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Pseudomonas aeruginosa flagellar antibodies in serum, saliva and sputum from patients with cystic fibrosis
Serodiagnosis
and immunotherapy
in Infectious Disease (1990) 4, 35 1-36 1
Pseudomonas aeruginosa flagellar antibodies in serum, saliva and sputum from patients with cystic fibrosis
James W. Nelson’*, John R. W. Govan’ and G. Robin Barclay’
‘Department of Medical Microbiology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG and 2Regional Blood Transfusion Centre and Department qf Transfusion Medicine, Royal Infirmary, Edinburgh, Scotland, U.K.
Flagellar preparationsfrom Pseudomonas aeruginosa strainswerecharacterizedand usedin ELISA andimmunoblot studiesto detectanti-P. aeruginosa flagellarantibodies in sera, saliva and sputum from patients with cystic fibrosis(CF). Serumantiflagellar IgG antibodieswere detected,particularly in those CF patients intermittently or chronically colonized by P. aeruginosa. Antibodies to both type-a and -b flagellaweredetected;however,in somepatientsa pronouncedantibody responseto only oneof the flagellartypeswasevident. Raisedlevelsof anti-flagellarantibodiesin intermittently and non-P. aeruginosa colonized patients may representan early antibody responseto P. aeruginosa colonization of the CF respiratory tract and implicateinstigation of early anti-pseudomonalantibiotic therapy. Keywords: Pseudomonas aeruginosa,
flagella,antibody, cystic fibrosis. Introduction
Respiratory colonization by mucoid P. aeruginosa presents the major microbial challenge in patients with CF’. Motility and chemotaxis have been shown to be important virulence determinants for non-mucoid P. aeruginosa- ‘A in the colonization of burn wounds. Recently, we have demonstrated chemotaxis of P. aeruginosa towards respiratory mucins and this property may well play a key role in the initial colonization of the CF respiratory tract5. Bacterial flagella are potent immunogens evoking both humoral and cellular immune responses’j.Pseudomonas aeruginosa flagella may be classified into two main types, a and b7. The heterologous a-types can be further subdivided into five partial antigens (a,, a,, a2, a3,a4) with molecular weights ranging from 45,000 to 52,0008. All a-types have the a, antigen with usually one or more of the additional subtypes. The b-types comprise an homologous group with a molecular weight of 53,0008. Antiflagellar antibodies are able to induce phagocytosis and killing of motile strains of P. aeruginosa9*‘0 and show protection in animal model experiments”-‘3. Serum IgG anti-P. aeruginosa flagellar antibodies have previously been demonstrated in patients with CF I4315 The aim of this study was to investigate the antibody responseto * Author to whom correspondence should be addressed. 0888-0786/90/05035
1 + 11 $03.00/O
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Press Limited
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P. aeruginosa flagella in serum, saliva and sputum samples obtained from CF patients chronically, intermittently or non-colonized by the organism. The possible significance of anti-P. aeruginosa flagellar antibodies is discussed.
Materials
and methods
Patients
Serum, saliva and sputum specimens were obtained from patients with CF attending the paediatric and adult CF clinics in Edinburgh. The CF patients (age range, l-27 years) included patients chronically colonized with P. ueruginosa as well as patients intermittently or non-colonized with the organism. Serum samples were stored at - 20°C and saliva and sputum specimens at - 70°C. Prior to storage, sputa were spun at 13,000 g for 15 min and the sol phase retained for analysis. Bacterial strains and growth conditions Pseudomonasaeruginosa strains included the well characterized
genetic strain PAOl, and three non-mucoid strains 51385, JN61 and JN62 isolated from the sputum of patients with CF. For flagellar antigen preparation 2-litre flasks containing 1 Iitre of nutrient broth with 0.5% (w/v) yeast extract (NYB) were inoculated with 2ml of an overnight preculture grown in NYB. The flasks were incubated in an orbital incubator (120 r.p.m.) at 37°C for 16 h. Preparation of flagella
Overnight bacterial cultures were harvested by centrifugation (10,000 g, 15 min, 4°C) and washed once in phosphate buffered saline (PBS). Each bacterial pellet was suspended in 20 ml of PBS and blended with a commercial blender for 2 min. Bacterial cells were removed by up to 12 cycles of centrifugation (10,000 g, 15 min, 4°C). Removal of cells was monitored at intervals by microscopy and culture of the supernatant. The cell-free supernatant was centrifuged at 100,000 g for 1.5 h at 4°C and the resultant pellet washed twice in distilled water. The pellet was then resuspended in 0.5 ml of distilled water, dialyzed overnight against distilled water at 4°C and stored at -20°C. The amount of protein in each sample was determined by the method of LowryI with bovine serum albumin as standard. The final flagellar preparations were visualized by electron microscopy after staining with 2% phosphotungstic acid. Flagella were classified into a and b flagellar types based on their migration in polyacrylamide gels, and ELISA and immunoblot analysis with rabbit anti-flagellar type-a and -b antisera. Absorption of specimens
To avoid immunological detection of anti-LPS antibodies” sera, saliva and sputa were mixed with an equal volume of LPS (1 mgml-‘) extracted from P. aeruginosa by the method of Westphal & Luderitz ‘* . Prior to analyses in ELISA and immunoblot studies, samples were incubated for 60 min at 37°C followed by overnight incubation at 4°C. The LPS used for absorption of LPS antibodies was from the strain used to prepare the corresponding flagella. Removal of LPS antibodies was confirmed by ELISA against each extracted LPS.
