Role of the Vpe Carbohydrate Permease in Escherichia coliUrovirulence and Fitness In Vivo
Vanessa Martinez-Jéhanne,a,b Christophe Pichon,a,c Laurence du Merle,a,c Olivier Poupel,c Nadège Cayet,d Christiane Bouchier,e andChantal Le Bouguéneca,c
Institut Pasteur, Unité Pathogénie Bactérienne des Muqueuses, Département de Microbiologie, Paris, Francea; Université Paris 7-Denis Diderot, Sorbonne Paris Cité, CellulePasteur, Paris, Franceb; Institut Pasteur, Biologie des Bactéries Pathogènes a Gram Positif, Département de Microbiologie, Paris, Francec; Institut Pasteur, Plate-forme deMicroscopie Ultrastructurale, Imagopole, Paris, Franced; and Institut Pasteur, Plate-forme de Génomique, Génopole, Paris, Francee
Uropathogenic Escherichia coli (UPEC) strains are a leading cause of infections in humans, but the mechanisms governing hostcolonization by this bacterium remain poorly understood. Previous studies have identified numerous gene clusters encodingproteins involved in sugar transport, in pathogen-specific islands. We investigated the role in fitness and virulence of the vpeoperon encoding an EII complex of the phosphotransferase (PTS) system, which is found more frequently in human strains frominfected urine and blood (45%) than in E. coli isolated from healthy humans (15%). We studied the role of this locus in vivo,using the UPEC E. coli strain AL511, mutants, and complemented derivatives in two experimental mouse models of infection.Mutant strains displayed attenuated virulence in a mouse model of sepsis. A role in kidney colonization was also demonstratedby coinfection experiments in a mouse model of pyelonephritis. Electron microscopy examinations showed that the vpeBC mu-tant produced much smaller amounts of a capsule-like surface material than the wild type, particularly when growing in humanurine. Complementation of the vpeBC mutation led to an increase in the amount of exopolysaccharide, resistance to serum kill-ing, and virulence. It was therefore clear that the loss of vpe genes was responsible for all the observed phenotypes. We also dem-onstrated the involvement of the vpe locus in gut colonization in the streptomycin-treated mouse model of intestinal coloniza-tion. These findings confirm that carbohydrate transport and metabolism underlie the ability of UPEC strains to colonize thehost intestine and to infect various host sites.
Escherichia coli is a normal resident bacterium of the intestinesof healthy humans. However, in certain circumstances, it may
also cause serious disease in humans. Uropathogenic E. coli(UPEC) strains are facultative pathogens that are present as com-mensal organisms in the normal flora of some healthy people.They are responsible for 80 to 90% of urinary tract infections(UTI) in humans (3, 17, 53). Many UTI are asymptomatic, butsome UPEC strains cause significant clinical symptoms, rangingfrom pain in uncomplicated cases of cystitis to sepsis in cases ofpyelonephritis. Antibiotic treatment may be difficult if the strainsconcerned are multiresistant (27). Despite tremendous advancesin our understanding of the genetic bases of pathogenicity and ofthe evolutionary diversity of UPEC strains over the last 10 years(28), the mechanisms by which UPEC strains colonize the humanintestine, which serves as their reservoir, and then travel to andpersist in the urinary tract (UT) remain poorly understood. Thesebacteria frequently express adhesin/invasin and toxin genes andare equipped with iron acquisition systems and mechanisms forevading the immune response, through the production of extra-cellular polysaccharides, for example. However, no virulence fac-tor or set of factors has yet been identified as essential for gutcolonization and infection of the bladder or kidney. This impliesthat a number of alternative factors may mediate each step in theinfection process and that each strain may have a unique combi-nation of such factors.
It also remains unclear how UPEC strains adapt their metabo-lism to derive carbon and energy from the environment, allowingthem to grow, to survive, and to colonize their hosts at both intes-tinal and extraintestinal sites. UTI generally develop followingcontamination of the UT by the intestinal or vaginal microflora(46). The UT constitutes a unique environment, in which UPEC
strains grow better than other bacteria. Several studies have shownthat the ability of UPEC strains to synthesize or to acquire aminoacids (arginine) and nucleosides (guanine) that are limiting inurine plays an essential role in maximizing virulence within theUT (43, 44). The impact of metabolic functions on urovirulencefirst became clear when effective iron acquisition systems wereshown to be required for urovirulence (21, 45), and associationwas demonstrated between D-serine metabolism and the intracel-lular accumulation and expression of virulence genes during as-cending UTI in a mouse model (20). Comparative and functionalanalyses have shown that the metabolic flexibility of E. coli reflectsgenetic diversity and the dynamic organization of the genome andhave highlighted the key role of carbohydrate metabolism in theadaptation of pathogenic strains to different ecological niches (30,51). Many genomic islands specific to UPEC strains harbor genesencoding proteins involved in the transport and use of carbohy-drates such as sucrose (50), cellobiose (33), and deoxyribose (6).The catabolism of deoxyribose is catalyzed by the products of thedeoK operon, which has been transferred from Salmonella entericato E. coli (6). Using a mouse model of intestinal colonization, we
recently demonstrated the involvement of this operon in gut col-onization by pathogenic E. coli strains, including a UPEC strain(32). The deoK operon is frequently associated with strains iso-lated from infected urine and blood, in which it is always locatedin large specific islands carrying genes contributing to the intrinsicvirulence and/or adaptive properties of the strain. The character-ization of PAI-I from the sepsis strain AL862, which carries thedeoK operon, identified and localized three genes encoding hypo-thetical proteins displaying 30, 27, and 35% identity to SgaT, -B,and -A, respectively, the components of the tripartite L-ascorbate-specific permease of E. coli (29) (GenBank accession numberGQ497943). Based on this sequence similarity and the location ofthe genes in a PAI, the locus was named vpe for virulence-associ-ated phosphotransferase (PTS) in E. coli. This locus contains thevpeA, vpeB, and vpeC genes, which encode the EIIA, EIIB, andEIIC constituents, respectively, of a putative carbohydrate-spe-cific permease of the SgaTBA family (23, 54).
The aim of this work was to explore the role of carbohydratemetabolism in urovirulence by investigating the vpe locus. We firstdocumented the association of this locus with strains from urineand blood. The UPEC AL511 strain is used as a model organismfor studies in our laboratory (10, 36). A clear link between expres-sion of the vpe locus and the intrinsic virulence of AL511 wasdemonstrated in a standardized mouse model of lethal infection.Competition experiments on human urine and in the mousemodel of ascending pyelonephritis provided information aboutthe role of vpe genes in the development of UTI. Finally, a role forvpe genes in intestine colonization was also suggested in the strep-tomycin mouse model of gut colonization.
MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. We studied 235well-characterized clinical isolates from our laboratory collection (32),including 88 isolates obtained from the urine or blood of patients, 20enteroaggregative E. coli (EAEC) strains, and 127 E. coli isolates obtainedfrom the stool samples of healthy subjects. Isolate AL511 was used as theprototype for characterization of the genes of interest (Table 1). StrainAL511, from phylogenetic group A, adheres to mouse renal epithelialcollecting ducts both in vitro and in vivo. This interaction stimulates aninnate immune response (10), and the uptake of AL511 bacteria into thesecells is dependent on the flagella (36).
Bacterial strains were routinely cultured in Luria-Bertani (LB) broth,with shaking (140 rpm), or on LB agar plates at 37°C. In various experi-ments, the strains were cultured in human urine or serum. Urine compo-sition is highly variable. We therefore carried out assays with independenturine samples (including samples collected first thing in the morning)from several healthy volunteers (at least five) who had not been treatedwith antibiotics in the preceding 2 months. Each urine sample was filtersterilized (passed through a filter with 0.22-�m pores). The samples werepooled, and aliquots were frozen at �80°C before use. Three independenturine pools were constituted. The sera from healthy subjects were pro-vided by the ICAReB platform (Investigation Clinique et Accès aux res-sources Biologiques, Institut Pasteur, Paris, France). Pooled serum sam-ples from at least two donors were either used fresh or stored in aliquots at�20°C until required. Antibiotics were used, as required, at the followingconcentrations: kanamycin, 100 mg/liter; Zeocin, 60 mg/liter; apramycin,30 mg/liter; and carbenicillin, 100 mg/liter.
Carbohydrate fermentation. We routinely assessed the ability of thevarious strains to metabolize carbohydrates by culturing the bacteriaovernight on LB plates. For some experiments, bacteria were grown over-night on K5 minimal medium (16) plates supplemented with 50% humanserum or urine. API 50 CH carbohydrate fermentation strips (bioMéri-eux, France) were used in accordance with the manufacturer’s instruc-tions. Bacterial growth was also assessed on K5 plates containing 0.1% to0.4% concentrations of the carbohydrate of interest as the sole source ofcarbon. The plates were incubated at 37°C for 24 to 48 h.
Construction of deletion mutants. We generated mutants of AL511by the allelic exchange recombination method, using derivatives of thethermosensitive plasmid pKOBEG, which carries lambda red recombina-tion genes (Table 1). Deletion strains were obtained by a one-step (14) ora three-step (11) method, with primers targeting the 5= and 3= ends of thetarget genes and kanamycin resistance cassettes derived from either pKD4(14) or MG4100ybe::GB (40) as templates. All allelic exchanges were ver-ified by PCR with primers flanking the gene of interest. We checked thatallelic exchanges resulted in mutants with no growth defect phenotype, asassessed in independent cultures in LB broth, urine, and heated serum-containing media.
For complementation assays, the vpeBC sequence was amplified fromAL511 genomic DNA with the Expand high-fidelity PCR system kit(Roche Applied Science), with the vpeBC-KpnI and vpeBC-HindIIIprimers. The PCR product was inserted into pZEZeoGFP cut with theappropriate restriction enzymes and the resulting recombinant plasmidwas introduced, by electroporation, into vpeBC mutant strains. All theprimers used are listed in Table S1 in the supplemental material.
TABLE 1 Strains and plasmids used in this study
Strain or plasmid Relevant characteristicsa Reference or source
E. coli strainsAL511 Urine isolate (O9:H12:F11) from a patient with acute pyelonephritis; Apr Cmr Strr Spr Tcr 2, 18AL511 vpeBC vpeBC::Km, resistance cassette from MC4100ybeW::GB This studyAL511 vpeBC (Km-FRT) vpeBC::Km-FRT, resistance cassette from pKD4 This studyAL511 vpeBC (FRT) vpeBC::FRT, removal of the Km-FRT cassette This studyAL511 vpeR vpeR::Km, resistance cassette from MC4100ybeW::GB This studyAL862 Human blood isolate from a patient with both UTI and bacteremia 29MG1655 Nonpathogenic K-12 reference strain 7
PlasmidspKOBEGA Derivative of pKOBEG, Apr 11pKOBAPRA Unpublished derivative of pKOBEG, Aprar 11pCP20.Zeo Flipase (Flp) expression plasmid for removal of the Km-FRT cassette; Zeor This studypZEZeoGFP Zeor derivative of pZE21GFP (13) 32pZEZeovpeBC vpeB-C genes from AL511 inserted in place of the GFP gene in pZEZeoGFP This study
In vitro growth curves. We checked that the independent growth ofthe mutant was similar to that of the wild-type strain, using coloniesgrown from a freezer stock to initiate overnight cultures in 5 ml of appro-priate medium. The following morning, the culture was diluted 1:100 infresh medium and incubated at 37°C, with shaking at 140 rpm. Straingrowth was monitored at hourly intervals until stationary phase wasreached. Growth was monitored by both determining the optical densityat 600 nm (OD600) and counting the CFU.
For cocultures in LB broth and urine, overnight precultures of wild-type and mutant strains in the medium of interest were mixed 1:1 (vol/vol). Aliquots of these mixtures were diluted and plated on LB plates andon LB plates supplemented with kanamycin to determine the input, inCFU/ml, of the two strains. The numbers of mutants were determineddirectly from the kanamycin-containing plates. Counts for the parentalstrain were obtained by subtracting the number of CFU on kanamycin-containing selective plates from that on LB plates without antibiotic. Weused a toothpick to transfer 100 colonies from plates without antibiotic toplates with or without kanamycin to check these counts. The fraction ofcolonies susceptible to kanamycin was determined. These two methodsgave highly consistent estimates of the number of parental strain colonies.The mixtures diluted 1:100 in fresh medium (5 and 2 ml of LB mediumand urine, respectively) were incubated at 37°C with shaking. They werepassaged (dilution, 1:500) every 8 and 16 h for 2 weeks. At the end of each24-h period, we determined the number of viable CFU of parental andmutant strains by plating on LB and antibiotic-containing LB plates. Thedetection limit for plate counts was 102 CFU/ml. Each coculture experi-ment was carried out at least twice. At the end of the experiment, wechecked the identity of each strain by PCR analysis and determination ofan antibiogram.
RNA extraction and cDNA synthesis. Bacterial cultures were stoppedduring the exponential (OD600 of 0.6 or 0.8 for LB broth and 0.3 for urine)or stationary (after 9 or 24 h) phase. The cells were harvested by centrif-ugation and immediately frozen at �80°C. Total RNA was extracted withTRIzol reagent according to the manufacturer’s instructions (Invitrogen).Dried RNA pellets were resuspended in diethyl pyrocarbonate (DEPC)-treated water. RNA extraction was followed by DNase I treatment (Am-bion) to eliminate contaminating genomic DNA. RNA quality was deter-mined with RNANano chips and a Bioanalyzer (Agilent Technologies).
