli and Sal. enterica (Guasch et al., 1996 ; Regué et al., 2001
). In this study we present results showing that the K. pneumoniae waaE gene is responsible for the attachment of the branched Glcp residue to L,D-HeppI. In addition, the presence of waaE homologues is determined for representative species of Enterobacteriaceae, and we show by complementation studies that all the waaE homologues perform the same function in inner core biosynthesis. We also investigated waaE gene function in some pathogenic features, as well as its role in motility and biofilm formation.
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METHODS |
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LPS isolation and analysis.
Cultures for analysis of LPS were grown in TSB at 37 °C. LPS was purified by the method of Westphal & Jann (1965) . For screening purposes LPS was obtained after proteinase K digestion of whole cells (Hitchcock & Brown, 1983
). LPS samples were separated by SDS-PAGE or SDS-Tricine-PAGE and visualized by silver staining as described previously (Tsai & Frasch, 1982
; Pradel & Schnaitman, 1991
). For chemical analyses, purified LPS was deacylated by treatment with acetic acid (Süsskind et al., 1998
) and the core region was purified by column chromatography on Sephadex G50. The core fractions were hydrolysed with 1 M trifluoroacetic acid for 4 h at 100 °C. Monosaccharides were analysed as their alditol acetate derivatives by GLC on a Rtx-2330 column (Restek). Alditol acetate monosaccharides were obtained by a procedure described by Vinogradov et al. (1992)
. Colorimetric analysis of Kdo was performed by the method of Karkhanis et al. (1978)
. Methylation of carboxyl-reduced core oligosaccharides was performed according to the method of Ciucanu & Kerek (1984)
. Briefly, the methylated samples were hydrolysed in 2 M trifluoroacetic acid at 100 °C for 2 h, reduced, acetylated and analysed using GLC-MS.
DNA sequencing and computer analysis of sequence data.
Double-stranded DNA sequencing was performed by using the dideoxy-chain termination method (Sanger et al., 1977 ) with the ABI Prism dye terminator cycle sequencing kit (Perkin Elmer). The relevant PCR-amplified genomic DNAs were sequenced by using oligonucleotides used in the amplification reactions. Other sequence-derived oligonucleotides were used to extend the nucleotide sequence. Primers 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 ORFs were inspected. Deduced amino acid sequences were compared with those of DNA translated in all six frames from non-redundant GenBank and EMBL databases by using the BLAST (Altschul et al., 1990
, 1997
) and FASTA (Pearson & Lipman, 1988
) network service at the National Center for Biotechnology Information and the European Biotechnology Information, respectively. CLUSTAL W (Thompson et al., 1994
) was used for multiple sequence alignments.
DNA amplification.
Genomic DNA from different strains of representative Enterobacteriaceae was isolated and used as template in PCR experiments using primers designed to amplify DNA fragments containing the 3' end of the waaA gene and the 3' end of the fpg gene, respectively, at their ends. Oligonucleotide pairs Pr2 (5'-GATGGCGGGGAAC-3') and Fpg2 (5'-CATTGCCAACATGGTGAA-3'), kdtb1 (5'-GTCATGGGATCGAACGTC-3') and Yer3 (5'-CTGTTGACCGACGAAGACTA-3'), and Nuk9 (5'-TGCTTTATACCACCCTACT-3') and Nuk10 (5'-GATAAACGACCACTCTTTG-3') were used to amplify and to subclone the waaE homologues from P. mirabilis, Y. enterocolitica and Ent. aerogenes, respectively, in vector pGEMT.
Complementation studies.
Complementation analysis of relevant waaE mutants was performed by transformation of waaE genes from different Enterobacteriaceae cloned in pGEMT. Transformants were selected on LB agar containing ampicillin (100 µg ml-1) and LPS was isolated and analysed by Tricine-SDS-PAGE.
Serum killing.
The survival of exponential-phase bacteria in non-immune human serum was measured as described previously (Merino et al., 1992 ).
Cell surface hydrophobicity.
