Microbial Pathogenesis 1992 ; 13 : 371-379

Interaction of Klebsiella capsule type 7 with human polymorphonuclear leucocytes Rainer Podschun,* Iris Penner and Uwe Ullmann Institute of Medical Microbiology and Virology, University of Kiel, Brunswiker Str . 4, D-2300 Kiel, Germany (Received April 24, 1992 ; accepted in revised form August 15, 1992)

Podschun, R . (Institute of Medical Microbiology and Virology, University of Kiel, Brunswiker Str . 4, D-2300 Kiel, Germany), I . Penner and U . U!Imann . Interaction of Klebsiella capsule type 7 with human polymorphonuclear leucocytes . Microbial Pathogenesis 1992 ; 13 : 371-379 . Klebsiella serotype K7 is found among the capsule types that are most prevalent in respiratory tract isolates . To evaluate the significance of the K7 antigen in bacteria-leucocyte interactions, K7-encapsulated Klebsiella pneumoniae strains and their non-capsulate mutants were investigated . The K7 isolates were compared to K2-capsulate strains and their respective K derivatives . K7-capsulate bacteria were less hydrophilic, and more readily phagocytosed and killed by human polymorphonuclear leucocytes (PMNL) than K2 strains . Loss of the K7 antigen resulted in increased surface hydrophobicity but did not affect phagocytosis and killing, whereas loss of the K2 capsule caused greater susceptibility to the phagocytic and killing action of PMNL . Both the K7 and K2 antigen stimulated the extracellular release of lysozyme from neutrophils but not of myeloperoxidase, indicating degranulation of only secondary granules . All K - mutants induced the release of both lysozyme and myeloperoxidase . Our results suggest that, in contrast to the K2 antigen, the K7 capsular polysaccharide does not confer antiphagocytic properties on bacteria . However, the K7 antigen is able to impede the extracellular release of primary granule enzymes . Key words : Klebsiella pneumoniae ; capsular polysaccharide ; polymorphonuclear leucocytes ; phagocytosis; degranulation .

Introduction Klebsiella pneumoniae is one of the five Gram-negative pathogens most commonly encountered in nosocomial infections .' Usually, Klebsiella spp . produce welldeveloped capsules which are classified serologically into 77 different serotypes . 2 Capsular polysaccharides play an important role in the virulence of the bacteria .` The significance of capsules in pathogenicity has been explained by interference of the capsular (K) antigen with phagocytosis by neutrophils and with bacterial killing by serum, as has been shown for Klebsiella spp . and Escherichia coli. 47-1 The various K antigens, however, contribute to different degrees to the virulence of Klebsie/la spp . Strains belonging to the serotypes K1 and K2 are the most virulent, with respect to mouse lethality after intraperitoneal injection . 12,11 In experimentally induced skin lesions in mice, K1, K2, K4 and K5 strains were found to be more virulent than K6 and serotypes higher than K6 . 14 Correspondingly, strains of these serotypes *Author to whom correspondence should be addressed . 0882-4010/92/110371 +09 $08 .00/0

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stimulated human neutrophils to a much lesser extent than other capsular types investigated ." Serotypes K2 and K7 are found among the capsule types that are prevalent in bacteremic and respiratory tract isolates ." , " Whereas the role of the K2 antigen in virulence is well-documented, little is known about the significance of capsule type K7 in host-parasite interactions . We previously reported that K7-encapsulated isolates are significantly less toxic for mice than K2 strains ." Correspondingly, K7 strains were phagocytosed and killed by polymorphonuclear leucocytes (PMNL) more readily than K2 isolates . A considerable strain-to-strain variation, however, was observed among the K7 isolates examined . To more precisely evaluate the significance of the capsule antigen K7 in bacteria-leucocyte interactions, K7-encapsulated strains and their noncapsulate mutants were investigated . To compare the K7 antigen with the K2 antigen, capsule-type K2 isolates and their K - derivatives were included in this study .