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ELISA Diluents and buffers used in the ELISA were essentially as described previously except sodium azide was omitted19. Indirect ELISA was used to determine the presenceof IgA and IgG anti-P. ueruginosa flagellar antibodies in CF sera, saliva and sputa. Flagellar antigens of both a- and b-types were diluted in coating buffer to a concentration of 1 ug ml-’ and added to polystyrene microwell strips (Polysorb, Nunc) at 100 ul per well. Strips were coated overnight at room temperature and washed four times with wash buffer. Strips were then post-coated with post-coat buffer (containing 5% bovine serum albumin) at 100ul per well overnight at room temperature. After being washed four times with wash buffer, strips were stored at -20°C until used. Pre-absorbed sera were diluted 1:200, and pre-absorbed saliva and sputa 1:100 in dilution buffer and added to flagella coated microstrips at 100 ul per well in duplicate. Strips were incubated at 37°C for 90 min before washing four times with wash buffer. Peroxidase-conjugated sheep anti-human IgA and IgG (ICN Biomedicals) were each diluted 1:lOOOand added to the relevant strips at 100ul per well. Strips were incubated for a further 90 min at 37°C. Strips were washed four times and rinsed with distilled water before substrate at 100pl per well was added. Substrate consisted of 0.1 M sodium acetate/citrate buffer (pH 6.0) containing 1% tetramethyl-benzidine and 0.0 15% (vol/ vol) hydrogen peroxide. Strips were incubated for 60 min at room temperature and reactions stopped by adding 2 M sulphuric acid at 25 ul per well. The optical density (OD) of wells was read at 450 nm on an automated microplate reader (Titretek Multiskan, Flow Laboratories). Final results were expressedafter subtraction of the OD of negative control wells (coated only with BSA post-coat) for each serum, saliva and sputum sample. SDS-PAGE Flagellar preparations were analysed by SDS-PAGE with 12% acrylamide gels and the buffer system of Laemmli*O. Flagellar protein samples(10 ug protein per loading track) were detected with Coomassie blue R250 stain as described by Hancock & Poxton”. Immunoblotting For immunoblotting, flagella separated in acrylamide gels were electrophoretically transferred to nitrocellulose membranes (0.2 urn Schleicher & Schuell) by the method of Towbin et al.” The flagella were then probed with absorbed sera, saliva and sputa. Antiflagellar antibodies in these specimenswere visualized by anti-human IgA and IgG horse-radish peroxidase conjugates (ICN Biomedicals) and subsequentincubation with substrate. Results Flagellar preparations isolated from the three clinical P. aeruginosa strains were characterized by molecular weight determination in SDS-PAGE and by ELISA and immunoblot analysis with anti-flagellar type-a and -b anti-sera. Flagella isolated from PA0 1, classifiedas flagellar type-b8, were used as a standard. Flagella from P. aeruginosa strains JN61 (type-a) and 51385 (type-b) (Figure 1) were subsequently used to screen anti-P. aeruginosa flagellar antibodies in patients with CF.
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Figure 1. SDS-PAGE of flagella preparations from P. ueruginosa strains PA01 (Track 1). 11385 (Track Z), JN61 (Track 3) and JN62 (Track 4) were separated using 12% w/v acrylamide gels and visualized with Coomassie blue stain. Molecular weights of protein standards are indicated.
A range of levels of anti-flagellar antibodies were demonstrated in the sera, saliva and sputum from patients with CF. Anti-flagellar IgG antibodies were found in sera from patients with CF and in sera from healthy controls (Figure 2). Elevated levels of antiflagellar IgG antibodies above the control range were found in CF patients intermittently or chronically colonized with P. aeruginosa. However, a number of CF patients classed as non-P. aeruginosa colonized on the basis of sputum bacteriology also had increased levels of serum anti-flagellar IgG antibodies. For many of the CF patients a similar IgG antibody response to both a-type and b-type flagella was observed. A pronounced antibody response to either a- or b-type flagella was also observed for some CF patients. The difference in absorbance readings between chronically colonized patients and non-P. aeruginosa colonized CF patients or healthy controls was very highly significant (P< 0.001, by the Student t test) for type-a and -b flagella; the
Flagella TvDe-a
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Figure 2. Serum IgG anti-P. aeruginosa flagellar antibodies in 48 CF patients and 10 non-CF individuals measured by ELBA with flagellar type-a and type-b as coating antigens. A total of 23 CF patients chronically colonized with P. aeruginosa, five intermittently colonized. 20 non-colonized CF patients and 10 healthy adults are included.
