Pseudomonas aeruginosa PA-I lectin gene molecular analysis and expression in Escherichia coli

BB. ELSEVIER

Biochimica et Biophysica Acta 1218 (1994) 11-20

Biochi~ic~a etBiophysicaA~ta

Pseudomonas aeruginosa PA-I lectin gene molecular analysis and expression in Escherichia coli Dody Avichezer, Nechama Gilboa-Garber *, Nachman C. Garber, Don J. Katcoff Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel (Received 28 July 1993)

Abstract

This communication describes a Pseudomonas aeruginosa DNA fragment (cloned in hgt11) which contains the structural gene coding for the galactophilic PA-I lectin (pa-lL, 369 bp) and an additional downstream 237 bp sequence. This DNA is relatively rich in G + C (54%), and exhibits a strong codon preference biased for XXC and also for XXG. The Shine-Dalgarno site of the gene is preceded by an adjacent ATATAT sequence resembling the - 10 sequence of the Escherichia coli promoter. The stop codons are followed by a stem and loop structure - typical of the rho-independent transcriptional stop element. This h gt11-cloned DNA was expressed in E. coli Y1090 cells. The resulting cell lysates exhibited a galactose-specific hemagglutination and a protein with electrophoretic mobility similar to that of the native PA-I, which were both absent from E. coli lysates infected with ovalbumin gene-bearing bacteriophages. The recombinant PA-I, purified by gel filtration and affinity chromatography, was shown to be a galactophilic hemagglutinin resembling the native lectin in molecular weight and selective reactivity with rabbit anti native PA-I serum. These results are important for development of a safe Pseudomonas aeruginosa vaccine using recombinant DNA techniques, thus avoiding contamination with toxic products of this bacterium. Key words: Galactophilic lectin; Lectin gene; Nucleotide sequence; Recombinant lectin; Synthetic peptide; (P. aeruginosa)

1. Introduction

The bacterium Pseudomonas aeruginosa is a powerful producer of two lectins PA-I and PA-II exhibiting strong agglutinating activity towards papain-treated human and other animal erythrocytes [1-4]. These lectins, the first galactophilic and the second one fucose and mannose-binding, were purified by affinity chromatography [1]. Their properties, diverse biological effects and important uses in science and medicine have been

* Corresponding author. Fax: + 972 3 344766. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number X65933. Abbreviations: IPTG, isopropyl-fl-D-thiogalactopyranoside; ORF, open reading frame; PA-I, P. aeruginosa galactose-binding lectin; PA-II, P. aeruginosa fucose/mannose-binding lectin; pa-lL, P. aeruginosa PA-I lectin gene; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; rPA-I, recombinant PA-I protein; TBS, Tris-buffered saline. 0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 3 ) E 0 2 5 3 - K

widely described [2-5]. Both lectins were shown to resemble plant lectins in relative heat-resistance, insensitivity to extreme p H changes and to proteolysis, and also in cation-binding dependence [1-3]. Recent examination of their metal content analyzed by atomic absorption, has revealed that both lectins contain Ca 2÷ and Mg 2+ (PA-II also contains Zn 2+) but not Mn 2+ (which was reported to be essential for the activity of galactose-binding Leguminous lectins [6,7]). The hemagglutinating activities of these lectins and their spectrum of sugar specificity were thoroughly explored [8,9]. They were also found to stimulate human T lymphocytes [10,11] and murine splenocytes [11] and to exhibit antitumorigenic activity [12]. Their immunogenic activity has been shown to be exploitable as a vaccine for protection against Pseudomonas infections [13,14]. Recently, we have isolated a 624 bp D N A fragment containing the entire PA-I-coding gene (palL) inserted in a A g t l l phage from a P. aeruginosa A T C C 27853 genomic library [15]. The 369 bp open reading frame ( O R F ) of this clone was used for the

12

D. Avichezeret al. / Biochimicaet BiophysicaActa 1218 (1994)11-20

PA-I amino acid sequence deduction with a predicted translation product of 12 754 Da [15]. The present paper describes the nucleotide sequence of the entire genomic DNA fragment, including the additional 237 bp downstream of the pa-lL gene, the successful expression of this gene in E. coli, and examination of synthetic peptides, produced on the basis of highly antigenic epitope index [15] for their ability to interfere with the reactions of the native PA-I lectin.

