molecular analysis of sphingomyelinase c leptospira interrogans … · a thermolabile hemolysin...

Vol. 58, No. 7INFECTION AND IMMUNITY, JUlY 1990, p. 2177-21850019-9567/90/072177-09$02.00/0Copyright C 1990, American Society for Microbiology

Molecular Analysis of a Sphingomyelinase C Gene fromLeptospira interrogans Serovar hardjo

RUUD P. A. M. SEGERS,* ABRAHAM VAN DER DRIFT, ARNE DE NIJS, PAOLA CORCIONE,BERNARD A. M. VAN DER ZEIJST, AND WIM GAASTRA

Department of Bacteriology of the Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine,University of Utrecht, P. 0. Box 80.165, 3508 TD Utrecht, The Netherlands

Received 29 January 1990/Accepted 10 April 1990

A thermolabile hemolysin from Leptospira interrogans serovar hardjo, strain Sponselee, was shown tospecifically degrade sphingomyelin. Nucleotide sequence determination revealed that sphingomyelinase activitywas encoded by an open reading frame of 1,668 nucleotides. Although a putative signal sequence could beidentified, no evidence for protein export in either L. interrogans or Escherichia coli was obtained. Theapparent molecular mass of the expression product in E. coli minicells was 41.2 kilodaltons, whereas openreading frame 1 encoded a protein of 63,268 daltons. The observed difference may be explained by processingat the carboxy-terminal part of the hemolysin in E. coli. A high degree of similarity on the DNA and proteinlevels with Staphylococcus aureus ,-hemolysin and sphingomyelinase C from three Bacillus cereus strains wasobserved. The presence of various sphingomyelinase genes within the L. interrogans species is demonstrated.

Leptospira interrogans is the etiologic agent of leptospiro-sis, which is a worldwide zoonosis. The bacteria of thisspecies are divided into 19 serogroups and subdivided intomore than 180 serovars on the basis of a microscopicagglutination test (23). Members of the serovar hardjo (be-longing to the serogroup Sejroe) cause leptospirosis in dairycattle, resulting in serious economic losses due to agalactiaand abortion (11, 36). The infection can be transmitted by theurine to humans, resulting in dairy fever, characterized byheadache, severe fever, meningitis, and icterus. In spite ofthe medical and economical importance of leptospirosis,very little is known about the virulence factors involved inpathogenesis. Hemolysins are involved in the pathogenesisof infections by Escherichia coli (16), Staphylococcus au-reus (5), Listeria monocytogenes (7, 26), and Streptococcuspneumoniae (4) and have been claimed to be important inleptospiral infections (2, 35, 38, 39). Hemolysis by leptospi-rae is caused by phospholipases (38); both phospholipase Aand sphingomyelinase C activities have been demonstrated(3, 6). Whereas pathogenic L. interrogans and nonpatho-genic Leptospira biflexa strains have phospholipase A activ-ity, sphingomyelinase C activity has only been demonstratedin strains of L. interrogans. A DNA fragment containing asphingomyelinase C gene has been cloned from a Dutch fieldstrain, causing bovine leptospirosis (10). In this study themolecular properties of the gene and its expression productare analyzed.

MATERIALS AND METHODSBacterial strains, plasmids, media, and transformation. L.

interrogans strains were obtained from the World HealthOrganization/Food and Agricultural Organization Collabo-rating Center for Reference and Research on Leptospirosisat the Royal Tropical Institute in Amsterdam, The Nether-lands, and were grown as described previously (10). Blue-script SK M13+ (pBS) was used as plasmid cloning vehicle,and all experiments with pBS, unless stated otherwise, wereperformed with XL1-Blue (Stratagene, La Jolla, Calif.) asthe E. coli host strain. E. coli cells were grown in Luria-

* Corresponding author.

Bertani (LB) medium or on LB agar plates (28) containing100 ,ug of ampicillin per ml. Competent E. coli cells wereprepared by the CaCI2 method (28). Plasmid pHL2-B3 hasbeen described previously (10). Plasmid pHL2-B4 containsthe same DNA fragment as pHL2-B3 in the opposite orien-tation.

Production of mutants and sequence analysis. DNA fromclones pHL2-B3 and pHL2-B4 was digested with restric-tion enzymes XbaI and SstI, and unidirectional deletionmutants were produced by exonuclease III digestion withthe Erase-a-Base kit (Promega Biotec, Madison, Wis.).Single-stranded DNA from these clones was prepared asdescribed by the manufacturers of pBS (Stratagene). Nucle-otide sequences were determined by the dideoxy-chaintermination method of Sanger et al. (33). Nucleotide se-quences were analyzed with the Beckman Microgenie (re-lease 6.0; Beckman Instruments, Palo Alto, Calif.) and thePC/Gene (release 6.0; Genofit S.A., Geneva, Switzerland)computing programs.The FASTA program, release 1.0, April 1988 (30), was

used to compare nucleotide and amino acid sequences withthe following data bases: EMBL (release 19.0), NBRF/PIR(release 21.0), NBRF/NEW (release 39.0), Swiss-prot (re-lease 11.0), and Brookhaven (July 1989). Similar sequenceswere aligned by using the Clustal computer program (19, 20).Hemolysin plate assay. Colonies of E. coli harboring

recombinant plasmids were streaked on plates containingLB broth, 1% agar, 20% (vol/vol) fresh sheep erythrocytes(washed twice in 0.9% NaCl), 25 mM MgCl2, and 100 ,g ofampicillin per ml. Cells were grown for 18 h at 37°C, andhemolytic zones appeared after an additional incubation of24 h at room temperature. Omission of MgCl2 from theagarplates resulted in much smaller hemolytic zones.

