FEMS Microbiology Letters 21 (1984) 63-69 63 Published by Elsevier

A novel biological function of alginate in Pseudomonas aeruginosa and its mucoid mutants" stimulation of exolipase

(Pseudomonas aeruginosa; mucoid mutants; lipase, extracellular; lipase, stimulation of bacterial; alginate, chemical modification of; alginate, biological function of)

J o s t Wingender and Ulrich K. Winkler *

Lehrstuhl fiir Biologie der Mikroorganismen, Ruhr-Universiti~t, D- 4630 Bochum, F.R.G.

Received and accepted 12 September 1983

1. SUMMARY

Trea tmen t of Pseudomonas aeruginosa ATCC9027 with various commercial alginates from brown algae enhanced extracellular,lipase activi- ties in a time- and concentration-dependent manner ("exolipase stimulation"). Alginate iso- lated from Azotobacter vinelandii and mucoid mutants of P. aeruginosa was similarly effective. Several independently isolated mucoid (alginate- producing) mutants of P. aeruginosa showed higher spontaneous exolipase activities than the non- mucoid wild type. Alginate was chemically mod- ified by (i) reduction of carboxyl groups (removal of charge), (ii) oxidation of pyranoid rings (de- struction of tertiary structure), and (iii) reduction of reducing end groups. None of the chemical modifications resulted in total loss of the exo- lipase-stimulating ability of the alginate deriva- tives.

2. INTRODUCTION

Alginate is a linear copolymer consisting of 1,4-1inked a-L-guluronate (GulA) and fl-D-mann-

* To whom correspondence and reprint requests should be addressed.

uronate (ManA) residues. Some of the residues are arranged in homopolymeric segments (G- and M- blocks) whereas other sequences consist of alter- nating or otherwise distributed sugar residues of both types [1]. Azotobacter vinelandii [2] and Pseu- domonas aeruginosa [3] are known to produce par- tially O-acetylated alginate as a slime-forming exo- polysaccharide. In the latter species alginate pro- duction is restricted to so-called mucoid mutants frequently isolated from cystic fibrosis (CF) pa- tients with chronic pulmonary infection [4].

Possible biological functions of alginate in P. aeruginosa were deduced from phenotypic dif- ferences observed between mucoid mutants and corresponding wild-type strains. Mucoid mutants show resistance against various antibiotics [5], phages [6] and host phagocytes [7]. Furthermore, the adherence of mucoid mutants to tracheal epi- thelium is improved [8]. This paper will describe that alginate is also able to enhance the extracellu- lar lipase ('exolipase') activity of P. aeruginosa. This function was discovered by short-term in- cubation of wild-type cells in the presence and absence of alginate. The results obtained were confirmed by studying newly-isolated mucoid mutants. In addition to these experiments alginate was chemically modified and then biologically tested in order to elucidate structural requirements of alginate for its exolipase-stimulating capability:

0378-1097/84/$03.00 © 1984 Federation of European Microbiological Societies

64

(i) carboxyl groups were reduced to convert the native polyanion into an uncharged polysac- charide; (ii) pyranoid rings were completely peri- odate-oxidized in order to destroy the rigid struc- ture of native alginate; (iii) the reducing power of terminal hexuronic acid residues was abolished by treatment of alginate with sodium borohydride.

Some of the results of this paper have been presented at the ASM-meeting in Ttibingen, Germany, February 28-March 2, 1983.

3. MATERIALS AND METHODS

3.1. Bacterial strains and media

Pseudomonas aeruginosa ATCC 9027 was purchased from the American Type Culture Col- lection (Rockville, USA). Mucoid mutants of this strain were isolated by selection for carbenicillin resistance [5]. Azotobacter vinelandii AE IV was kindly provided by Dr. B. Larsen (Trondheim, Norway). Standard media were minimal glucose medium (M9glc) and Pseudomonas Agar P (PAP).

3.2. Testing of exolipase-enhancing ability of poly- saccharides

Bacteria were grown with shaking in M9glc- medium at 30°C as described previously [9]. After centrifugation the cells were suspended in 2 /3 volume of 67 mM Tris-HCl buffer (pH 7.5) to give a cell density of about 5 X 109/ml. Equal volumes (0.25 ml) of this bacterial suspension and polysaccharide dissolved in Tris-HC1 buffer were mixed and shaken at 30°C. After various periods of time samples were centrifuged for 4 rain at 8000 x g. The clear supernatants were immediately assayed for exolipase activity using emulsified p- nitrophenylpalmitate as substrate [10].

