Immunoblot fingerprinting Aspergillus fumigatus

Journal of Immunological Methods, 118 (1989) 179-186

179

Elsevier JIM 05107

Immunoblot fingerprinting Aspergillusfumigatus J.P. B u r n i e 1, R.C. M a t t h e w s 1, I. C l a r k 1 a n d L.J.R. M i l n e 2 Department of Medical Microbiology, St. Bartholomew'sHospital, West Smithfield, London ECIA 7BE, U.K.., and 2 Central Microbiological Laboratories, Western GeneralHospital, Crewe Road Edinburgh EH4 2XU, U.K.

(Received 14 September1988, revised received4 November 1988, accepted 9 November 1988)

A new technique for typing Aspergillus fumigatus is presented. This is based on immunoblot fingerprinting each isolate against a rabbit hyperimmune antiserum raised against A. fumigatus N C T C 2109. All isolates were typable and reproducibility for the 16 antigenic bands which formed the basis of the system was excellent. Discrimination was better than silver staining and revealed 11 types among the 21 isolates from eight patients with an aspergilloma. Each aspergilloma could be due to either a single or multiple types. Key words: Aspergillusfumigatus; Aspergilloma; Immunoblot fingerprinting

Introduction

Invasive aspergillosis has become an important clinical problem due to the increase in the number of immunocompromised patients (Fraser et al., 1979). The disease is most commonly due to the mould A. fumigatus and carries a mortality in excess of 90%. In recent years there has been a marked increase in the number of outbreaks of disseminated aspergillosis occurring within hospitals (Opal et al., 1986; Perraud et al., 1987). These outbreaks have been seen associated with the building of new hospitals, although the lack of a typing method has made it impossible to prove this connection. Conventional techniques such as phage typing, antibiograms and plasmid profiles are clearly inappropriate with fungal moulds. However, techniques such as crossed immunoelectrophoresis and crossed radioimmunoelectro-

Correspondence to: J.P. Burnie, Department of Medical Microbiology, St. Bartholomew's Hospital, West Smithfield, London EC1A 7BE, U.K.

phoresis have demonstrated considerable antigenic variability between different isolates of A. fumigatus (Wallenbeck et al., 1984) and this suggests that immunoblot fingerprinting may be a suitable typing method. A. fumigatus can form a fungal ball in the lungs of patients who have had a previous cavitating chest lesion such as tuberculosis. This fungal ball or aspergilloma results in a strong immune response which can be detected as multiple bands by either simple double diffusion or countefirnmunoelectrophoresis (Schaefer et al., 1976). The reason for the extensive sensitisation to A. fumigatus in these patients is at present unclear. It may reflect the chronicity of the disease with a gradual release of aspergillar antigens with time. This would lead to a prolonged immune stimulation and high levels of circulating antibody. An alternative explanation is that each aspergilloma contains several antigenically distinct strains. These would all possess their own immunodominant antigens which elicit antibody responses in the host. When this reaction is monitored by a simple technique such as double diffusion multi-

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180 p i e p r e c i p i t i n lines are observed. These lines represent cross-reacting a n t i b o d i e s p r o d u c e d against several isolates r a t h e r t h a n a n intensive a n t i b o d y response against a single strain. This p a p e r examines isolates f r o m eight p a t i e n t s w h o h a d chest X - r a y a n d s p u t u m evidence of a n aspergilloma. Single isolates were available f r o m two o f the patients, b u t in six cases m u l t i p l e isolates were available. These isolates h a d p r e v i o u s l y b e e n differentiated b y m o r p h o l o g i c a l characteristics such as their rate of g r o w t h o n m a l t extract agar (Leslie et al., 1988). T h e y were e x a m i n e d b o t h b y silverstained SDS-PAGE electrophoresis and imm u n o b l o t f i n g e r p r i n t i n g with a r a b b i t h y p e r i m m u n e a n t i s e r u m r a i s e d against A. fumigatus N C T C 2109.

c r o s c o p y a p p e a r a n c e . D e t a i l s of the isolates f r o m each i n d i v i d u a l case a r e given in T a b l e I. A. fumigatus N C T C 2109 was o b t a i n e d f r o m the c u l t u r e collection at C o l i n d a l e .

