INFECTION AND IMMUNITY, Mar. 1993, p. 852-8600019-9567/93/030852-09$02.00/0Copyright ©) 1993, American Society for Microbiology

Clq Binding and Activation of the Complement ClassicalPathway by Klebsiella pneumoniae Outer Membrane Proteins

SEBASTIAN ALBERTI,1 GUILLERMO MARQUES,2 SILVIA CAMPRUBI,3 SUSANA MERINO,3JUAN M. TOMAS,3 FERNANDO VIVANCO,2 AND VICENTE J. BENEDI1*

Universidad de las Islas Baleares and Instituto de Estudios Avanzados (UIB-CSIC), Departamento deBiologia Ambiental, Area de Microbiologia, Crtra. de Valldemosa Km. 7.5, E-07071 Palma deMallorca,1 Fundacion Jimenez Diaz, Depto. de Inmunologia, and Universidad Complutense,Departamento de Bioquimica y Biologia Molecular I, E-28040 Madrid,2 and Universidad

de Barcelona, Depto. de Microbiologia, E-08071 Barcelona,3 Spain

Received 19 August 1992/Accepted 16 December 1992

The mechanisms of killing of Klebsiella pneumonwiae serum-sensitive strains in nonimmune serum by thecomplement classical pathway have been studied. The bacterial cell surface components that bind Clq more

efficiently were identified as two major outer membrane proteins, presumably the porins of this bacterialspecies. These two outer membrane proteins were isolated from a representative serum-sensitive strain. Wehave demonstrated that in their purified form, they bind Clq and activate the classical pathway in an

antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum totalhemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum andagammaglobulinemic serum (both depleted in factor D). Binding of Clq to other components of the bacterialouter membrane, in particular the rough lipopolysaccharide, could not be demonstrated. Activation of theclassical pathway by this lipopolysaccharide was also much less efficient than activation by the two outermembrane proteins. The antibody-independent binding of Clq to serum-sensitive strains was independent ofthe presence of capsular polysaccharide, while strains possessing lipopolysaccharide 0 antigen bind less Clqand are resistant to complement-mediated killing.

The complement system plays a key role in the defenseagainst microorganisms. Its importance is clearly seen inindividuals with complement deficiencies because they havean increased risk to develop severe and recurrent microbialinfections (14). Resistance to the complement action is thena requisite for pathogenic microorganisms, which have de-veloped a variety of mechanisms to ensure survival innonimmune serum (9, 23). Gram-negative bacteria activatecomplement via the classical or the alternative pathway, andmore frequently both pathways are required for the effectiveelimination of serum-sensitive strains (53). Activation of theclassical pathway usually requires the presence of antibodiesbound to bacterial antigens, whereas the alternative pathwayis activated by certain bacterial surfaces by an antibody-independent mechanism (24).Blood isolates of Klebsiella pneumoniae (6), as well as

nearly all of the gram-negative bacteria tested (44), areresistant to complement-mediated killing in nonimmune se-rum.K pneumoniae is an important opportunistic pathogen,particularly in patients under stress (8, 18, 56), that, unlikeother enterobacteria, has both capsule and lipopolysaccha-ride (LPS) in its cell surface. We have previously demon-strated that the 0 side chain of the LPS confers resistance tocomplement in this bacterial species, whereas the capsuledoes not play an important role in this phenomenon (11, 47).Results in these works showed that strains with rough LPSwere sensitive to the action of complement and that sensi-tivity was increased in LPS deep-rough mutants. This findingsuggested the existence of molecules binding and activatingcomplement in the cell surface of those bacterial strains.

In the present study, we have focused on defining the

* Corresponding author.

852

mechanisms of complement sensitivity in this bacterium.Both the classical and alternative complement pathwayswere effective in the elimination of K pneumoniae serum-sensitive strains in nonimmune serum, although bacterialkilling was more effective when both pathways were active.Activation of the classical pathway by these strains wasstudied in more detail, and we have identified two bacterialouter membrane proteins (OMP) that bind Clq and activatethis pathway in nonimmune serum and in agammaglobuline-mic (agamma) serum.

MATERIALS AND METHODS

Bacteria, bacteriophage, and media. The serum-sensitiveK pneumoniae strain KT793 (0-:K-) used in this study is adouble mutant obtained from the serum-resistant K pneu-moniae strain C3 (serotypes O1:K66). It was made unencap-sulated (K-) after UV mutagenesis and selection with anti-capsular serum, as described before (5). Spontaneous LPS-deficient mutants (O-) were obtained from the K- mutantstrain by selection with the LPS-specific bacteriophageFC3-2, as described previously (48). The other serum-sensi-tive strains used were KT701, KT702, and KT707, whichwere derived from the wild-type C3 strain and which were

serotypes 0-:K66 (47). Strain KT707 shows only one OMPwith an Mr of about 36,000 in the range of 35,000 to 40,000(47). Strains KT717 (47) and KT791 (an isogenic mutant fromstrain C3) (5) are serum-resistant O1:K- strains. Luria brothor nutrient broth was used for bacterial growth and phagepropagation (33).Human sera. A pool of nonimmune human sera (NHS) was

obtained from healthy volunteers. NHS diluted 1/100 did notreact with OMP from K pneumoniae KT793 in Western blot(immunoblot) experiments. NHS was made deficient in Clq

Vol. 61, No. 3

on August 14, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

Clq BINDING BY K PNEUMONIAE PROTEINS 853

(a classical pathway component) or factor B (an alternativepathway component) as described previously (20, 25). TheClq titer in Clq-deficient serum was 6.8 x 105 hemolyticallyactive molecules per ,ul; in NHS, it was 1.9 x 109 hemolyt-ically active molecules per ,ul. Factor B-deficient serumcontained less than 0.5% of the original amount of factor B.Classical and alternative pathway activities were less than 1and 5%, respectively, in Clq-deficient serum and factorB-deficient serum, as measured by hemolytic assays detailedin previous works (41, 42). Serum depleted in Clq and factorD was obtained from NHS or agammaglobulinemic serum byBioRex 70 chromatography (39) and reconstituted with pu-rified Clq to a final concentration of 80 ,ug/ml. Beforereconstitution, the amounts of Clq and factor D werequantitated (41, 42), and they were less than 0.5% of theiroriginal amounts. After reconstitution with purified Clq, theCH50 was more than 80% of the original serum, while theAH50 was less than 1%. Agammaglobulinemic serum wasobtained from a patient receiving chemotherapy for a non-Hodgkins lymphoma. This serum contained 0.38, 0.07, 0.08,1.26, 0.21, and 0.30 mg of immunoglobulin G (IgG), IgM,IgA, C3, C4, and factor B, respectively, per ml; the totalcontent of proteins in this serum was 57.40 mg/ml.Clq purification and labeling. Clq was purified from NHS