Total RNA was used as a template for cDNA synthesis with the AMVreverse transcriptase (Roche). Reaction mixtures (20 �l) containing 200ng of random hexamers (Roche), 0.5 mM deoxynucleotide triphosphate,and 4 �g of total RNA were heated at 65°C for 5 min to denature thenucleic acid and placed on ice. Reverse transcription was carried out withAMV reverse transcriptase (25 U) and the 1� buffer supplied in the kit,according to the manufacturer’s protocol.
Quantitative reverse transcription-PCR (qRT-PCR). Specific primerpairs were designed with BEACON designer software, version 4.02 (Pre-mier Biosoft International, Palo Alto, CA) (see Table S1 in the supplemen-tal material). PCR amplification was performed in a reaction volume of 25�l containing 5 �l of a 1:100 dilution of cDNA, 1 �l of gene-specificprimers (10 �M), and 12.5 �l of iQ SYBR green Supermix (Bio-Rad),according to the manufacturer’s instructions. Control reactions with noreverse transcriptase were also performed. Amplification, detection, andanalysis were performed with the MyiQ single-color real-time PCR detec-tion system (Bio-Rad). Each assay was performed in triplicate and re-peated with at least two independent RNA samples. All data were normal-ized with respect to an internal standard (16S rRNA). The fold changeratio between growth conditions for each gene was calculated by the cyclethreshold (��CT) method (31).
Experimental mouse models. All animal experiments were per-formed according to protocols approved by the institutional animal careand use committee. Groups of at least five mice per coinoculation wereused to determine defects in the fitness and virulence of mutants. Thecoinfection was repeated whenever a defect was apparent. At the end ofthe experiments, the identity of the bacteria recovered from the biological
samples was checked by bacteriological and genetic approaches. The datawere plotted and analyzed with Prism 5.0a (GraphPad software). P valuesbelow 0.05 were considered significant.
Lethality model. The intrinsic virulence of the E. coli strains was eval-uated in a previously described model of systemic infection (35). Briefly,bacterial strains were grown overnight from frozen glycerol stocks in LBbroth. The cells were harvested by centrifugation, washed, and suspendedin Ringer solution to obtain inocula of about 2 � 108 CFU in 200 �l,which were injected into the peritoneal cavities of 8-week-old femaleSwiss mice (Janvier Laboratories, France). The mice were observed 6 hafter inoculation to detect whether rapid death was linked to endotoxinicshock; no mouse died within the first 6 h. The infected mice were thenobserved daily for up to 1 week. Death and the time of death were notedfor each mouse. Lethality was evaluated on the basis of at least two inde-pendent series, with 4 to 10 mice studied for each strain. Each experimen-tal series included the negative-control strain MG1655, which, like a typ-ical commensal E. coli strain, killed no mice during the 7 days of the assay(51).
Urinary tract infection model. Kidney and bladder colonization bythe AL511 strain and its vpe derivatives was assayed in a model of ascend-ing infection, essentially as previously described (10), except that 6- to8-week old female inbred CBA mice were used within a week of deliveryfrom Charles River Laboratories (France). No bacteria were recoveredfrom the plating of urine from the mice used in the study on Drigalski orMueller-Hinton agar plates on the day before the experiment. Before in-fection, the bladders of the mice were voided by gentle compression of theabdomen, and the sterility of the urine was again tested. The mice wereanesthetized by intraperitoneal injection of a mixture of ketamine andxylazine and infected with 50 �l of a bacterial suspension. The inoculumwas prepared from E. coli strains grown overnight from frozen stocks instatic LB broth; it contained approximately 108 bacteria suspended inphosphate-buffered saline (PBS). For competitive infection experiments,the parental and mutant strains were grown independently and thenmixed in a 1:1 ratio to obtain inocula. Bacteria were delivered to thebladder via the transurethral route through a polyethylene catheter (In-syte Autoguard soft catheter, 0.7-mm external diameter; Vygon, France).We monitored the colonization dynamics of strains by sequential urinecultures during the first 2 days. Four days after infection, mice were killedhumanely by CO2 asphyxiation, and the bladders and kidneys were ex-cised, weighed, and homogenized in PBS supplemented with 0.025% Tri-ton X-100. Bacterial counts were determined by plating serial dilutions onLB agar plates with and without kanamycin, to differentiate between thetwo strains in coinfections. Raw data were adjusted for the number ofCFU per gram of organ. We excluded animals with sterile kidney culturesfrom strain comparisons. The minimum thresholds of detection for in-fection were 30 CFU/g for the kidney and 300 CFU/g for the bladder. Thedegree of attenuation of the mutant was estimated by determining thecompetitive index (CI). The CI was calculated as the ratio of mutant towild-type CFU recovered from the organs on day 4 divided by the initialratio of mutant to wild-type CFU. The results are expressed as log CI in thefigures. A log CI of zero indicates that the two strains were recovered in thesame ratio as that in which they were injected; negative values indicate thatthe parental strain outcompeted the mutant strain, and positive valuesindicate that the mutant outcompeted the parental strain. Mutants wereconsidered attenuated if the log CI was below �0.3 (26).
Intestinal colonization model. The intestine-colonizing abilities ofthe AL511 strain and its vpe derivative were compared in a previouslydescribed competition-based model (32) in streptomycin-treated BALB/cfemale mice (7 weeks old; Janvier Laboratories). After 18 to 20 h of foodand water starvation, the mice were fed 200 �l of a sucrose (20%)-bicar-bonate (2.6%) solution supplemented with 105 to 106 CFU of bacteriagrown overnight in LB broth with shaking. The mice were then returnedto their normal diet, including streptomycin (5 g/liter). After the bacterialsuspension had been ingested, mice were housed individually in cageswithout bedding that were cleaned daily, and the food and streptomycin-
treated water supply was reestablished. Fresh fecal samples collected after24 h and at the times indicated in the figures were weighed, homogenized,and diluted in 1� PBS. Bacterial counts were determined by plating serialdilutions of homogenates on LB agar plates. Raw data were adjusted ac-cording to the number of CFU per gram of feces. The threshold for thedetection of infection was 6 � 102 CFU/g of feces. For competitive infec-tion experiments, the parental and mutant strains were grown indepen-dently and then mixed in a 1:1 ratio to obtain inocula. Bacteria werecounted by plating biological samples on LB agar plates with and withoutkanamycin to differentiate between the two strains. The degree of atten-uation of the mutant was estimated by determining log CI.
Exopolysaccharide staining and electron microscopy. We visualizedpolysaccharide material at the surface of the bacteria by staining withcationized ferritin (39). Bacteria were grown overnight in 2 ml of LB brothor urine at 37°C with shaking, collected by centrifugation, and washed in2 ml of 1� PBS. They were then fixed and stained with polycationicferritin (Sigma) as previously described (4). Thin sections of samples em-bedded in epoxy resin were stained with 2% uranyl acetate and lead ci-trate. They were observed in a JEOL JEM 1010 transmission electronmicroscope operating at 80 kV, and images were recorded with a KeenView camera (Eloise Ltd.).