difference in OD readings between intermittently colonized CF patients and healthy controls was very highly significant (P < 0.00 1) for type-a flagella and highly significant (P
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Figure 3. Sputum IgA anti-P. ueruginosa flagellar antibodies in 39 CF patients measured by ELBA with flagellar type-a and -b as coating antigens. A total of 18 CF patients chronically colonized with P. aeruginosu, seven intermittently colonized patients and 14 non-colonized CF patients are included. Flagella Type-a
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antibodies in 27 CF patients measured by ELISA A total of 10 CF patients chronically colonized and 12 non-colonized CF patients are included.
with with
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Figure 5. Strip immunoblots of isolated P. ueruginosa flagella (type-a and/or type-b) reacted with serum from patients with CF and analysed for the presence of anti-tlagellar IgG antibodies. Strip 1, healthy control; Strip 2, CF non-col.; Strip 3, CF non-co].; Strip 4, CF co].; Strip 5, CF col.; Strip 6, CF int. col.; Strip 7. CF int. CO].; Strip 8. CF int. col.; Strip 9, chron. col.: Strip 10, chron. co].; Strip 11, chron. co].; Strip 12, chron. col. (CF non-col. = CF patient non-P. aeruginosa colonized; CF col. = CF patient colonized with non-mucoid P. aeruginosa; CF int. col. = CF patient intermittently colonized with non-mucoid P. aeruginosa; CF chron. col. = CF patient chronically colonized with mucoid P. aeruginosa).
J. W. Nelson et al.
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Figure 6. Strip immunoblots of isolated P. aemginosa flagella (type-a and/or type-b) reacted with saliva or sputum from patients with CF and analysed for the presence of anti-flagellar IgA antibodies. Strip 1, CF noncol. (Sputum); Strip 2, CF int. col. (Sputum); Strip 3, CF int. col. (Saliva); Strip 4, CF int. col. (Sputum); Strip 5, CF int. col. (Sputum); Strip 6, chron. col. (Sputum); Strip 7, chron. col. (Sputum); Strip 8, chron. col. (Saliva). (CF non-col. = CF patient non-P. aeruginosa colonized; CF int. col. = CF patient intermittently colonized with non-mucoid P. aeruginosa; CF chron. col. = CF patient chronically colonized with mucoid P. aeruginosa).
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Discussion The observation of similar levels of anti-flagellar type-a and type-b antibodies observed in many of the CF patients could be due to infections with at least two different P. aeruginosastrains. Alternatively, the presence of the b or a, epitope in all 17 flagellar serotypes7 as well as a common N-terminal amino acid sequence in the flagellin proteinI may account for some of the cross-reactions observed in the ELISA and immunoblot studies. Another possible explanation is diphasic variation in the flagellar antigens27 although no such variations were reported by Montie & Anderson24. A pronounced antibody response to either type-a or -b flagella found in some CF patients indicate either infection by P. aeruginosa strains of one flagellum type or perhaps by only one single strain25. Shand et al.‘4 reported that many CF patients infected with P. aeruginosa show antibodies against flagellar preparations early after the onset of colonization. The raised levels of anti-flagellar antibodies observed in intermittently and sometimes noncolonized patients adds support to this report. ELISA screening of anti-flagellar IgA and IgG antibodies in patients with CF may provide a useful indication of the early onset of pulmonary colonization by P. aeruginosaand provide a suitable parameter for instigation of anti-pseudomonal antibiotic therapy. Przyklenk & Bauernfeind26 demonstrated the presence of P. ueruginosuantibodies in non-P. aeruginosacolonized CF patients and concluded that secretory IgA antibodies correlated better with the clinical status of the patients than the serum response. The anti-P. aeruginosaflagellar antibodies observed in saliva and sputum specimens from patients with CF may thus reflect a current exacerbation with P. aeruginosu. Our future studies will focus on longitudinal monitoring of anti-flagellar antibodies in sputum and saliva from CF patients classified as nonP. aeruginosacolonized (on the basis of sputum bacteriology) and the use of such studies for prediction of P. ueruginosacolonization. Absence or reduced levels of anti-flagellar IgA antibodies in saliva or sputum specimens obtained from CF patients, confirmed by bacterial culture to be colonized by P. aeruginosa, may be caused by involvement of the antibodies in immune complex formation27, or degradation by P. aeruginosa elastase**. Despite an apparently competent antibody response to P. aeruginosa cell surface antigens29,30 including the anti-flagellar IgA and IgG antibodies reported in this study, the organism is able to persist and indeed, is seldom eradicated from the lungs of CF patient$‘. Experimental animal models involving vaccination with P. aeruginosaflagellar antigens and protection against subsequent infection, have been reported”-13. Antiflagellar antibodies may act by simple inhibition of the motility of P. aeruginosa’0,32 or by opsonophagocytosis of the organism9.‘0. The role of mucosal immunity and the protective capacity of IgA antibodies is not well understood. In patients with CF an antibody response to flagella is likely to be manifested at an early stage of colonization/ infection since motility, chemotaxis and expression of flagellar appendages appears to be less important during chronic infection33. Since motility of P. aeruginosamay be important in early lung colonization of patients with CF, vaccination with a bivalent vaccine consisting of a- and b-type flagella or passive treatment with flagellar antibodieslO would appear rational, but as yet unproven, strategies for prevention of colonization. Vaccination of patients already harbouring Pseudomonasmay exacerbate pulmonary deterioration34 as a result of immune complex mediated tissue damage3’. However, strategies to improve pulmonary clearance via the
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muco-ciliary escalator and the presence of pre-existing mucosal antibodies induced by immunization, may combined, present a more formidable barrier to initial colonization by P. aeruginosa.