Results Isolation of U mutant strains K- derivatives from five encapsulated parent strains were obtained without mutagenic treatment. Loss of the K antigen was confirmed by the capsular swelling reaction . While the parent strains strongly reacted with homologous antisera, no positive reaction could be observed in the mutant bacteria . No differences in biotype could be detected between mutant and parent strains . All strains are listed in Table 1 . Surface hydrophobicity The surface hydrophobicity of the strains was measured by two different techniques (Table 1) . In the salt aggregation test, both the K+ and the K - strains aggregated at ammonium concentrations of >, 1 .6 M, indicating that all strains were hydrophilic . As the SAT assay is not satisfactory for detecting hydrophilic microorganisms, the HIC method was used alternatively . Determination of the R F values revealed K2 strains to be much more hydrophilic than K7 isolates . In all cases mutant strains showed higher R F values than their respective parent strains, indicating that K - mutants were markedly more hydrophobic than K+ bacteria .

Table 1 Surface hydrophobicity and phagocytic killing of encapsulated K/ebsie/la strains and their non-capsulate mutants . Results of killing experiments are expressed as percentage of bacteria surviving after incubation with PMNL Killing (% survival) by' Strain no . 23.K+ 23 .K11 5.K+ 115.K37 . K+ 37X52 .K+ 52 .K58.K+ 5&K-

K antigen type 2 2 7 7 7

SAT value >1 >1 1 1 >1 1 1 1 >1 1

.6 .6 .6 .6 .6 .6 .6 .6 .6 .6

M M M M M M M M M M

HIC R F valuea 13 .5+1 .0 25 .2+1 .7 31 .5+2 .4 38 .5+1 .6 71 .7+2 .5 92 .9±1 .6 74 .0+4 .6 94 .6+2 .9 59 .2+2 .4 79 .8+1 .4

PMNL after 30 min 60 min 122+16 45+5 144+31 52+4 47+2 45+3 62+12 56+8 51+6 41+16

218+27 39+7 183+28 37+10 24+4 56+6 54+23 45+6 38+12 40+10

'Median ±standard error of the median of at least three separate experiments .

NHS alone after 60 min 233+28 218+22 158+27 146+5 107+4 232+11 194+14 191+28 229+36 221+28



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0 23

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Fig . 1 . CL of human PMNL after stimulation with K2 and K7 capsulate Klebsiella isolates and their noncapsulate mutant strains . Values are expressed as percentage of CL obtained with opsonized zymosan . Results are expressed as the median±standard error of the median of at least three separate experiments using PMNL from different donors .

Chemiluminescence (CL) of PMNL The ability of the Klebsiella strains to stimulate polymorphonuclear leucocytes was determined by measuring the chemiluminescence (CL) response of the granulocytes . The results are shown in Fig . 1 . Incubation of leucocytes with K2-encapsulated bacteria resulted in very low CL values compared with those induced by K7-capsulate bacteria (P < 0 .001) . Loss of the K2 antigen markedly increased CL responses (P < 0 .001) . In contrast, loss of the K7 capsule did not result in better activation of PMNL metabolism, since K- mutants and K parent strains did not differ significantly in CL induction . Killing of bacterial strains by PMNL Phagocytic killing of the strains is shown in Table 1 . To exclude the possibility of bacterial killing by normal human serum, serum controls (10% NHS) without leucocytes were run within each experiment . K2 capsulate bacteria were resistant to phagocytic destruction, and cell counts increased to approximately 200% within 1 h . Noncapsulate mutant strains, however, were killed by PMNL, and after 60 min only 37-39% of the bacteria were viable . In contrast to K2 bacteria, viable counts of K7encapsulated strains decreased to 40-50% within 60 min . No differences in phagocytic killing could be observed between the K7 parent strains and their respective K mutants . L ysosomal enzyme re/ease Degranulation of PMNL in response to bacterial stimulation was assayed by measuring the release of granule enzymes . Myeloperoxidase (MPO) was used as marker of primary (azurophil) granules, and lysozyme was chosen as marker of both primary and secondary granules . The results are shown in Figs 2 and 3 . Parental and mutant strains of both capsule types caused a significant release of lysozyme, compared to the amount released by non-stimulated control PMNL (P < 0 .001) . MPO release, on the other hand, was caused only by non-capsulate bacteria (P < 0 .002), while capsulate strains did not promote significant release of MPO . No differences in



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O

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P N .. 0

0 Control

23

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37

K2

52

58 Strain

K7

Fig . 2 . Extracellular release of lysozyme from 5x106 PMNL after incubation for 60 min with K2 and K7 capsulate Klebsiella isolates and their non-capsulate mutant strains . Results are expressed as the median±standard error of the median of at least three separate experiments using PMNL from different donors .