Phospholipase assay and analysis by thin-layer chromatog-raphy. Sphingomyelinase activity was tested in a biphasicsystem, essentially as described previously (24), consistingof an ether-methanol (9:1, vol/vol) organic phase containing2 mg of sphingomyelin isolated from bovine brain (SigmaChemical Co., St. Louis, Mo.) per ml and a water phasecontaining 10 mM Tris hydrochloride (pH 7.4), 25 mMMgCl2, and (sonicated) bacteria and/or culture medium. The

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2178 SEGERS ET AL.

10 20 30 40 50 60 910 920 930 940 950 960(5'CH)

70 s0 90 100 110 120

130 140 150 160 170 180

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Q N E R A Q R I V S S N Y I Q N Q D V I

970 980 990 1000 1010 1020

V F D E A F D T D A R K I L L D G V R S

1030 1040 1050 1060 1070 1080

E Y P Y Q T D V I G R T K K G W D A T L

250 260 270 280 290 300 1090 100 1. 120 1130 1140

G L Y R T D A F T N G G V V I V S K W P310 320 330 340 350 360

A ____AA1~2AAATOIT7mATA1AT~TTA1~AAATT1t1TATTTA& 1130 1160 1170 L1a1 1190 1200370 380 390 400 410 420 I E E K I Q L V F K E K G C G A D V F S

1210 1220 1230 1240 1230 1260430 440 450 460 470 480_

TATC8rIAAA1'rGA0G11GIT80QAnoc8TIrAAn~ATTAco NR G F A Y V R I DN N G RR P H I I G

490 500 510 520 530 540 1270 1280 1290 1300 1310 1320

-35 T H V Q A Q D S G C A N L G V V S R V N

550 560 570 580 590 600 1330 1340 1350 1360 1370 1380

-10 8 .D. IM R I K K Y TF N E I R D F I D SR R I P K N E H V(start 011)

610 620 630 640 650 660

K V R L L V N C C L L L F P L I D C G

670 680 690 700 710 720~~M80ATT06I~~TMTTT~-ATTACATTCAATMTAA

D R Q S L Y K D L L A S L I Y I S D N K

730 740 750 760 770 780

N I G S T N S D L T G S G S V S SS P A

790 800 810 820 830 840

D A A P E N S I L A N S I P E N M G I K

850 860 870 880 890 900

I L T H N V F L L P K T L P G W G N W G

111

1390 1400 1410 1420 1430 1440

L I A G D L N V I K G S R E Y H Q M L C

1450 1480 1470 1480 1490 1500

I L N V N N P I Y V G V P F T W D T I T

1510 1520 1530 1340 1550 1560

N E I A A F Y Y K K V 15 P A Y L D Y I F

1570 1580 1590 1600 1610 1620

V S K S N F Q P P I W Q N L A Y D P I S

1630 1640 1650 1660 1670 1680

A K T W T A K G Y T S D E F S D H Y P V

1690 1700 1710 1720 1730 1740

Y G F I Y A D S S T P T K S G R K R K Y2

1750 1760 1770 1780 1790 0

D R V S F V S V A T G K K I Q A N S E K113

181 1820 1830 1840 1850 1860

S N A W L K V N A T T E T D L T K F N L

1870 l880 1890 1900 1910 1920

V Q T N D P D S N P S C MI S G H V R I

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E S S H S L N Y F W N WV L G G G K G N

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G G C L Q D G S R V A F K D Y D T I S R

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R Q Y F L T V W E G G N W D K Y L Y L W

2170 2180 2190 2200 2210 2220

R S H I G L R E I F Y L K L D S S P E M

2230 2240 2250 2260 2270 2280

N W S K K L I Y R(end 0911l)

2290 2300 2O31030 2340

IR4 1R

2350 2360 2370 2380 2390 2400mA GMAMTT AT 8

FIG. 1. Nucleotide and derived amino acid sequences from the 3,987-bp BamHI insert in pHL2-B3. The putative signal sequence is boxed,and -10 and -35 transcription signals and putative ribosome-binding sites (RBS) are underlined. Inverted repeats (IRs) are numbered andoverlined by arrows. Gibbs free energy differences were calculated by using the Microgenie program and are indicated in kilocalories (1 kcalis equal to ca. 4.184 kJ) per mole: IR1, -9.6; IR2, -8.8; IR3, -9.6; IR4, -25.4; IR5, -10.8; IR6, -8.0. Sequences similar to the potentialregulatory sequence in L. biflexa (42) are overlined with a boldface bar. These data have been submitted to the EMBL Data Library underaccession no. X52176.