by high-pH precipitation, ion-exchange chromatography onBioRex 70, and gel filtration on Bio-Gel A5m (27, 45) asdescribed previously (2). The purity of the isolated Clq wasverified by polyacrylamide gel electrophoresis (PAGE). To-dination of purified Clq was carried out with lactoperoxi-dase-glucose oxidase as described previously (45). Clq wasbiotinylated with sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate (Pierce) at a Clq-to-biotin molar ratio of 1to 25 for 4 h at room temperature (19). After extensivedialysis at 4°C against phosphate-buffered saline (PBS),labeled Clq was stored at -70°C. Purified and labeled Clq(both biotinylated and iodinated) were hemolytically activeand able to interact with immune complexes (2).

Bacterial cell surface isolation and analysis. Bacterial cellenvelopes, containing cytoplasmic and outer membranes,were obtained by French press cell lysis and centrifugation.OMP were isolated as sodium lauryl-sarcosinate-insolublematerial (15, 47). Clq-binding OMP were isolated and iden-tified by electrophoresis and Western blot analysis (seebelow). For their isolation, cell envelopes of strain KT793were suspended in 10mM Tris-HCl (pH 8.0) and treated withtrypsin (36) for 24 h at 37°C at a protein-to-enzyme ratio of 10to 1. After digestion, treated cell envelopes were subjectedto a standard method for the isolation of bacterial porinproteins (35). Briefly, 2% sodium dodecyl sulfate (SDS) wasadded to the trypsin-treated material, and it was incubated at32°C for 1 h. Insoluble material was pelleted by centrifuga-tion at 100,000 x g for 1 h, treated again with 2% SDS, andpelleted. After solubilization in 50 mM Tris (pH 7.7) con-taining 1% SDS, 0.4 M NaCl, 5 mM EDTA, and 0.05%,B-mercaptoethanol, proteins were separated from LPS bySephacryl S-200 chromatography equilibrated in the solubi-lization buffer. This chromatography was carried out in acolumn (1.6 by 90 cm), and fractions (2 ml) were monitoredat 280 nm and analyzed as detailed below. Fractions con-taining Clq-binding proteins were pooled and then dialyzedagainst 3 mM sodium azide, first for 1 day at room temper-ature and then for 7 to 9 days at 4°C. They were finallytreated with phenol to remove LPS contamination (21). TheLPS content of the purified Clq-binding proteins was as-sessed by electrophoresis in 15% polyacrylamide gels andsilver staining (50, 51) and by the Limulus amoebocyte lysate

assay (46) with purified Escherichia coli 055:B5 LPS (Sig-ma) as the standard. The protein content was determined bythe method of Lowry et al. (31) with bovine serum albuminas the standard.The K pneumoniae high-molecular-mass Clq-binding

protein was isolated from the mutant strain KT793 thatexpresses only this protein (OmpK36) in the molecular massrange of 35 to 40 kDa. The isolation and purification ofOmpK36 were performed exactly as described above for theClq-binding proteins of strain KT793. The N-terminal se-quence of OmpK36 was determined in an Applied Biosys-tems 470A gas-phase sequencer as previously described (4).

Protein components of the bacterial cell surface andchromatography fractions were analyzed by electrophoresisin an 11% acrylamide-0.1% SDS resolving gel with 25 mMTris (pH 8.3)-192 mM glycine-0.1% SDS as the runningbuffer (SDS-PAGE). Samples (about 5 p,g of OMP or 2 ,ug ofClq-binding proteins) were boiled for 5 min in Laemmli'ssample buffer before analysis. For the identification ofbacterial Clq-binding proteins, after SDS-PAGE separation,electroblotting to polyvinylidene difluoride (Millipore) mem-branes was carried out at 1 A for 1 h (49). After blocking inPBS containing 1% bovine serum albumin (PBS-BSA) for 2h, membranes were incubated with purified biotin-labeledClq (0.2 mg in PBS-BSA, 37°C, for 1 h) and alkalinephosphatase-labeled avidin (Sigma; 0.1 ,ug in PBS-BSA,37°C, for 1 h). Alternatively, membranes were sequentiallyincubated with NHS (diluted 1/10 in PBS, 5 min), rabbitanti-Clq, and alkaline phosphatase-labeled goat anti-rabbitIgG. Incubations with the antisera were carried out at 37°Cfor 1 h, and washing steps with PBS were included after theincubations. Alkaline phosphatase was visualized on theblots with BCIP-NBT (5-bromo-4-chloro-3-indolylphosphatedisodium-nitroblue tetrazolium) (7).LPSs from strain KT793 and from a smooth-LPS Salmo-

nella typhimunum strain were isolated by the phenol-watermethod of Westphal and Jann (54). These LPSs were ana-lyzed by SDS-PAGE in 15% polyacrylamide gels and bysilver staining (50, 51).

Antisera. Antisera against Clq were raised in NewZealand White rabbits by immunization with 1 ml containing50 to 100 pg of purified Clq in complete Freund's adjuvant(Difco). Rabbits were injected intramuscularly once a weekfor a 6-week period and bled 10 to 14 days after the lastinjection.

Binding of Clq to bacterial cells. Mid-logarithmic-phasebacterial cells were recovered by centrifugation, washedwith PBS, and incubated sequentially with biotin-labeledClq (0.2 mg in PBS, 1 h) and colloidal gold-labeled avidin(Sigma; diluted 1 to 20 in PBS, 1 h). Washing steps with PBSwere included after each incubation period. Controls (KT793cells) to show the specifity of the Clq binding to the bacterialcells were treated only with the gold-labeled reagent. Cellswere observed with a Hitachi H600 electron microscope at75 kV.