2. Materials and methods

2.1. Isolation and sequencing of the P. aeruginosa genomic DNA fragment containing the pa-lL gene Polymerase chain reaction (PCR) was performed for DNA amplification, using two degenerate synthetic oligonucleotide primers and P. aeruginosa chromosomal DNA. The oligonucleotides were prepared according to the reverse translation of the N-terminal amino acid sequence of the purified lectin resolved on SDSpolyacrylamide gel electrophoresis (PAGE [16]). The chromosomal DNA was obtained from P. aeruginosa (ATCC 33347) cells grown in 500 ml of Trypticase Soy Broth (Biolife) medium at 28°C with shaking for 20 h. The harvested (10000 × g for 10 min) cells were washed with 0.15 M NaC1 solution, suspended in 10 ml of 10% (w/v) sucrose solution in Tris-HCl (50 mM, pH 8.0) and mixed with 2 ml containing 20 mg lysozyme (Sigma) and 8 ml of 0.25 M EDTA (pH 8.0) at 0°C for 10 min. Bacteriolysis was performed by adding 4 ml of 10% (w/v) SDS solution followed by vigorous shaking with a glass rod and sonication. Nucleic acids were isolated from the lysates by several phenol/chloroform extractions and ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.2) [17]. The amplified specific DNA was purified, radiolabeled [15] and used for the screening (by the plaque hybridization method [17]) of a P. aeruginosa ATCC 27853 genomic library constructed in Agtll phages and propagated in E. coli Y1090 ceils [15]. One positive clone (out of 5-105 screened) was isolated, subcloned in pBluescript KS vector, introduced into E. coli K12-HB101, and used for nucleotide sequencing in the Taq Track Sequencing System (Promega) with the classical T3 and T7 oligonucleotide primers [15]. In order to improve the sequencing, two synthetic internal 20 bp oligonucleotides; GGCCAGTTACGGACCTACCC (20306, located about 100 bp downstream from the initiation codon) and AGCCCAGGAGTFCGATACCC (20305, complementary to positions 512-532 located about 120 bp upstream from the TAA stop codon) were used (Fig. 1).

2.2. Expression of pa-lL gene inserted in Agtll phages propagated in E. coli Y1090 cells E. coli Y1090 cells grown (at 37°C for 18 h) in LB medium [17] containing 0.2% maltose were harvested (4000 × g for 10 min at room temperature) and suspended in 0.01 M MgSO4 to a density of A600 = 2 (~ 1.6-109 cells/ml). 200 /xl of this bacterial cell suspension were inoculated into 50 ml of NZCYM medium [17], placed in a 250 ml flask, and incubated at 37°C with shaking. When the culture reached A600 = 0.6, the bacteriophages (one stock bearing the pa-lL gene and the other one chicken ovalbumin gene as a negative control) were added (at a titer of 2.108 pfu), each either with or without 0.5 mM isopropyl-/3-D-thiogalactopyranoside (IPTG, Sigma), to the bacterial culture. After 6 h incubation at 37°C with vigorous shaking, the lysates were centrifuged at 10000 × g at 4°C for 15 min and lyophilized (to 1/5 of the original volume). 2.3. Assays of hemagglutination and its inhibition by sugars Hemagglutinating activity was examined by serial dilutions of the samples in a total volume of 100 /xl saline (0.15 M NaCl) solution and addition of half a volume of papain-treated human type O erythrocytes (2% (v/v) suspension in saline solution, [1]). In assays of inhibition by sugars, mixtures of the samples with either D-galactose or D-mannose (0.15 M), incubated at room temperature for 30 min, were serially diluted with saline solution and assayed for residual hemagglutinating activity, as described above. 2.4. Purification of the native and recombinant PA-I lectins Native PA-I was purified from P. aeruginosa (ATCC 33347) cell extracts by heating to 65°C, ammonium sulfate precipitation and affinity chromatography on Sepharose 4B, as previously described [1]. The recombinant PA-I protein (rPA-I) was purified from E. coli lysates infected by Agtll phages bearing the pa-lL gene as described above. The culture (800 ml) supernatant (10000 × g for 15 min) was concentrated by lyophilization to 1/100 of the original volume and dialyzed against Tris-HCl (0.001 M, pH 8.4). The exdialysate was loaded on a Biogel P-10 (Bio-Rad) column (2.5 × 14 cm) equilibrated with Tris-buffered saline (TBS, 0.01 M, pH 8.4), and fractions of 3 ml were collected. Those exhibiting hemagglutinating activity were pooled and applied, following concentration by lyophilization, onto a Sepharose 4B column (3 × 21 cm) equilibrated and washed with TBS. Fractions of 8 ml, eluted with D-galactose (0.3 M in water), were

D. Avichezer et al. / Biochirnica et Biophysica Acta 1218 (1994) 11-20

13

2.6. Immunoprecipitation in agar

extensively dialyzed against large volumes of Tris-HC1 (0.001 M, pH 8.4) and those exhibiting hemagglutinating activity were pooled and lyophilized.

