Vol. 59, No. 6
INFECTION AND IMMUNITY, June 1991, p. 2006-2011
0019-9567/91/062006-06$02.00/0 Copyright © 1991, American Society for Microbiology
Surface Exposure of 01 Serotype Lipopolysaccharide in Klebsiella pneumoniae Strains Expressing Different K Antigens JUAN M.
TOMAS,'*
SILVIA CAMPRUBI,' SUSANA MERINO,' MICHAEL R. DAVEY,2 AND PAUL WILLIAMS3 Departamento de Microbiologia, Facultad de Biologia, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain,' and Departments of Botany2 and Pharmaceutical Sciences,3 University of Nottingham, Nottingham NG7 2RD, United Kingdom Received 20 August 1990/Accepted 4 March 1991
Surface exposure of the 01 serotype lipopolysaccharide in encapsulated Klebsiella pneumoniae strains belonging to different serotypes was examined by using the 01 antigen-specific bacteriophages FC3-1 and FC3-2 in conjunction with immunogold electron microscopy and enzyme immunoassays with specific antisera. Despite the presence of the capsular polysaccharide, the 01 antigen was exposed at the cell surface in strains producing K2, K7, K8, K12, K19, K21, K22, K34, K35, K42, K45, K55, K57, K62, K66, K69, and K70 capsular polysaccharides. However, in strains producing KI, K10, and K16 capsular polysaccharides, the 01 antigen was masked by the K antigen. These results suggest that, since the 01 antigen is surface exposed in many different strains of K. pneumoniae with different capsular serotypes and is also able to immunoprotect, its potential as a useful vaccine component should not be overlooked.
Klebsiella species are important human pathogens which infect many different body sites and have become a leading cause of morbidity and mortality in hospital-acquired infections (2, 4, 12, 15). The majority of clinical Klebsiella isolates possess a well-defined capsule (4, 13, 20), and the genus Klebsiella has been classified serologically into some 77 capsular (K) types (18, 20). In addition, Klebsiella pneumoniae produces lipopolysaccharides (LPS) bearing complete 0-antigen chains, and eight chemically distinct 0 antigens (01, 03, 04, 05, 07, 08, 09, and 012) have been identified (19). Among clinical isolates, the 01 antigen is the most common 0 antigen encountered (18, 19). Also, there seem to be differences in the frequencies of capsular polysaccharides among clinical isolates (6). Both the 0 and K antigens contribute significantly to the virulence of K. pneumoniae (4, 7, 18, 19), by conferring protection against complementmediated serum killing (25, 31) and phagocytosis (31, 32), respectively. The emergence of multiply resistant K. pneumoniae strains and the high mortality rates associated with Klebsiella infections (2, 4, 12, 15) have highlighted the need to develop immunoprophylactic and immunotherapeutic agents (4, 5). In animal models, serotype-specific protection has been achieved in both murine pneumonia and burn wound models following vaccination with the homologous capsular polysaccharide (5). However, with antibodies directed against 24 different K serotypes which make up approximately 70% of all the bacteremic isolates, a capsular polysaccharide-based vaccine against Klebsiella organisms is now undergoing clinical evaluation (6), but alternatives for such a complex vaccine still need to be evaluated. As K. pneumoniae produces few 0 serotypes (18, 19, 24), knowledge of the distribution and surface exposure of this cell envelope antigen is required. We have previously reported that the 01 LPS molecules are not exposed in the different 01:K1 strains examined (27). In the present study, we examined a variety of different K. pneumoniae strains from different capsular serotypes to *
determine whether the 01 antigen was exposed at the cell surface or masked by the exopolysaccharide capsule. MATERIALS AND METHODS
