Antigenicity and immunogenicity of the polyagglutinable antigen of Pseudomonas aeruginosa

FEMS Microbiology Immunology 47 (1988) 103-108 Published by Elsevier

103

FIM 00017

Antigenicity and immunogenicity of the polyagglutinable antigen of Pseudomonas aeruginosa A. M a r x a, A u r o r a Salageanu

b,

Eugenia Meitert b a n d H. M a y e r a

a Max-Planck-Institutpftr Immunbiologie, Freiburg i. Br., F.R.G. and b Cantacuzino Institute, Bucharest, Rumania

Received 10 February 1988 Revision received and accepted 15 April 1988

Key words: Antigenicity; Immunogenicity; Polyagglutinable antigen; Pseudomonas aeruginosa; Cystic fibrosis; Haemagglutination

1. SUMMARY Pseudomonas aeruginosa strains isolated from cystic fibrosis patients agglutinate in antisera against anti-polyagglutinable antigen (PA). AntiPA antibodies were formed in rabbits when immunization was carried out with bacteria possessing core-bound PA, independently of whether the strains were of S or R phenotype. For bacterial agglutination with anti-PA antibodies two prerequisites are essential: the bacterial cell must be of R phenotype and must possess the core-linked PA. In contrast, the PA in the isolated LPS's can be demonstrated in passive haemagglutination for both (S or R) phenotypes, provided the PA is core-linked. Two PA forms have been recognized, one found only in P. aeruginosa species, both in free and bound form. The other one is shared by all members of Pseudomonas genus but is present only in a free, unbound form.

Correspondence to: A. Marx, Max-Planck-Institut fiir Immunbiologie, D-7800 Freiburg i. Br., F.R.G.

2. I N T R O D U C T I O N In chronic bronchopulmonary infections the onset of P. aeruginosa colonization often represents the starting point of a fatal evolution [1]. Strains, isolated from these patients, usually have no serological specificity and express, instead, a new polyagglutinable character [2,3,4]. The polyagglutinability and the poor prognostic for the patient may be related, as these strains no longer engender antibodies against O-specific chains and the core region [5]. In the preceding paper it has been described that, although only strains isolated from cystic fibrosis patients are agglutinable in anti-polyagglutinable antigen (PA) antisera, PA is synthesized, in fact, by all P. aeruginosa strains [5]. The question, however, remained unanswered, what is the structural pecufiarity that only these strains agglutinate in anti-PA antisera? Another puzzling question is why do these strains only agglutinate in some distinct standard antisera and not in others, that is, why are some strains immunogenic for PA and others not? It has been shown [5] that PA can be detected by passive haemagglutination using LPS as coating antigen, provided PA is

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104 linked to the core. It will be tested whether there are cross-reactions among LPS preparations that are positive in P H A (anti-PA serum). The distribution of PA in various Pseudomonas genus and whether PA represents a unique structure in every strain will be reported.

cells/ml and the addition of 0.1 ml antiserum to the sediment. It was incubated at 3 7 ° C for 2 h and then kept overnight at 4 °. The supernatant was used after centrifugation.

3.6 Bacterial agglutination This was carried out according to a micromethod described by Petcovici et al. [8].

3. M A T E R I A L S A N D M E T H O D S

3.1 Strains For immunization and isolation of LPS Pseudomonas aeruginosa standard strains were used (Meitert-Meitert classification). Strains belonging to Pseudornonas genus were: P. diminuta, P. stutzeri, P. maltophilia, P. pseudoalcalinogenes, P. mendocina, P. fluorescens and P. picketti. Two polyagglutinable strains (designated as P. aeruginosa 100 and P. aeruginosa 200) were isolated from patients with chronic pulmonary infection. All strains originate from the E. Meitert collection, Bucharest.

3.7 Passive haemagglutination and its inhibition Passive haemagglutination (PHA) was carried out by using a Takatsy microtitration system. To coat erythrocytes, 0.1 mg LPS was kept at 100 ° C for 1 h, then mixed with 25 btl human erythrocytes of blood group O in 5 ml buffered saline. In haemagglutination inhibition (HAI), 25 btl PA antiserum (in twice the end point titre dilution) were incubated for 1 h at 3 7 ° C with 25 ~1 inhibiting LPS serially diluted, starting from a concentration of 1 m g / m l . After this preincubation, 50 /~1 of erythrocytes coated by LPS 100 or LPS 200 and suspended in buffered saline were added to each dilution. After 1 h at 37 ° C titres were read.

3.2 Media Strains of Pseudomonas were grown on agar using a tryptic digest of casein with p H 7.2. After 24 h of incubation at 3 7 ° C the bacteria were harvested.