Precipitation of the recombinant PA-I lectin (1 ~ g / 1 0 / x l per well) by the rabbit antisera (15/zl/well) was examined using the double diffusion in agar (1% special Difco Noble agar in 0.15 M NaC1 solution supplemented with 1 m g / m l sodium azide) on microscope slide.

2.5. Preparation of rabbit antisera Immune rabbit anti PA-I and anti PA-II sera were produced by injecting rabbits six times during 2 weeks with the purified native lectin preparations (1 mg protein), alternately in Freund's complete adjuvant (Difco)-intramuscularly and without the adjuvant-subcutaneously. 5 days after the last injection the rabbits were bled. The immune sera, separated from the blood clots, were heated to 56°C for 30 min (for complement inactivation).

2. 7. Construction of synthetic peptides according to the predicted lectin antigenic regions Two peptides (681 and 682) were synthesized and lyophilized at the Peptide Synthesis Unit of the Biological Services of the Weizmann Institute of Science,

A T A T A T C G G A G A T C A A T C A T G G C T T G G A A A G GT G A G G T T C T G G C T A A T A A C G A A G C A G G G C A G A W K G E V L A N N E A G Q

GTAAC GT CG V T S

20306

ATTATCTACAATCCGGGCGATGT CATTACCATCGTCGCCGCCGGTTGGGO__CLAGTT_ACG_ _C~CCS_ACC__CLAGAAA I

I

Y

N

P

G

.......

145

D

V

I

T

I

V

A

A

G

W

A

S

Y

G

P

T

Q

K

.,,,,,.,,,,,,

TGGGGGCCGCAGGGC W G P Q G

GATC GGGAGCATC CGGACCAAGGGCTGAT CTGCCACGAT GCGTTTTGTGGT D R E H P D Q G L I C H D A F C G

GCGCTG A L

681

217

2a9

GT C A T G A A G A T C G G C A A C A G C G G A A C C A T T V M K I G N S G T I

CCGGT CAATAC CGGGTTGTTC CGTTGGGTT GCAC C CAATAAT P V N T G L F R W V A P N N

GT C C A G G G T G C A A T C A C T C T T A T C T A C A A C G A C G T G C C C G G A A C C T A T G G C A A T A A C T C C V Q G A I T L I Y N D V P G T Y G N N S .......

GGCTCGTTCAGT G S F S

E"

::~

682

3Ex

GT CAATATT GGAAAGGAT CAGT CCTGATAACTTGT V N I G K D Q S

433

ATACAAATAAAGT

sos

A T G A C A A GG_G_T_ATC_G_A~A_C_T_C_CT_G_G_G_G_C_TG C C S C G G T C G T G G G A A A C C G A G C G A G G G G G S G C G G G G A A C T G C T

s~

TCAACACGCTT

CT C G G A A A A A A A G G G C C

CGAAT GGGCT CTTTTTTTAA > <

G A A G T T G C C C GT GT G G C C G T T A T G A A C G G A C A G G C A G C G C T T C G C A G T T G

-_[:

C GACTAC CA

20305

C G GT CT G A A C G G G A A T A T C

GATT CCTGACCCGATTCA

Fig. 1. Sequence of the 624 bp P. aeruginosa D N A fragment containing the structural pa-lL gene. The amino acid sequence is listed under the nucleotide sequence. The numbers of the nucleotides and amino acids are indicated on the left and right sides, respectively. The asterisks indicate the initiation and stop codons and the bold letters mark the potential glycosylation site. The underlined sequence upstream of the start codon represents the Shine-Dalgarno ribosome binding site and the converging arrows downstream of the termination TAA codon - the presumptive transcriptional terminator signal. The dashed lines indicate the location of the two (20305 and 20306) synthetic oligonucleotides used to complete the sequence, the double underlines - the two (681 and 682) synthetic peptides. The wavy underlining denotes minor repeat units and the dotted line indicates the V I T I V symmetry.

D. Avichezer et al. / Biochimica et Biophysica Acta 1218 (1994) 11-20

14

Rehovot, using an automatic (430A Applied Biosysterns) peptide synthesizer (cysteine residue was included at the carboxy-terminus of the peptides for conjugation purposes). These peptides were purified by gel filtration on Sephadex G-25 column.

gram (Centre d'Etudes Nucleaires de Saclay, Cedex, France). The CODONFREQUENCY [19] and FOLDRNA [19,20] programs (University of Wisconsin Genetics Computer Group (UWGCG)) were used for generation of the codon usage table and the pa-lL mRNA structure analysis, respectively.

2.8. Examination of the synthetic peptide effects on the native PA-I hemagglutinating activity 2-Fold dilutions of the synthetic peptides in 100/xl saline solutions were mixed with an equal volume of the native PA-I lectin (both the peptide and lectin final concentrations are indicated in the text). Then half a volume of papain-treated human erythrocytes (5% (v/v) suspension in saline solution, [1]) was added. After 1 h incubation at ambient temperature, the hemagglutination intensity [18] was determined and compared to that of PA-I lectin alone.