Cell surface hydrophobicity was determined by two different methods. The first method used was hydrophobic interaction chromatography (HIC) on phenylsepharose as described by Källenius et al. (1985) . Briefly, bacteria were suspended in 10 mM PBS (pH 7·4) to an OD470 of 1·0, applied to a phenylsepharose column and eluted with 4 M NaCl. The eluate was collected and its OD470 determined. The second method used was bacterial adherence to hydrocarbons (BATH) as described previously by Rosenberg et al. (1980)
. Briefly, cells were washed twice in phosphate/urea/magnesium buffer (pH 7·1), suspended in the same buffer at an OD400 of 1·0 and vortexed with various volumes of hydrocarbon. The OD400 of the aqueous phase was expressed as a percentage of the OD400 of a standard volume of untreated cells.
Bacterial adherence assay.
The adherence assay using K. pneumoniae strains to uroepithelial cells (UEC) was done as described previously (Falkowski et al., 1986 ; Merino et al., 1997
).
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 resuspended in PBS to the appropriate concentration. In each experiment, 12 female Wistar rats (200250 g; strain CFHB; Interfauna UK) were used. Ten animals were infected and two were used as controls. The infections were established and quantified as described previously (Camprubí et al., 1993 ).
Swimming and swarming motility assay.
Freshly grown bacterial colonies were transferred with a sterile toothpick into the centre of motility agar (1% tryptone, 0·5% NaCl, 0·25% agar) or swarm agar (LB base, 0·6% agar). The plates were incubated at 37 °C for 1624 h and motility was assessed by examining the migration of bacteria through the agar from the centre towards the periphery of the plate.
Transmission electron microscopy.
Bacterial suspensions were placed on Formvar-coated copper grids and negatively stained with a 2% solution of potassium phospho-tungstate. Bacteria were then viewed on a Phillips EM 400 transmission electron microscope.
Biofilm formation.
Quantitative biofilm formation was performed in a microtitre assay as described by Pratt & Kolter (1998) with minor modifications. Briefly, bacteria were grown on trypticase soy agar (TSA) and several colonies were gently resuspended in trypticase soy broth (TSB) with or without the appropriate antibiotics; 100 µl aliquots were placed in a microtitre polystyrene plate and incubated for 48 h at 30 °C without shaking. After the bacterial cultures were poured out, the plate was washed extensively with water, fixed with 2·5% glutaraldehyde, washed once with water and stained with 0·4% crystal violet solution. After solubilization of the crystal violet with ethanol/acetone (80/20, v/v) the absorbance was determined at 570 nm.
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RESULTS |
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An alignment of the known Enterobacteriaceae WaaA proteins was used to design degenerate oligonucleotide MRI (5'-ATGGRACYGARCTITGGCC-3'). The oligonucleotide pair MRI/Fpg1 allowed the amplification of the waaA-fpg region in all the strains of Enterobacteriaceae tested in this study, using genomic DNA as template. Amplified fragments of about 1·7 kb were obtained when using genomic DNA from strains of different serovars of Sal. enterica, E. coli, Shi. dysenteriae, Shi. flexneri and Shi. sonnei as template. In addition, amplified fragments of about 2·7 kb were obtained when using genomic DNA from different strains of K. pneumoniae, Ser. marcescens, P. mirabilis, Y. enterocolitica and Ent. aerogenes as template. Representative results of such amplification experiments are shown in Fig. 4. Thus, this oligonucleotide pair can be used to detect the presence of the waaE gene in members of the Enterobacteriaceae and to predict the presence of a Glcp residue attached via a ß1,4 linkage to the L,D-HeppI residue in the inner-core LPS (Fig. 1
).
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Cross complementation among waaE homologues
Alignment of the known Enterobacteriaceae WaaE proteins revealed that all these proteins share a significant level of similarity (76%) and identity (53%). This similarity strongly suggests that these proteins perform the same function in the biosynthesis of the inner-core LPS. To confirm this functional relationship, the waaE gene homologues from Ent. aerogenes, Y. enterocolitica and P. mirabilis were cloned in vector pGEM-T. These recombinant plasmids were introduced into the waaE mutant strains K. pneumoniae 889-1 and NC16 (Regué et al., 2001 ), Ser. marcescens N28b-16 and P. mirabilis CECT170-8. The LPS from mutant strains 889-1 and N28b-16 transformed with pGEMT-WaaEKp, pGEMT-WaaESm, pGEMT-WaaEPm, pGEMT-WaaEYe and pGEMT-WaaEEa showed a wild-type migration pattern (Fig. 2
). Similar results were obtained for K. pneumoniae NC16 and P. mirabilis CECT170-8. These results confirm that the waaE gene homologues perform the same role in inner-core LPS biosynthesis.