samples were vigorously shaken for 4 h at 37°C, and 2 ,lI ofthe organic phase was applied on a silica gel-60-coated glassplate (E. Merck AG, Darmstadt, Federal Republic of Ger-many). When MgCl2 was omitted from the reaction mixture,sphingomyelinase activity was much lower. The chromato-gram was developed with a chloroform-methanol-water-25% ammonia (58:35:3.5:3.5, vol/vol) mixture as the mobilephase. (Phospho)lipids were visualized by spraying theplates with 30% sulfuric acid, followed by heating at 110°Cfor 5 min. Purified sphingomyelinase C (0.08 U) from S.aureus (Sigma) was used as a positive control. Sonicated E.coli cells containing pBS were used as a negative control.Degradation of other phospholipids was performed as de-scribed above, chromatograms were developed with mobile-phase mixtures as previously described (24, 25), and reactionproducts were visualized as described above. Degradation ofL-a-lysophosphatidylcholin (egg yolk; Sigma), L-a-phospha-tidylethanolamine (bovine brain; U.S. Biochemical Corp.,Cleveland, Ohio), phosphatidyl-L-serine (bovine brain; U.S.Biochemical), L-a-phosphatidic acid (egg yolk; U.S. Bio-chemical), and L-a-lecithin (U.S. Biochemical) as substrateswas tested.

RESULTS

Nucleotide sequence analysis. The nucleotide sequence of acloned 3,987-base-pair (bp) BamHI DNA fragment, whichwas shown to code for sphingomyelinase activity (10), wasdetermined (Fig. 1; data submitted to EMBL Data Libraryunder accession no. X52176). For this purpose, a number ofdeletion clones (Fig. 2C) were produced. After translation ofthe nucleotide sequence, three open reading frames (ORFs)were identified (Fig. 2A). The percentage of G+C in thewhole fragment (35.9%) as well as in the individual ORFs(Fig. 2A) was within the range of 34.1 to 39.1%, whichcorresponds to the known G+C percentage ofgenomic DNAfrom L. interrogans (23) and from L. interrogans serovarhardjo strains (27).Upstream of ORFi and ORF2, putative -10 and -35

transcription and Shine-Dalgarno translation-initiation se-quences similar to those in E. coli were identified. In ORF2a second putative translation initiation site was identified atbp 3264 (Fig. 1). Sequences upstream of ORFi and ORF2and the internal transcription initiation site in ORF2 weresimilar to the previously reported potential regulatory se-

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L. INTERROGANS SEROVAR HARDJO SPHINGOMYELINASE C GENE

2410 2420 2430 2440 2450 2460

(300H) GIMArMWWXGGCI!AL K L G G G I M(.nd 0112)

2470 2480 2490 2500 2510 2520

F I P E N F H Y S Q T A G I L D T K R Y

2530 2540 2550 2560 2570 2580

Q L A I N F F Y S E L E W Q I G T G V F

2590 2600 2610 2620 2630 2640

L V T Q V K S H V G S E I S K G V G L G

2650 2660 2670 2680 2690 2700

V Q T F W F I S E S L P F Y Y K M G P L

2710 2720 2730 2740 2750 2760

L S L T E I G F K D N G Q K D F N F N G

2770 2780 2790 2800 2810 2820

E F L F L N I SI S S L T Q Y E P T S N

2830 2840 2850 2860 2870 2880

F N S N M L I G V L P S I K R D P F S Y

2890 2900 2910 2920 2930 2940

S N L E I G F L F S L N L N Y R Y E G N

2950 2960 2970 2980 2990 3000

F S T T K T N V T Y RK E I Y P D I Y I

3010 3020 3030 3040 3050 3060

V G T I L D T I N Y G R N F N M K E T V

3070 3080 3090 3100 3310 3312

K T S W S S L N W S F Y F K K I N N H E

3130 3140 3150 3160 3170 3180

K Q K E E G E P K K I E S V L I K E V L

3190 3200 3210 3220 3230 3240

T L D I K E V K G T E D E V L V H D E R

3250 3260 3270 3240 3290 3300

L S T R V N G H K R G D K L I L T D G FS.D. -10 -35

3310 3320 3330 3340 3350 3360

T_A_TAGATTA TAGTT TA GAGGI W I P D L L L C I C F I E M 6 S.D.