Acknowledgements We thank Dr G. H. Shand (Statens Seruminstitut, Copenhagen) for providing the rabbit anti-flagellar type-a and -b antisera.
References 1. Govan JRW, GlassS. The microbiology and therapy of cystic fibrosislung infections. Rev Med Microbial 1990;1: 19-28. 2. Craven RC, Montie TC. Motility and chemotaxisof three strainsof P. aeruginosa usedfor virulence studies.Can J Microbial 1981;27: 458-60. 3. Montie TC, Doyle-Huntzinger D, Craven RC, Holder IA. Lossof virulence associatedwith absenceof flagellumin an isogenicmutant of Pseudomonas aeruginosa in the burned-mouse model. Infect Immun 1982;38: 1296-8. 4. Drake D, Montie TC. Flagella, motility and invasivevirulence of Pseudomonas aeruginosa. J Gen Microbial 1988;134:43-52. 5. NelsonJW, Tredgett MW, SheehanJK, Thornton DJ, Notman D, Govan JRW. Mucinophilit and chemotacticpropertiesof Pseudomonas aeruginosa in relation to pulmonary colonization in cystic fibrosis.Infect Immun 1990;58: 148995. 6. Newton SMC, JacobCO, Stocker BAD. Immuneresponse to choleratoxin epitopeinsertedin Salmonella flagellin. Science1989;244: 70-2. 7. Ansorg R. Flagellaspezfisches H-antigenschemavon Pseudomonas aeruginosa. Zentralbl Bakteriol Mikrobiol Hyg (A) 1978;242: 228-38. 8. Allison JS, Dawson M, Drake D, Montie TC. Electrophoretic separationand molecular weight characterization of Pseudomonas aeruginosa H-antigen flagellins.Infect Immun 1985; 49: 770-4. 9. Anderson TR, Montie TC. Flagellar antibody stimulatedopsonophagocytosis of Pseudomonas aeruginosa associatedwith responseto either a- or b-type flagellar antigen. Can J Microbial 1989;35: 890-4. 10. Rotering H, Domer F. Studieson a Pseudomonas aeruginosa flagellarvaccine. In: Hoiby N, PedersenSS,ShandGH, Doring G, Holder IA, eds.Antibiot Chemother42. Base]:Karger, 1989:218-38. 11. Holder IA, Wheeler R, Montie TC. Flagellar preparationsfrom Pseudomonas aeruginosa: Animal protection studies.Infect Immun 1982;35: 27680. 12. Drake D, Montie TC. Protection against Pseudomonas aeruginosa infection by passive transfer of anti-flagellarserum.Can J Microbial 1987;33: 755-63. 13. Montie TC, Drake D, SellinH, Slater0, EdmondsS. Motility, virulenceand protection with a flagella vaccineagainstinfection. Antibiot Chemother1987;39: 23348. 14. ShandGH, FomsgaardA, PedersenSS,Hoiby N. Antibody responsein CF serumto protein antigensof Pseudomonas aeruginosa outer membrane.Pediatr Pulmonol 1988;Suppl2: 125. 15. Anderson TR, Montie TC, Murphy MD, McCarthy VP. Pseudomonas aeruginosu flagellar antibodiesin patientswith cystic fibrosis.J Clin Micro 1989;27: 2789-93. 16. Lowry OH, RosenbroughNJ, Farr AL, Randall RJ. Protein measurementwith the folin phenol reagent.J Biol Chem 1951;193:265-75. 17. Poxton IR, Bell GT, Barclay GR. The associationon SDS-polyacrylamidegelsof lipopolysaccharideand outer membraneproteinsof Pseudomonas aeruginosa asrevealedby monoclonal antibodiesand Westernblotting. FEMS Microbial Lett 1985;27: 247-51. 18. Westphal 0, Luderitz 0. Chemischeerforschung von lipopolysaccharidengramnegative bacterien.Angew Chem 1954;66: 407-17.