Binding was also studied with radiolabeled Clq as de-scribed previously (3). Briefly, early-stationary-phase bacte-rial cells were recovered and washed by centrifugation withDGVB2" (5 mM barbital [pH 7.51-70 mM NaCl-0.15 MCaCl2-0.5 mM MgCl2-2.5% dextrose-0.5% gelatin). Tubescontaining 108 bacterial cells (in 50 ,ul of DGVB2+) anddifferent amounts of 125I-labeled Clq (in 150 ,ul of DGVB2+)were incubated at 0°C for 45 min. After centrifugation,pellets (cell-bound "2I-labeled Clq) and supernatant fluid(unbound 1"I-labeled Clq) were counted. Control tubeswithout bacteria were handled as described above. The

VOL. 61, 1993

on August 14, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

854 ALBERTI ET AL.

g ,01

,001

,0001- . . . . .0 20 40 60

Time (min)

FIG. 1. Survival ofK pneumoniae KT793 in nonimmune serum(closed circles), heat-inactivated (30 min, 56°C) serum (open cir-cles), Clq-deficient serum (open squares), factor B-deficient serum(open triangles), and Clq-deficient serum reconstituted with Clq(closed squares). The results are the averages of at least fiveindependent experiments.

binding of Clq to different 0 and K mutant strains was

studied as described above, except that 109 bacterial cells, a

fixed amount of '25I-labeled Clq (500 ng), and an incubationtime and temperature of 30 min and 4°C were used.

Bacterial survival in human serum. Bacterial cells (108CFU) of the serum-sensitive strains in the logarithmic phasewere suspended in 10% serum-PBS and incubated at 37°C.Viable counts were made at different times by dilution andplating.Complement assays. Human sera were incubated for 45

min at 37°C with Clq-binding proteins or LPS purified fromstrain KT793. After incubation, CH50 (37, 52) and C4 con-

sumption (1, 16) were measured. AH50 was determined byusing rabbit erythrocytes as described previously (37).

RESULTS

Complement sensitivity of K. pneumoniae. Killing of Kpneumoniae serum-sensitive strains KT701, KT702, andKT793 was mediated by both the classical and alternativepathways of complement. An example of this type of exper-iment, performed with strain KT793, is shown in Fig. 1.Figure 1 shows that after incubation in nonimmune serum for1 h, there is a decrease of 6 orders of magnitude in bacterialviability. Control incubations in heat-inactivated serum

showed that complement was responsible for the loss ofviability observed. To further assess the role of the classicaland alternative pathways, we incubated bacteria in Clq- or

factor B-deficient serum. After incubation in either serum,there was a decrease of 3 orders of magnitude in viability,demonstrating that neither of the two pathways alone ac-

counts for the decrease in viability observed with completeserum. As a supplementary control, we also incubatedbacteria in Clq-deficient serum reconstituted with purifiedClq. There was a decrease of almost 5 orders of magnitudein bacterial viability after incubation in Clq-reconstitutedserum.

Binding of Clq to bacterial cells. Accessibility and bindingof Clq to bacterial cells were studied by electron microscopywith biotin-labeled purified Clq and gold-labeled avidin. A

A

C

B

{Jir

Frt-tClq fn.kt D

FIG. 2. Binding of Clq to K pneumoniae cells demonstrated byelectron microscopy (A, B, and C) and by direct binding of 125-labeled Clq (D). Cells were incubated with biotinylated Clq andavidin-colloidal gold (A and C) or with avidin-colloidal gold alone(B). Cells in panels A and B correspond to K pneumoniae KT793,and those in panel C correspond to strain C3. Colloidal gold particleswere 20 nm in diameter. Panel D shows the binding of "NI-labeledClq to cells from strains C3 (open circles) and KT793 (closedcircles).

representative example of this type of experiment is depictedin Fig. 2. Cells of the serum-sensitive strain KT793 treatedwith biotinylated Clq and colloidal gold-labeled avidinbound Clq, and Clq was visualized as gold beads associatedwith the cells (Fig. 2A). Control experiments with KT793cells incubated only with the gold-labeled reagent (Fig. 2B)or with cells from the serum-resistant strain C3 (Fig. 2C)treated as described for Fig. 2A showed the absence of goldbeads bound to these cells, i.e., biotinylated Clq was notbound by these cells.

Binding of Clq was more precisely quantitated for strainsKT793 and C3 by using 251I-labeled Clq. As shown in Fig.2D, cells of the serum-sensitive strain KT793 bind Clq muchmore efficiently than cells from the serum-resistant strainC3.

Identification and isolation of bacterial structures involvedin Clq binding. We then identified the molecules of thebacterial cell surface responsible for the observed Clqbinding. Since strain KT793 is devoid of LPS 0 antigen andcapsular polysaccharide, candidate molecules were OMPand rough LPS. Figure 3, lane 1, shows the electrophoreticprofile of strain KT793 OMP after SDS-PAGE andCoomassie blue staining. Only a few proteins were visual-ized, as is typical for enterobacterial outer membranes. Thebinding of Clq to OMP and identification of the OMPresponsible for this binding were studied by Western blot.Filters were incubated with either biotinylated Clq andavidin-alkaline phosphatase (lane 2) or with NHS, rabbitanti-human Clq, and alkaline phosphatase-labeled goat anti-rabbit IgG (lane 4). In both cases, two bands with molecular

INFECT. IMMUN.

I

on August 14, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

Clq BINDING BY K PNEUMONIAE PROTEINS 855

A

66*

.445

36,-

29- 40 60Fraction

24

D

20.1 0

FIG. 3. Binding of Clq to K pneumoniae KT793 OMP. OMPwere separated by PAGE and stained with Coomassie blue (lane 1)or electroblotted and probed with either biotinylated Clq andalkaline phosphatase-labeled avidin (lane 2) or NHS, rabbit anti-Clq, and alkaline phosphatase-labeled anti-rabbit IgG (lane 4). BSA(lane 3) was also electroblotted from the gel and incubated as

described for lane 4. Molecular masses indicated on the left of thefigure are in kilodaltons.

masses of approximately 36 and 35 kDa were stronglystained, and a faint band with a higher molecular mass wasalso detected. Lane 3, containing blotted BSA, was treatedin the manner described for lane 4 to demonstrate thespecificity of the analysis. An additional indirect proof forthis specificity is deducted from the fact that a low-molecu-lar-weight major OMP was not detected in the Westernblots. Finally, since lane 2 was analyzed with purified Clq, itis concluded that Clq binding to these OMP is antibodyindependent. Identical results, i.e., binding of Clq to twomajor OMP of strains KT`701 and KT702 (one protein instrain KT707), was observed in Western blot experimentscarried out as described above for strain KT793 (data notshown).To further characterize the binding phenomenon, we

isolated and purified the bacterial OMP previously shown tobind Clq. For this purpose, we took KT793 as a represen-tative strain of the serum-sensitive strains studied in previ-ous experiments. The molecular masses of the two Clq-binding proteins suggested that they were porins. We thentried to isolate them by following protocols for E. coli porinisolation. The peptidoglycan-associated material ofK pneu-

moniae KT793 was obtained in this way and subjected to gelpermeation chromatography (Fig. 4A). Three groups offractions (I, II, and III) were individually pooled and sub-jected to further analysis.