3.3. Polysaccharides

Commercial sodium alginates from brown algae (Manucol LB and LH, Manugel D J) and pro- pylene glycol alginate (Manucol ester E / R E ; esterified 83%) were kindly provided by Alginate Industries Ltd. (Hamburg). Bacterial alginates were

isolated from A. vinelandii AE IV [11] and from mucoid mutants of P. aeruginosa ATCC 9027 [5]. For most experiments alginate was dialyzed against demineralized water and freeze-dried. Mannan from baker's yeast was purchased from Sigma (Munich).

3.4. Identification of alginate

Alginate was hydrolyzed with sulfuric acid [12]. After neutralization hydrolysates were applied to thin-layer chromatography (TLC)-plates (10 x 10 cm) coated with silica gel 60 (Merck, Darmstadt). The solvent system was ethyl acetate-acetic acid-pyridine-water (5 : 1 : 5 : 3) [13]. The plates were developed 3 times each for 1 h and the spots were visualized with p-anisidine reagent. Reference substances were ManA and GulA obtained by the method of Haug and Larsen [12] and ManA-lac- tone (Sigma, Munich). Infra-red spectra were re- corded with a Perkin-Elmer model 983 spec- trophotometer using alginate samples in KBr-pel- lets.

3.5. Chemical modifications of alginate (Manucol LB)

3.5.1. Reduction of carboxyl groups Carboxyl groups of sodium alginate were re-

duced by reaction with 1-cyclohexyl-3-(2-morpho- linoethyl) carbodiimide method-p-toluenesulfonate (CMC) and subsequent treatment with sodium borohydride [14]. The product obtained was di- alyzed against demineralized water, freeze-dried, dissolved in 67 mM Tris-HC1 buffer (pH 7.5) and further purified by ion-exchange chromatography on a DEAE-Sephadex A-25 column (2.5 x 7 cm). Elution was carried out stepwise with 80 ml Tris buffer, 100 ml 0.3 M NaC1 and 200 ml 0.6 M NaC1. The flow rate was 45.7 m l / h and the frac- tion volume was 4 ml. Fractions containing carbohydrate were pooled, dialyzed against de- mineralized water and freeze-dried. Commercial propylene glycol alginate was purified by the same procedure.

3.5.2. Oxidation of alginate Sodium alginate was completely oxidized by

alternating oxidation with sodium metaperiodate and reduction with sodium borohydride [15]. The equivalent weight of all reaction products was determined by titration with cetylpyridinium chlo- ride [16].

3.5.3. Reduction of reducing end groups To aliquots of a 1% (w/v) aqueous solution of

sodium alginate were added different amounts of solid sodium borohydride (final conc., 0.5, 1 and 2 M). After various periods of time at room temper- ature the mixtures were neutralized with con- centrated acetic acid, dialyzed against demineral- ized water and freeze-dried.

3.6. Chemical determinations

Carbohydrate was determined by the phenol- sulfuric acid method [17]. Reducing sugars were determined with the 2,2'-bicinchoninate reagent [18] using mannuronic acid as a standard.

65

4. RESULTS

4.1. A lginate stimulates formation of exolipase in the wild-type

Treatment of P. aeruginosa ATCC 9027 with commercial alginates from brown algae greatly enhanced exolipase activities in a time- and con- centration-dependent manner. After 30 min of in- cubation exolipase activity was increased about 23-fold (Fig. 1A). At low concentrations alginate of high-M r had a stronger stimulatory effect than the same type of alginate of low-M r (Fig. 1B). The latter result might indicate that alginate molecules possess more than a single 'site' for being biologi- cally active.

Bacterial alginates isolated from A. oinelandii AE-IV and mucoid mutant M J-2 of P. aeruginosa ATCC 9027 were identified by TLC of acid hydro- lysates revealing GulA and ManA as uronic acid components (Fig. 2). Infra-red spectra of bacterial

~ 6 0 0 >- 1-

400 <

LLI O9 < n 200 ._1

A w ! II

~ o / • ~ • . I , 0 ~

o/e + ALGINATE

/

/ /

p e~e--e~e -- ALGINATE .o / , - o - - - - , o • •

20 40 60

TIME (min)

APPROX. PREDOMINANT ALGINATE M E A N M W URONIC ACID

Manucol LB 18000 M a n A

Manucol LH 88000 M a n A

Manugel DJ 106000 Gul A

/ ~ eLB

10 30 50

C O N C . ( ~ M )

Fig. 1. Stimulation of extracellular lipase activity of P. aeruginosa ATCC 9027 by alginate. (A) Time-dependent stimulation by Manucol LB (1 mg/ml); (B) concentration-dependent stimulation by various alginates (t = 30 min).

66

.,==.

1 2 3 4 5 6 7 8

Fig. 2. Thin-layer chromatography (TLC) of acid-hydrolyzed alginate preparations. Hydrolysates: (1) Manucol LB; (2) Manucol LH; (3) Manugel DJ; (4) P. aeruginosa alginate; (5) A. vinelandii alginate. Controls: (6) GulA; (7) ManA; (8) ManA-lactone.