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19. Nelson JW, Barclay GR, Govan JRW. Diagnosis of chronic Pseudomonus ueruginosu infection in cystic fibrosis by ELISA for anti-Pseudomonas LPS IgG antibodies. Serodiag Immunother Infect Dis 1990; 4: 9-16. 20. Laemmli UK. Cleavage of structural proteins during the assembly of the head protein of bacteriophage T4. Nature 1970; 277: 680-8. 21. Hancock IC, Poxton IR. Bacterial cell surface techniques. Chichester: John Wiley and Sons Ltd, 1988: 279-80. 22. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat Acad Sci USA 1979: 76: 4350-4. 23. Pitt TL. Diphasic variation in the flagellar antigens of Pseudomonas aeruginosa. FEMS Microbial Lett 1980; 9: 301-6. 24. Montie TC. Anderson TR. Enzyme-linked immunosorbent assay for detection of Pseudomonas aeruginosa H (flagellar) antigen. Eur J Clin Microbial Infect Dis 1988; 7: 256-60. 25. Ogle JW, Janda JM, Woods DE, Vasil ML. Characterization and use of a DNA probe as an epidemiological marker for Pseudomonas aeruginosa. J Infect Dis 1987; 155: 119-26. 26. Przyklenk BK, Bauernfeind A. Significance of immunologic factors in cystic fibrosis. Stand J Gastroenterol 1988; 23 (Suppl. 143): 103-9. 27. Kronborg G, Fomsgaard A, Shand GH, Hoiby N. Determination of the components of immune complexes made in vitro with antigens derived from Pseudomonas aeruginosa. J Immunol Methods 1989; 122: 51-7. 28. Heck LW, Alarcon PG, Kulhauy RM, Morihara K, Russell MW, Mestecky JF. Degradation of IgA proteins by Pseudomonas aeruginosu elastase. J Immunol 1990; 144: 2253-7. 29. Hoiby N. Microbiology of lung infections in cystic fibrosis patients. Acta Paediatr Stand Suppl 1982; 301: 3%54. 30. Speert DP. Host defences in patients with cystic fibrosis: modulation by Pseudomonas ueruginosu. Surv Synth Path01 Res 1985; 4: 14-33. 31. Pier GB. Pulmonary disease associated with Pseudomonas aeruginosu in cystic fibrosis: current status of the host-bacterium interaction. J Infect Dis 1985; 151: 575-80. 32. Montie TC, Craven RC, Holder IA. Flagellar preparations from Pseudomonus aeruginosa isolation and characterization. Infect Immun 1982; 35: 281-8. 33. Luzar MA, Thomassen MJ, Montie TC. Flagella and motility alterations in Pseudomonas ueruginosu strains from patients with cystic fibrosis: relationship to patient’s clinical condition. Infect Immun 1985; 50: 577-82. 34. Langford DT. Hiller J. Prospective, controlled study of a polyvalent pseudomonas vaccine in cystic fibrosis-three year results. Arch Dis Child 1984; 59: 11314. 35. Moss RB. Lewiston NJ. Immune complexes and humoral response to Pseudomonas aprugi~OSU in cystic fibrosis. Am Rev Resp Dis 1980: 121: 23-9.
and immunotherapy
in Infectious Disease (1990) 4, 35 1-36 1
Pseudomonas aeruginosa flagellar antibodies in serum, saliva and sputum from patients with cystic fibrosis
James W. Nelson’*, John R. W. Govan’ and G. Robin Barclay’
‘Department of Medical Microbiology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG and 2Regional Blood Transfusion Centre and Department qf Transfusion Medicine, Royal Infirmary, Edinburgh, Scotland, U.K.