I.

B

66to

4513 ,

36? a

291

241

20.1 *

1 2 3 4 5

C

f

i.i1 2 3 4 5 1 2 3 4 5FIG. 4. Isolation of Clq-binding proteins from K. pneumoniae

KT793. Cell envelopes were treated with trypsin and subjected to aporin isolation method. (A) Material isolated by this method (LPSand peptidoglycan-associated proteins) was separated by gel perme-ation chromatography on Sephacryl S-200. Fractions were scannedat 280 nm, and three pools were made (I, II, and III). (B) Pools wereanalyzed by SDS-PAGE and Coomassie blue staining. Lanes: 1,total OMP from strain KT793; 2, fraction pool I; 3, fraction pool II;4, fraction pool III; 5, BSA. The numbers on the left are molecularmass markers in kilodaltons. (C) Western blot analysis with bio-tinylated Clq. Lanes correspond to those described for panel B. (D)SDS-PAGE and silver staining of LPS. Lanes: 1, LPS purified fromstrain KT793; 2, chromatography pool I (1 jig of protein); 3,chromatography pool II (1 jig of LPS); 4, chromatography pool III;5, smooth LPS from S. typhimurium (2 ,ug of LPS).

Analysis by SDS-PAGE and Coomassie blue stainingshowed that only fraction I (Fig. 4B, lane 2) contained OMPor, specifically, two proteins with molecular masses coincid-ing with those of the previously shown Clq-binding proteins.Fractions II and III contained no appreciable amounts ofproteins (lanes 3 and 4). Lane 1 shows the unfractionatedOMP profile ofK pneumoniae KT793 as a reference, andlane 5 contained BSA as a control for the Western blotexperiment shown in Fig. 4C.The chromatographic fractions were also analyzed by

Western blot with biotinylated Clq (Fig. 4C). The results ofthis analysis show that the two major Clq-binding proteinsof theK pneumoniae KT793 outer membrane (lane 1) were

VOL. 61, 1993

on August 14, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

856 ALBERTI ET AL.

A .**-B

1 234567FIG. 5. Dot blot analysis with purified Clq labeled with biotin.

Purified Clq-binding proteins (row A), LPS from strain KT793 (rowB), and BSA (row C) were dot blotted starting at a concentration of10 ,ug per ml (column 1) and then at twofold dilutions (columns 2through 7). The volume used in each instance was 100 pLl. Binding ofbiotinylated Clq was detected with alkaline phosphatase-labeledavidin.

recovered in fraction I after chromatography (lane 2). Theother fractions (lanes 3 and 4) and BSA (lane 5; included as

a control in the Western blot) did not bind Clq.Finally, we identified by SDS-PAGE and silver staining

(Fig. 4D) that the material contained in fraction II (lane 3)was the rough LPS of strain KT793. This can be deducted bycomparison of the material in fraction II with LPS purifiedfrom this strain (lane 1) and smooth S. typhimunum LPS(lane 5). Fractions I and III in the chromatogram (lanes 2 and4, respectively) did not contain material detectable by thistype of analysis. After phenol treatment to further remove

LPS contamination, the amount of LPS in fraction I, deter-mined by the Limulus amoebocyte lysate assay, was about1.5 pg per 10 ,ug of protein.

In summary, the Clq-binding proteins of strain KT793with a very reduced content of LPS could be isolated. Afterisolation, they bind Clq in Western blot experiments,whereas binding of Clq to LPS could not be demonstrated.The isolation of a protein equivalent to the high-molecular-mass Clq-binding protein of strain KT793 was accomplishedfrom the mutant strain KT707 expressing only this protein(OmpK36). The purified OmpK36 protein also bound Clq inWestern blot experiments and contained an amount ofcontaminating LPS similar to the amount contained in theClq-binding proteins purified from KT793 (data not shown).The N-terminal sequence of OmpK36 (one-letter code foramino acids) was as follows: AEIYNKDGNKLDLYGKIDGLHYFS. Therefore, OmpK36 contains in its N termi-nus a typical sequence of enterobacterial porins (22).

Contribution of cell surface components to Clq binding andclassical complement pathway activation. Since the cell sur-face ofK pneumoniae, unlike that of other enterobacteria,is a rather complex structure constitutively containing cap-sular polysaccharide, LPS, and OMP, it was important tostudy the relative contribution of these components to theClq binding phenomenon. We have studied this point by dotblot analysis of the purified Clq-binding OMP isolated asdescribed above for Fig. 4 and of purified LPS from strainKT793. The results of this analysis are shown in Fig. 5. RowA contained twofold dilutions of the two purified OMP(starting at 1 mg/ml), and rows B and C contained purifiedLPS and BSA (as a control) at the same concentrations.After incubation with biotinylated Clq and avidin-alkalinephosphatase, only the OMP containing dots (row A) werevisualized. We conclude that Clq binding is due to OMP,

and, although Clq binding to the purified KT793 rough LPScannot be ruled out, it is at least 32-fold less efficient thanClq binding to OMP (Fig. 5, compare row A, lane 6, withrow B, lane 1). It is important to note that this binding wasobserved in an antibody-free experiment, as was the bindingobserved in Fig. 2, 3, and 4. Also, in contrast to the proteinsof SDS-PAGE and Western analysis, the proteins used inthis dot blot assay had not been boiled, indicating that Clqbinds to these proteins in their native state.The ability of the Clq-binding proteins purified from strain