Serum bactericidal assay. We evaluated changes in cell viability fol-lowing the exposure of E. coli strains to human serum. Bacteria from anovernight culture in LB broth (containing antibiotics when required)were washed twice in 1� PBS. For the assay, we mixed 100 �l of 1� PBScontaining approximately 106 bacteria with 100 �l of 40% serum. Wewithdrew 5-�l samples at 30 min, 1 h, 2 h, 3 h, and 4 h of incubation at37°C for the counting of viable bacteria. All assays were carried out intriplicate at least twice. When required, the complement was inactivatedby incubating the serum for 30 min at 56°C before mixing it with bacteria.
Nucleotide sequence accession numbers. The GenBank accessionnumbers for the sequence of the 9,092-bp chromosomal region fromAL511 carrying the deoK and vpe loci and the 59,151-bp region of PAI-IAL862 reported here are FJ999998 and GQ497943, respectively. The PAI-IAL862 sequence was deduced from the sequence of three cosmid clones(pILL1255, pILL1269, and pILL1270) as described in a previous study(29).
RESULTSThe vpe locus is associated with pathogenic E. coli strains.BLAST analyses showed that the vpe locus from PAI-IAL862 wasstrongly conserved in the genomes of four pathogenic E. coli iso-lates (UPEC isolate CFT073, EAEC isolates 55989 and O42, andShiga toxin-producing isolate 4797/97). The vpe cluster was alsoidentified in the genome of one commensal isolate (SE15). Com-parative genomic analysis revealed that the region of PAI-IAL862
conserved in these five genomes spanned 3,318 bp, with 100%coverage and 99% identity. In addition to vpeA, -B, and -C, itincludes a divergent transcriptional unit, vpeR, encoding a puta-tive 343-amino-acid protein (Fig. 1). BLAST analysis also showedthe sequence of VpeR to be very similar to those of transcriptionalregulators of the LacI family (33% identity to GntR), with a pre-dicted N-terminal helix-turn-helix motif coinciding with a DNA-
binding domain at residues 16 to 67, a putative dimerization in-terface at residues 72 to 313, and a ligand-binding domain atresidues 72 to 342. The vpe and deoK operons were found to belinked, in a conserved 9-kb region, in all genomes studied otherthan that of the 4797/97 isolate. The two loci are separated byabout 700 bp in length, the features of this region being reminis-cent of an insertion sequence.
We carried out PCR analyses with primers derived from thePAI-IAL862 sequence to determine whether the vpe locus was pres-ent in another of the 233 UPEC, sepsis, EAEC, and commensalstrains. Carbohydrate permease specificity is strongly linked to theintegral membrane transporter (EIIC) component. We thereforeused the vpeC gene as the target for amplification. The AL862isolate and the E. coli MG1655 strain were used as positive andnegative controls, respectively. Our results strongly suggested anassociation of the vpe locus with strain pathogenicity, despite thepresence of the vpeC gene in both pathogenic and commensalstrains. This gene was present in 48.15% of pathogenic isolates(52/108) and 15% of commensal strains (19/127); this differencewas found to be significant (P � 0.0001) in �2 analysis. The pres-ence and expression of the deoK operon had previously been in-vestigated in all the strains of the collection (32). For confirmationof the linkage of the two loci observed during genome compari-son, we amplified the region between the vpeC and deoM genes byPCR with the vpeC-F and deoM-F primers (see Tables S1 and S2in the supplemental material and Fig. 1). These loci were found tobe linked in 76.1% of the vpe-positive isolates, regardless of theorigin of the strain.
We selected E. coli AL511, a pyelonephritic isolate carryingonly one copy of the vpe locus (data not shown), for analysis of theexpression of this locus and its role in virulence. The nucleotidesequence of the vpe-deoK region from AL511 was 99% identical tothose of the AL862 and CFT073 strains over a stretch of 9,092 bp.
The vpe genes and virulence in mice. We evaluated the linkbetween vpe locus expression and the experimental virulence ofAL511 in a standardized mouse lethality assay (35). Reproducibledata were obtained for the commensal MG1655 isolate and theparental AL511 strain in 10 independent experiments, indicatingthat the experimental conditions were well standardized and thatthe model was not impaired by variability of host susceptibility.MG1655 was innocuous, whereas AL511 killed all of the mice ineach experiment (Table 2). The intrinsic virulence of AL511 wasstrongly attenuated by the vpeBC mutations. The AL511 vpeBCmutant remained lethal but killed only 33% of the mice (mean ofnine independent experiments on a total of 74 mice). This modelwas considered appropriate for tackling this question, because themean percentage of mice killed remained fairly constant. We in-vestigated four other independent vpeBC mutants (two indepen-dent AL511 vpeBC [Km-FRT] and two independent AL511 vpeBC[FRT] clones), each of which killed �20% of the mice. We alsodemonstrated, in this model, that it was possible to restore thevirulence of the mutant to wild-type levels by introducingpZEZeovpeBC into AL511 vpeBC. This complementation resultedin a strain that killed more than 90% of the mice (Table 2). Thus,there is clearly a link between expression of the vpe locus fromAL511 and lethality in mice.
Analysis of vpe gene expression in human urine. The AL511strain was cultured in human urine, and the levels of transcriptsfor the vpeA, -B, -C, and -R regions were evaluated by qRT-PCRexperiments. Transcript levels for the vpeA, -B, and -C genes
FIG 1 Genomic organization of the vpe gene cluster, which is frequentlylinked to the deoK gene cluster, in a conserved 9-kb region.
halved in stationary phase, with no significant difference in tran-scription level observed between the three genes, strongly suggest-ing that these genes, which are separated by only a few nucleotides(15 and 12, respectively), were cotranscribed (Fig. 2A). The struc-ture of the transcriptional unit was confirmed by RT-PCR on thesame cDNA sample with the vpeA-F and vpeC-R primers, whichamplified a 1,210-bp fragment, consistent with the production ofa single transcript corresponding to all three genes (data not
shown). A stronger effect of the change of growth phase was ob-served for the expression of vpeR, which decreased by a factor ofabout 10 in the stationary phase (Fig. 2A). We carried out qRT-PCR assays on total RNA extracted from cultures in LB broth tocontrol for the specificity of the changes in vpe gene transcriptionin response to urine. We observed no significant effect of growthphase in LB broth on mRNA levels (data not shown). Due todifferences in the rates of growth of the bacterium in urine and LBbroth, significant results were obtained only for comparisons ofgene expression during the stationary phase. The induction ratefor vpeABC expression was about three times higher in urine thanin LB broth during the stationary growth phase, and that for vpeRin urine was 1/10 that in LB broth (Fig. 2B).