Table 1

Exolipase activities of mucoid mutants in comparison with the wild-type of P. aeruginosa in the absence and presence of exogenous alginate (final conc. 1 mg/ml)

Strain Stimulation factor a

no. - alginate + alginate

Wild-type &l.0 b 3.2 c MJ-2 4.2 8.8 MJ-8 2.6 8.4 MJ-9 2.2 6.0

a The stimulation factor is a value for enzyme activities mea- sured in the presence of a given polysaccharide as a multiple of spontaneous enzyme activity.

b The stimulation factor of 1.0 corresponds to 69.0 enzyme units (EU). The relatively low stimulation factors were due to culture conditions different from the standard procedure described under MATERIALS AND METHODS.

and algal alginates were nearly identical. Two ad- ditional absorption bands at 1732 and 1252 c m - I in the spectra of both bacterial alginates indicated the presence of O-acetyl groups [11]. When tested for exolipase-enhancing ability in P. aeruginosa both bacterial alginate preparat ions also proved to be very effective. Further experiments showed that alginate could not be utilized by P. aeruginosa as a sole carbon and energy source and that alginate did not significantly alter the exolipase activity in cell-free supernatants.

4.2. Over-production of exolipase by mucoid mutants

Several spontaneous mucoid mutants were iso- lated independently. Mutants and wild-type were compared with respect to their exolipase activities. Since the mutants did not form slime when grown in liquid medium M9glc the following growth con- ditions were chosen: PAP-plates were inoculated in the form of lines and incubated for 24 h at 37°C. Then the cells were suspended in Tris buffer (pH 7.5), incubated for a further 60 min at 30°C and removed by centrifugation. As shown in Table 1 the spontaneous extracellular lipase activity of all mutants tested was well above that of the wild-type. Therefore it seemed that in vivo synthe- sis of alginate was accompanied by higher exo-

lipase activities. Ramia et al. [19] also reported on increased lipolytic breakdown of Enterobacterial C o m m o n Antigen (ECA) by mucoid strains com- pared to that of non-mucoid wild-type of P. aeru- ginosa. The results suggest that exogenous alginate when added to wild-type cells (Fig. 1) merely simulated physiological conditions characteristic of mucoid mutants (phenocopy effect).

4.3. Biological effectivity of chemically modified al- ginate

4.3.1. Uncharged derivatives Carboxyl-reduced alginate was prepared and

biologically tested in order to see whether the polyanionic character of native alginate is a prere- quisite for being able to enhance bacterial exo- lipase activities. The carboxyl groups of alginate (Manucol LB) were activated with C M C and sub- sequently reduced with sodium borohydride. Fully carboxyl-reduced alginate was separated from par- tially or non-reduced molecules by anion-exchange chromatography. Uncharged alginate could be eluted from the column by buffer alone whereas partially or fully charged alginate required NaCI solutions for elution (Fig. 3). Commercial pro- pylene glycol alginate was purified by the same procedure.

35

E c -

O o'3

~2~ u,.I o Z < m n- O O9 15 ¢n .<

5

0

I

O.3 J.

0.6 M NaCI

10 30 50 70 FRACTION NUMBER

Fig. 3. Elution profile of fully (I), partially (II) and non (Ill) carboxyl-reduced alginate (Manucol LB) on DEAE-Sephadex A-25. The elution diagram represents two separate experi- ments, one performed with carboxyl-reduced (o) and the other with native (©) alginate. Due to differences in absorption maxima of hexoses (~ = 490 nm, peaks I and II) and hexuronic acids (~, = 480 nm, peak III) A480-values of peak II1 were multiplied by a factor of 3.11 to obtain corrected A49o-values.

u I

• " • T -

D 600

• ~ o ~

400 0

LLI 09 •

2 o o -

I I

1 2 CONC. (mg/ml)

Fig. 4. Extracellular lipase activity of P. aeruginosa ATCC 9027 as a function of different concentrations of alginate (Manucol LB) (1), propylene glycol alginate (II), fully carboxyl-reduced alginate (III) and mannan from baker's yeast (IV). All test-tubes were incubated for 30 min.

67

Both carboxyl-reduced and propylene glycol al- ginate showed diminished exolipase-enhancing ca- pability when compared with the native polymer (Fig. 4). The residual effectivity of uncharged al- ginate preparations, however, indicated that the carboxyl groups of native alginate are not the inevitable key structures of the polymer for being biologically active in our test system.

The neutral polysaccharide mannan partially resembling the structure of carboxyl-reduced M- blocks of alginate also enhanced exolipase activity, but was less effective than carboxyl-reduced and propylene glycol alginate (Fig. 4).