KT793 to activate the classical complement pathway wasstudied by hemolytic assays. A dose-dependent reduction ofthe C4 and CH50 hemolytic activities was observed afterincubation of a serum depleted in Clq and factor D (andreconstituted with Clq) with different amounts of the twoisolated OMP (Fig. 6A).We then investigated the ability of the isolated proteins to

activate complement in different sera. After incubation ofNHS, serum depleted in Clq and factor D (reconstitutedwith Clq), or agamma serum for 45 min at 37°C with 25 ,ug ofprotein, drastic reductions of both CH50 and C4 levels wereobserved (Fig. 6B). After these two experiments (Fig. 6), itcould be concluded that the two isolated OMP bind Clq andthat this binding leads to activation of the classical comple-ment pathway, with the subsequent consumption of C4 andreduction of the total hemolytic activity of complement.Importantly, Clq binding and complement activation havebeen shown in the antibody-free experiments demonstratedin Fig. 2 through 6.The ability of the two isolated OMP and LPS to activate

the classical pathway was studied in an agamma serumwithout alternative pathway activity. For this purpose, ag-amma serum was depleted in Clq and factor D and wasreconstituted with purified Clq. This serum was then incu-bated with different amounts of protein or LPS. As can beseen in Fig. 7, a dose-dependent reduction of the totalhemolytic activity of this serum was observed after incuba-tion with either component of the outer membrane. A 50%reduction of the CH50 was obtained by incubation with 0.7,ug of protein, whereas 3 jig of LPS was necessary to obtainthe same reduction. The difference in the amounts of OMPneeded to obtain a 50% reduction of CH50 in this agammaserum and in the NHS used in Fig. 6A (both sera depleted infactor D) is most probably due to differences in their initialcomplement activities, with nearly 10-fold less hemolyticactivity in the agamma serum than in the NHS. The CH50reduction shown in Fig. 7 must be attributed to an antibody-independent activation of the classical pathway since theassay serum is an agamma serum depleted in both Clq andfactor D and reconstituted with purified Clq.

Since the structure of the core LPS is not known inKlebsiella species, it is not possible to accurately express thelatter result on a molar basis. Nevertheless, assuming thatthe molecular weight of the Kiebsiella LPS used here isabout 5,000, i.e., on the order of that of a chemotype RaSalmonella minnesota strain (53), the 3 ,ug of LPS needed toreduce the CH50 by 50% may represent a concentration of

z6 x 10-10 M. The Mr of the two OMP used in thisexperiment is approximately 36,000, so 0.7 ,ug represents aconcentration of =2 x 10-1 M. Clearly, on a molar basis,the two OMP would be about 30 times more efficient thanLPS for activation of the classical pathway. These differ-ences could be even greater considering that the two OMPare, by the tested criteria, porins, and that in their nativeform porins typically exist as trimers.

Finally, having identified two OMP as the major targets for

IN ECT. IMMUN .

on August 14, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

VOL. 61, 1993

@0

.9

9i

S.a

.9

a9

100

80

60

40

20

0

100

80

60

40

20

0 4,5 9 133,5 18 22,5Porin Added (jig)

IEThzIWzMRQD+Q Agamma NHS

SeraFIG. 6. Activation of the classical pathway by OMP. (A) Tubes

containing 40 ,ul of serum depleted in Clq and factor D andreconstituted with Clq were incubated with different amounts (0 to22.5 ,ug) of the isolated Clq-binding proteins in a final volume of 120p.1 of DGVB2+. After 45 min at 37°C, the remaining C4 (empty bars)and CH50 (stripped bars) hemolytic activities were determined. Theresults are the averages of six independent experiments. (B) NHS,NHS depleted in Clq and factor D and reconstituted with Clq(RQD+Q), and agamma serum were incubated for 1 h at 37°C with25 ,ug of the isolated Clq-binding proteins. After incubation, theremaining C4 (empty bars) and CH50 (stripped bars) hemolyticactivities were assayed as described for panel A. The results are theaverages of three independent experiments.

Clq binding and classical complement pathway activation,we studied the relative contributions of LPS and capsularpolysaccharide to the binding of Clq by bacterial cells. Forthis purpose, K pneumoniae strains with different combina-tions of 0 and K antigens were used. As shown in Fig. 8,binding of Clq depends on the absence of the LPS 0 antigen.Strains with smooth LPS (such as C3, KT717, and KT791)are all serum resistant and bind less Clq than serum-

sensitive rough-LPS strains, independent of the presence(strains KT701 and KT702) or absence (strain KT793) of a

capsule.

DISCUSSION

The mechanisms that pathogenic bacteria use to evade thecomplement action have been extensively studied and re-

Clq BINDING BY K PNEUMONIAE PROTEINS 857

75i

50 IX.- I ji1L10 1 10 2 10 3 10 4 10 5

Amount of protein or lipopolysaccharide (ng)FIG. 7. Antibody-independent activation of the classical path-

way by outer membrane components. An agamma serum depleted inClq and factor D and reconstituted with purified C1q was incubatedwith different amounts of Clq-binding proteins (open circles) or LPS(closed circles). After 45 min at 37°C, the remaining CH50 wa'sdetermined. The results are the averages of three independentexperiments.

viewed (23, 40). It is also important to define the bacterialmolecules acting as targets for complement activation anddeposition, since this probably represents the basis of thenatural immune state against bacterial infections. K pneu-moniae rarely causes infections in healthy individuals,whereas it is a common pathogen for immunodepressedpatients. Most people, then, are successfully dealing withKpneumoniae strains through the action of the complementsystem, a fact which stresses the need for a better definitionof the molecules involved in complement activation by thisspecies.We have previously shown that the classical and alterna-

*1-

Is

I.

aU

KT701 KT702 KT793 KT717 KT791 C3

Strains

FIG. 8. Binding of "2I-labeled Clq toK pneumoniae cells fromdifferent 0 and K mutant strains. Strains KT701 and KT702 are

O-:K+ derived from strain C3 (O1:K66), strains KT791 and KT793are isogenic mutants (O+:K- and O-:K-, respectively) derivedfrom C3, and strain KT791 is a nonisogenic O1:K- mutant strain.The results are the averages of at least four independent experi-ments.