(str 0RB2)

3370 3380 3390 3400 3410 3420M8Tl M 5G M A hl

-10

(3'0C) GSAGMMMTI80CGAZ4TAAA-35 F S I S E I N

(end ORF3)

3430 3440 3450 3460 3470 3480

T S E I I R K K E N E E L I Y Q S G Y Q

3490 3500 3510 3520 3530 3540

H A N G K L I T G D K L Q I T K N G L R

3610 3620 3630 3640 3650 3660

E Q R K E I E S L K I P E S N G L Q N S

3670 3680 3690 3700 3710 3720

I N E L D K L Y L E N L P A Q K K Q S V

3730 3740 3750 3760 3770 3780

N K I I Q Q Q I E K L I N L N E Q A S K

3790 3800 3810 3820 3830 3840

A D L R D E K L T K F L S K E R S G S L

3850 3860 3870 3880 3890 3900

N L P K I E F Y S N S I D E S S T D V W

3910 3920 3930 3940 3950 3960

V K E L E T K Q G F N S G T Q K N L S H

3970 3980

(5'03)E I L E E K Q P D (0813)

3550 3560 3570 3580 3590 3600

L P K E Q N S E T N T T T S T K R E I P

FIG. 1-Continued

quence TCAAAAT/CGAAT, found upstream of L. biflexatrpE and trpG genes (42) (Fig. 1). In ORFi and ORF2 thissequence was closer to the putative translation initiation sitethan was found with the trp genes. Inverted repeats thatcould function as rho-independent transcription terminationsignals were located directly downstream of ORFi andORF3. The first inverted repeat downstream of ORF2 wasafter 80 bp. The initiation methionine codon and the regula-tory sequences of ORF3 were not located on clone pHL2-B3. Therefore we only sequenced the coding region for thecarboxy-terminal part of the protein.

Localization of region encoding hemolytic and sphingomy-elinase activity. A number of deletion mutants (Fig. 2C) weretested for hemolytic activity in blood agar plates and forsphingomyelinase C activity (Fig. 3). In all clones tested inboth assays, the presence of sphingomyelinase activity co-incided with the presence of hemolytic activity. In bothexperiments, pHL2-B4-51 was the clone with the smallestDNA insert, still expressing hemolytic and sphingomyeli-nase C activities at the same level as clones pHL2-B3 andpHL2-B4. E. coli containing recombinant pHL2-B4-53, cod-ing for 45.6 kilodaltons (kDa) of the amino-terminal part ofthe protein, lost both enzymatic activities. RecombinantpHL2-B4-06 contained the coding region of ORFi except forthe last 10 amino acids; E. coli cells harboring this recombi-nant still contained both enzymatic activities, but at lowerlevels (data not shown). Therefore the whole coding regionof ORFi is required for full expression of hemolytic andsphingomyelinase activities in E. coli. Since E. coli contain-

ing pHL2-B3-05 was not hemolytic and had no sphingomy-elinase activity, expression also required the presence of atleast a portion of the 580-bp noncoding DNA region up-stream of ORF1. A DNA sequence controlling the expres-sion of sphingomyelinase activity may be located on thisDNA region.Temperature sensitivity and substrate specificity. Sphingo-

myelinase activity in lysates from E. coli cultures harboringpHL2-B3 and L. interrogans Sponselee cultures was de-stroyed by heating the samples for 10 min at 56°C. Lysatesfrom clone pHL2-B3 were also tested for the ability todegrade L-a-lysophosphatidylcholin, L-x-phosphatidyletha-nolamine, phosphatidyl-L-serine, L-a-phosphatidic acid, andL-a-lecithin, but no activity was detected.

Protein sequence analysis and homology with other sphin-gomyelinases. At the amino terminus of ORF1, the first 27amino acids have the consensus of a procaryotic signalsequence (40): a basic amino-terminal region, central hydro-phobic region, and a polar carboxy-terminal region. Thecleavage site of signal peptides is always preceded by a smallamino acid residue at the -1 position and a small unchargedamino acid residue at the -3 position (40). Therefore,cleavage is most likely to occur after the alanine residue atposition 27.Comparison of nucleotide and amino acid sequences of

ORF1 with those in data bases revealed homology with a,-hemolysin from S. aureus (32) and three sphingomyeli-nases from different B. cereus strains (14, 22, 41) on both theDNA level (data not shown) and the protein level (Fig. 4).

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pHL2 -B3

A:length (nt)length (aa)Mol. wt.

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FIG. 2. Restriction map of pHL2-B3 and sequence strategy. (A) Properties of the three ORFs deduced from the nucleotide sequenceanalysis. The hatched bar indicates a putative signal peptide. (B) Restriction map of the 3,987-bp BamHI insert in pHL2-B3 (pHL2-B4contains the same insert in the opposite orientation) (B, BamHI; C, ClaI; E, EcoRI; H, HindIII; P, PvuI). (C) Deletion clones generated byexonuclease III digestion and used for nucleotide sequence analysis and localization of hemolytic and sphingomyelinase activity. Arrowspointing to the right indicate clones derived from pHL2-B3; the 3,987-bp BamHI insert was digested by exonuclease III exclusively from theside of bp 1, and the remaining insert DNA extends from the base of the arrow up to bp 3987. Arrows pointing to the left are derived frompHL2-B4; the 3,987-bp BamHI insert was digested by exonuclease III exclusively from the side of bp 3987, and the remaining insert DNAextends from the base of the arrow up to bp 1. The length of the arrow represents the part of which the nucleotide sequence was determined.Deletion clones are named after the clone from which they were derived (i.e., pHL2-B3 or pHL2-B4), followed by the number indicatedabove. A number of clones were tested for hemolytic activity; boldface and broken-line arrows indicate the presence and absence of hemolyticactivity, respectively. The other arrows indicate clones that were not tested.