Bacteria, bacteriophages, and media. We used 157 different K. pneumoniae strains belonging to serotype 01 and with different K antigens belonging to different culture collections (18, 10; International Center for Reference of Klebsiella; this laboratory) and also some clinical isolates from different hospitals in the Barcelona, Spain, area (Hospital de la Esperanza, Hospital del Mar, and Hospital de Sant Joan de Deu). The distribution of the strains for K serotypes is as follows: KI (6 strains), K2 (24 strains), K7 (5 strains), K8 (7 strains), K10 (12 strains), K12 (8 strains), K16 (8 strains), K19 (4 strains), K21 (14 strains), K22 (6 strains), K27 (5 strains), K34 (3 strains), K35 (6 strains), K42 (5 strains), K45 (8 strains), K55 (5 strains), K57 (3 strains), K62 (6 strains), K64 (9 strains), K66 (7 strains), K69 (4 strains), and K70 (2 strains). Bacteriophages FC3-1 and FC3-2 are LPS-specific phages (26, 28). FC3-9 and 44 are bacteriophages showing capsule depolymerase activity against strains from serotypes K66 and Kl, respectively (10, 27). Luria broth (LB; 17) was routinely used for bacterial cultures and phage propagation. Solid medium (LB agar) for plates and soft agar (LB soft) for phage titrations were obtained by the addition of 1.5 and 0.6% agar, respectively. In some cases, the strains were grown on Worfel-Ferguson broth (Difco Laboratories). Phage adsorption and sensitivity. Bacteriophage adsorption to bacterial cells was studied by incubating 103 PFU with 107 bacteria for 15 min at 37°C. Chloroform (2 to 3 drops) was added, and the mixture was vortexed for 1 min. After centrifugation at 12,000 x g for 10 min, the titers of the remaining unadsorbed phages in the supernatant were determined for K. pneumoniae C3 (bacteriophages FC3-1, FC3-2, and FC3-9) or K. pneumoniae A5054 (bacteriophage 41). Phage sensitivity was assayed by a spot test. Cell surface isolation and analyses. Cell envelopes were prepared by lysis of whole cells in a French press at 16,000 lb/in2. Unbroken cells were removed by centrifugation at 10,000 x g for 10 min, and the envelope fraction was
Corresponding author. 2006
VOL. 59,
1991
SURFACE EXPOSURE OF K. PNEUMONIAE 01 SEROTYPE LPS
collected by centrifugation at 100,000 x g for 2 h. Cytoplasmic membranes were solubilized twice with sodium N-lauryl-sarcosinate (9), and the outer membrane fraction was collected as described above. Outer membrane proteins were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) by a modification (1) of the Laemmli procedure (14). Protein gels were fixed and stained with Coomassie blue. LPS was isolated and purified by using the methods of Westphal and Jann (30) as modified by Osborn (21). LPS was analyzed by SDS-PAGE and silver stained by the method of Tsai and Frasch (29). Also, LPS from strain C3 (O1:K66) was fractionated by the method of Ciurana and Tomas (3), and the high-molecular-weight LPS (HMW-LPS) fractions were pooled after extensive dialysis against distilled water. Isolation of K- mutants. Isogenic unencapsulated mutants were isolated as previously described (16), by using diethylsulfate as the mutagenic agent. Antisera. Anti-LPS serum (serotype 01) was obtained as previously described (25). Anti-K66 and anti-Kl sera were also obtained as previously described (25, 27). Anti-KlO and anti-K16 sera were obtained from adult New Zealand White rabbits with whole cells of formalin-killed K. pneumoniae KT759 and KT762, respectively, by the method of Edwards and Ewing (8). The serum was rendered capsular polysaccharide specific by extensive adsorption with strain KT717 (O1:K-, Frield 204) as described by 0rskov and 0rskov (19). Isogenic K- mutant strains were tested by different enzyme-linked immunosorbent assays (ELISAs), by using whole cells as antigens, as previously described (25). Accessibility of the 01 antigen to specific antibodies. K. pneumoniae strains were grown overnight