0\'0

0

on August 14, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

858 ALBERTI ET AL.

tive complement pathways are involved in the killing ofserum-sensitive K pneumoniae strains (11). Also, Loos etal. have reported that serum-sensitive strains of E. coli andK pneumoniae interact with Cl and activate the classicalpathway (30). They have also reported that this activationprocess requires the interaction between unidentified bacte-rial outer membrane components and complement compo-nent Cl. The results reported here confirm the importance ofthe classical pathway in the killing ofK pneumoniae strainsand extend the previous knowledge of the interaction be-tween this bacterium and the classical complement pathway.Activation was largely mediated by Clq binding to bacterialouter membrane components. Our experiments using puri-fied OMP are clearly consistent with the notion that Clqbinding to intact K pneumoniae cells incubated in serumoccurs on OMP that by all the criteria tested seem to beporins. However, a direct definitive proof that Clq bindingto porin-type OMP occurs during classical complementpathway activation would require the development of a Clqcapture system or cross-linking experiments.

Porin proteins from other bacterial species, such as S.typhimurium (17) and S. minnesota (43) have also beenshown to activate the classical complement pathway after Cl(or Clq) binding. One major difference between these twospecies andK pneumoniae is that the latter contains a largecapsular polysaccharide involved in colonization and prolif-eration in its host (10, 55). Thus, our description that thecapsular polysaccharide is not a barrier for Clq binding toOMP and complement activation through the classical path-way (for instance, in strains KT701 and KT702) describes anew phenomenon.

Binding of Clq to other outer membrane components oftheK pneumoniae strain used in this study, in particular therough LPS, could not be demonstrated. The lack of bindingof Clq to LPS is consistent with our previous observationthat the rough LPS from differentK pneumoniae strains isunable to activate the classical complement pathway (11).This result may seem contradictory to those obtained for therough LPSs of E. coli and different Salmonella species (3,13, 28, 34) that have been shown to activate the classicalpathway after Clq binding. We do not have any definitiveexplanation for this discrepancy from other reports atpresent, we have just the suggestion that the structure of thecore LPS and lipid A of K pneumoniae may be differentfrom those of the other species cited before. Alternatively, italso has to be noted that not all the rough LPSs used in thosereports were equally effective in terms of activation of theclassical pathway and that, actually, at least one of the roughLPSs studied was not at all able to activate this pathway(28). After these early and more recent works (26), it is clearthat, among the rough LPSs, it is the lipid A componentwhich, in an antibody-independent manner, interacts withClq, with a subsequent activation of the classical comple-ment pathway (12). This binding and activation phenomenonhas been shown to be not just dependent on a rough-typeLPS but restricted to chemotype Re and isolated lipid A (53).In this respect, the LPSs from strains KT701 and KT702have been described as Rb or Rc (47). Strain KT793, whichhas been used here as a representative serum-sensitivestrain, is an LPS mutant obtained by selection with bacte-riophage FC3-2, and strains selected in this way are alwayschemotype Rb or Rc (47, 48).

Strain KT793 and two other K pneumoniae strains withchemotype Rb or Rc are sensitive to complement-mediatedkilling through the classical pathway. Because the killingtakes place without an apparent or significant binding of Clq

to the rough LPS, it suggests a major role of the porins in theactivation of the classical complement pathway. This isfurther supported by the results described here, demonstrat-ing that purified OMP were more effective than purified LPSin terms of the binding of Clq, the consumption of C4, andthe reduction of the total hemolytic activity of serum. Porinproteins from other bacteria have also been shown to bemore effective, in terms of Clq and Cl consumption, thanrough LPSs extracted from the same strains (43).The binding of Clq toK pneumoniae OMP is an antibody-

independent process. This has been proven in the presentwork by direct binding of purified Clq to the bacterial cellsand OMP and also by the ability of purified Clq-bindingproteins (porins) to activate the classical pathway both innonimmune and agamma sera. The present results coincidewith those reported by other authors for different bacterialspecies, describing the interaction between bacterial porinsand Clq as an antibody-independent phenomenon (26, 29,43). The molecular basis for the direct, antibody-indepen-dent interaction between Clq and bacterial porins is notknown, but in S. minnesota the negative charge of the porins(pI 4.5 to 5.0) may explain their ability to bind the positivelycharged Clq (pI > 9.0) (43). This hypothesis remains to betested for K pneumoniae, although the fact that Clq binds inWestern blots only to porin-like proteins suggests that theabove explanation may be valid in this case as well.The antibody-independent interaction between Clq and

OMP, with the subsequent activation of the classical path-way, is a relevant finding in the case of K pneumoniae.First, it is relevant because this occurs without an apparentimpediment due to the capsular polysaccharide. Second,since K pneumoniae is an opportunistic pathogen of partic-ular importance for immunocompromised patients, the anti-body-independent bactericidal mechanisms of these individ-uals presumably play an important role in the defense againstinfections caused by this bacterium. Also, in a larger con-text, since the enterobacterial porins studied to date sharehomology in their primary amino acid sequence (22), thebinding of Clq to porins may represent a mechanism for theactivation of the classical complement pathway common toenterobacteria and which acts independently of the presenceof a capsular polysaccharide layer. Restriction of the anti-body-independent classical complement pathway activationin these species may then be dictated mainly by the LPS 0antigen, by impeding the access of Clq to porins. In othercases, such as in E. coli Kl (38) and in someK pneumoniaeserotypes (32), both the capsular polysaccharide and theLPS 0 antigen contribute to the shielding of classical com-plement pathway activator structures on the cell surface.

This work raised some interesting questions that we arecurrently trying to address. Of these, we have demonstratedthat the effective killing of K pneumoniae strains requiresthe activation of both the classical and alternative comple-ment pathways. It is clear, then, that a better definition ofthe activation of the alternative pathway inK pneumoniae isneeded, and we are now trying to identify the bacterialmolecules that bind C3 in Clq-depleted serum.

In summary, we have demonstrated that the effectivekilling of K pneumoniae serum-sensitive strains in nonim-mune serum requires the action of both complement path-ways. The classical pathway is activated after the binding ofClq to two major bacterial OMP, presumably porins, in anantibody- and capsule-independent manner. The binding ofClq to the rough LPS of this strain could not be demon-strated, and activation of the classical pathway by purifiedLPS was less efficient than activation by the two OMP. The

INFECT. IMMUN.

on August 14, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

Clq BINDING BY K. PNEUMONIAE PROTEINS 859

role of these proteins, and that of other outer membranecomponents, in the activation of the alternative pathwayremains to be investigated.

ACKNOWLEDGMENTS

This work was supported by CICYT grant PB91-0233-CO2 toV.J.B. and J.M.T. and CICYT grant PM90-101 and grant FIS90-0224 to F.V.