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ca) U

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Fig . 3 . Extracellular release of MPO from 5x106 PMNL after incubation for 60 min with K2 and K7 capsulate Klebsiella isolates and their non-capsulate mutant strains . Results are expressed as the median±standard error of the median of at least three separate experiments using PMNL from different donors.

lysozyme and MPO release were observed between K2- and K7-capsulate bacteria . Regardless of the serotype, loss of the capsule resulted in significantly increased MPO and lysozyme release (P < 0 .002) . Discussion The importance of Klebsiella capsular polysaccharides as virulence factors has been stressed by a number of studies . However, it is not the capsule per se but rather particular capsule types that confer enhanced virulence on bacteria, as has been shown



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14,17-21 We previously reported that K7for Klebsiella as well as for other bacteria . encapsulated Klebsiella strains were significantly less virulent for mice and were more readily phagocytosed than K2 strains . 17 As this investigation, however, was based on the comparison of two sets of completely different strains, the possibility that other surface components (e .g . lipopolysaccharides) interfered could not be excluded . In the present study, K7- and K2-encapsulated strains were compared with their respective non-capsulate mutants . Since these K - derivatives were obtained without mutagenic treatment, it is likely that the spontaneously occurring K - variants are-apart from the lack of a capsule-otherwise isogenic to the parental strains as has been described by several authors .' 22 Such K - variations occur spontaneously and are caused by mutational blocks in capsular polysaccharide synthesis . 23 They are easy to detect because mutant colonies are morphologically different from colonies of the parent strains . Capsule polysaccharides are mostly hydrophilic and confer a negative net charge on the bacterial cell . By means of hydrophobic interaction chromatography, K2capsulate bacteria could be demonstrated to be strongly hydrophilic . In contrast to the capsular polysaccharide K2, the K7 antigen does not seem to confer strong hydrophilic surface properties on bacteria, since K7-encapsulated cells showed markedly greater hydrophobicity than K2-capsulate strains . Loss of both types of capsules resulted in increased surface hydrophobicity of the mutant strains . Bacterial surface hydrophobicity plays an important role in phagocytosis . 24 Bacteria with hydrophobic exteriors are rapidly phagocytosed by PMNL, whereas bacteria possessing hydrophilic capsules resist ingestion by phagocytes . 2526 Correspondingly, the strongly hydrophilic K2 isolates were relatively resistant to phagocytic destruction, and loss of the K2 antigen rendered K - mutant strains susceptible to ingestion by PMNL . The markedly less hydrophilic K7-capsulate strains, on the other hand, were readily killed intracellularly by PMNL . Loss of the K7 antigen did not result in increased phagocytic susceptibility . Thus, the K7 antigen apparently does not act as an antiphagocytic barrier . During activation, neutrophils undergo the respiratory burst and degranulation . Stimulation of the PMNL respiratory burst is determined as the CL response of neutrophils and has been regarded as an indirect measure of phagocytosis . In correspondence to the killing data, K7-capsulate strains induced a much higher CL response than K2 isolates . Loss of the K2 capsule resulted in greatly increased PMNL stimulation, whereas no differences in CL responses could be observed between K7 strains and their K - derivatives . In contrast to the different degrees of respiratory burst activation, the degranulation patterns of PMNL were similar in response to both K7- and K2-capsulate strains . Each capsule type triggered the release of significant amounts of Iysozyme, an enzyme found in both primary and secondary granules ." MPO, a marker of primary granules, was not released in appreciable amounts . This suggests that mainly secondary granule enzymes are released during the interaction of the capsulate bacteria with neutrophils . Loss of both types of K antigen, however, resulted in significantly increased release of MPO and lysozyme . During the interaction of phagocytes with bacteria, enzymes of both primary and secondary granules are typically released extracellularly . 28 Our results thus indicate that the K7 and the K2 antigen prevent the degranulation of primary granules and decrease the release of enzymes from secondary granules . Enzyme release from secondary granules has only been described for non-phagocytic stimuli, such as the calcium ionophore A23187, concanavalin A (ConA), and phorbol myristate acetate (PMA) . 293° Escherichia coli strains bearing MR(P) fimbriae stimulate the release of secondary granule enzymes only, demonstrating the selectivity of PMNL degranulation