Contribution of the waaE gene to some pathogenic features
Since all the waaE homologues studied appear to have the same function, we decided to investigate the contribution of waaE genes to some pathogenic features involving surface molecules by using the set of isogenic mutants obtained. The waaE mutant strains K. pneumoniae 889-1 and NC16, Ser. marcescens N28b-16 and P. mirabilis CECT170-8 were sensitive to the bactericidal activity of NHS (non-immune human serum). The respective wild-type strains, as well as the mutants complemented with the waaE gene homologues, were resistant to this activity. As an example, Table 3 shows the results obtained for K. pneumoniae strains. Surface hydrophobicity of K. pneumoniae waaE mutant strain 889-1, measured by both the HIC and the BATH methods, was higher than the wild-type strain or the waaE mutant complemented with the waaE gene homologues. The Ser. marcescens and P. mirabilis waaE mutants were also more hydrophobic than their respective wild-type strains and the complemented waaE mutants.
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The role of the waaE gene in swarming motility and formation of biofilms
The results obtained in colonization of the rat urinary tract prompted us to examine if the lack of waaE was able to alter other surface molecules involved in bacterial colonization besides the LPS, for instance flagella. Using only the waaE mutants from Ser. marcescens and P. mirabilis, we studied their motility either on swimming or swarming agar plates. No differences could be observed in swimming plates, but a large swarming reduction of the waaE mutants in comparison to their wild-type strains or the mutants complemented with the different waaE gene homologues was found on the appropriate plates (see Fig. 5 for P. mirabilis results). However, when we studied the swarming cells using light or electron microscopy no differences were found between the waaE mutants and the wild-types or the waaE-complemented mutants. All the strains showed characteristic elongated and hyperflagellated cells.
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DISCUSSION |
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Studies on the genetic organization of the Enterobacteriaceae genes involved in core LPS biosynthesis have showed that the waa gene cluster contains two conserved regions. One is in the proximal end of the cluster with three conserved genes, gmhD, waaF and waaC, encoding the last enzyme in the biosynthesis of L,D-Hepp, L-glycero-D-manno-heptosyltransferase II and L-glycero-D-manno-heptosyltransferase I, respectively (Heinrichs et al., 1998 ; Regué et al., 2001
). There are two different models for the distal end of the waa gene cluster. In the five known E. coli core LPS types and in Sal. enterica serovar Typhimurium the waa clusters end with the waaA gene encoding the bifunctional Kdo transferase (Sofia et al., 1994
; Heinrichs et al., 1998
). In K. pneumoniae and Ser. marcescens waa clusters the waaE gene is found just downstream from the waaA gene (Guasch et al., 1996
; Regué et al., 2001
). In both models the coaD and fpg genes are found just downstream from the last waa gene, either waaA or waaE. A PCR assay for waaE homologues among clinical isolates of several species of Enterobacteriaceae suggested the presence of an extra gene in strains of P. mirabilis, Y. enterocolitica and Ent. aerogenes. No such waaE extra gene was detected among clinical isolates of E. coli, Sal. enterica serovars Typhimurium, Enteritidis and Infantis, and Shi. dysenteriae, Shi. flexneri and Shi. sonnei (Figs 3
, 4
). Furthermore, nucleotide sequence determination for a representative strain for each species confirmed the presence of waaE homologues flanked by the waaA gene and the coaD-fpg genes in P. mirabilis, Y. enterocolitica and Ent. aerogenes (Fig. 3
). Analysis of the corresponding sequence strongly suggests that the waaA, waaE and coaD genes are cotranscribed and that fpg is transcribed in the opposite direction; these two features have also been found in K. pneumoniae (Regué et al., 2001
). Genetic complementation studies showed that the identified waaE homologues are able to complement non-polar K. pneumoniae and Ser. marcescens waaE mutant strains, thus proving that all the waaE homologues perform the same function in inner-core LPS biosynthesis. These results agree with core LPS structural studies in P. mirabilis and Y. enterocolitica where a substitution of L,D-HeppI at the O-4 position by a Glcp [ß-D-Glcp-(1
4)-
-L,D-HeppI] has been found (Radziejewska-Lebrecht et al., 1994
; Vinogradov et al., 2000
). From the Enterobacteriaceae waa gene cluster distal end sequence data a set of primers was designed to amplify genomic DNA containing the 3' end of the waaA gene up to the 3' end of the fpg gene. These primers allow rapid screening for the presence of waaE homologues and can be used to predict the presence of a Glcp residue attached by a ß1,4 linkage to L,D-HeppI in the inner-core LPS of members of the Enterobacteriaceae, such as Ser. marcescens and Ent. aerogenes.
