Microbial Pathogenesis 1997; 23: 49–53

PATHOGENESISMICROBIAL

The role of O1-antigen in the adhesion touroepithelial cells of Klebsiella pneumoniaegrown in urineSusana Merino, Xavier Rubires, Alicia Aguilar & Juan M. Tomas∗

Departamento de Microbiologıa, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain

(Received July 30, 1996; accepted in revised form January 23, 1997)

We obtained mutants devoid of the O1-antigen, the capsular polysaccharide (K antigen) or bothfrom Klebsiella pneumoniae clinical isolates (urinary infection). These mutants were grown inurine, and their ability to fimbriate and to adhere were studied. Mutants lacking the O1-antigen,independently of the other surface molecules (capsule and fimbriae), showed a great decrease inadhesion to these cells. 1997 Academic Press Limited

Key words: Klebsiella, lipopolysaccharide, capsule, fimbriae, adhesion to uroepithelial cells.

growing in vivo are known to differ markedlyIntroductionfrom cells cultured in conventional laboratorymedia, with important consequences for the ex-

Klebsiella pneumoniae is a naturally encapsulated pression of virulence determinants [8]. We de-bacterium and an important opportunistic scribe here the isolation of mutants devoid ofpathogen [1]. The presence of a smooth lipo- the O1-antigen, the capsule, or both from K.polysaccharide (LPS) in its cell wall provides a pneumoniae isolated from urinary infection. Wegood model to study the contribution of both the also characterized their ability to fimbriate whenO-antigen and capsule (K antigen) to bacterial they grow in urine and studied the role of thesevirulence. K. pneumoniae is one of the most com- surface molecules (O-antigen, capsular poly-mon organisms isolated in urinary tract in- saccharide and fimbriae) in adhesion to uro-fections (UTI) in man [1] and animals [2, 3]. In epithelial cells (UEC).other organisms involved in UTI, like Escherichiacoli, a number of virulence factors have beenrecognized, including some O-antigens [4], someK-antigens [5], some fimbrial antigens [6], or the Resultsability to produce haemolysin [5] or aerobactin[7]. Fimbrial expression

The physiology and biochemistry of bacteriaNo differences between fimbrial expression werefound on cells growing on CFA plates or in∗Author to whom correspondence should be addressed.

0882–4010/97/070049+05 $25.00/0 1997 Academic Press Limitedmi970132

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Figure 1. Competitive ELISA using purified type 1 and 3 fimbriae as antigen (10 lg) with different dilutionsof pooled specific antiserum against type 1 and 3 of fimbriae absorbed by whole cells (107) of K. pneumoniaestrains grown in urine as competitors. (a) Strain KT1134 (O1:K1) and their isogenic mutants; Β: wild-type;Χ: KT1136 (O1:K−); Α: KT1138 (O−:K1); Μ: KT1139 (O−:K−). (b) Strain KT1085 (O1:K2) and their isogenicmutants; Β: wild-type; Χ: KT1094 (O1:K−); Α: KT1095 (O−K2); Μ: KT1096 (O−:K−).

Table 1. The adhesion of different K. pneumoniae and only a partial competition (always clearlystrains growing in urine to UEC cells. inferior to that seen with the unencapsulated

mutants) was observed for the K2 strains [Fig.Strain Percentage of 1(b)].bacteria adhereda

Bacterial adherence to UEC cells136-94 (O1:K2) 45.6±4.2KT1085 (O1:K2) 45.9±4.0KT1094 (O1:K−) 48.3±4.1 The wild-type strains (136-94 and 204-14), theKT1095 (O−:K2) 16.8±3.2 same strains harbouring pTROY11 plasmidKT1096 (O−:K−) 9.6±2.9 (KT1085 and KT1134, respectively), and their

isogenic unencapsulated mutants (KT1094 and204-94 (O1:K1) 41.0±3.8 KT1136, respectively) showed a strong ad-KT1134 (O1:K1) 40.8±4.0 herence to UEC cells, always to >40% (Table 1).KT1136 (O1:K−) 48.2±4.3

However, isogenic mutants devoid of the O-KT1138 (O−:K2) 15.3±3.3antigen (with or without capsule) showed a veryKT1139 (O−:K−) 10.1±2.7low adherence to UEC cells, always to <17%;

a The percentage of bacteria adhered to UEC cells±SD was the double mutants (O−:K−) showed the lowestcalculated as described in Materials and methods. All the adherence (to <11%). It is also interesting toassays were done in triplicate. Student’s t-test, P<0.005. point out that the percentage of adherence of

the unencapsulated mutants was slightly higherthan that of their wild-type parents (O+:K+),urine. The unencapsulated mutants (either O+this difference was more evident in the lineageor O−) were clearly agglutinated by anti-type 1in which the K1 capsular polysaccharide com-and anti-type 3 fimbriae serum, while the wild-pletely covered the O1-antigen [9]; and less evid-type strain O1:K2 was poorly agglutinated, andent in the K2 lineage.the wild type strain O1:K1 was not agglutinated.