2.9. Computer analyses Nucleic acid and deduced amino acid sequence analyses were performed with the DNA-Strider pro-

3. Results

3.1. The nucleotide sequence of the entire P. aeruginosa genomic DNA fragment containing the pa-lL gene A Agtll recombinant phage containing a 624 bp EcoRI fragment of P. aeruginosa (ATCC 27853) DNA was isolated by screening a genomic library with the 32p-labeled PCR product (see Materials and methods). The nucleotide sequence of the entire DNA fragment (subcloned into the Eco RI site of pBluescript KS plasmid) is presented in Fig. 1. As shown in this figure, the DNA fragment was found to contain a major open reading frame (ORF) of 369 bp, encoding the entire

Table 1 Codon usage in the Pseudomonas aeruginosa p a - l L gene a

Codon

A m i n o acid

Number b

Fraction c

Codon

A m i n o acid

Number b

Fraction c

GGG GGA GGT GGC GAG GAA GAT GAC GTG GTA GTT GTC GCG GCA GCT GCC AGG AGA AGT AGC AAG AAA AAT AAC ATG ATA ATT ATC ACG ACA ACT ACC

Gly Gly Gly Gly Glu Glu Asp Asp Val Val Val Val Ala Ala Ala Ala Arg Arg Ser Ser Lys Lys Asn Asn Met Ile Ile lie Thr Thr Thr Thr

4 4 4 5 2 1 4 2 1 1 2 6 2 3 2 3 0 0 2 1 2 2 7 4 2 0 4 6 1 0 1 5

0.24 0.24 0.24 0.29 0.67 0.33 0.67 0.33 0.10 0.10 0.20 0.60 0.20 0.30 0.20 0.30 0.00 0.00 0.29 0.14 0.50 0.50 0.64 0.36 1.00 0.00 0.40 0.60 0.14 0.00 0.14 0.71

TGG TGA TGT TGC TAG TAA TAT TAC TTG TTA TTT TTC TCG TCA TCT TCC CGG CGA CGT CGC CAG CAA CAT CAC CTG CTA CTT CTC CCG CCA CCT CCC

Trp End Cys Cys End End Tyr Tyr Leu Leu Phe Phe Ser Set Ser Ser Arg Arg Arg Arg Gin Gin His His Leu Leu Leu Leu Pro Pro Pro Pro

4 1 1 1 0 1 1 3 1 0 1 2 2 0 0 2 1 0 1 0 5 1 1 1 3 0 1 0 4 0 1 2

1.00 0.50 0.50 0.50 0.00 0.50 0.25 0.75 0.20 0.00 0.33 0.67 0.29 0.00 0.00 0.29 0.50 0.00 0.50 0.00 0.83 0.17 0.50 0.50 0.60 0.00 0.20 0.00 0.57 0.00 0.14 0.29

a Total number of codons 124. h The number of times a codon occurred in the examined sequence. c The ratio of the number of occurrences of a specific codons to that of all codons in the same synonymous codon group.

15

D. Al,ichezer et aL /Biochimica et Biophysica Acta 1218 (1994) 11-20

PA-I protein. The O R F begins with an A T G codon at nucleotide 19 and ends with two consecutive stop codons T G A and T A A at nucleotides 385 and 388, respectively. Analysis of the 5' region of the O R F revealed a Shine-Dalgarno ribosome-binding site with a G G A G A sequence located 7 bp upstream of the initiation codon and an A + T-rich area ( A T A T A T ) preceding it (Fig. 1). Analysis of the 3' region downstream of the O R F revealed the presence of an inverted repeat beginning 11 bp downstream of the T A A stop codon, which can form a stable stem (13 bp length) and loop structure ( z i G = - 1 3 . 2 kcal/mol) characteristic of a rho-independent transcriptional terminating signal [21,22]. A low free energy hypothetical secondary structure of the entire p a - l L m R N A was predicted using the computer assisted programming algorithm of Zucker et al. [20]. It predicts a AG energy of - 1 3 8 . 9 k c a l / m o l for an R N A molecule 435 nucleotide long, and contains a stern and loop structure adjacent to the p a - l L stop codons. Another A T G sequence found 115 bp downstream of the T A A stop codon (Fig. 1), was not shown to be preceded by a typical ribosome-binding site. Several additional terminator codons were found in each of the three reading frames downstream of the ORF, which indicates that this region is probably not translated. The coding sequence of the p a - l L gene exhibits a high overall G + C content (54.5%) and a frequent utilization of codons that have a cytidine or guanine in their third position (Table 1), features which are characteristic of other Pseudomonas chromosomal genes [23]. The major 369 bp O R F encodes a polypeptide of 12754 Da containing 121 amino acids [15] without any characteristics of a typical signal sequence. T h e amino acid sequence does not contain extensive repeats or an R G D region [24]. A short symmetric V I T I V sequence is present at residues 25-29, but its significance is unknown.