Both on the DNA and protein levels, the similarity waspresent in the middle part of the gene and protein, respec-tively. The amino termini differed considerably, and thecarboxy termini could not be aligned because of the differ-ence in molecular weight. No sequences homologous toORF2 or ORF3 were detected during the homologysearches.

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FIG. 3. Assay of sphingomyelinase activity of various recombi-nant DNA clones to localize the region on pHL2-B3 encodingenzyme activity. Sonicated E. coli cells containing the plasmidsindicated at the top were tested for sphingomyelinase activity; thereaction products were analyzed by thin-layer chromatography.Purified sphingomyelinase C from S. aureus and E. coli containingthe pBS vector were used as positive and negative controls, respec-tively. The sphingomyelin substrate and ceramide degradation prod-ucts are indicated on the left by SP and C, respectively.

Codon usage. The codons used in L. interrogans for theexpression of the ORFs of pHL2-B3 are shown in Table 1and compared with the codon usage in L. biflexa trpE andtrpG genes (42), E. coli (average values for 407 genescalculated from the data from Aota et al. [1]), S. aureus,-hemolysin (32), and B. cereus sphingomyelinase C genes(average values compiled from three published sequences[14, 22, 41]). Codon usage in the L. interrogans genes wasquite different from the codon usage in E. coli genes for theamino acids arginine, asparagine, cysteine, glutamine, gly-cine, proline, and leucine. The codon usage was more similarto that of L. biflexa, although there were differences forasparagine, arginine, isoleucine, and proline. Comparison ofthe codon usage in the different sphingomyelinase genesrevealed the frequent use of the arginine codon AGA and theproline codon CCC in the leptospiral sphingomyelinasegene, whereas these codons were hardly or not at all used inthe other four genes. The frequencies of bases in the thirdposition of a codon were calculated and were similar to whathas been shown for L. biflexa (42), reflecting the overall basecomposition of the organism.

Sphingomyelinase activities of different Leptospira strains.Total culture, pellet, and supernatant fractions of four L.interrogans strains belonging to different serogroups werecompared for the ability to degrade sphingomyelin (Fig. 5).The results indicated that all four strains contained sphingo-myelinase activity. In strains Sponselee and Mus 127, sphin-gomyelinase activity was associated with the cellular frac-tion, whereas the sphingomyelinase activity from strains

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*.* ***. .**-* . ****..-- *370 380 390 400 410 420

LI AKG----YTSDEFSDHYPVYGFIYADSSTPTKSGRKRKYDRVSFVSVATGKKIQANSEKSSA VYAFPYYYVYNDF DHYPIKAYSP-------------------------------------BC SE1 VTSWLKKYTYDDYDHYPVAATIT-------------------------------------BC IAM1208 VTSWFQKYTYNDY SDDYPVEATI-------------------------------------BC GP4 VTSWF RNIRIMITLIIIQVEATI-------------------------------------




430 440 450 460 470 480NAWLKVNATTETDLTKFNLVQTNDPDSNPSCMKSGHVRIESSHSLNYFWNWWLGGGKGNY-_______K--------------------------------------------SMK--------------------------------------------------------.SNK.--______________________

490 500 510 520 530 540AYYPKFNDGSNRIQIINLDGGCLQDGSRVAFKDYDTISRRQYFLTVWEGGNWDKYLYLWR

550 560SHIGLREIFYLKLDSSPEMNWSKKLIYR

FIG. 4. Alignment of the amino acid sequences from sphingomyelinases from L. interrogans (LI) Sponselee (this work), S. aureus1-hemolysin (SA) (32), and B. cereus (BC) SEI (22), IAM1208 (41), and GP4 (14). Identical residues are marked with an asterisk, andconservative mutations are marked with a dot, according to the log-odds amino acid similarity matrix of Dayhoff (9). Strokes indicate a gapintroduced into the sequence for alignment purposes.

Pomona and Hond Utrecht IV seemed to be secreted. Whensonication was omitted, the cellular fraction of strain Spon-selee still had sphingomyelinase activity (data not shown butidentical to those in Fig. 5). Therefore the enzyme isprobably located in the outer envelope. No sphingomyeli-nase activity could be demonstrated in strains Wijnberg andM20 (belonging to serogroup Icterohaemorrhagiae) and theapathogenic Patocl strain (L. biflexa).