4. R E S U L T S

3.3 Antigen isolation LPS was isolated from wet bacteria by the method of Westphal et al. [6] or by the PCP method of Galanos et al. [7] from previously dried bacteria in P. aeruginosa 100 and P. aeruginosa 200. 3.4 Preparation of antisera Rabbits, weighing 2 kg, were immunized intravenously on days 0, 3, and 7, respectively, with 0.2, 0.5, and 1.0 ml of bacterial suspension of 10 l° cells/ml (heat killed for 1 h at 100°C). The animals were bled on day 14. For immunization with LPS, each animal was given 0.2, 0.5, 1.0 mg innoculate at days 0, 3, and 7, respectively. 3.5 Absorption of antisera This was achieved by centrifugation of a 0.5 ml bacterial suspension at a concentration of 10 a°

Some PA preparation of P. aeruginosa can be investigated by P H A (anti-PA antiserum) due to a linkage existing between PA and the LPS core, thus the capacity to coat erythrocytes is secured by the lipid A moiety of LPS [5]. The question was posed whether a core-PA linkage also infers a more exposed sterical position of PA which could influence its immunogenicity. Five bacterial strains with core-bound PA were used for immunization, as well as five that produce PA exclusively in a free form [5] (Table 1). All strains with core-bound PA were found to be highly immunogenic, in contrast to those strains harbouring only free PA, which were all non-immunogenic for this antigen (Table 1). The two types of strains could, possibly, produce PA in non-comparable amounts, therefore, to exclude a quantitive phenomenon, immunization was repeated by using LPS's of both types. With a view to equalize the injected amounts of PA, every LPS preparation had previously been

105 Table 1

Table 3

Imrnunogenicity of bacterial cells, in rabbits tested in PHA by LPS 100 (PA + )

Structural conditions for serological reactions of PA

Strains with bound PA used for immunization

Strains with free PA used for immunization

Strain serotype

PHA titre

Strain serotype

PHA titre

III v IX xI xxI

160 2560 10240 10240 2560

II vI xvII xvIIl xIx

< 10 < 10 < 10 < 10 < 10

investigated on its P A a m o u n t s b y H A I ( c o a t i n g with LPS 100 a n d its antiserum). I n this w a y for i m m u n i z a t i o n it was used in m e d i u m 3.8 m g L P S c o n t a i n i n g the free P A a n d we o b t a i n e d a n t i s e r a with 20 P H A m e d i u m titer. F o r the c o r e - b o u n d P A o n l y 0.4 m g were used for i m m u n i z a t i o n a n d a m e a n titer of 320 was observed. Hence, the b a c t e r i a l strains as well as their L P S p r e p a r a t i o n s are highly i m m u n o g e n i c for P A p r o v i d e d it is p r e s e n t in the c o r e - l i n k e d form. A s the strains m e n t i o n e d a b o v e are i m m u n o genic for PA, the existence of c r o s s - r e a c t i o n s a m o n g all L P S ' s of the various serotypes b e a r i n g the s a m e b o u n d P A h a d been a s s u m e d . T a b l e 2 shows that identical titers in P H A were o b t a i n e d for every s m o o t h a n d r o u g h L P S with b o u n d PA. F o l l o w i n g a b s o r p t i o n s with the r o u g h p o l y a g g l u t i n a b l e strain P. aeruginosa 100, all titres decreased to < 10, i n d i c a t i n g that in b o t h g r o u p s (S-LPS's a n d R - L P S 100) the P H A h a d b e e n Table 2 Absorption of anti-PA antiserum (Serotype XI) Coating antigen

PHA titre in untreated antiserum

PHA titre after absorption

LPS VIII LPS IX LPS XIII

2560 2560 2560 P. aeruginosa

< 10 < 10 < 10

LPS XX LPS XI LPS 100

2560 5120 2560

< 10 5120 < 10

100

Bacterial agglutination LPS PHA LPS HAl

S

S*

R

R*

free PA

bound PA

free PA

bound PA

+

+ +

+

+ + +

* S and R indicate the phenotype.

c a u s e d solely b y PA. W h e n erythrocytes, however, were c o a t e d b y the h o m o l o g o u s L P S 11, the P H A was secured b y b o t h the P A a n d the O-antigen. T h a t is, following a b s o r p t i o n with P. aeruginosa 100 ( P A + O - ) the P H A titer was n o t decreased. T h e s e results clearly d e m o n s t r a t e d that s m o o t h strains with b o u n d P A e n g e n d e r both, a n t i - O a n d anti-PA antibodies. T h e a n t i g e n i c i t y o f b a c t e r i a l cells h a d been tested in a similar e x p e r i m e n t a l scheme, using an anti-P, aeruginosa 11 a n t i s e r u m . N o n e of the s m o o t h cells (with b o u n d o r free PA), a g g l u t i n a t e d in the a n t i - P A a n t i s e r u m , except the h o m o l o g o u s s e r o t y p e 11 (via its O-antigen). These a n t i b o d i e s (anti-011) c o u l d n o t be r e m o v e d b y the r o u g h P. aeruginosa strain 100. H o w e v e r , the p o l y a g g l u t i n a b l e P. aeruginosa 100 d i d a g g l u t i n a t e in a n t i - O l l s e r u m (via P A ) a n d in its h o m o l o g o u s a n t i s e r u m as well, p r o b a b l y d u e to the lack of an O-specific c h a i n which c o u l d h i n d e r the r e a c t i o n b e t w e e n P A o n the cell surface a n d its specific a n t i b o d i e s . T a b l e 3 shows the r e l a t i o n s h i p s existing between L P S p h e n o t y p e (S, R) a n d structural forms o f P A in e x p r e s s i n g v a r i o u s serological reactions. T h e q u e s t i o n arises h o w w i d e s p r e a d is P A in the Pseudomonas genus a n d does o n l y one single P A t y p e exist. T h e e x a m i n a t i o n of seven p o l y a g g l u t i n a b l e strains led to the i d e n t i f i c a t i o n of two d i s t i n c t types, with the collective n a m e of P. aeruginosa 100 a n d P. aeruginosa 200. H A l exp e r i m e n t s i n d i c a t e d a p a r t i a l c r o s s - r e a c t i o n between the L P S ' s o f the two strains. T h e two antisera ( a n t i - L P S 100 a n d a n t i - L P S 200) were tested with L P S ' s i s o l a t e d f r o m different Pseudomonas species ( T a b l e 4).