Materials and methods

Isolates 21 isolates were o b t a i n e d f r o m eight cases of aspergilloma. T h e y were identified as A. fumigatus b y their s t a n d a r d cultural characteristics a n d mi-

Immunoblotting Preparation of antigen. T h e A. fumigatus isolate was s u b c u l t u r e d o n t o S a b o u r a u d ' s d e x t r o s e a g a r a n d g r o w n at 30 ° C for 48 h. It was t h e n inoculated into Sabouraud's dextrose broth and g r o w n for a p e r i o d of 3 - 5 d a y s at 3 0 ° C in an o r b i t a l s h a k e r r o t a t i n g at 3000 rpm. T h e m y c e l i u m was h a r v e s t e d b y f i l t r a t i o n a n d the solid culture f r a g m e n t e d in a n ' X p r e s s (LKB, Bromma, Sweden). T h e p r e s s was l o a d e d a n d frozen at -35°C overnight. It was t h e n p l a c e d in the ' X p r e s s ' a n d r u p t u r e d at 200 m P a . T h e process was r e p e a t e d three times to o b t a i n 90% or m o r e d i s r u p t i o n of cells. T h e resulting extract was m e l t e d a n d s p u n at 12000 r p m for 20 min. T h e s u p e r n a t a n t was used in s u b s e q u e n t experiments. 10 g l were l o a d e d into each well o f the gel. B e t w e e n e x p e r i m e n t s the s u p e r n a t a n t was s t o r e d at - 7 0 ° C .

TABLE I DETAILS OF THE 21 ISOLATES FROM THE EIGHT CASES OF ASPERGILLOMA no. of isolates 1 2 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Case no.

Silver stain type

1 2 3 3 4 4 4 5 5 5 6 6 6 7 7 8 8 8 8 8 8

A A B B A A A C D D C C D E E A A A F A A

Illustration

Fig. 1 track 1

Fig. 1 track 4 Fig. 1 track 5 Fig. 1 track 7 Fig. 1 track 2

Fig. 1 track 6 Fig. 1 track 3 Fig. 1 track 8

Immunoblot type

Illustration

1 2 3 3 4 4 4 5 6 6 7 7 6 8 8 9 9 10 11 9 9

Fig. 2 track 2 Fig. 2 track 1 Fig. 2 track 4 Fig. 2 track 5 Fig. 3 track 1 Fig. 3 track 2 Fig. 2 track 6

Fig. 2 track 7 Fig. 3 track 3 Fig. 3 track 4 Fig. 2 track 8 Fig. 3 track 5 Fig. 3 track 7 Fig. 3 track 6 Fig. 2 track 9

181

Preparation of the antibody probe. The protein content of the supernatant of A. fumigatus NCTC 2109 was determined by the Lowry method and 25 mg were then diluted in 1 ml of sterile distilled water and mixed with 1 ml of complete Freund's adjuvant. 0.5 ml of this mixture was injected subcutaneously into New Zealand White rabbits. This was repeated every 14 days until the animals produced a high titre of antibody as measured by Western blotting. The animals were then bled out and the serum pooled. Gel electrophoresis. Each supernatant was solubilised in sodium dodecyl sulphate (SDS) 2.6% and 2 mercaptoethanol 1.3% at 100 °C for 5 min. Electrophoresis of this preparation was performed with a 10% polyacrylamide gel in a discontinuous buffer system (Laemmli, 1970). The following molecular weight standards were applied: myosin 200000; fl-galactosidase 116 250; phosphorylase B 92 500; bovine serum albumin 66 200; ovalbumin 45 000; carbonic anhydrase 31000; soy bean trypsin inhibitor 21500; lysozyme 14400. Gels were either developed by silver staining (Amersham International) or blotted onto nitrocellulose membrane (Bio-Rad Laboratories, Richmond, CA, U.S.A.) in 25 mmol Tris, 192 mmol glycine buffer pH 8.3, containing 20% methanol. Transfer was performed at 25 °C with a current of 0.5 A for 45 min in an LKB transblot cell. Protein sites were saturated by incubation in bovine serum albumin (Sigma) 3% in buffered saline (sodium chloride) 0.9% and 10 mmol iris, pH 7.4 at 4 ° C overnight. The nitrocellulose membrane was then incubated at 25 °C for 2 h with the rabbit hyperimmune antiserum diluted 1/10 in bovine serum albumin 3% and Tween 20 0.05%. After washing 5 times in saline 0.9% and Tween 20 0.05% the nitrocellulose was incubated for 1 h at 25 °C with alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma) at a 1/1000 dilution in bovine serum albumin 3%. After washing again, the membrane was incubated for 15 min at 25°C with 100 ml buffer (100 mmol Tris hydrochloride, pH 9.5, 100 mmol sodium chloride, 5 mmol magnesium chlofide) containing a mixture of 660 /xl nitroblue tetrazolium (NBT 15 mg/ml in 70% N,N-dimethylformamide) and 330 #1 of 5-bromo-4-chloro-3indolyl phosphate (BCIP 50 mg/ml in 70% N, Ndimethylformamide). The reaction was stopped by

washing in water and the results for each antigenic band were determined as absent ( - ) , present ( + ) or double band (DB). Faint and inconsistent bands were ignored.