at 37°C, and the cells (A6. = 0.1) were suspended in phosphate-buffered saline (PBS) buffer and washed three times with the same buffer by centrifugation in an Eppendorf microcentrifuge for 1 min. The cells were resuspended in anti-O1-specific serum (1: 100 dilution in PBS plus 1% of bovine serum albumin) and incubated for 1 h at 37°C, washed three times again with PBS buffer, and resuspended in protein A-alkaline phosphatase conjugate (Sigma P9650) (1:100 dilution in PBS plus 1% bovine serum albumin). After incubation for 1 h at 37°C, the cells were washed extensively with PBS buffer, resuspended in 1 ml of 4-nitrophenylphosphate (Sigma 104; 1 mg/ml) in diethanolamine buffer (0.95 M diethanolamine, 0.5 mM MgCl2 [pH 9.5]), and incubated at 37°C for 30 min. The reaction was stopped by adding 50 RI of 3 M NaOH, and the A405 was recorded. Controls not washed with PBS, with no added anti-O1-specific serum, or without cells were treated as described above. Immunogold electron microscopy. Immunogold electron microscopy was performed by the method of Godfrey et al. (11), a slight modification of the Robinson et al. procedure (23). Briefly, 20 RI of undiluted anti-Ol serum was mixed with 50 ,lI of bacteria resuspended in PBS (A6. = 1.0), and this mixture was incubated for 30 min at 37°C. After three washes in PBS, bacteria were resuspended in 20 ,u of protein A-10-nm gold particles (1:20 dilution in PBS) if not indicated otherwise and incubated for 30 min at 37°C prior to washing as described above. For serotypes Ki, K1O, K16, and K66, the entire process was also repeated by using, in addition to anti-Ol serum linked to protein A-20-nm gold particles, antisera to the different K antigens in conjunction with protein A-5-nm gold particles. Controls were cells treated with protein A-gold particles in the absence of specific antibodies. Bacterial suspensions were placed on 1Pormvarcoated copper grids, air dried, and examined ip a JEOL
2007
TABLE 1. K. pneumoniae strains from different serotypes, their phage sensitivity, and their adsorption to different bacteriophages Sensitivity to phage (% adsorbed phage)a:
Serotype
O1:Kl O1:K-
O1:K2 O1:K7 O1:K8 Ol:K10 O1:KOl:K12 Ol:K16 O1:KOl:K19 O1:K21 O1:K22 O1:K27 O1:K34 O1:K35 O1:K42 O1:K45 O1:K55 Ol:K57 O1:K62 O1:K64 O1:K66 O1:KO1:K69 O1:K70 a
FC3-1
FC3-2
FC3-9
4i1
R (
INFECTION AND IMMUNITY, June 1991, p. 2006-2011
0019-9567/91/062006-06$02.00/0 Copyright © 1991, American Society for Microbiology
Surface Exposure of 01 Serotype Lipopolysaccharide in Klebsiella pneumoniae Strains Expressing Different K Antigens JUAN M.
TOMAS,'*
SILVIA CAMPRUBI,' SUSANA MERINO,' MICHAEL R. DAVEY,2 AND PAUL WILLIAMS3 Departamento de Microbiologia, Facultad de Biologia, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain,' and Departments of Botany2 and Pharmaceutical Sciences,3 University of Nottingham, Nottingham NG7 2RD, United Kingdom Received 20 August 1990/Accepted 4 March 1991
Surface exposure of the 01 serotype lipopolysaccharide in encapsulated Klebsiella pneumoniae strains belonging to different serotypes was examined by using the 01 antigen-specific bacteriophages FC3-1 and FC3-2 in conjunction with immunogold electron microscopy and enzyme immunoassays with specific antisera. Despite the presence of the capsular polysaccharide, the 01 antigen was exposed at the cell surface in strains producing K2, K7, K8, K12, K19, K21, K22, K34, K35, K42, K45, K55, K57, K62, K66, K69, and K70 capsular polysaccharides. However, in strains producing KI, K10, and K16 capsular polysaccharides, the 01 antigen was masked by the K antigen. These results suggest that, since the 01 antigen is surface exposed in many different strains of K. pneumoniae with different capsular serotypes and is also able to immunoprotect, its potential as a useful vaccine component should not be overlooked.