Flagellar preparationsfrom Pseudomonas aeruginosa strainswerecharacterizedand usedin ELISA andimmunoblot studiesto detectanti-P. aeruginosa flagellarantibodies in sera, saliva and sputum from patients with cystic fibrosis(CF). Serumantiflagellar IgG antibodieswere detected,particularly in those CF patients intermittently or chronically colonized by P. aeruginosa. Antibodies to both type-a and -b flagellaweredetected;however,in somepatientsa pronouncedantibody responseto only oneof the flagellartypeswasevident. Raisedlevelsof anti-flagellarantibodiesin intermittently and non-P. aeruginosa colonized patients may representan early antibody responseto P. aeruginosa colonization of the CF respiratory tract and implicateinstigation of early anti-pseudomonalantibiotic therapy. Keywords: Pseudomonas aeruginosa,
flagella,antibody, cystic fibrosis. Introduction
Respiratory colonization by mucoid P. aeruginosa presents the major microbial challenge in patients with CF’. Motility and chemotaxis have been shown to be important virulence determinants for non-mucoid P. aeruginosa- ‘A in the colonization of burn wounds. Recently, we have demonstrated chemotaxis of P. aeruginosa towards respiratory mucins and this property may well play a key role in the initial colonization of the CF respiratory tract5. Bacterial flagella are potent immunogens evoking both humoral and cellular immune responses’j.Pseudomonas aeruginosa flagella may be classified into two main types, a and b7. The heterologous a-types can be further subdivided into five partial antigens (a,, a,, a2, a3,a4) with molecular weights ranging from 45,000 to 52,0008. All a-types have the a, antigen with usually one or more of the additional subtypes. The b-types comprise an homologous group with a molecular weight of 53,0008. Antiflagellar antibodies are able to induce phagocytosis and killing of motile strains of P. aeruginosa9*‘0 and show protection in animal model experiments”-‘3. Serum IgG anti-P. aeruginosa flagellar antibodies have previously been demonstrated in patients with CF I4315 The aim of this study was to investigate the antibody responseto * Author to whom correspondence should be addressed. 0888-0786/90/05035
1 + 11 $03.00/O
351
ic 1990 Academic
Press Limited
352
J. W. Nelson et al.
P. aeruginosa flagella in serum, saliva and sputum samples obtained from CF patients chronically, intermittently or non-colonized by the organism. The possible significance of anti-P. aeruginosa flagellar antibodies is discussed.
Materials
and methods
Patients
Serum, saliva and sputum specimens were obtained from patients with CF attending the paediatric and adult CF clinics in Edinburgh. The CF patients (age range, l-27 years) included patients chronically colonized with P. ueruginosa as well as patients intermittently or non-colonized with the organism. Serum samples were stored at - 20°C and saliva and sputum specimens at - 70°C. Prior to storage, sputa were spun at 13,000 g for 15 min and the sol phase retained for analysis. Bacterial strains and growth conditions Pseudomonasaeruginosa strains included the well characterized
genetic strain PAOl, and three non-mucoid strains 51385, JN61 and JN62 isolated from the sputum of patients with CF. For flagellar antigen preparation 2-litre flasks containing 1 Iitre of nutrient broth with 0.5% (w/v) yeast extract (NYB) were inoculated with 2ml of an overnight preculture grown in NYB. The flasks were incubated in an orbital incubator (120 r.p.m.) at 37°C for 16 h. Preparation of flagella
Overnight bacterial cultures were harvested by centrifugation (10,000 g, 15 min, 4°C) and washed once in phosphate buffered saline (PBS). Each bacterial pellet was suspended in 20 ml of PBS and blended with a commercial blender for 2 min. Bacterial cells were removed by up to 12 cycles of centrifugation (10,000 g, 15 min, 4°C). Removal of cells was monitored at intervals by microscopy and culture of the supernatant. The cell-free supernatant was centrifuged at 100,000 g for 1.5 h at 4°C and the resultant pellet washed twice in distilled water. The pellet was then resuspended in 0.5 ml of distilled water, dialyzed overnight against distilled water at 4°C and stored at -20°C. The amount of protein in each sample was determined by the method of LowryI with bovine serum albumin as standard. The final flagellar preparations were visualized by electron microscopy after staining with 2% phosphotungstic acid. Flagella were classified into a and b flagellar types based on their migration in polyacrylamide gels, and ELISA and immunoblot analysis with rabbit anti-flagellar type-a and -b antisera. Absorption of specimens
To avoid immunological detection of anti-LPS antibodies” sera, saliva and sputa were mixed with an equal volume of LPS (1 mgml-‘) extracted from P. aeruginosa by the method of Westphal & Luderitz ‘* . Prior to analyses in ELISA and immunoblot studies, samples were incubated for 60 min at 37°C followed by overnight incubation at 4°C. The LPS used for absorption of LPS antibodies was from the strain used to prepare the corresponding flagella. Removal of LPS antibodies was confirmed by ELISA against each extracted LPS.