Results

Silver staining and immunoblotting detected multiple antigenic bands with molecular weights ranging from 39000 to 200000. 16 of these were selected for the development of the fingerprinting system on the basis of their reproducibility. This was both by silver staining and immunoblotting. Fingerprinting systems can be assessed by three standards: typability, reproducibility and discrirnination. All isolates of A. fumigatus were typable by both techniques. Reproducibility between gels was assessed by repeating each test at least three times on different gels. Previous fingerprinting methods had advocated the sequential analysis of isolates with one representative from each type on each subsequent gel (Lee et al., 1986). In the case of A. fumigatus the reproducibility of both immunoblots and silver gels was sufficient that the direct comparison of isolates was possible. The final criterion is discrimination and this was higher for immunoblotting than for silver staining. Silver staining generated six separate types (A-F, Table II) and eight of the isolates are illustrated in Fig. 1. These came from cases 3, 5, 6 and 8 (Table 1). When the 21 isolates from the patients with aspergillomas were immunoblot fingerprinted 11 separate types were generated (Table I) Ten of these are illustrated in Figs. 2 and 3 and their details are given in Table I. Each type was distinguished from the others by a difference in at least three bands. Bands which were prominent by silver staining could be faint by immunoblotting. All isolates had bands at 84, 73, 63, 51, 49 and 40 kDa by silver staining and 73 kDa immunoblotting. Most had an immunoblot band at 145, 88, 84, 82, 63, 49 and 33 kDa whilst bands at 37, 77 and 185 kDa were only found in a single type. The bands at 40, 51, 101, 135 and 200 kDa were especially valuable in typing as they occurred in only a few types.

182 TABLE II DETAILS

OF THE SIX TYPES GENERATED

Band molecular

BY SILVER STAINING

SDS-PAGE

GELS

P r e s e n c e o f t h e b a n d in t y p e

weight (kDa)

A

B

C

D

E

F

200

-

-

+

-

-

-

185

-

-

+

+

+

-

145

+

-

+

+

+

-

135

-

-

+

+

-

-

10l

-

-

+

+

+

-

84

+ +

DB

+ DB

+ +

+ +

+

82

+

DB

DB

+

+

-

77

+

+

+

+

+

-

73 63 51 49 40

+ + + + +

+ + + + +

+ + + + +

+ + + + +

DB + + + +

+ + + + +

37 33

--

--

+ +

+ +

-+

--

88

Key: - = absent; + = present; DB ~ double band,

200 185 145 135 101 88 84 82 77 73

63 51

49 40 1

2

3

4

5

6

7

8

Fig. 1. Silver s t a i n e d S D S - P A G E p r o f i l e s o f i s o l a t e 3 ( t r a c k 1), i s o l a t e 14 ( t r a c k 2), i s o l a t e 19 ( t r a c k 3), i s o l a t e 8 ( t r a c k 4), i s o l a t e 9 ( t r a c k 5), i s o l a t e 18 ( t r a c k 6), i s o l a t e 11 ( t r a c k 7) a n d i s o l a t e 21 ( t r a c k 8). T h e m o l e c u l a r w e i g h t s o f t h e b a n d s s h o w n in k D a w e r e derived from the migration positions of the various standards.

200 185 145 '130: 88 Bs8 7';

63

Fig. 2. Immunoblot fingerprints of isolate 2 (track I), isolate 1 (track 2), NCTC 2109 (track 3), isolate 3 (track 4), isolate 4 (track 5), isolate 9 (track 6), isolate 13 (track 7), isolate 16 (track 8) and isolate 21 (track 9). The molecular weights of major bands are shown in kDa at left.

The results from the aspergilloma patients demonstrated that both by SDS-PAGE and immunoblotting (Fig. 2, track 3) the isolate from case no. 1 was identical to the NCTC 2109 strain. When the multiple isolates were examined by immunoblotting these could either be identical to each other as in cases 3, 4 and 7 or different as in cases 5, 6 and 8. Patients 5 and 6 demonstrated two separate immunoblot types with conserved antigenic bands at 84, 73, 63, 51 and 33 kDa. Patient 8 had three separate aspergillar types with bands at 145,73,63 and 49 kDa in common. This pattern of results was confirmed by silver staining with the exception of case 8. Here, five of the six isolates were silver type A shared by isolates from cases 1, 2 and 4. The isolates from case 8 pro-

duced unique immunoblot I).

types 9 and 10 (Table

Discussion

This paper describes the first immunologically based typing system for A. fumigutus. All strains were typable by the technique and reproducibility between immunoblots was excellent provided the antibody probe was standardised and the analysis restricted to the 16 antigenic bands described. This was in contrast to C. albicans (Lee et al., 1986) where isolates had to be examined on the same gel. In this system organism breakdown was by enzyme (a-methylmannosidase) followed by

184

20O 1 85 145 135 101 88 84 82 73

63 51 49 40 37

1 F i g . 3. l m m u n o b l o t

fingerprints

2 of isolate

3

6 (track isolate

TABLE

4

1), isolate

8 (track

19 (track

5 2), isolate

6) and

isolate

6

14 (track

18 (track

7

3), isolate

15 (track

4), isolate

17 (track

9

10

11

-

-

+

+

+

-

7).