The contribution of the waaE gene to the different pathogenic features studied could be explained by the fact that these mutants showed a clear reduction in O-antigen LPS molecules (Fig. 2). It seems clear, at least in K. pneumoniae, that the O-antigen LPS is critical for serum resistance (Reid & Sobel, 1984
; Figueroa & Densen, 1991
; Merino et al., 2000
), changes in surface hydrophobicity (Marshall, 1984
; Merino et al., 2000
), adhesion to UEC cells (Merino et al., 1997
, 2000
), and urinary tract infection and colonization in rats (Nevola et al., 1987
; Finlay & Falkow, 1989
; Licht et al., 1996
; Russo et al., 1996
; Merino et al., 2000
). We may conclude also that these facts could be extended to other enterobacteria, such as Ser. marcescens and P. mirabilis.
It has been reported that some Sal. enterica serovar Typhimurium mutants unable to show swarming motility (efficient colonization of the growth surface) with functional flagella (essential for this form of motility), showed defects in LPS biosynthesis (Toguchi et al., 2000 ). These authors suggested that the O-antigen LPS improves surface wettability required for swarm colony expansion. The role of the waaE gene in P. mirabilis and Ser. marcescens swarming phenotype could be attributed to O-antigen LPS reduction (surface wettability reduction) observed in the waaE mutants, while the flagella in both bacteria remain functional. Furthermore, we suggest that this altered swarming phenotype with a reduction of surface wettability could explain the reduced biofilm formation ability of the waaE mutants. Recently, Watnick et al. (2001)
demonstrated that at least exopolysaccharide synthesis and flagella are needed for the efficient formation of a three-dimensional biofilm in Vibrio cholerae O:139.
All these roles that we attribute to the waaE gene are supported by the fact that well genetically characterized isogenic mutants were used, because all the waaE gene homologues were able to complement the waaE mutants, independently of their origin, and because the complemented mutants recovered the wild-type strain characteristics.
We cannot exclude that the inner-core change observed in the waaE mutants, the lack of substitution of L,D-HeppI at the O-4 position by ß-D-Glcp, could also play a role in swarming motility, biofilm formation and deficient pathogenic phenotypes showed by these mutants. Interestingly, both the WaaP and WaaE proteins are involved in substitution of L,D-HeppI at the O-4 position by a phosphate or pyrophosphoethanolamine (Yethon et al., 1998 , 2000
) and by a ß-D-Glcp residue, respectively. The phenotype of the waaE mutants shares some similarities to E. coli F470 and Sal. enterica serovar Typhimurium waaP mutants. In all these cases a faster migrating LPS band is seen in Tricine-SDS-PAGE, the core LPS appears to be capped with O-antigen and the amount of O antigen is reduced in the mutants. In addition, in Sal. enterica serovar Typhimurium a waaP mutant showed reduced virulence in vivo (Yethon et al., 2000
).
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ACKNOWLEDGEMENTS |
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Received 21 March 2002;
revised 16 July 2002;
accepted 16 July 2002.