DISCUSSION

The nucleotide sequence analysis of a 3,987-bp DNAfragment of L. interrogans, encoding sphingomyelinase,revealed the presence of three ORFs. These are the first L.interrogans protein-encoding genes for which the nucleotidesequence has been determined. The molecular mass of theproduct of ORF1 (63,268 Da), corresponds to the apparent

molecular mass of 64 kDa from a hemolysin cloned from L.interrogans serovar pomona (8; A. A. Dain, M. N. Rozinov,and Y. G. Chernukha, VI Joint Meeting of LeptospiraWorkers, abstr. no. 7.7, 1988). It has previously been shownthat from the DNA insert of pHL2-B3, only one smallerprotein of 39.2 kDa (41.6 kDa, including the 2.4-kDa signalpeptide) is expressed in E. coli (10). Indeed, ORF1 has aputative signal peptide with a calculated molecular mass of3,175 Da. Moreover, ORF1 is the only reading frame largeenough to encode such a protein and has been shown here tocode for hemolysin and sphingomyelinase activities. Wetherefore conclude that the 39.2-kDa protein is the matureexpression product of ORF1 in E. coli minicells and proba-bly has sphingomyelinase activity. However, we cannotexclude the possibility that sphingomyelinase activity isexpressed as a larger, short-lived protein, and that the

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TABLE 1. Codon usage in L. interrogans, L. biflexa, E. coli, S. aureus, and B. cereusaFrequency of codon usage (%) in:

Amino acid Codon L. interrogansL. biflexa E. coli S. aureus B. cereus

ORF1 ORF2 ORF3

AlaAlaAlaAla

ArgArgArgArgArgArg

AsnAsn

AspAsp

CysCys

GlnGln

GluGlu

GlyGlyGlyGly

HisHis

IleIleIle

LeuLeuLeuLeuLeuLeu

GCTGCCGCAGCG

CGTCGCCGACGGAGAAGG

AATAAC

GATGAC

TGTTGC

CAACAG

GAAGAG

GGTGGCGGAGGG

CATCAC

ATTATCATA

TTATTGCTTCTCCTACTG

LysLys

Met

PhePhe

ProProProPro

SerSerSerSerSerSer

8 (28)4 (13)9 (31)8 (28)

4 (16)0 (0)3 (12)1 (4)

13 (52)4 (16)

20 (51)19 (49)

32 (86)5 (14)

7 (88)1 (12)

15 (83)3 (17)

15 (75)5 (25)

10 (25)4 (10)22 (55)4 (10)

7 (70)3 (30)

16 (40)18 (45)6 (15)

10 (24)10 (24)8 (20)6 (15)4 (10)3 (7)

AAAAAG

ATG

TTTTTC

CCTCCCCCACCG

TCTTCCTCATCGAGTAGC

35 (80)9 (20)

6 (100)

16 (64)9 (36)

4 (20)8 (40)3 (15)5 (25)

15 (33)11 (24)6 (13)4 (9)8 (17)2 (4)

1 (50)0 (0)0 (0)1 (50)

1 (13)2 (25)1 (13)1 (13)3 (37)0 (0)

13 (62)8 (38)

7 (58)5 (42)

1 (50)1 (50)

8 (100)0 (0)

18 (78)5 (22)

1 (4)6 (25)14 (58)3 (13)

3 (75)1 (25)

13 (48)9 (33)5 (19)

7 (22)6 (19)8 (25)2 (6)5 (16)4 (12)

19 (83)4 (17)

6 (100)

18 (78)5 (22)

3 (33)0 (0)0 (0)6 (67)

9 (38)5 (21)3 (13)1 (4)3 (12)3 (12)

1 (25)0 (0)3 (75)0 (0)

1 (20)0 (0)1 (20)1 (20)2 (40)0 (0)

13 (76)4 (24)

5 (71)2 (29)

0 (0)0 (0)

14 (93)1 (7)

19 (76)6 (24)

1 (13)0 (0)6 (74)1 (13)

1 (100)0 (0)

5 (28)3 (33)7 (39)

2 (10)9 (43)3 (14)3 (14)4 (29)0 (0)

20 (83)4 (17)

1 (100)

3 (75)1 (25)

3 (50)1 (17)0 (0)2 (33)

5 (20)1 (5)3 (15)6 (30)2 (10)3 (15)

16 (45)3 (9)10 (29)6 (17)

7 (25)2 (7)10 (36)2 (7)5 (18)2 (7)

24 (89)2 (11)

24 (83)5 (17)

3 (60)2 (40)

19 (90)2 (10)

49 (86)8 (14)

20 (32)2 (3)

31 (50)9 (15)

12 (75)4 (25)

32 (64)12 (24)6 (12)

18 (29)14 (22)17 (28)9 (13)5 (8)0 (0)

39 (81)9 (19)

15 (100)

32 (84)6 (16)

11 (31)8 (22)

13 (36)4 (11)

13 (28)7 (14)8 (16)9 (18)11 (22)1 (2)

(20)(23)(22)(35)

(59)(37)(4)(6)(2)(2)

(37)(63)

(57)(43)

(42)(58)

(30)(70)

(71)(29)

(41)(41)(7)

(11)

(49)(51)

(44)(51)(5)

(10)(11)(9)(9)(3)

(58)

(76)(24)

(100)

(47)(53)

(14)(8)

(18)(60)

(21)(18)(10)(13)(11)(27)

3 (18)3 (18)8 (46)3 (18)

2 (40)1 (20)1 (20)0 (0)1 (20)0 (0)