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in response to different phagocytic stimuli ." Although degranulation is associated with ingestion, phagocytosis is not essential for extracellular granule enzyme release . 32 This is illustrated by our finding that K2 strains which were resistant to phagocytosis stimulated secondary granule exocytosis as well as did K7 strains which were susceptible to phagocytic destruction . The observed differences between the capsulate parent strains and their K mutants indicate that, in contrast to capsule type 2, the K7 antigen does not prevent phagocytosis and subsequent killing by PMNL . Both types of capsular polysaccharide, however, showed a similar inability to stimulate primary granule secretion . The molecular mechanism responsible for the antiphagocytic activity of the K2 antigen is unknown . Apart from differences in hydrophobicity, specific interactions between capsular polysaccharides and surface carbohydrates of phagocytes are likely ." Very recently, a specific surface lectin commonly found in tissue macrophages has been reported to bind a Mana2/3Man repeating structure in certain capsular polysaccharides of K/ebsie//a pneumoniae . 34 Binding of the bacteria was followed by ingestion and killing . The K7 capsular polysaccharide is known to contain such a Mana2/3Man repeating structure whereas the sequence is not present in the K2 antigen ." However, considering that the Man/GIcNAc-specific surface lectin is not expressed on PMNL, this kind of recognition should not be responsible for interactions between K7 bacteria and neutrophils . On the other hand, mannose-specific interactions between bacteria and neutrophils are commonly found as has been demonstrated for type 1 fimbriae . Further investigations have to clarify whether the differences in composition of the K2 and the K7 capsules are responsible for the differences we found in the interaction of these bacteria with PMNL . Detailed binding and phagocytosis measurements are needed to evaluate the capsule effects .

Materials and methods

Bacterial strains . Five clinical Klebsiella pneumoniae isolates of capsule types K7 (three strains) and K2 (two strains) which have been described previously, 77 were used in this study (Table 1) . These strains were chosen because they had been proven to be serum resistant . The strains were biotyped and serotyped by previously described methods . 17 Non-capsulate mutants were derived without mutagenic treatment by the method of Simoons-Smit et al. 7 Briefly, serial dilutions of the parent strains were plated on brain-heart-infusion agar . Unencapsulated mutants appeared as slightly more translucent colonies or sectors of colonies . K - mutants were subcultured and tested for the absence of capsule antigen . Biotyping by the API 20E system confirmed that they were biochemically identical to the parent strain . Growth of bacteria . Strains were cultured overnight in brain-heart-infusion broth at 37°C . Cells were harvested by centrifugation, washed twice in PBS (pH 7 .2) and resuspended in the medium appropriate to the methods described below . Hydrophobicity studies . For the salt aggregation test (SAT) the method of Lindahl et al." was used . Bacteria were adjusted to 109 cells/ml in 0 .002 M phosphate buffer (pH 6 .8) . Ten microliters of bacterial cell suspension were mixed with 10 µl of ammonium sulphate of varying molarity (in 0 .002 M phosphate buffer, pH 6 .8) on glass slides (giving final concentrations of 0 .1-1 .6 M ammonium sulphate) . The slides were rocked for 3 min at room temperature and observed for aggregation . The results are expressed as the lowest concentrations of ammonium sulphate causing macroscopically visible aggregation . Hydrophobic interaction chromatography (H IC) was performed as described by Smyth et a/. 37 with phenyl Sepharose CL-4B (Pharmacia) as column packing . Adsorption and desorption of bacteria were determined by measuring the absorbance (A600-) of the suspensions according