The results of a competitive ELISA using puri-fied fimbriae from strain KT1094 (K−) an antigen Discussionand pooled antiserum against type 1 and type3 of fimbriae, whole cells grown in urine ascompetitor, showed that K− cells (either O+ or K. pneumoniae 136-94 and 204-94 were recent

clinical isolates from human urinary tract in-O−) competed efficiently, while no competitionwas observed with the K1 strains [Fig. 1(a)], fection. From these isolates we obtained mutants

Adherence of K. pneumoniae to uroepithelial cells 51

unable to synthesize K-antigens (un- identical surface molecules studied as their wildtypes.encapsulated), LPS mutants devoid of the O1-

antigen, and double mutants lacking both these We isolated mutants from the wild-typestrains using lambda Tn5 insertion mutagenesis.surface antigens. When these strains were grown

in urine and studied for their adherence to UEC, K. pneumoniae wild-type strains were first trans-formed with pTROY11 plasmid DNA [15] andwe found that:selected on ampicillin-containing LB agar (2 mg/

(1) The O1-antigen is important for the ad- ml) to obtain bacteria expressing the lamB re-hesion; mutants lacking the O1-antigen ceptor prior to infection with lambda Tn5 (b21,showed a drastic reduction in their adhesion Oam, Pam, rex:Tn5, c11857) [16]. Tn5-inducedto UEC cells, independently of the other mutants were selected on kanamycin-containingsurface molecules. It has been shown in en- LB agar (25 lg/ml) and K− or O− were pre-teric bacteria that the O-antigen LPS is an liminary identified by colony blotting using spe-important colonization factor [10], but some- cific antisera raised against LPS, K1 and K2times is not known in which step of the capsular polysaccharide. O− mutants (KT1095colonization is implicated. It seems clear that and KT1138, respectively) were reconfirmed byin our case it is in the primary step of col- LPS gels and phage sensitivity while K−mutantsonization, i.e. the adhesion. We suggest that (KT1094 and KT1136, respectively) were re-the lack of the O1-antigen increased the neg- confirmed as previously indicated [17].ative surface charge of the bacteria [11] and We obtained double mutants (O−:K−) as spon-this influenced the bacterial association to taneous mutants of K− strains by resistancethe UEC cells. to bacteriophage FC3-1 (bacteriophage whose

(2) Unencapsulated mutants (O− or O+) showed receptor is the O-antigen of LPS9) as we pre-no differences on fimbrial expression (Fig. viously reported [9]. They were devoid of the1) and neither in the proportion of type 1/ high molecular weight LPS (O1-antigen) by LPStype 3 (data not shown). gels and as all the O− mutants unable to react

(3) Because K1 completely masked the O1-an- with Mab 2A4 (specific for the O1-antigen [18]).tigen [9] no fimbriae was detected in these All the strains showed identical studied surfacestrains and only a minor amount of fimbriae molecules when they grow in LB or urine.was detected on K2 (coexpressed in the sur- Bacteriophages FC3-1 and FC3-2 were par-face together with O1-antigen [9] strains. It tially characterized previously by us, and wereseems that high expression of one adhesin propagated on K. pneumoniae C3 [9]; and bac-like K antigens [12] impedes or decreases teriophages U1 and U2 on K. pneumoniae DL1expression of other adhesin like fimbriae (O1:K1) and 52145 (O1:K2), respectively. The[12]. There are some examples about the basal medium used for bacterial growth andinterference on the expression of both ad- phage propagation was Luria broth (LB [19]) orhesins [13]. LB with 1.5% agar. To prepare soft-agar we

added 0.6% agar to LB. Bacteria were also grownin pooled human urine as previously described[14].