HEMAGGLUTINATING ACTIVITY (Log z Dilution-1) 1 2 3 4 5 6 7

$

A B C

"

D E F G H I J

Fig. 2. tlemagglutination of papain-treated human O erythrocytes by lysates of E. coli Y1090 cells infected with Agtl 1 phages bearing the PA-I-coding gene (rows A to F) or the chicken ovalbumin-coding gene (rows G and H). The cells were grown either in the absence (rows A to C and G) or presence (rows D to F and H) of IPTG. In rows I and J the culture supernatants of E. coli Y1090 cells grown without or with IPTG, respectively,were examined. Inhibition of the hemagglutinating activity by D-galactose (rows B and E) but not by D-mannose (rows C and F) confirms that the sugar specificityof the recombinant hemagglutinin is similar to that of the native PA-I lectin.

trophoretic mobility (on SDS-PAGE) similar the native PA-I (Fig. 3). Such a protein was the control lysates of E. coli cells infected ovalbumin gene-bearing bacteriophages (Fig.

to that of absent in with the 3).

3.2. Expression of the Agtll-cloned p a - i L gene in E. coil cells

3.3. Purification of the recombinant PA-I lectin

Using the h g t l l bacteriophage as a vector, the cloned Pseudomonas DNA, encompassing the p a - l L gene, was expressed in E. coli Y1090 host cells. The lysates obtained from cells grown either in the presence or absence of IPTG, exhibited a remarkable hemagglutinating activity, specifically inhibited by Dgalactose but not by D-mannose (Fig. 2). No hemagglutinating activity was detected with the E. coli cells grown either alone or with h g t l l bacteriophages bearing a chicken ovalbumin-coding gene (as a negative control). The hemagglutinating lysates derived from E. coil cells infected with the p a - l L gene-bearing bacteriophages were found to contain a protein with elec-

The recombinant PA-I was purified by the twostep-procedure involving gel filtration and affinity chromatography as described in Materials and methods. S D S - P A G E (15% acrylamide) analysis of this recombinant lectin under reducing (dithiothreitol 100 mM) conditions showed that the recombinant protein migrated slightly slower than the purified native PA-I lectin preparations isolated from different P. aeruginosa A T C C 33347 and PAO strains (Fig. 4). Its apparent molecular mass of 13 500 Da was compatible with that of 12754 Da predicted from the deduced amino acid sequence. The level of the recombinant protein in lysates of E. coil cells carrying the recombinant phages

16

D. Avichezer et al. / Biochirnica et Biophysica Acta 1218 (1994) 11-20

Si, ~ -'7

~-::'

~a

:~

~

:4~:-: . i I ~t,<~),g:-.'~-.~.,. ~ ,.~-~ .:-;

~:

~

~'."

:;. . . . . "r., :-~:-..,

,,,~

;

~C~..,;~

.?~,~..... fill ~ ~"~ .~:.

" : ~;~.'~" £3%

~:. !

'

l.::i.~::P ~ •

Fig. 5. Immunoprecipitation in agar (1%) of the recombinant PA-I lectin (central well) by rabbit anti native PA-I serum (trough A). Rabbit anti PA-II serum (trough B) did not react with this PA-I lectin.

was estimated to be 1 2 / z g / 1 culture medium, and was not significantly affected by growing the E. coli-infected cells at lower temperatures ( 2 5 - 2 8 ° 0 or by increasing the time of infection to 12 h. Fig. 3. SDS-polyacrylamide (18.5%) gel electrophoresis of lysates obtained from E. coli Y1090 cells infected with bacteriophages bearing either the PA-I lectin-coding gene (lane 1) or the ovalbumin-coding gene (lane 2) as compared to the purified native PA-I lectin (lane 3).

3.4. Immunoprecipitation of the purified recombinant PA-I lectin by rabbit anti native PA-I serum The recombinant PA-I lectin was specifically immunoprecipitated by rabbit anti native PA-I serum (Fig. 5). Rabbit antiserum raised against the Lfucose/D-mannose binding PA-II lectin derived from the same P. aeruginosa strain did not precipitate it (Fig. 5).