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to Mozes and Rouxhet . 38 Results are expressed as percentages of retention (R F values) which allow characterization of hydrophilic microorganisms . Serum. Human blood was obtained from healthy individuals . After clotting at room temperature, serum was separated by centrifugation . Sera from approximately 100 donors were pooled and stored in aliquots at -80°C until use . Preparation of human leucocytes . Human polymorphonuclear leucocytes (PMNL) were obtained from heparinized blood of healthy volunteers . Leucocytes were separated by a Ficoll gradient (Pharmacia), followed by dextran sedimentation as described previously ." Residual erythrocytes were removed by hypotonic lysis . PMNL were washed twice in Hanks' balanced salt solution (HBSS) and adjusted to 1 x10' cells/ml . Viability of the cells as determined by trypan blue exclusion was always >95% . Contamination of the preparations by lymphocytes and monocytes were always < 1 % . Chemiluminescence (CL) of PMNL . Stimulation of leucocytes by bacteria was determined in a luminol-enhanced CL assay as described previously ." Ten microliters of PMNL suspension (1 x 10'/ml), 10 pl luminol (2 mg/ml), and 500 yl HESS containing normal human serum (NHS, final concentration 10%) were mixed in Lumac" polystyrene tubes (Bio-Vials, USA), and the background CL was measured over a 5-min period in a luminometer (Biolumat LB 9505, Berthold, Wildbad, Germany) at 37°C . CL was induced by adding 10 yl of bacteria (1 x 10 9 /ml) or zymosan (50 mg/ml) preopsonized with NHS . The bacteria : leucocyte ratio was approximately 200 :1 . The CL response was measured every 20 sec over a period of 60 min . After curve-area integration, the results were expressed as percentage of CL obtained with zymosan . All tests were run in duplicate, and each strain was tested at least three times with leucocytes from different donors . Killing by PMNL . 0 .1 ml bacterial suspension (4x10'/ml), 0 .4 ml HESS, 0 .4 ml leucocytes (1 x10' cells/ml), and 0 .1 ml normal human serum (final concentration 10%) were mixed in siliconized glass tubes . HESS was substituted for leucocytes in the controls . Vials were incubated in a shaking waterbath at 37°C . After 0, 30 and 60 min samples (0 .1 ml) were taken and diluted in 9 .9 ml of ice-cold distilled water to lyse PMNL . Viable counts of bacteria were determined by plating serial dilutions on BHI agar . Each test was run in duplicate, and each strain was tested three times . Measurement of lysozyme and MPO . Lysozyme and MPO release was used as a marker of degranulation of PMNL after incubation with bacteria . Bacterial cells were preopsonized in HBSS/10% NHS for 30 min, washed twice in HBSS and adjusted to 1 .5x10 9/ml . A mixture of 0 .75 ml bacterial suspension and 0 .75 ml of PMNL (7 .5x106 /ml) were incubated for 1 h at 37°C in a shaking waterbath and then placed in crushed ice for 5 min . Granulocytes were pelleted by centrifugation at 250xg for 10 min at 4°C . Supernatants were recentrifuged at 11 000xg for 5 min in an Eppendorf Centrifuge (Eppendorf, Hamburg, Germany) to pellet the bacteria and assayed for enzyme activity . Controls consisted of leucocytes incubated with HBSS under identical conditions . Enzyme release from PMNL was calculated as percentage of the total activity released in mixtures containing 0 .75 ml PMNL and 0 .75 ml of 0 .4% Triton X-100 after subsequent sonication for two 60-s periods on ice (Heat Systems-Ultrasonics, Plainview, NY) . Lysozyme was determined by measuring the decrease in A5ao of a suspension of Micrococcus lysodeikticus (0 .15 mg/ml, Sigma Chemical Co .) in HBSS . 39 MPO was assayed as described by Steadman et al . with o-dianisidine as substrate . 31 After the reaction was stopped with 3 .25 M perchloric acid the A405 was measured in a spectrophotometer (Pye Unicam, Cambridge, UK) . Each test was run in triplicate, and each experiment was repeated twice . Statistics. The significance of differences between particular capsule types and mutants was evaluated by the U test of Mann and Whitney .

We thank Miss Andrea Holzgen for her expert technical assistance .



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Interaction of Klebsiella capsule type 7 with human polymorphonuclear leucocytes.

Klebsiella serotype K7 is found among the capsule types that are most prevalent in respiratory tract isolates. To evaluate the significance of the K7 ...
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