Materials and methodsLPS isolation and analyses

Bacteria, bacteriophages and mediaLPS was purified by the method of Westphal &Jann [20]. Purified LPS was further analysedThe K. pneumoniae strains used are listed on

Table 2. Urinary tract isolates 136-94 and 204-94 by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and silver stainedwere serotyped in our laboratory, unable to

produce haemolysin, and growing in urine by the method of Tsai & Frasch [21].showed the expression of five outer membraneproteins iron regulated as we previously re- Antiseraported for other Klebsiella strains [14]. StrainsKT1085 and KT1134 were obtaining after trans- Anti-LPS (serotype O1), anti-K1 and anti-K2 sera

was obtained as previously described [9]. Mono-forming wild type-strains 136-94 and 204-94 withpTROY11 plasmid DNA, in order to allow Tn5 clonal antibody (MAb) 2A4 specific for the O1-

antigen LPS of K. pneumoniae [18] was a giftinsertion mutagenesis (Table 2) and their showed

S. Merino et al.52

Table 2. Strains of K. pneumoniae used and their relevant characteristics and origin.

Strain Relevant characteristics Origin

136-94 O1:K2; wild type Isolated from urine infectionKT1085 136-94 harboring pTROY11 plasmid This workKT1094 O1:K−; Tn5 unencapsulated mutant This work

derived from KT1085KT1095 O−:K2; Tn5 LPS mutant (O−) derived This work

from KT1085KT1096 O−:K; FC3-1 resistant mutant This work

derived from KT1094204-94 O1:K1; wild type Isolated from urine infectionKT1134 204-94 harbouring pTROY11 plasmid This workKT1136 O1:K−; Tn5 unencapsulated mutant This work

derived from KT1134KT1138 O−:K1; Tn5 LPS mutant (O−) derived This work

from KT1134KT1139 O−:K−; FC3-1 resistant mutant This work

derived from KT1136DL1 O1:K1; bacterial host for U1 [14]52145 O1:K2; bacterial host for U2 [9]

from E. Mandine. Antisera against type 1 and Falkowski et al. [22]. Briefly, samples containing0.2 ml of 107 of K. pneumoniae cells/ml growntype 3 of fimbriae were kindly provided by T.

K. Korhonen. in urine and 0.2 ml of 105 UEC cells/ml werecombined and gently swirled at 37°C for 1 h.After incubation, 0.2 ml of each sample waswashed extensively and filtered under vacuumImmunoscreening of Tn5 mutantsthrough a polycarbonate membrane filter (poresize, 5 lm; Nucleopore Corp., Pleasanton, CA).Mutants grown on LB agar plates (plus 25 lg/The filters were solubilized with Hank’s bal-ml of kanamycin) were overlayed with precutanced salt solution prior to lysing the UEC cellsnitrocellulose membranes (Millipore Corp., Bed-with 0.01% Triton X-100 and enumeration offord, MA). Dried nitrocellulose membranes weretotal bacteria by plate count. The percentage ofthen blocked, immunostained with rabbit anti-bacteria adhered to UEC cells was calculated byserum (1:1000) and stained with goat anti-rabbitdividing the number of cfu of filters with theimmunoglobulin G alkaline phosphatase con-mixture of bacteria and UEC minus the numberjugate (Bio-Rad). The colonies that gave negativeof cfu of filters with bacteria in the absencesignals were purified and rescreened by theof UEC (non-specific adherence) by the totalsame procedure.number of cfu of the unfiltered bacteria. Ad-herence was also examined in some experimentsby direct visualization of gram-stained speci-Bacterial adherence assaymens in which a minimum of 40 UEC cells wereexamined. All assays were done in triplicate.Uroepithelial cells (UEC) were obtained from

the first morning urine of healthy adult femaleswithin 2 h of collection by centrifugation,washed twice with cold phosphate-buffered-sa-

Acknowledgementsline (PBS) and resuspended in minimal essentialmedium containing Earle salts (GIBCO Laborat-ories, Grand Island, NY) at pH 7.2 to a con- This work has been supported by a research grantcentration of 2×105 cells per ml. Cell viability PB94-0906 from DGICYT (Mintiserio de Education ydetermined by triptan blue exclusion was always Ciencia), X.R. and A.A. for a fellowship from the>80%. same institution. We thank I. Ørskov for serotyping

some strains, E. Mandine for the MAb 2A4 and T. K.The adherence assay was done as described by

Adherence of K. pneumoniae to uroepithelial cells 53

11 Camprubı S, Merino S, Benedı VJ, Williams P, TomasKorhonen for the antisera against type 1 and 3 ofJM. Physicochemical surface properties of KlebsiellaK. pneumoniae fimbriae. We also acknowledge thepneumoniae. Curr Microbiol 1992; 24: 31–3.SGR00414 grant from Generalitat de Catalunya.

12 Favre-Bonte S, Darfeuille-Michaud A, Forestier C. Ag-gregative adherence of Klebsiella pneumoniae to humanIntestine-407 cells. Infect Immun 1995; 63: 1318–28.

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