21 (81)5 (19)

19 (73)7 (27)

2 (100)0 (0)

9 (90)1 (10)

13 (87)2 (13)

11 (52)5 (24)3 (14)2 (10)

8 (89)1 (11)

5 (33)6 (40)4 (27)

11 (52)3 (14)4 (19)1 (5)2 (10)0 (0)

36 (88)5 (12)

4 (100)

5 (50)5 (50)

7 (50)0 (0)7 (50)0 (0)

5 (19)1 (4)9 (35)1 (4)6 (23)4 (15)

14 (21)4 (6)37 (54)13 (19)

16 (100)0 (0)0 (0)0 (0)0 (0)0 (0)

63 (70)27 (30)

57 (92)5 (8)

1 (17)5 (83)

27 (75)9 (25)

25 (60)17 (40)

22 (33)5 (7)

27 (40)13 (20)

13 (76)4 (24)

37 (62)7 (12)16 (26)

43 (61)14 (20)3 (6)1 (1)8 (11)1 (1)

61 (74)21 (26)

19 (100)

17 (63)10 (37)

9 (23)0 (0)

25 (62)6 (15)

25 (28)1 (1)

25 (28)6 (7)

20 (22)13 (14)

Continued on following page

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TABLE 1-Continued

Frequency of codon usage (%) in:

Amino acid Codon L. interrogansL. biflexa E. coli S. aureus B. cereus

ORF1 ORF2 ORF3

Thr ACT 9 (32) 7 (35) 3 (25) 5 (25) (21) 6 (38) 23 (34)Thr ACC 4 (14) 3 (15) 4 (33) 5 (25) (46) 1 (6) 0 (0)Thr ACA 5 (18) 3 (15) 2 (17) 7 (35) (10) 8 (50) 24 (35)Thr ACG 10 (36) 7 (35) 3 (25) 3 (15) (22) 1 (6) 21 (31)

Trp TGG 15 (100) 5 (100) 1 (100) 2 (100) (100) 4 (100) 18 (100)

Tyr TAT 20 (67) 9 (64) 3 (75) 15 (60) (50) 15 (71) 45 (87)Tyr TAC 10 (33) 5 (36) 1 (25) 10 (40) (50) 6 (29) 7 (13)

Val GTT 11 (31) 4 (25) 0 (0) 12 (31) (31) 14 (52) 19 (28)Val GTC 6 (17) 5 (31) 1 (33) 4 (11) (18) 3 (11) 1 (2)Val GTA 9 (26) 5 (31) 1 (33) 13 (33) (18) 7 (26) 26 (38)Val GTG 9 (26) 2 (13) 1 (33) 10 (25) (33) 3 (11) 22 (32)

a Codons used in the three ORFs of L. interrogans were compared with those of L. biflexa trpE and trpG genes (41), E. coli (average values for 407 genescalculated from the data from Aota et al. [1]), S. aureus 3-hemolysin (32), and B. cereus sphingomyelinase C genes (average values compiled from three publishedsequences [14, 22, 41]). The frequency of codon usage is indicated, followed by a percentage, representing the number of times this codon is used to encode itsamino acid.

39.2-kDa protein, generated by degradation or processing,has no enzymatic activity. The molecular mass of 39.2 kDais comparable to the observed molecular mass of the fourhomologous sphingomyelinases and corresponds very wellto the calculated molecular mass of 38,660 Da of the N-terminal part of the protein, starting after the signal se-quence, up to the point where homology with the othersphingomyelinases ends (Fig. 4). How do we explain thediscrepancy between the size of ORF1 and the experimen-tally detected product? Inverted repeats IR2 and IR3, whichcould be involved in transcription or translation termination,are located immediately downstream of the DNA regioncoding for this part of the protein (Fig. 1). Prematuretranscription or translation termination, however, is unlikelyto occur, since the whole coding region of ORF1 is neededfor optimal sphingomyelinase activity in E. coli. More likely,the 39.2-kDa protein would be the result of posttranslationalprocessing of the complete 63-kDa expression product ofORF1. Since in E. coli minicells processing occurs beforecleavage of the amino-terminal signal sequence, the formerprocessing can only take place at the carboxy terminus of theprotein (10). This is supported by the homology data pre-sented in Fig. 4. Similar posttranslational processing at thecarboxy terminus of a protein has previously been reportedfor the immunoglobulin A protease from Neisseria gonor-rhoeae (31), serine protease from Serratia marcescens (29),and activation of aerolysin from Aeromonas hydrophila (21).The codon usage in the L. interrogans sphingomyelinase

gene is quite different from what is normally observed in E.coli for genes with an average expression level. Since theexpression rate of genes in E. coli is known to be related totheir codon usage (15, 34), the leptospiral genes will proba-bly have a low level of expression in E. coli. Indeed, nodifference could be detected between Coomassie-stainedpolyacrylamide gels containing lysates from E. coli contain-ing pBS or pHL2-B3 (data not shown). Alternatively, thelow expression level of the enzyme could be the result ofweak promoter activity. On both the DNA and proteinlevels, the middle part of the leptospiral sphingomyelinaseshares a high degree of similarity with sphingomyelinasesfrom the distantly related bacteria S. aureus and B. cereus.Obviously this part is important for enzyme activity. The