4.3.2. Oxidized alginate Native alginate possesses a relatively rigid ter-

tiary structure [15] which is labile to oxidation by sodium metaperiodate. Alginate (Manucol LB) was completely oxidized and the product was then biologically tested in order to get information about the importance of the tertiary structure of alginate for its exolipase-enhancing capability.

Full oxidation could not be achieved in a single step because of hemiacetal formation of cleaved pyranoid rings with intact neighbouring sugar units protecting them from further oxidation [15]. Re-

25

n- O I-- O

z 15 0

_.1

~ - 5 u)

I NATIVE 'Ak~INATE

1" 01

I I

R2

T T 02 03

I I I

2 4 6 REACTION NUMBER

Fig. 5. Decrease of exolipase-stimulating ability of alginate in P. aeruginosa after stepwise oxidation with sodium metaperio- date. Degree of oxidation (O,): approx. 44% (Ol) , 88% (02) and 100% (03). Reductions ( R , ) were performed with sodium borohydride.

68

duction of these interresidue hemiacetals with sodium borohydride allowed further oxidation. For cleavage of all hexuronic acid residues several al- ternating oxidation and reduction steps were re- quired. After total oxidation of alginate, titration with cetylpyridinium chloride ensured that no change in charge density (i.e., number of carboxyl groups) had taken place.

The exolipase-enhancing ability of periodate- treated alginate in P. aeruginosa decreased as the degree of oxidation increased (Fig. 5). This ability diminished even further after each reduction of interresidue hemiacetal rings rising again after the next oxidation step. About 20% of the original biological activity remained even when alginate was completely oxidized.

4. 3.3. Terminally reduced alginate Reducing end groups of native alginate

(Manucol LB) were treated with various con- centrations of sodium borohydride. Maximum re- duction of about 87% was achieved with 2 M sodium borohydride after 1 day treatment. The biological activity of various reduced alginate pre- parations did not significantly differ from that of native alginate.

5. DISCUSSION

The main results of our study are the following: (i) alginate when exogenously applied to P. aeru- ginosa ATCC 9027 caused a rapid and strong increase in exolipase activity; (ii) alginate-produc- ing (i.e., mucoid) mutants showed higher sponta- neous exolipase activities than their non-mucoid parental strain; (iii) none of the chemical modifi- cations of alginate we performed resulted in total loss of exolipase-stimulating activity.

From the experimental findings mentioned above several conclusions can be drawn. One of the natural functions of alginate or alginate-like polysaccharides in P. aeruginosa is the ability to mediate or facilitate the release of cell-bound lipase and possibly other exoenzymes into the surround- ing medium (see 'detachment hypothesis', [10]). The presumed presence of preexisting lipase in the cell envelope has recently been proved by detect-

ing high lipase activities in inner and outer mem- brane fractions prepared from P. aeruginosa ATCC 9027 (L. Bohne, S. Reifenberg, G. Schulte and U. Winkler, 15th FEBS Meeting, Brussels, Belgium, 1983).

Native alginate is known to interact with vari- ous proteins [20,21]. So it is possible that exolipase molecules themselves might be the 'targets' for exogenous alginate at the outer cell membrane. Supporting evidence for direct exolipase-alginate interactions comes from the observation that al- ginate increased the heat stability of partially purified pseudomonad lipase (J. Wingender, un- published results). It was recently discovered that chemical derivatives of mannuronate and guluronate were components of the O-specific side chain of lipopolysaccharide (LPS) in P. aeruginosa [22,23] thus rendering LPS partially alginate-like in structure. So LPS could be another target struc- ture on the bacterial cell surface. Preliminary ex- periments with purified LPS from P. aeruginosa ATCC 9027 revealed that it also strongly en- hanced exolipase activity (not shown).

It could be possible that lipase molecules in the outer membrane are associated with LPS, this interaction would be favoured by the amphipathic nature of both exolipase and LPS. One could imagine that alginate which is a weak surfactant [24] displaces lipase molecules by forming water- soluble alginate-lipase complexes. This assump- tion agrees well with the observation that even drastic chemical modifications of alginate such as removal of charge or destruction of intact tertiary structure did not completely abolish its exolipase- enhancing ability. Furthermore, it has been re- ported that polysaccharides of very different chemical structure stimulated the exolipase of P. aeruginosa [9,25].

ACKNOWLEDGEMENTS

We gratefully acknowledge the excellent techni- cal assistance of Miss Petra DrOse. We thank Mr. W. Schrttler of Kelco-AIL-International GmbH, Hamburg, for supplying us with various samples of alginate. We appreciate the help of Mr. B. Bartylla, Bochum, who kindly performed the IR-

spec t roscopy . Th is r e sea rch was s u p p o r t e d by a

g r a n t f r o m the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t

(Wi 1 7 2 / 4 ) .

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