III

DETAILS

OF

THE

11 TYPES

GENERATED

of the band

BY THE

RABBIT

HYPERIMMUNE

ANTISERUM

Band

Presence

molecular weight

1

2

3

in type 4

5

6

7

200

-

-

+

-

-

+

-

185

-

-

-

+

-

+

.

145

-

,+

+

+

-

-

135

-

-

+

+

+

+/

101

+

-

-

+

-

-

+

.

88

+

+

--

+

+

--

--

+

+

+

--

84

+

+

--

+

+

+

+

+

+

--

+

82

+

+

--

+

+

--

+

+

+

--

+

77

+

.

73

+

+

+

+

+

+

+

+

+

+

+

63

+

+

-

+

+

+

+

-

+

+

+

51

+

-

-

+

+

+

+

-

-

+

-

49

+

+

-

+

DB

--

-

+

+/DB

+

DB

40

+

-

+

-

37

+/

+

.

33

-

+

+

+

-

8

(kDa)

Key:

-

= absent;

.

.

.

. -

+ = present;

.

. DB = double

.

band.

.

.

-

+

.

. +

. +

. .

.

+ .

.

+ / .

.

+ .

+ .

.

.

.

. .

+

.

.

. .

.

.

. . +

5),

185

sonication. The reproducibility of this kind of procedure has been shown to be inferior to mechanical techniqes such as Xpressing (Lind, 1960). Silver stained SDS-PAGE profiles generated six groups whilst the degree of discrimination with immunoblotting was much higher with 11 types. This supports the idea that immunoblot fingerprinting will be valuable in the investigation of outbreaks of nosocomial aspergillosis. The degree of serological heterogeneity revealed by this study helps explain the poor results obtained when serology is used to diagnose invasive aspergillosis (Gerson et al., 1985). Clearly antigen or antibody detecting systems based on one isolate cannot encompass all the potentially important immunodominant aspergillar antigens. The results with the antibody probe (Table III) did not always match those obtained by silver staining each individual band (Table II). This underlines the problem that, although two isolates may have a band in the same place, that does not mean that these two bands are identical. Previous work (Matthews et al., 1985) identified a band at 40 kDa representing an antigen to which patients who recovered from invasive aspergillosis made an antibody response. This band was present in all six silver types but in only two of the 11 immunoblot types. One explanation for this is that these two immunoblot types predominate in clinical lesions. This is unlikely in view of the results from the patients with aspergillomas. A more plausible explanation is that the rabbit antibody probe only detects those aspergillar epitopes which are not observed within the band whereas the human response is mainly to a conserved epitope. The results with the immunoblot fingerprinting suggest that there are conserved bands at 145, 88, 84, 82, 73, 63, 149 and 33 kDa. Other workers have described important antigenic bands at 70 kDa and 30 kDa (Leung et al., 1988), and 36-38 kDa (Brouwer, 1988). Our own work demonstrated antibody to bands at 88 and 63 kDa in both normal controls, patients with invasive aspergillosis and in two patients with an aspergilloma (Matthews et al., 1985). This is consistent with the finding that nine of the 11 immunoblot types demonstrated a band at 63 kDa and seven showed a band at 88 kDa.

The patients with multiple isolates from an aspergilloma demonstrated two kinds of results. In the first (cases 3, 4 and 7) the isolates were identical so that the pronounced antibody response must have been due to the chronic release of aspergillar antigens. In the second (cases 5, 6 and 8) there was a multiplicity of types. This will lead to antibody against both strain specific immunodominant aspergillar antigens and those aspergillar antigens which are constant between all the isolates present. The inter-relationship of these antigens can only be dissected by examining the antibody response of individual patients with an aspergilloma against all the strains isolated from this lesion. This will be examined in a further study. In conclusion, immunoblot fingerprinting provides a method of typing isolates of A. fumigatus. This will be important in furthering an understanding of the pathogenesis of the diseases caused by the mould. It will also be of value when studying outbreaks of nosocomial invasive aspergillosis.

Acknowledgements J.B. is the recipient of grants from the Peel and Nuffield Trusts. R.C.M. is a Wellcome Senior Research Fellow.

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