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ELISA Diluents and buffers used in the ELISA were essentially as described previously except sodium azide was omitted19. Indirect ELISA was used to determine the presenceof IgA and IgG anti-P. ueruginosa flagellar antibodies in CF sera, saliva and sputa. Flagellar antigens of both a- and b-types were diluted in coating buffer to a concentration of 1 ug ml-’ and added to polystyrene microwell strips (Polysorb, Nunc) at 100 ul per well. Strips were coated overnight at room temperature and washed four times with wash buffer. Strips were then post-coated with post-coat buffer (containing 5% bovine serum albumin) at 100ul per well overnight at room temperature. After being washed four times with wash buffer, strips were stored at -20°C until used. Pre-absorbed sera were diluted 1:200, and pre-absorbed saliva and sputa 1:100 in dilution buffer and added to flagella coated microstrips at 100 ul per well in duplicate. Strips were incubated at 37°C for 90 min before washing four times with wash buffer. Peroxidase-conjugated sheep anti-human IgA and IgG (ICN Biomedicals) were each diluted 1:lOOOand added to the relevant strips at 100ul per well. Strips were incubated for a further 90 min at 37°C. Strips were washed four times and rinsed with distilled water before substrate at 100pl per well was added. Substrate consisted of 0.1 M sodium acetate/citrate buffer (pH 6.0) containing 1% tetramethyl-benzidine and 0.0 15% (vol/ vol) hydrogen peroxide. Strips were incubated for 60 min at room temperature and reactions stopped by adding 2 M sulphuric acid at 25 ul per well. The optical density (OD) of wells was read at 450 nm on an automated microplate reader (Titretek Multiskan, Flow Laboratories). Final results were expressedafter subtraction of the OD of negative control wells (coated only with BSA post-coat) for each serum, saliva and sputum sample. SDS-PAGE Flagellar preparations were analysed by SDS-PAGE with 12% acrylamide gels and the buffer system of Laemmli*O. Flagellar protein samples(10 ug protein per loading track) were detected with Coomassie blue R250 stain as described by Hancock & Poxton”. Immunoblotting For immunoblotting, flagella separated in acrylamide gels were electrophoretically transferred to nitrocellulose membranes (0.2 urn Schleicher & Schuell) by the method of Towbin et al.” The flagella were then probed with absorbed sera, saliva and sputa. Antiflagellar antibodies in these specimenswere visualized by anti-human IgA and IgG horse-radish peroxidase conjugates (ICN Biomedicals) and subsequentincubation with substrate. Results Flagellar preparations isolated from the three clinical P. aeruginosa strains were characterized by molecular weight determination in SDS-PAGE and by ELISA and immunoblot analysis with anti-flagellar type-a and -b anti-sera. Flagella isolated from PA0 1, classifiedas flagellar type-b8, were used as a standard. Flagella from P. aeruginosa strains JN61 (type-a) and 51385 (type-b) (Figure 1) were subsequently used to screen anti-P. aeruginosa flagellar antibodies in patients with CF.
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Figure 1. SDS-PAGE of flagella preparations from P. ueruginosa strains PA01 (Track 1). 11385 (Track Z), JN61 (Track 3) and JN62 (Track 4) were separated using 12% w/v acrylamide gels and visualized with Coomassie blue stain. Molecular weights of protein standards are indicated.
A range of levels of anti-flagellar antibodies were demonstrated in the sera, saliva and sputum from patients with CF. Anti-flagellar IgG antibodies were found in sera from patients with CF and in sera from healthy controls (Figure 2). Elevated levels of antiflagellar IgG antibodies above the control range were found in CF patients intermittently or chronically colonized with P. aeruginosa. However, a number of CF patients classed as non-P. aeruginosa colonized on the basis of sputum bacteriology also had increased levels of serum anti-flagellar IgG antibodies. For many of the CF patients a similar IgG antibody response to both a-type and b-type flagella was observed. A pronounced antibody response to either a- or b-type flagella was also observed for some CF patients. The difference in absorbance readings between chronically colonized patients and non-P. aeruginosa colonized CF patients or healthy controls was very highly significant (P< 0.001, by the Student t test) for type-a and -b flagella; the
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Figure 2. Serum IgG anti-P. aeruginosa flagellar antibodies in 48 CF patients and 10 non-CF individuals measured by ELBA with flagellar type-a and type-b as coating antigens. A total of 23 CF patients chronically colonized with P. aeruginosa, five intermittently colonized. 20 non-colonized CF patients and 10 healthy adults are included.
difference in OD readings between intermittently colonized CF patients and healthy controls was very highly significant (P < 0.00 1) for type-a flagella and highly significant (P
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J. W. Nelson et al. Flagella Type-a I.5
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Figure 3. Sputum IgA anti-P. ueruginosa flagellar antibodies in 39 CF patients measured by ELBA with flagellar type-a and -b as coating antigens. A total of 18 CF patients chronically colonized with P. aeruginosu, seven intermittently colonized patients and 14 non-colonized CF patients are included. Flagella Type-a
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antibodies in 27 CF patients measured by ELISA A total of 10 CF patients chronically colonized and 12 non-colonized CF patients are included.
with with
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Figure 5. Strip immunoblots of isolated P. ueruginosa flagella (type-a and/or type-b) reacted with serum from patients with CF and analysed for the presence of anti-tlagellar IgG antibodies. Strip 1, healthy control; Strip 2, CF non-col.; Strip 3, CF non-co].; Strip 4, CF co].; Strip 5, CF col.; Strip 6, CF int. col.; Strip 7. CF int. CO].; Strip 8. CF int. col.; Strip 9, chron. col.: Strip 10, chron. co].; Strip 11, chron. co].; Strip 12, chron. col. (CF non-col. = CF patient non-P. aeruginosa colonized; CF col. = CF patient colonized with non-mucoid P. aeruginosa; CF int. col. = CF patient intermittently colonized with non-mucoid P. aeruginosa; CF chron. col. = CF patient chronically colonized with mucoid P. aeruginosa).