We investigated the potential regulatory role of VpeR by com-paring levels of expression of the vpeA, -B, and -C genes in theparental isolate and the AL511 vpeR derivative. As expected, novpeR transcription was detected in the mutant, whether it wasgrown in urine or LB broth (Fig. 3). In LB broth cultures, inacti-vation of the vpeR gene induced a large increase in expression ofthe vpeA, -B, and -C genes, regardless of the growth phase, pro-viding clear evidence of an inhibitory effect of VpeR on the expres-sion of the vpeABC operon (Fig. 3A and B). In contrast, in urine,the induction of vpeABC operon expression in the vpeR mutantdepended on growth phase, with little or no effect (an increase ofabout 1.5 times) during the exponential growth phase and an ef-fect similar to that observed in LB broth in stationary phase (in-duction by a factor of more than 15) (Fig. 3C and D). These dataconfirm that VpeR belongs to the GntR superfamily of transcrip-tional regulators, which typically respond to metabolite effectormolecules. Putative effectors present in human urine seem to in-duce the repression of vpeR transcription. These data also indicatethat the regulation of vpeABC operon expression is not purelydependent on VpeR. Instead, it is complex and controlled at mul-tiple levels, as suggested by the presence of binding sites for the
TABLE 2 Virulence of strains evaluated in the mouse lethality model
a Mice die 24 to 48 h postinfection. No deaths occur after this period.b The significance of differences in the lethality phenotype between mutants and the wild type was estimated by Fisher’s exact test; P values of �0.05 were considered significant.c The presence of pZEZeoGFP has no significant effect on the lethality phenotype of AL511 or AL511 vpeBC.
FIG 2 Effect of growth phase and urine on the transcription of the vpe genesfrom AL511. Transcription of the vpeA, -B, -C, and -R genes was analyzed byqRT-PCR after growth in either LB broth or urine. (A) A growth phase-depen-dent effect of urine on gene expression was demonstrated. Fold change indi-cates the ratio of growth in stationary phase to that in exponential phase. (B)The induction of transcription by urine is illustrated for bacteria in the station-ary growth phase. Fold change refers to the ratio of growth in urine to that inLB broth.
CRP and Mlc transcriptional regulators in the promoter region ofvpeABC genes (data not shown).
Our data suggest that the vpeABC operon encodes an EII per-mease specific for a substrate present in human urine. The use ofthis substrate as an additional carbon source might increase thefitness of the bacterium in urine. We tested this hypothesis bycarrying out coculture experiments with the AL511 strain andisogenic vpeBC mutants. The growth rates of the two strains didnot differ significantly in LB broth cocultures (Fig. 4A), but theAL511 vpeBC mutant was less competitive than the parental strainin urine cocultures. The parental strain reached a density of 109
CFU/ml after 24 h, this density then being maintained through the10 days of coculture, whereas the density of the vpeBC mutant didnot exceed 108 CFU/ml after 24 h and subsequently decreaseduntil the end of the experiment. The mutant strain was eliminatedfrom the culture after 8 to 10 days (Fig. 4B). Similar results wereobtained for coculture experiments with a mutant constructedwith a different kanamycin cassette (AL511 vpeBC [Km-FRT])(Fig. 4C). Similar results were also obtained in experiments withthree independent urine pools, indicating that the substrate spe-cifically transported by the EII permease is a constituent of humanurine in general.
As two independent mutants displayed the same growth defectin urine cocultures, we were able to exclude the hypothesis thatunknown mutations were responsible for the phenotype. Wedemonstrated that the vpeBC gene deletions, rather than expres-sion of the kanamycin cassettes, were responsible for the lowerfitness of the mutants in cocultures in urine, by introducingpZEZeovpeBC into the AL511 vpeBC mutant to complement thegrowth defect and restore fitness. qRT-PCR analysis of transcriptlevels in the resulting strain confirmed that expression of the vpeCgene in urine resulted in levels of vpeC expression about 10 timeshigher than those of the parental AL511 strain, regardless of thegrowth phase (data not shown). Despite this expression, we wereunable to demonstrate complementation of the deficiency of thevpeBC mutant in cocultures in urine. This suggests that overex-
pression of the vpeBC genes inhibits the complementation processin urine. We therefore set up mixed cultures of two mutant strainssimultaneously (AL511 vpeBC [Km-FRT] and AL511 vpeBC[FRT]). We found that the two strains displayed similar patternsof growth and maintenance over the 10 days of the experiment(Fig. 4D). These experiments confirmed that the kanamycin cas-settes used had no significant effect on the results obtained.
Various assays were carried out to identify substrates trans-ported and phosphorylated by the Vpe permease. We comparedthe abilities of the parental and mutant strains to metabolize car-bohydrates present on API 50 CH strips, but no metabolic changeattributable to the mutation could be identified. There are severalpossible reasons for this: the carbohydrate of interest was nottested, it was not present in large enough amounts to induce ex-pression of the vpe genes in the parental strain, or it has an affinityfor AL511 transporters other than Vpe. In addition, no change incell physiology associated with the loss of vpe gene function wasobserved in analyses of the various strains on phenotypic microar-rays (Biolog plates), in which we investigated carbon sources to-gether with the metabolism of nitrogen, phosphorus, and sulfur.
The vpe genes and colonization of the mouse urinary tract.We investigated the association between the growth advantage inurine conferred by expression of the vpeABC operon and uroviru-lence by comparing the survival of vpeBC mutants with that of thewild type in a mouse model of UTI. Preliminary experiments wereperformed with the parental AL511 strain to optimize the proto-col and achieve efficient colonization of the bladder and kidneys.With an inoculum of 108 CFU per mouse, significant organ colo-nization (104 CFU/g) was observed 4 days after the challenge in
FIG 3 Regulatory effect of vpeR on transcription of the vpeABC operon. Rel-ative expression of the genes in the AL511 vpeR and AL511 strains was analyzedby qRT-PCR after growth in either LB broth or urine. Downregulation wasobserved for cells grown in LB broth (A and B) and in urine (C and D), in theexponential growth and stationary phases.
FIG 4 Survival of independent AL511 vpeBC mutants in coculture experi-ments. The parental AL511 strain and the AL511 vpeBC mutant were cocul-tured in both LB broth (A) and human urine (B) for 2 weeks. The mutant wasoutcompeted by the wild-type strain only in urine. Similar results were ob-tained when AL511 was cocultured with an independent AL511 vpeBC deriv-ative (vpeBC [Km-FRT]) in urine (C). No competition was observed in urinecocultures of two independent AL511 derivatives (vpeBC [Km-FRT] andvpeBC [FRT]) (D). Similar results were obtained in at least two independentexperiments.