3.5. Analysis of the synthetic peptides, constructed according to the predicted lectin antigenic regions, for their ability to compete with the natural PA-I lectin

/

IIII W

W

O

Fig. 4. SDS-polyacrylamide (15%) gel electrophoresis of the purified recombinant PA-I lectin (lane 2) as compared to the purified native PA-I lectin preparations derived from P. aeruginosa ATCC 33347 (lane 3) and PAO (lane 4) strains. Lane 1 represents the migration of the following BioRad low molecular mass standards (from top to bottom): phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa) and lysozyme (14.4 kDa).

The two synthetic peptides: 681 and 682 (residues 40-53 and 101-113, respectively) depicted by double underlines in Fig. 1, which were chosen on basis of their potential as antigenic epitopes (according to Jameson and Wolf [25] prediction analysis), have been tested for their ability to compete with the native PA-I lectin in the hemagglutination test. Incubation of the 682 peptide (at 0.125 mM concentration) with the PA-I lectin (0.1/zM) led to a 37% inhibition (n = 4, P < 0.01 using the Student's t-test) of the hemagglutination score [18]. Higher peptide concentrations (0.5-1 mM) did not increase the inhibitory effect. With the 681 peptide no inhibition of PA-I hemagglutinating activity, but rather a slight increase in it was observed.

4. Discussion

In the first part of the present study we have analyzed the total nucleotide sequence of the D N A fragment containing the p a - l L gene (isolated from a genomic library in Agtll phages) of Pseudomonas aerugi-

D. Avichezer et aL / Biochimica et BiophysicaActa 1218 (1994) 11-20 nosa ATCC 27853. In addition to the 369 bp ORF encoding the PA-I structure (121 amino acids; 12754 Da) a 237 bp sequence downstream of the gene was elucidated (Fig. 1). The total sequence was found to be relatively rich in G + C content (54%), which is higher than that found in bacteria such as E. coli [23] but somewhat lower than that reported (60.1-69.5%) in other chromosomal genes of P. aeruginosa [23]. The codon usage of the gene represented in Table 1 resembles that described in other P. aeruginosa genes [23]; exhibiting a strong codon preference for C and G in the third wobble codon position. Generally, C usage was found to be higher than that of G, but for serine equal (TCG and TCC) and for leucine (CTG), proline (CCG) and glutamine (CAG) higher G usage was demonstrated. Another interesting feature was that certain codons regarded as rare in P. aeruginosa genes [23] are used in pa-lL gene, including AAT for asparagine and ATI" for isoleucine (Table 1). Although no intact promoter was found upstream of the pa-lL ORF (probably due to DNA fragmentation during construction of the genomic library) there is an ATATAT sequence 13 bp upstream of the initiation (ATG) codon. This sequence which precedes the potential Shine-Dalgarno site by one nucleotide, resembles the - 1 0 sequence of the promoter (ATAAT) of E. coli [26] and that (TATATA) of the progenitor gene of type C toxin of Clostridium botulinum [27]. The 3' region downstream of the pa-lL gene probably does not code for a protein since it lacks typical ribosomebinding sites and contains several terminator codons in all of the three reading frames. This region contains a presumed stable stem (13 bp, Fig. 1) and loop structure typical of the rho-independent transcriptional termination signal [21,22]. This stem and loop structure, closely following the pa-lL stop codons may contribute to the stabilization of the pa-lL mRNA transcript, acting as a protective barrier against 3' exoribonucleases as was recently described in the extremely stable mRNA of the galactose-binding lectin of Myxococcus xanthus [28]. The P. aeruginosa DNA fragment containing the pa-lL gene isolated and characterized in this study, in its subcloned (to the pBluescript KS plasmid) form, was recently provided to Prof. B.W. Holloway (Monash University, Clayton, Victoria, Australia) for chromosomal mapping on the P. aeruginosa PAO genome. It was found that it hybridized (by the Southern hybridization method) to the 150 kb SpeI digest fraction containing Q and R fragments (Carey, E. and Krishnapillai, V., unpublished results), which are separated on the physical map by about 1 mb [29]. Interestingly, the Q fragment is adjacent to the J in which the structural alkaline proteinase (apr) gene has been located, and the R fragment is adjacent to the L in which the LasA gene (also associated with proteolytic activity) and the exoenzyme S regulatory gene have been mapped [29].