106 Table 4 H A l and PHA of various Pseudomonas LPS's in PA systems

P. diminuta P. stutzeri P. maltophilia P. pseudoalcalinogenes P. rnendocina P. fluorescens P. picketti

PA 100 system (LPS 100 and its antiserum)

PA 200 system (LPS 200 and its antiserum)

HAI (In PA 100 system)

PHA (In anti LPS 100 antiserum)

HAl (In PA 200 system)

PHA (In anti LPS 200 antiserum)

no no no no no no no

< < < < < < <

0.1 0.5 2.0 2.0 1.0 2.0 2.0

< < < < < < <

reaction reaction reaction reaction reaction reaction reaction

10 10 10 10 10 10 10

No inhibition was observed in the LPS 100 system, that is, PA 100 type is not synthesized in the genus Pseudomonas, except in P. aeruginosa species, as described in this paper and earlier [5]. Type 200 is, however, produced by each Pseudomonas species tested and exists in a free form (without coating properties). It has been found that LPS isolated from some mucoid forms of Escherichia coli and Klebsiella strains inhibited system 200, indicating that type 200 PA is perhaps related to the mucoid material formed by Pseudomonas species.

5. DISCUSSION P. aeruginosa, if involved in respiratory failure, leads to the death of almost all cystic fibrosis patients [1]. Nevertheless, patients with cystic fibrosis apparently do not show any of the immunodeficiency phenomena. They even manifest a pronounced humoral immune response and have increased numbers of circulating T and B cells [9]. If the host's immune response is unimpaired, the poor prognostic for cystic fibrosis patients may be due to a defective interaction between antibody and bacterial cell or to an intense adherence of bacterial cells to tracheal cells through their mucoid exopolysaccharide, often produced in large amounts. This mucoid exopolysaccharide could simply act as a protective layer or by interfering with the binding of antibodies to LPS [10]. In a

mg mg mg mg mg mg mg

10 10 10 10 10 10 10

preceding paper [5] it has been demonstrated that the polyagglutinable strains isolated from cystic fibrosis patient only stimulate the formation of anti-PA antibodies but not of anti-core or anti-O antigen antibodies. Therefore, anti-PA antibodies might be neutralized by the mucoid layer, surrounding the bacterial cells, and which contains PA in large amounts, so that PA antibodies can either not or only partly reach the target P. aeruginosa cells. It is known that strains isolated from cystic fibrosis patients agglutinate only in some but not in all of the standard antisera. The reason is that some of the standard strains are immunogenic for PA whilst others are not. It has been shown here that only bacteria that produce the core-bound PA are capable to engender anti-PA antibodies, indifferently of their respective phenotype (S, R). Similar structural restrictions have been observed with the enterobacterial common antigen (ECA) [11,12,13], where, however, only rough strains with complete core possess the core-linked type of ECA, whereas smooth cells do not contain core-linked ECA and which are all non-immunogenic [13]. Concerning the antigenicity of PA, there is a fundamental difference between the PA-containing LPS preparations and bacterial cells. In the first case, PA can reach its specific antibodies and express a haemagglutination reaction, provided PA is linked to the core. With the bacterial cells, however, they can only agglutinate in the anti-PA

107

antiserum when the O-specific chains are lacking, and when they possess the core-linked PA. The same structural conditions are required for bacterial agglutination with ECA-antibodies, as described by Marx et al. [14]. The question whether PA is always built up with an identical structure in all strains, represents a fundamental theoretical and clinical question. It has been found that there are at least two PA types which show partial cross-reactivity. One PA (designated here as P. aeruginosa 100) is only present in strains of the P. aeruginosa species. The other PA (designated here as P. aeruginosa 200) is present in each species of the Pseudomonas genus tested so far and even cross-reacts with some mucoid forms of Klebsiella and E. coli, but is produced only in a free form, except with some P. aeruginosa strains. Further studies are needed to establish the common and the differing features among the two PA types which lead to the partial cross-reaction in HAI and in the Ouchterlony gel precipitation test (Marx, unpublished data).

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

ACKNOWLEDGEMENTS The authors gratefully acknowledge the experienced help of Mrs. I. Dima, R. Tudorache, C. Bulumac and A. Wambach.

[11]

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