animals for which urine samples tested positive for the presence ofE. coli 2 days after inoculation (104 CFU/ml). Five of six micewith AL511-infected urine displayed colonization of both thebladder (1.7 � 104 to 2.6 � 108 CFU/g of organ) and the twokidneys (6 � 104 to 9.1 � 106/g of organ). One mouse with nodetectable bladder infection had bacteria in only one kidney (6 �104 CFU/g of organ). Variability in tissue titers between mice, asobserved in our experiments, has been reported in several otherstudies (19, 22) and may be accounted for by various factors, suchas differences in the administration of the inoculum, the degree ofvesicoureteral reflux, and the susceptibility of individual animals,even for mice of the same genetic background. In analyses of thesurvival of the AL511 vpeBC mutant in the UT, bladder and/orkidney colonization was also observed, and survival in the UT didnot differ significantly from that of the wild type. Only two of sixmice in which the UT was colonized by the mutant displayedbladder colonization, with 107 CFU/g of organ. However, five ofthese six mice displayed colonization of one or both kidneys, with104 to 106 CFU/g of organ (data not shown). We corrected for thevariations inherent to the model by carrying out experiments in-volving mixed infections for estimation of the relative persistenceof the strains in the UT for each mouse (Fig. 5). The wild typecolonized the bladder and kidneys to an extent similar to that
observed in single infections (from 104 to 109 CFU/g of bladdertissue and 104 to 5 � 106 CFU/g of kidney tissue). AL511 vpeBCsurvived almost as well as the wild type in the bladder (P 0.212),but its survival was markedly lower in the kidneys (P 0.0014)(Fig. 5A). The mutant was outcompeted by the wild type in thekidneys of 58% of the mice, with a mean log CI value of �0.7 (P 0.038), indicating strong attenuation of the strain. No trend to-ward a disadvantage of the mutant in the bladder was detected inCI analysis (mean log CI of �0.2, P 0.483) (Fig. 5B). No com-plementation of the poor competitiveness of the mutant was ob-tained in urine cocultures. We therefore did not carry out in vivocomplementation experiments. However, we carried out two se-ries of coinfections (with five mice in each case), which providedevidence for a specific role of the vpeBC deletion in the poor sur-vival of the mutant in the kidneys. Like the AL511 vpeBC mutant,the AL511 vpeBC (Km-FRT) strain colonized the bladder, but itssurvival in the kidneys was lower than that of the wild type. In-deed, four mice had approximately 104 CFU of each strain/g ofbladder tissue, and two mice had only the wild type in the kidneys(104 CFU/g of organ). Assuming that no cost was associated withthe introduction of the kanamycin cassette in place of the vpeBCgenes in the mutant, we expected AL511 vpeBC (Km-FRT) and itskanamycin-sensitive derivative (AL511 vpeBC [FRT]) to havesimilar colonizing abilities. Mice were infected with the twostrains simultaneously, and the two strains were recovered at sim-ilar levels from both the bladder and kidneys: four mice had 103 to104 CFU of each strain/g of bladder tissue, and three mice had 103
to 105 CFU of each strain/g of kidney tissue (data not shown).Exopolysaccharide production in urine and vpe genes. E. coli
strains produce many cell surface polysaccharides with biologicalfunctions. We investigated whether differences in exopolysaccha-ride production reflected differences in the virulence of the AL511and AL511 vpeBC strains by culturing bacteria overnight in LBbroth or human urine and labeling them with cationic ferritin.Under both sets of growing conditions, the vpeBC deletion signif-icantly decreased the amounts of cell surface-associated polysac-charides. These effects were stronger in urine than in LB broth. Inurine, substantial amounts of polymer were clearly released fromthe surface. Complementation of the vpeBC mutation with thepZEZeovpeBC plasmid significantly increased the production offerritin-labeled material at the cell surface, as observed by electronmicroscopy (Fig. 6).
The exopolysaccharides produced by E. coli strains include theO-specific polysaccharide (or O antigen) and the capsular poly-saccharide. The AL511 stain has been reported to express the O9antigen. Four capsule groups (groups 1 to 4) have been defined inE. coli (52). We determined the capsular serotype of AL511 in aPCR-based assay (24, 25, 37, 38). AL511 had typical group 4 cap-sules (data not shown). This classification was based on the am-plification of gfcC and etk, two genes of the gfc operon involved inassembly and export at the cell surface of the capsule (34) (seeTable S1 in the supplemental material). We compared the relativeexpression of gfc genes and O9 antigen (wbdB, wzt, and wzm)genes by qRT-PCR after stationary-phase culture of AL511 inurine and LB broth. The levels of expression of gfcC and etk weresimilar in the two media, but the rate of induction of wbdB, wzt,and wzm expression was more than eight times higher in urinethan in LB broth, suggesting an overproduction of the O9 antigenin urine (Fig. 7A). We then investigated the cross-regulation of vpegenes and the exopolysaccharide genes in assays comparing rela-
FIG 5 Competitive colonization of the urinary tract (UT) by AL511 andAL511 vpeBC. Two independent colonization experiments were performed ona total of 24 mice displaying UT colonization in the bladder 4 days after thesimultaneous administration of the two strains (1:1 ratio). (A) The results arereported as log CFU/g of organ for AL511 (}) and AL511 vpeBC (�). Thehorizontal bars represent the mean values. The asterisks indicate significantdifferences, with P values of �0.05 in a paired one-tailed t test. (B) The resultsare reported as log CI values. Each point (o) corresponds to a single mouse,and the horizontal bars represent the mean values. The asterisk indicates meanvalues of less than �0.3, indicating significant attenuation of the mutant.
tive expression in AL511, AL511 vpeBC, and AL511 vpeR in urine.The vpeR mutation had no significant effect on mRNA levels (datanot shown), suggesting that VpeR plays no role in exopolysaccha-ride production. Interestingly, the level of expression of the fivegenes was significantly modified (P 0.0056) in the vpeBC mu-tant. However, this modification was an increase (by a factor of 4to 9), rather than the expected decrease (Fig. 7B). These data in-dicate that the expression of vpe genes is linked to that of exopoly-saccharide genes in urine and suggest that the carbohydrate trans-
ported by the Vpe permease may affect the production ofexopolysaccharides by AL511.
Exopolysaccharide production in serum and vpe genes. Exo-polysaccharides contribute to the virulence of many bacterialpathogens, partly by protecting against complement-mediatedkilling. We therefore compared the survival, in the presence ofhuman serum, of AL511, two independent vpeBC mutants, and acomplemented mutant strain (AL511 vpeBC � pZEZeovpeBC)(Fig. 8). No significant difference in survival was observed be-tween the parental and complemented strains. In contrast, 30 minafter the start of the incubation period, we recovered significantly
FIG 6 Transmission electron micrographs of exopolysaccharide production by the wild type and vpe mutants of the AL511 strain grown in urine. Surface-expressed polysaccharides are visible as electron-dense material on the surface of bacteria stained with ferritin and examined by transmission electron micros-copy. Representative images of wild-type AL511 (A), AL511 vpeBC (B), and complemented AL511 vpeBC (C) are shown. Thick, dark, circumferential staining(solid arrow) was observed at the surface of most (89%) AL511 cells. In contrast, only a thin layer of irregular staining was identified at the surface of AL511 vpeBCcells. Labeling was discrete, with detached material visible (dashed arrow). The expression of pZEZeovpeBC restored capsule production to wild-type levels, interms of the number of stained bacteria and the thickness of the ferritin-stained layer. This thickness was estimated by measurements taken at five different pointson seven bacteria. The mean scores were 129, 27, and 84 nm for AL511, AL511 vpeBC, and complemented strains, respectively. Bars, 1 �m.