Klebsiella species are important human pathogens which infect many different body sites and have become a leading cause of morbidity and mortality in hospital-acquired infections (2, 4, 12, 15). The majority of clinical Klebsiella isolates possess a well-defined capsule (4, 13, 20), and the genus Klebsiella has been classified serologically into some 77 capsular (K) types (18, 20). In addition, Klebsiella pneumoniae produces lipopolysaccharides (LPS) bearing complete 0-antigen chains, and eight chemically distinct 0 antigens (01, 03, 04, 05, 07, 08, 09, and 012) have been identified (19). Among clinical isolates, the 01 antigen is the most common 0 antigen encountered (18, 19). Also, there seem to be differences in the frequencies of capsular polysaccharides among clinical isolates (6). Both the 0 and K antigens contribute significantly to the virulence of K. pneumoniae (4, 7, 18, 19), by conferring protection against complementmediated serum killing (25, 31) and phagocytosis (31, 32), respectively. The emergence of multiply resistant K. pneumoniae strains and the high mortality rates associated with Klebsiella infections (2, 4, 12, 15) have highlighted the need to develop immunoprophylactic and immunotherapeutic agents (4, 5). In animal models, serotype-specific protection has been achieved in both murine pneumonia and burn wound models following vaccination with the homologous capsular polysaccharide (5). However, with antibodies directed against 24 different K serotypes which make up approximately 70% of all the bacteremic isolates, a capsular polysaccharide-based vaccine against Klebsiella organisms is now undergoing clinical evaluation (6), but alternatives for such a complex vaccine still need to be evaluated. As K. pneumoniae produces few 0 serotypes (18, 19, 24), knowledge of the distribution and surface exposure of this cell envelope antigen is required. We have previously reported that the 01 LPS molecules are not exposed in the different 01:K1 strains examined (27). In the present study, we examined a variety of different K. pneumoniae strains from different capsular serotypes to *
determine whether the 01 antigen was exposed at the cell surface or masked by the exopolysaccharide capsule. MATERIALS AND METHODS
Bacteria, bacteriophages, and media. We used 157 different K. pneumoniae strains belonging to serotype 01 and with different K antigens belonging to different culture collections (18, 10; International Center for Reference of Klebsiella; this laboratory) and also some clinical isolates from different hospitals in the Barcelona, Spain, area (Hospital de la Esperanza, Hospital del Mar, and Hospital de Sant Joan de Deu). The distribution of the strains for K serotypes is as follows: KI (6 strains), K2 (24 strains), K7 (5 strains), K8 (7 strains), K10 (12 strains), K12 (8 strains), K16 (8 strains), K19 (4 strains), K21 (14 strains), K22 (6 strains), K27 (5 strains), K34 (3 strains), K35 (6 strains), K42 (5 strains), K45 (8 strains), K55 (5 strains), K57 (3 strains), K62 (6 strains), K64 (9 strains), K66 (7 strains), K69 (4 strains), and K70 (2 strains). Bacteriophages FC3-1 and FC3-2 are LPS-specific phages (26, 28). FC3-9 and 44 are bacteriophages showing capsule depolymerase activity against strains from serotypes K66 and Kl, respectively (10, 27). Luria broth (LB; 17) was routinely used for bacterial cultures and phage propagation. Solid medium (LB agar) for plates and soft agar (LB soft) for phage titrations were obtained by the addition of 1.5 and 0.6% agar, respectively. In some cases, the strains were grown on Worfel-Ferguson broth (Difco Laboratories). Phage adsorption and sensitivity. Bacteriophage adsorption to bacterial cells was studied by incubating 103 PFU with 107 bacteria for 15 min at 37°C. Chloroform (2 to 3 drops) was added, and the mixture was vortexed for 1 min. After centrifugation at 12,000 x g for 10 min, the titers of the remaining unadsorbed phages in the supernatant were determined for K. pneumoniae C3 (bacteriophages FC3-1, FC3-2, and FC3-9) or K. pneumoniae A5054 (bacteriophage 41). Phage sensitivity was assayed by a spot test. Cell surface isolation and analyses. Cell envelopes were prepared by lysis of whole cells in a French press at 16,000 lb/in2. Unbroken cells were removed by centrifugation at 10,000 x g for 10 min, and the envelope fraction was