17

These genetic findings reinforce the previous report on the close physiological coregulation between the lectin production and extracellular proteolytic activities in P. aeruginosa [3]. Moreover, they may indicate that the Pseudomonas lectins are involved in the pathogenicity of the bacterium, since genes and operons that encode bacterial virulence factors are often subject to coordinated regulation [30]. If the final mapping of the pa-lL gene would indicate its linkage to those coding for proteolytic a n d / o r toxic activities, it would fit GilboaGarber and Garber's postulate and model on the general functional association between lectins and key (lytic or other reaction cascade initiating) enzyme activities [3,5]. The deduced product of the pa-lL gene ORF fits a protein composed of 121 amino acids with molecular mass of 12 754 Da [15] confirmed by SDS-PAGE analysis (Fig. 3 lane 3) and matches perfectly the quantitative amino acid analysis. Analysis of the N-terminal region of the PA-I-deduced polypeptide revealed that it does not contain a typical signal peptide (Fig. 1). Hydropathy analysis [15] showed that it contains two predominant hydrophobic stretches (residues 20-34 and 56-72 in Fig. 1), which are separated by a hydrophilic one, that may be associated with membrane (anchor-like) interactions. Although localization studies on the Pseudomonas lectins indicated that these lectins are present mainly in the intracellular compartment and in the periplasm (attached to the inner membrane), these lectins may also be partially externalized to the outer cell membrane [3,4]. The external exposure of these lectins was shown by the use of rabbit antibodies against the purified P. aeruginosa lectins. These antibodies agglutinated intact P. aeruginosa cells which produce the lectins, but not those lacking them [31]. It is suggested that the lectins may be translocated via alternative transport pathways. Such pathways were recently described in a number of bacterial proteins which lack the classical N-terminal signal peptide. They involve C-terminal domain of the molecule a n d / o r protein secretion systems encoded by a cluster of accessory genes. These include the alkaline proteinase of P. aeruginosa, the proteinases B and C of Erwinia chrysanthemi, the metalloproteinase of Serratia marcescens, the ot haemolysin of E. coli and the cytotoxins of Bordetella pertussis and Pasteurella hemolytica [32]. The lectin protein is rich in glycine, aspartic acid/ asparagine, alanine, isoleucine and valine which (except for isoleucine) have been reported to be involved in hydrogen bonding with carbohydrates in several protein-saccharide complexes [7,33]. Alanine has also been described to be involved in hydrophobic interactions [7]. Another common feature of carbohydratebinding domains is a central /3-pleated sheet region bounded by helices [33]. Analysis of the PA-I protein

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D. Al,ichezer et al. /Biochimica et Biophysica Acta 1218 (19~14) 11-20

secondary structure [15] according to the Chou and Fasman algorithm [34] indicated that this protein is composed of an extensive network of /3 sheets and contains two major helical regions in the initial Nterminal and in the central domains of the protein. The two synthetic peptides (681 and 682, Fig. 1) lacked, both in soluble and hemocyanin (KLH)-conjugated forms, the hemagglutinating ability possessed by both the native and recombinant lectins. However, the KLH-conjugated forms induced cell aggregation of intact P. aeruginosa cells to a certain degree - a property also shared by the native lectin (unpublished results). Incubation of the 682 peptide (which coincides with the domain containing the potential Asn-Asn-Ser glycosylation site, Fig. 1) at submolar concentration (0.125 mM) with the PA-I lectin (0.1 txM) led to a significant inhibition (by approx. 37%) of the hemagglutinating activity. Higher peptide concentrations (0.5-1 mM) did not inhibit the PA-I lectin-induced hemagglutination more strongly. Therefore, whether this inhibition is due to competitive or allosteric inhibition is not clear. With the 681 peptide (which coincides with the predominant hydrophilic core of the protein) no inhibition but rather a slight increase of the PA-I lectin hemagglutinating activity was observed. In addition to the classical carbohydrate-binding sites, the microbial lectins and hemagglutinins may exhibit other adhesion binding sites. Such an example is the Arg-Gly-Asp (RGD) sequence which is usually identified as an eukaryotic cell-binding site and was identified in the Bordetella pertussis [24] and Haemophilus influenzae [35] hemagglutinins. This sequence which was reported to play a central role in the pathogenic Bordetella adherence [24,35,36] is absent in the P. aeruginosa PA-I protein (Fig. 1). Though its gignificance is not known, the p a - l L product has a three-amino acid sequence: Gln-Gly-Asp (QGD, residues 45-47 from the amino-terminus, Fig. 1) which was noted to be commonly conserved in Leguminous lectins [6]. QPD (Gin-Pro-Asp) sequence, which is similar to it contains the conserved glutamine and aspartic acid residues which were recently suggested to play an important role in the carbohydrate-binding domain of the galactose-binding animal C-type lectins [37]. PA-I lectin does not contain extensive repeating elements (except for a few minor repeat units and the short VITIV symmetry noted in Fig. 1) such as those described in the glucan-binding protein of Streptococcus mutans [38], which possess two sets of repeats (possibly resulting from gene duplication) which are presumed to be involved in its glucan-binding. Four internal homologous repeating domains (each of 67 amino acid long) have also been reported in the galactose-binding hemagglutinin of M. xanthus [39] and shown to confer upon the protein a multivalent structure which is required for the lectin hemagglutinating