differences in the amino termini of the proteins could reflectdifferences in transport, since B. cereus and S. aureussphingomyelinase are extracellular enzymes, whereas thesphingomyelinase C activity of L. interrogans Sponseleeseems to be cell bound. ORF2 and ORF3 are not necessaryfor sphingomyelinase activity (Fig. 3); no similar nucleotideor amino acid sequences were found in the data banks, andthe functions of the proteins encoded by ORF2 and ORF3remain unknown. Unlike the case in B. cereus GP-4 andIAM1208 (14, 41), in which a phospholipase C gene is locateddirectly downstream of the sphingomyelinase C gene, nosuch gene was found downstream of the leptospiral sphin-gomyelinase gene. Since it has previously been shown thatfive different clones, containing over 10 kilobases of genomic

SEROVAR: ballum canicola pomona hardjo

STRAIN: Mus 127 Hond Utrecht IV Pomona Sponselee :

PS T PS T P1S TP'S T n

C - - * * * *

SP - d d99 9 I I 1.

FIG. 5. Sphingomyelinase activity of four different L. interro-gans strains. Bacterial cultures were harvested in the logarithmicgrowth phase, and a total (T) culture sample was separated intopellet (P) and supernatant (S) fractions by centrifugation for 10 minat 6,000 x g. After mild sonication, the samples were tested for theirability to degrade sphingomyelin, which was monitored by thin-layer chromatography. The sphingomyelin substrate and ceramidedegradation products are indicated on the left by SP and C,respectively. Purified sphingomyelinase C from S. aureus and E.coli containing the pBS vector were used as positive and negativecontrols, respectively. The following L. interrogans strains weretested; Sponselee (serogroup Sejroe, serovar hardjo); Mus127 (sero-group Ballum, serovar ballum); Hond Utrecht IV (serogroup Cani-cola, serovar canicola); Pomona (serogroup Pomona, serovarpomona).

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DNA on which the sphingomyelinase gene is located, werenegative in a phospholipase C assay (10), it is unlikely thatsuch a gene is located nearby on the L. interrogans genome.Among strains of L. interrogans, various sphingomyeli-

nases are produced. Strain Mus127 (serovar ballum) seemsto contain a gene similar to that present in pHL2-B3 (10).Strains Hond Utrecht IV and Pomona, however, which donot cross-hybridize with the sphingomyelinase gene fromstrain Sponselee under stringent conditions (10), do degradesphingomyelin. Moreover, the sphingomyelinase activitiesof strains Hond Utrecht IV and Pomona are predominantlyfound in the supernatant, whereas those of strain Sponseleeand Mus127 are cell associated. Possibly, the sphingomyeli-nase is a contact hemolysin in strains Mus127 and Sponseleeand an excreted hemolysin in strains Pomona and HondUtrecht IV. Strains belonging to the pomona and ballumserovars were also reported to have different hemolyticproperties (37). Bovine erythrocytes are preferentially lysedby pomona strains, and hamster erythrocytes are preferen-tially lysed by ballum strains. It is not known whether thisdifference is caused by a difference in substrate specificity ofthe sphingomyelinase. Contrary to the Dutch field strainSponselee, L. interrogans strains of the serovar hardjo,isolated in New Zealand, do not lyse ovine erythrocytes (18).Therefore genetic variation seems to occur within the sero-var hardjo. This is supported by the presence of differentgenotypes within the serovar hardjo, based on DNA restric-tion endonuclease analysis (12), and differences in the G+Cpercentage of the genomic DNA (27). Although the presenceof multiple sphingomyelinase genes within the L. interro-gans species indicates the importance of this enzyme for thebacterium, the involvement of the sphingomyelinase inpathogenesis is not known. However, several speculationscan be made. The homologous P-hemolysin from S. aureussignificantly increases recovery of bacteria from experimen-tally infected mice, compared with that of the 3-hemolysin-negative mutant, and therefore contributes to virulence invivo (5). Second, leptospires cannot grow without iron (13)and use free fatty acids as the main carbon and energysource (23). The sphingomyelinase could therefore play arole in obtaining iron and fatty acids from lysed erythro-cytes. Third, sphingomyelinase may be an important factorin pathogenesis without lysing erythrocytes; sphingolipidslike sphingomyelin and their degradation products affectmany pharmalogical responses, growth factor action, recep-tor functions, and phorbolester-induced responses and havebeen implicated as second messengers (17).

ACKNOWLEDGMENTS

R.P.A.M.S. thanks Intervet International B.V. (Boxmeer, TheNetherlands) for a research grant. Use of the services and facilitiesof the Dutch National NWO/SURF Expertise Center CAOS/CAMM, under grant numbers SON 326-052 and STW NCH99.1751,is gratefully acknowledged.

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molecular analysis of sphingomyelinase c leptospira interrogans … · a thermolabile hemolysin...

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