FIG 7 Transcription of genes involved in exopolysaccharide production inAL511. Transcription of genes specific for the assembly and export of group 4capsule at the cell surface (gfcC and etk) and of genes specific for the ABC-dependent mechanism of O9-antigen biosynthesis (wbdB, wzt, and wzm) wasanalyzed by qRT-PCR after growth to stationary phase in either LB broth orurine. (A) An effect of urine on the expression of genes involved in O9-antigensynthesis is illustrated. The indicated fold change is the ratio of growth in urineto that in LB broth. (B) The relative expression of the genes in the AL511 vpeBCand AL511 strains was analyzed after growth in urine. The expression of all thegenes was induced. The relative expression of the genes in the AL511 vpeR andAL511 strains was set to 1 in urine (data not shown).
FIG 8 Kinetics of the killing of AL511 wild type and vpe mutants by humanserum. Changes in cell viability were estimated after exposing bacteria to 20%normal (A) or heat-treated (B) human serum. All points are the means of atleast three independent determinations in two independent experiments. Sim-ilar data were obtained with serum samples from two other healthy people.
fewer CFU of the mutant strains than of the parental or comple-mented strains. The mutants were no longer viable after 2 h ofincubation (Fig. 8A). The inactivation of complement by heatingprevented the serum-mediated killing of the vpeBC mutants (Fig.8B). We therefore suggest that the link between vpe gene expres-sion and the virulence of AL511 may reflect a link between vpegene expression and the production of exopolysaccharides.
The vpe genes and colonization of the mouse intestine.Pathogenic E. coli infections in humans and animals are transmit-ted by the orofecal route. Adaptation to the intestinal environ-ment is therefore the first step in infection for UPEC strains. Weinvestigated the possible dependence of intestinal colonization onthe expression of vpe genes in the streptomycin-treated mousemodel of gut colonization. We fed the AL511 and mutant strainsindependently to mice, and these strains were found to have col-onized the mouse intestine to similar levels (107 to 109 CFU/g offeces) 1 day postinfection. Both strains persisted at this levelthroughout the 18 days of the assay (data not shown). The relativefitness of the AL511 and vpeBC mutant strains was evaluated intwo sets of mixed-infection experiments on a total of 15 mice.Fecal colonization by the parental strain occurred at a level similarto that in single infections, and the bacterium remained present, ata density of 107 to 109 CFU/g of feces, throughout the 18 days.However, the counts of the two parental and mutant strains weresimilar in feces after 24 h in only 11 of the 15 mice, and in these 11mice, mutant levels decreased over time (Fig. 9). For both strains,the numbers of CFU per gram of feces differed significantly be-tween 5 days after challenge (P 0.044) and the end of the exper-iment (P 0.032, P 0.016, P 0.017, and P 0.016 on days 8,12, 15, and 18 postinfection, respectively). Similarly, significantdifferences in the mean log CI were observed from 8 days afterinoculation (�1.08, �1.61, �1.56, and �1.56 on days 8, 12, 15,and 18 postinfection, respectively). Therefore, although theAL511 vpeBC mutant persisted in the intestine when fed to theanimals alone, it did not grow well enough to maintain its popu-lations in the intestine when fed to mice simultaneously with theparental strain. We checked that there was no cost associated withthe introduction of the cassette replacing the vpeBC genes inAL511 vpeBC by also carrying out a set of mixed-infection exper-iments with the AL511 vpeBC (Km-FRT) and AL511 vpeBC (FRT)mutant strains. Both these strains increased in number to 107 to108 CFU/g of feces after infection; colonization was maintained atthis level throughout the 12 days of the experiment (data notshown).
DISCUSSION
Studies are increasingly demonstrating a link between the trans-port and metabolism of various carbohydrates and virulence inenterobacteria (30). However, in most cases, the precise role ofsuch metabolic traits in the expression and regulation of virulenceremains to be determined. They may play a fundamental role infacilitating the adaptation of the bacteria to their environment, byenabling the bacteria to use the carbohydrates present in the hostfor growth. This may promote host colonization and increase thepathogenic potential of the bacteria. It has also been suggested thatthe enzymes involved in carbohydrate transport may control geneexpression in response to nutrient availability (12). Extraintestinalpathogenic E. coli (ExPEC) strains, including UPEC, are uniqueamong pathogenic E. coli strains in being able to adapt their me-tabolism first to the intestine, their reservoir, in which they must
outcompete members of the normal microflora for nutrients, andthen to the various normally sterile sites at which they need tosurvive and grow. Many operons involved in carbohydrate metab-olism have been identified in the genomes of ExPEC strains, butonly the fos, frz, and deoK operons have been reported to play arole in gut colonization (30, 41, 48). We investigated the role of thePTS system in the development of E. coli-associated infectionsoutside the gastrointestinal tract.
The PTS system is a sensing and transport system present inmany bacteria that allows substrates to cross the inner membrane.
FIG 9 Competitive colonization of the mouse intestine by AL511 and AL511vpeBC. Two independent colonization experiments were performed. Elevenmice had intestinal colonization 1 day after the simultaneous administrationof the two strains (1:1 ratio) by oral force-feeding. At the times indicated, fecalsamples were plated on LB agar with and without kanamycin. (A) The resultsare reported as log CFU/g of feces for AL511 (}) and AL511 vpeBC (�). Wearbitrarily attributed a value of 102 CFU/g of feces to a strain if no bacteria wererecovered on plates. The horizontal bars represent the mean values. The aster-isks indicate significant differences, with P values of �0.05 in a paired one-tailed t test. (B) The results of the experiments are reported as log CI values.The CI was calculated as the ratio of colony counts for the mutant to those forthe wild type recovered from feces at the various times divided by the initialratio of mutant to wild-type CFU. Each point (Œ) corresponds to a singlemouse, and the horizontal bars represent the mean values. The asterisks indi-cate mean values of less than �0.3, reflecting significant attenuation of themutant.
It uses phosphoenolpyruvate (PEP) as a source of energy. It con-sists of two general cytoplasmic components, both of which areenergy-coupling proteins required for the transfer of phosphatefrom PEP to any of a number of membrane-associated sugar per-mease complexes, also referred to as enzymes II (EII), each specificfor a substrate (15). Comparative genomics studies of E. colistrains led to identification of the vpe locus, which encodes thethree components of an EII complex (VpeA, VpeB, and VpeC)and a putative transcriptional regulator (VpeR). These genes arepresent in large strain-specific islands that frequently harbor othergenes related to carbohydrate transport and metabolism. In mostcases, the vpe and deoK loci are strongly linked (75% associa-tion). We previously showed that the deoK operon encodes pro-teins involved in deoxyribose utilization and was acquired by hor-izontal transfer from Salmonella enterica (6, 32). The high level ofsimilarity between the vpe locus and a chromosomal sequence inYersinia (75% identical to the sequence of Y. enterocolitica strain8081) and its G�C content of 46.8% suggested that E. coli ac-quired this sequence from Yersinia. The 9-kb region carrying thesetwo loci seems to have resulted from a stepwise acquisition ofDNA sequences from foreign sources.