J. W. Nelson et al.
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Figure 6. Strip immunoblots of isolated P. aemginosa flagella (type-a and/or type-b) reacted with saliva or sputum from patients with CF and analysed for the presence of anti-flagellar IgA antibodies. Strip 1, CF noncol. (Sputum); Strip 2, CF int. col. (Sputum); Strip 3, CF int. col. (Saliva); Strip 4, CF int. col. (Sputum); Strip 5, CF int. col. (Sputum); Strip 6, chron. col. (Sputum); Strip 7, chron. col. (Sputum); Strip 8, chron. col. (Saliva). (CF non-col. = CF patient non-P. aeruginosa colonized; CF int. col. = CF patient intermittently colonized with non-mucoid P. aeruginosa; CF chron. col. = CF patient chronically colonized with mucoid P. aeruginosa).
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Discussion The observation of similar levels of anti-flagellar type-a and type-b antibodies observed in many of the CF patients could be due to infections with at least two different P. aeruginosastrains. Alternatively, the presence of the b or a, epitope in all 17 flagellar serotypes7 as well as a common N-terminal amino acid sequence in the flagellin proteinI may account for some of the cross-reactions observed in the ELISA and immunoblot studies. Another possible explanation is diphasic variation in the flagellar antigens27 although no such variations were reported by Montie & Anderson24. A pronounced antibody response to either type-a or -b flagella found in some CF patients indicate either infection by P. aeruginosa strains of one flagellum type or perhaps by only one single strain25. Shand et al.‘4 reported that many CF patients infected with P. aeruginosa show antibodies against flagellar preparations early after the onset of colonization. The raised levels of anti-flagellar antibodies observed in intermittently and sometimes noncolonized patients adds support to this report. ELISA screening of anti-flagellar IgA and IgG antibodies in patients with CF may provide a useful indication of the early onset of pulmonary colonization by P. aeruginosaand provide a suitable parameter for instigation of anti-pseudomonal antibiotic therapy. Przyklenk & Bauernfeind26 demonstrated the presence of P. ueruginosuantibodies in non-P. aeruginosacolonized CF patients and concluded that secretory IgA antibodies correlated better with the clinical status of the patients than the serum response. The anti-P. aeruginosaflagellar antibodies observed in saliva and sputum specimens from patients with CF may thus reflect a current exacerbation with P. aeruginosu. Our future studies will focus on longitudinal monitoring of anti-flagellar antibodies in sputum and saliva from CF patients classified as nonP. aeruginosacolonized (on the basis of sputum bacteriology) and the use of such studies for prediction of P. ueruginosacolonization. Absence or reduced levels of anti-flagellar IgA antibodies in saliva or sputum specimens obtained from CF patients, confirmed by bacterial culture to be colonized by P. aeruginosa, may be caused by involvement of the antibodies in immune complex formation27, or degradation by P. aeruginosa elastase**. Despite an apparently competent antibody response to P. aeruginosa cell surface antigens29,30 including the anti-flagellar IgA and IgG antibodies reported in this study, the organism is able to persist and indeed, is seldom eradicated from the lungs of CF patient$‘. Experimental animal models involving vaccination with P. aeruginosaflagellar antigens and protection against subsequent infection, have been reported”-13. Antiflagellar antibodies may act by simple inhibition of the motility of P. aeruginosa’0,32 or by opsonophagocytosis of the organism9.‘0. The role of mucosal immunity and the protective capacity of IgA antibodies is not well understood. In patients with CF an antibody response to flagella is likely to be manifested at an early stage of colonization/ infection since motility, chemotaxis and expression of flagellar appendages appears to be less important during chronic infection33. Since motility of P. aeruginosamay be important in early lung colonization of patients with CF, vaccination with a bivalent vaccine consisting of a- and b-type flagella or passive treatment with flagellar antibodieslO would appear rational, but as yet unproven, strategies for prevention of colonization. Vaccination of patients already harbouring Pseudomonasmay exacerbate pulmonary deterioration34 as a result of immune complex mediated tissue damage3’. However, strategies to improve pulmonary clearance via the
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muco-ciliary escalator and the presence of pre-existing mucosal antibodies induced by immunization, may combined, present a more formidable barrier to initial colonization by P. aeruginosa.
Acknowledgements We thank Dr G. H. Shand (Statens Seruminstitut, Copenhagen) for providing the rabbit anti-flagellar type-a and -b antisera.
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