S. Alberti is the recipient of a predoctoral fellowship from CICYT(Programa 07, Salud). G. Marques received a fellowship fromFundaci6n Conchita Rabago.We thank M. L6pez-Trascasa and F. Ortiz-Masllorens for provid-

ing us with agamma serum, and J. Imperial and M. Kearns forhelpful suggestions and style corrections, respectively. S.A. andV.J.B. thank J. Lalucat for his continuous support.

REFERENCES1. Alcolea, J. M., L. C. Ant6n, G. Marques, P. Sinchez-Corral, and

F. Vivanco. 1987. Formation of covalent complexes between thefourth component of human complement and IgG immuneaggregates. Complement 4:21-32.

2. Anton, L. C., J. M. Alcolea, P. Sinchez-Corral, G. Marques,M. A. Sanchez, and F. Vivanco. 1989. C3 binds covalently to theCg3 domain of IgG immune aggregates during complementactivation by the alternative pathway. Biochem. J. 257:831-837.

3. Aubert, B., S. Chesne, G. J. Arlaud, and M. G. Colomb. 1985.Antibody-independent interaction between the first componentof human complement, Cl, and the outer membrane of Esch-enchia coli D31m4. Biochem. J. 232:513-519.

4. Barrio, E., L. C. Ant6n, G. Marques, A. Sinchez, and F.Vivanco. 1991. Formation of covalently linked C3-C3 dimers onIgG immune aggregates. Eur. J. Biochem. 21:343-349.

5. Benedi, V. J., B. Ciurana, and J. M. Tomis. 1989. Isolation andcharacterization of Kiebsiella pneumoniae unencapsulated mu-tants. J. Clin. Microbiol. 27:82-87.

6. Benge, G. R. 1988. Bactericidal activity of human serum againststrains of Kiebsiella from different sources. J. Med. Microbiol.27:11-15.

7. Blake, M. S., K. H. Johnston, G. J. Russell-Jones, and E. C.Gotschlich. 1984. A rapid, sensitive method for detection ofalkaline phosphatase-conjugated anti-antibody on Westernblots. Anal. Biochem. 136:175-179.

8. Bodey, G. P., L. S. Elting, S. Rodriguez, and M. Hernindez.1989. Klebsiella bacteremia-a 10-year review in a cancerinstitution. Cancer 64:2368-2376.

9. Brown, E. J., and K. A. Joiner. 1988. Mechanisms of hostresistance to bacterial infection, p. 715-736. In M. Samter (ed.),Immunological diseases. Little, Brown & Co., Boston.

10. Bryan, C. S., K. L. Reynolds, and E. R. Brenner. 1983. Analysisof 1,186 episodes of gram-negative bacteremia in non-universityhospitals. The effects of antimicrobial therapy. Rev. Infect. Dis.5:629-638.

11. Ciurana, B., and J. M. Tomas. 1987. Role of lipolysaccharideand complement in susceptibility of Klebsiella pneumoniae tononimmune serum. Infect. Immun. 55:2741-2746.

12. Cooper, N. R. 1985. The classical complement pathway: activa-tion and regulation of the first complement component. Adv.Immunol. 37:151-216.

13. Cooper, N. R., and D. C. Morrison. 1978. Binding and activationof the first component of human complement by the lipid Aregion of lipopolysaccharides. J. Immunol. 120:1862-1868.

14. Figueroa, J. E., and P. Densen. 1991. Infectious diseases asso-ciated with complement deficiencies. Clin. Microbiol. Rev.4:359-395.

15. Filip, C., G. Fletcher, J. L. Wulf, and C. F. Earhart. 1973.Solubilization of the cytoplasmic membrane of Eschenchia coliby the ionic detergent sodium-lauryl sarcosinate. J. Bacteriol.115:717-722.

16. Gaither, T. A., D. W. Alling, and M. M. Frank. 1974. A newone-step method for the functional assay of the fourth compo-nent (C4) of human and guinea pig complement. J. Immunol.113:574-583.

17. Gaildiero, F., M. A. Tufano, L. Sommese, A. Folgore, and F.Tedesco. 1984. Activation of complement system by porins ex-tracted from SalnoneUa typhimurium. Infect. Immun. 46:559-563.

18. Garcia de la Torre, M., J. Romero-Vivas, J. Martinez-Beltran,A. Guerrero, M. Meseguer, and E. Bouza. 1985. Kiebsiellabacteremia: an analysis of 100 episodes. Rev. Infect. Dis.7:143-150.

19. Ghebrehiwet, B., S. Bossone, A. Erdei, and K. B. M. Reid. 1988.Reversible biotinylation of Clq with a cleavable biotinyl deriv-ative. J. Immunol. Methods 110:251-260.

20. Gotze, O., and H. J. Muller-Eberhard. 1971. The C3 activationsystem: an alternate pathway of complement activation. J. Exp.Med. 134:90S-108S.

21. Hancock, R. E. W., and H. Nikaido. 1978. Outer membranes ofgram-negative bacteria. XIX. Isolation of Pseudomonas aerug-inosa PAO1 and use in reconstitution and definition of thepermeability barrier. J. Bacteriol. 136:381-390.

22. Jeanteur, D., J. H. Lakey, and F. Pattus. 1991. The bacterialporin superfamily: sequence alignment and structure prediction.Mol. Microbiol. 5:2153-2164.

23. Joiner, K. A. 1988. Complement evasion by bacteria and para-sites. Annu. Rev. Microbiol. 42:201-230.

24. Joiner, K. A., E. J. Brown, and M. M. Frank. 1984. Comple-ment and bacteria: chemistry and biology in host defense.Annu. Rev. Immunol. 2:461-491.

25. Kolb, W. P., L. M. Kolb, and E. R. Podack 1979. Clq: isolationfrom human serum in high yield by affinity chromatography anddevelopment of a highly sensitive hemolytic assay. J. Immunol.122:2103-2111.

26. Latsch, M., J. Mollerfeld, H. Ringsdorf, and M. Loos. 1990.Studies on the interaction of Clq, a subcomponent of the firstcomponent of complement, with porins from Salmonella min-nesota incorporated into artificial membranes. FEBS Lett.276:201-204.

27. Liberti, P. A., D. M. Bausch, and R. D. Baillie. 1981. Theincorporation of high pH euglobulin precipitation in the isolationof Clq. J. Immunol. Methods 40:243-245.