Previous studies have reported UPEC strains to have some vir-ulence characteristics in common with diarrheagenic E. coli—par-ticularly those associated with the EAEC pathotype—suggestingthat some EAEC strains may be potential uropathogens or thatsome UPEC strains might infect the gastrointestinal tract (1). Theoccurrence of the deoK-vpe region not only in strains isolatedfrom infected urine and blood but also in EAEC isolates providessupport for this hypothesis and raises questions about the role ofthis region in intestinal and extraintestinal infections. The geneticlinkage of the deoK and vpe clusters suggests possible complemen-tary functions. The recent demonstration that deoxyribose catab-olism is involved in colonization of the intestine by pathogenic E.coli is consistent with a role for the vpe operon in host coloniza-tion. Carbohydrate metabolism has been shown to be importantfor colonization of the mammalian intestine by E. coli (9). Wetherefore investigated the role of the VpeABC EII permease in theprovision of an additional source of nutrients. We found that itincreased bacterial fitness during in vivo assays of competition incolonization of the mouse gut. Interestingly, similar results wereobtained for the UPEC AL511 and EAEC 55989 (5) strains (datanot shown).
We demonstrated that expression of the vpe cluster also plays arole in the virulence of AL511, increasing resistance to killing bycomplement and favoring the development of systemic infection.We suggest that the vpe locus responds to the availability of nutri-ents present in human urine, and we have identified the UT as asite at which the vpe-specific PTS system may also be involved inthe expression of bacterial virulence. As the induction of expres-sion in urine increased bacterial fitness during coculture experi-ments, we initially investigated the involvement of the PTS systemin fitness in vivo and UT colonization. We developed an ascendingUTI model of infection in mice, in which we showed that the vpelocus was involved in kidney colonization.
Exopolysaccharides (lipopolysaccharides [LPS], O antigens,and capsular polysaccharides) play a crucial role in bacterial sur-vival in hostile environments. Many studies have been carried out,but conflicting conclusions have been drawn regarding the role ofexopolysaccharides in urovirulence, based on the quantificationof UT colonization and mortality due to urosepsis in various
mouse models (42, 47). The K2 capsule of the CFT073 strain wasrecently shown to play a role in such infections. It was shown to beimportant for kidney colonization in a mouse model of UTI andto provide protection against complement-mediated killing (8).The AL511 strain expresses the O9 antigen and is the first UPECisolate reported to carry the gfc operon required for the produc-tion of a group 4 capsule (BLAST analysis on 3 January 2012). Wedemonstrated cross-regulation of the expression of vpe genes andproduction of exopolysaccharides in biological assays on strainscultured in human fluids (urine and serum). A cross-regulation ofvpe and exopolysaccharide gene expression was also detected inqRT-PCR assays on RNA samples extracted from overnight cul-tures in urine. In summary, an absence of vpeBC gene expressionin the mutant was found to be correlated with a significantlyhigher level of expression of both group 4 capsule and O9-antigengenes and with the excretion of polysaccharide chains retainingonly a limited association with the cell surface, suggesting poten-tial defects in the final stages of assembly or polymer export whentoo many of these polysaccharides are produced. No such modi-fications were detected in the vpeR mutant, suggesting that thesubstrate of the Vpe permease may play a role in exopolysaccha-ride expression and production. Further investigations of the in-teraction of the phosphorylated carbohydrate with polysaccharidegene expression are required and will be facilitated by determina-tion of the complete genome sequence of the AL511 strain. Indiarrhea-associated E. coli strains, group 4 capsule polysaccha-rides are produced and assembled from the same O-antigen repeatunit as used to produce LPS (34). We need to determine whethera similar process occurs in AL511.
UPEC isolates classically belong to phylogenetic group B2 andhave group 2 and 3 capsules. AL511 is, thus, an atypical UPECstrain, because it belongs to phylogenetic group A and has a group4 capsule. Preliminary studies determined the potential uroviru-lence of this strain (10, 36). We provide here the first evidence of arole for O9 antigen and/or the group 4 capsule in urovirulence andserum resistance and suggest that the production of these exopo-lysaccharides may be under metabolic control. A previous analysisof the transcriptome of a well-characterized group B2 UPECstrain, CFT073, provided evidence for upregulation of the vpelocus in vitro, during growth in human urine, and in vivo, duringUTI (49). We now need to determine whether the group 2 and 3capsules produced by classical group B2 UPEC isolates are alsounder the control of the PTS system, to improve our understand-ing of the development of urinary tract infections. Interestingly,characterization of the PAI-V from the UPEC strain 536 revealedthat the K15 (group 2/3) capsule locus involved in the uroviru-lence of this strain was genetically linked to a region of about 10 kbin length carrying the deoK operon and an EII complex-encodinglocus (47).
Pathogens require nutrients provided by the host to grow andcause disease. A comprehensive understanding of pathogen nutri-tion and metabolism may therefore provide a rational basis for thedevelopment of new pathogen identification tools and therapeuticstrategies. It is becoming increasingly apparent that there is a linkbetween the transport and metabolism of carbohydrates and vir-ulence in enterobacteria. We identified a new metabolic trait in-fluencing the expression of full virulence in a uropathogenic E. colistrain. However, as in most previous studies, the carbohydratemetabolized has yet to be identified, perhaps because it is notcommercially available or is transported by another system that
complements the vpeBC mutation. The recent massive develop-ment of equipment, software, and powerful analytical methodsshould facilitate more global studies of the link between metabo-lism and virulence. In particular, additional studies on the metab-olism of carbohydrates present in newly developed foods and di-etary supplements are required, because the catabolism of suchmolecules by pathogenic bacteria might have major implicationsfor human health.
ACKNOWLEDGMENTS
This work was supported by grants PTR-165 from the Institut Pasteur andANR-06-PATHO-002-03 from the Agence Nationale de la Recherche“ERA-NET PathoGenoMics.” V.M.-J. held a fellowship from theMinistère de l’Enseignement Supérieur et de la Recherche. The fundershad no role in study design, data collection and analysis, decision to pub-lish, or preparation of the manuscript.
We thank I. Lequeutre and L. Ma (Institut Pasteur) for expert techni-cal assistance. We thank A. M. Gilles (Institut Pasteur) and Y. Guérardel(Université des Sciences et Technologies de Lille, France) for identifyingthe carbohydrate transported by the product of vpe genes and for helpfuldiscussions. We also thank P. Trieu-Cuot for critical reading of the man-uscript.
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