activity. The results presented in Fig. 2 demonstrating the considerable hemagglutinating activity of PA-I recombinant lectin (expressed in E. coli cells with Agtl 1 as a vector), against human papain-treated erythrocytes could be explained on the basis of: (1) Self-association of the PA-I lectin subunits (which are presumed to be monovalent with regard to galactose-binding according to Scatchard analysis of the native lectin [9]), yielding a multimeric form of the protein. The high glycine content in this molecule may contribute to this self-association process, since glycinc- and proline-rich repetitive sequences have been shown to promote self-association of molecules [40,41], and highly conserved glycine residues were reported to play a critical role in the polymerization process of the Neisseria gonorrhoeae and other related pill [42]. (2) Lectin-binding to the erythrocyte surface components via noncarbohydrate hydrophobic or electrostatic domains in conjunction with thc saccharidebinding sites. (3) Formation of intermolecular disulfide bonds via the cysteine residues located at positions 57 and 62 of the protein (Fig. 1), like the monomeric Mac-2 animal lectin [43]. Another notable finding is the expression (shown by the hemagglutination test) of the p a - l L gene inserted in the Agtl 1 phages, apparently independently of the LacZ promoter control (Fig. 2). In the IPTG-supplemented cultures, even a lower hemagglutination titer was observed (Fig. 2), which could be attributed to the previously reported outstandingly high sensitivity of PA-I to inhibition by this thiogalactopyranoside derivative [9]. The hemagglutinating rPA-l-harboring lysates contain a protein with electrophoretic (SDS-PAGE) mobility similar to that of the native PA-I lectin (Fig. 3). Control lysates derived from E. coli cells infected with ovalbumin gene-bearing bacteriophages did not contain such a protein (Fig. 3), nor did they exhibit any detectable hemagglutinating activities (Fig. 2). An earlier example of a similar expression of a protein placed under the control of the LacZ promoter in Agtll vector in noninducible conditions was reported by Ycung and Fernandez [44]. In our case, however, the possibility that the promoter for the p a - l L gene is contained within the Pseudomonas DNA fragment cloned in Agtll is unlikely, since extracts and supernatants of E. coli HB101 cells transformed with the pBluescript KS plasmid bearing the p a - l L gene did not exhibit hemagglutinating activity. Moreover, the 5' region adjacent to the p a - l L O R F in the isolated DNA insert is substantially shorter (18 nucleotides) than those of promoters described in E. coli and other prokaryotes [26]. It is possible that a certain nucleotide region in the a g t l l vector may serve as a cryptic promoter for the p a - l L gene or that the p a - l L expres-

D. Auichezer et al. / Biochimica et Biophysica Acta 1218 (1994) 11-20

sion is due to basal level expression of the LacZ promoter. The recombinant lectin behaved in the two-step purification procedure (involving gel filtration on a Biogel-P10 column, followed by affinity chromatography on a Sepharose 4B column) like the native lectin. The purified rPA-I was found to be a functionally stable protein (in lyophilization and at -20°C) exhibiting a galactose-sensitive hemagglutinating activity (Fig. 2) and molecular mass (13.5 kDa) almost equivalent to that of the native lectin subunit (13 kDa) of either homologous (ATCC 27853) or heterologous (ATCC 33347, PAO) P. aeruginosa cells (Fig. 4). Its reactivity with rabbit antiserum raised against the native purified PA-I lectin (Fig. 5), but not with rabbit antiserum raised against the L-fucose/o-mannose-binding PA-II lectin, derived from the same P. aeruginosa strain nicely confirmed its antigenic similarity to the native PA-I. The possibility of rPA-I production in E. coli cells as a fusion protein (linked either to a Agtll or LacZ portion) which undergoes a peptide cleavage as described for several LacZ-fusion proteins expressed in E. coli [45-47], is not excluded. In our partially purified preparations of the rPA-I there were also additional protein bands of higher molecular masses (ranging from about 18 to 60 kDa), some of which exhibited hemagglutinating activity. The results presented in this study describing the expression, purification and immunoreactivity of the recombinant PA-I lectin may be of clinical importance for vaccination against Pseudomonas infections [13,14] using recombinant D N A methods. Such a vaccine would have several advantages, by excluding the need to work with P. aeruginosa cells in the manufacturing process and eliminating potential contaminations of the vaccine with toxic products of Pseudomonas (such as exotoxins A and S, proteinases, cytotoxin, lipopolysaccharide, etc.).

Acknowledgments The authors thank Mrs. Avrille Goldreich for the preparation of the manuscript. This research was supported in part by funds from the Health Science Research Center of Bar-Ilan University. The synthetic peptides were prepared for another research project, supported by the USA-Israel Binational Science Foundation (BSF) Grant 89-00454.

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