Corresponding author. 2006
VOL. 59,
1991
SURFACE EXPOSURE OF K. PNEUMONIAE 01 SEROTYPE LPS
collected by centrifugation at 100,000 x g for 2 h. Cytoplasmic membranes were solubilized twice with sodium N-lauryl-sarcosinate (9), and the outer membrane fraction was collected as described above. Outer membrane proteins were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) by a modification (1) of the Laemmli procedure (14). Protein gels were fixed and stained with Coomassie blue. LPS was isolated and purified by using the methods of Westphal and Jann (30) as modified by Osborn (21). LPS was analyzed by SDS-PAGE and silver stained by the method of Tsai and Frasch (29). Also, LPS from strain C3 (O1:K66) was fractionated by the method of Ciurana and Tomas (3), and the high-molecular-weight LPS (HMW-LPS) fractions were pooled after extensive dialysis against distilled water. Isolation of K- mutants. Isogenic unencapsulated mutants were isolated as previously described (16), by using diethylsulfate as the mutagenic agent. Antisera. Anti-LPS serum (serotype 01) was obtained as previously described (25). Anti-K66 and anti-Kl sera were also obtained as previously described (25, 27). Anti-KlO and anti-K16 sera were obtained from adult New Zealand White rabbits with whole cells of formalin-killed K. pneumoniae KT759 and KT762, respectively, by the method of Edwards and Ewing (8). The serum was rendered capsular polysaccharide specific by extensive adsorption with strain KT717 (O1:K-, Frield 204) as described by 0rskov and 0rskov (19). Isogenic K- mutant strains were tested by different enzyme-linked immunosorbent assays (ELISAs), by using whole cells as antigens, as previously described (25). Accessibility of the 01 antigen to specific antibodies. K. pneumoniae strains were grown overnight at 37°C, and the cells (A6. = 0.1) were suspended in phosphate-buffered saline (PBS) buffer and washed three times with the same buffer by centrifugation in an Eppendorf microcentrifuge for 1 min. The cells were resuspended in anti-O1-specific serum (1: 100 dilution in PBS plus 1% of bovine serum albumin) and incubated for 1 h at 37°C, washed three times again with PBS buffer, and resuspended in protein A-alkaline phosphatase conjugate (Sigma P9650) (1:100 dilution in PBS plus 1% bovine serum albumin). After incubation for 1 h at 37°C, the cells were washed extensively with PBS buffer, resuspended in 1 ml of 4-nitrophenylphosphate (Sigma 104; 1 mg/ml) in diethanolamine buffer (0.95 M diethanolamine, 0.5 mM MgCl2 [pH 9.5]), and incubated at 37°C for 30 min. The reaction was stopped by adding 50 RI of 3 M NaOH, and the A405 was recorded. Controls not washed with PBS, with no added anti-O1-specific serum, or without cells were treated as described above. Immunogold electron microscopy. Immunogold electron microscopy was performed by the method of Godfrey et al. (11), a slight modification of the Robinson et al. procedure (23). Briefly, 20 RI of undiluted anti-Ol serum was mixed with 50 ,lI of bacteria resuspended in PBS (A6. = 1.0), and this mixture was incubated for 30 min at 37°C. After three washes in PBS, bacteria were resuspended in 20 ,u of protein A-10-nm gold particles (1:20 dilution in PBS) if not indicated otherwise and incubated for 30 min at 37°C prior to washing as described above. For serotypes Ki, K1O, K16, and K66, the entire process was also repeated by using, in addition to anti-Ol serum linked to protein A-20-nm gold particles, antisera to the different K antigens in conjunction with protein A-5-nm gold particles. Controls were cells treated with protein A-gold particles in the absence of specific antibodies. Bacterial suspensions were placed on 1Pormvarcoated copper grids, air dried, and examined ip a JEOL
2007
TABLE 1. K. pneumoniae strains from different serotypes, their phage sensitivity, and their adsorption to different bacteriophages Sensitivity to phage (% adsorbed phage)a:
Serotype
O1:Kl O1:K-
O1:K2 O1:K7 O1:K8 Ol:K10 O1:KOl:K12 Ol:K16 O1:KOl:K19 O1:K21 O1:K22 O1:K27 O1:K34 O1:K35 O1:K42 O1:K45 O1:K55 Ol:K57 O1:K62 O1:K64 O1:K66 O1:KO1:K69 O1:K70 a
FC3-1
FC3-2
FC3-9
4i1
R (