28. Loos, M., D. Bitter-Suermann, and M. Dierich. 1974. Interactionof the first (Cl), the second (C2) and the fourth (C4) componentof complement with different preparations of bacterial lipopoly-saccharides and with lipid A. J. Immunol. 112:935-940.

29. Loos, M., and F. Clas. 1986/1987. Antibody-independent killingof gram-negative bacteria via the classical pathway of comple-ment. Immunol. Lett. 14:203-208.

30. Loos, M., B. Wellek, R. Thesen, and W. Opferkuch. 1978.Antibody-independent interaction of the first component withgram-negative bacteria. Infect. Immun. 22:5-9.

31. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

32. Merino, S., S. Camprubi, S. Alberti, V. J. Benedi, and J. M.Tomas. 1992. Mechanisms of Kiebsiella pneumoniae resistanceto complement-mediated killing. Infect. Immun. 60:2529-2535.

33. Miller, J. H. 1977. Experiments in molecular genetics. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

34. Morrison, D. C., and L. F. Kline. 1977. Activation of theclassical and properdin pathways of complement by bacteriallipopolysaccharides (LPS). J. Immunol. 118:362-368.

35. Nikaido, H. 1983. Proteins forming large channels from bacterialand mitocondrial outer membranes: porins and phage lambdareceptor proteins. Methods Enzymol. 97:85-100.

36. Nurminen, M. 1978. A mild procedure to isolate the 34K, 35Kand 36K porins of the outer membrane of Salmonella typhimu-num. FEMS Microbiol. Lett. 3:331-334.

37. Phimister, G. M., and K. Whaley. 1990. Measurement of com-plement, p. 81-109. In H. C. Gooi and H. Chapel (ed.), Clinicalimmunology: a practical approach. Oxford University Press,Oxford.

38. Pluschke, G., J. Mayden, M. Achtman, and R. P. Levine. 1983.Role of the capsule and the 0 antigen in resistance of 018:K1Escherichia coli to complement-mediated killing. Infect. Im-mun. 42:907-913.

39. Praz, F., M. C. R. A. Barreira, and P. Lesavre. 1982. A one-step

VOL. 61, 1993

on August 14, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

860 ALBERT! ET AL.

procedure for preparation of classical pathway (Clq) and alter-native pathway (factor D) depleted human serum. J. Immunol.Methods 50:227-231.

40. Roberts, I. S., F. K. Saunders, and G. J. Boulnois. 1989.Bacterial capsules and interactions with complement and phago-cytes. Biochem. Soc. Trans. 17:462-464.

41. Sanchez-Corral, P., L. C. Ant6n, J. M. Alcolea, G. Marques, A.Sanchez, and F. Vivanco. 1989. Separation of active and inactiveforms of the 3rd component of human complement, C3, by fastprotein liquid chromatography (Fplc). J. Immunol. Methods122:105-113.

42. Sanchez-Corral, P., L. C. Ant6n, J. M. Alcolea, G. Marques, A.Sanchez, and F. Vivanco. 1990. Proteolytic activity of thedifferent fragments of factor B on the third component ofcomplement (C3). Involvement of the N-terminal domain of Bbin magnesium binding. Mol. Immunol. 27:891-900.

43. Stemmer, F., and M. Loos. 1985. Evidence for direct binding ofthe first component of complement, Cl, to outer membraneproteins from Salmonella minnesota, p. 73-84. In M. Loos(ed.), Bacteria and complement. Springer-Verlag, Berlin.

44. Taylor, P. W. 1983. Bactericidal and bacteriolytic activity ofserum against gram-negative bacteria. Microbiol. Rev. 47:46-83.

45. Tenner, A. J., P. H. Lesavre, and N. R. Cooper. 1981. Purifica-tion and radiolabeling of human Clq. J. Immunol. 127:648-653.

46. Thye Yin, E., C. Galanos, S. Kinski, R. A. Bradshaw, S. Wessler,0. Luderitz, and M. F. Sarmiento. 1972. Picogram-sensitiveassay for endotoxin: gelation of Limulus polyphemus blood celllysate induced by purified lipopolysaccharide and lipid A fromGram-negative bacteria. Biochim. Biophys. Acta 261:284-298.

47. Tomas, J. M., V. J. Benedi, B. Ciurana, and J. Jofre. 1986. Roleof capsule and 0 antigen in resistance of Klebsiella pneumoniaeto serum bactericidal activity. Infect. Immun. 54:85-89.

48. Tomas, J. M., V. J. Benedi, and J. T. Jofre. 1987. Identificationof the cell surface receptor for FC3-2, FC3-3 and FC3-6 bacte-riophages from Klebsiella pneumoniae. FEMS Microbiol. Lett.41:223-228.

49. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

50. Tsai, C.-M. 1986. The analysis of lipopolysaccharide (endotox-in) in meningococcal polysaccharide vaccines by silver stainingfollowing SDS-polyacrylamide gel electrophoresis. J. Biol.Stand. 14:25-33.

51. Tsai, C.-M., and C. E. Frasch. 1982. A sensitive silver stain fordetecting lipopolysaccharides in polyacrylamide gels. Anal.Biochem. 119:115-119.

52. Vivanco, F., R. Bragado, J. P. Albar, C. Juarez, and F.Ortiz-Masllorens. 1980. Chemical modification of carboxylgroups in human Fc fragment: structural role and effect on thecomplement fixation. Mol. Immunol. 17:327-336.

53. Vukajlovich, S. W., J. Hoffman, and D. C. Morrison. 1987.Activation of human serum complement by bacterial lipopoly-saccharides: structural requirements for antibody independentactivation of the classical and alternative pathways. Mol. Im-munol. 24:319-331.

54. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides:extraction with phenol-water and further applications of theprocedure. Methods Carbohydr. Chem. 5:83-91.

55. Williams, P., P. A. Lambert, M. R. W. Brown, and R. J. Jones.1983. The role of the 0 and the K antigens in determining theresistance of Kiebsiella aerogenes to serum killing and phago-cytosis. J. Gen. Microbiol. 129:2181-2191.

56. Williams, P., and J. M. Tomas. 1990. The pathogenicity ofKlebsiella pneumoniae. Rev. Med. Microbiol. 1:196-204.

INFECT. IMMUN.

on August 14, 2019 by guest

http://iai.asm.org/

Dow

nloaded from