Mol cular Mlcrob ology (199 ) 5(5), 1135-1 43

E. Winte d. Hacker•

e , U. Ade t 8. Ludwi , A.. Debe

Institut fQr Genetik und Mikrobiologie, Unlversttat Würzburg_. Röntgenring 11 f W-8100 Würzburg, Germany.

mmary

Irrttod on

nd

L.egionella pneumophlla is a Gram-nega lve, rod-shaped bacterium and ts the causatJve agent of Legionnaires dtsease(McDadeeta/., 1977; Winn 1988; Horwitz, 1988). The organism is a facultative antraceltular parasite and is able to survive and muftiply in human monocytes and atveolar macrophages (Horwitz, 1983: 1984). L. pneumo-

Recetved 5 November. 1990; revlsed 1 J nuary. 199 • FOf es nd· • T . {931}31378/31575; F (93 ) 571954.

A IS 0950382X91 00126P

phlla lnh bit quatic biotops and hot wa , y where the bacteria are able to I ve and mul iply within protoz.oa or anism uc a envtro mental amo b e (W1nn, 1988).

Th cytopathology of destroyed Jung tis u 1nf t d by pecim ns of L pneumophlla sugge t an lnvot m t of

cytotoxins in the pathogenic processe of L gio n r s' disesse (Wdliams et at., 1987; Winn, 1988). S veraf cytotoxic or h em.olytic factors produced by L pneumo­phila have been described in the last decade Baine e al •• 1979; Fdedman et af., 1980; Thorpe and Miller 98 ; Berdal et al. 1982; Baine 1984) but so far only a prot as that exhibi s ha molytic activity has been char c nzed accur tety and studl d at the molecular tevel (Dreyf n lglew kl, 1986· Qu nn and Tompkins 1989; Keen and Hoffm n 1989· Black et af. 1990). f w s al o shown that Legionella stralns exhiblt oth r properties such as produc .. tion of catafase, superoxid dismu se per xi extracellular and intracellul r ftuorescence ctivitl

roduction of a brown metanin-like pigmen (Ba1n an Rashe 1979· Vick rs and Vu 1984· Pin et al.. 984; Berg et sl. 1985). The role of th se propert' es ln the pathogene is of L lonelfl or in 1 s abiUty to survtve in aquat c biotops rema ns to establlshed.

Here we de cribe the cloning of a DNA fr gment rom the chromosome·of L. pneumoph/fa wh eh i able o confer haemoly is and whlch is responsible for the product•on ot

brown pigment-lik colour and fluo escenc activi y but does not. exhibtt any proteolytic or cytolytic ac tvities. Furthermore, our studies show th t t corres . nd ng gene produot. a 39kDa proteln termed legiolysrn ( ly). is produced by L. pneumophi/a wiJd .. typ straln an i not dentical to t e haemolytic protease descri d r ly.

Detection of haemoly ts of pneumophila strains and reoomb;nant Escherichia coli K12 clones

As recently shown (Keen and Hoffm nn, 1989· Born eta/ .• 1988), L.pneumophilaisolate conferh emoly i on blood agar plates. Stralns Philadelphia I and 685 pro uce lysts zone on plates contatning can·ne, human, or p erythrocytes, respectlvely (see F1g. 1).1n order to clon th determ nant r spon tble for the haemolytic phenoty

136 E Wint tmeyeret I.

B

of Leglonella we constructed a genomic library. For this. chromosomal 0 was asoJat from L.. ptJSUmophila

r n Phil lphaa t. Sau3A D fragment of arly 20kb were Iigated lnto the vector motecule pLAFR2 (Knapp and MetUllanos, 1988) d, fol tng packag1ng aod transduc Ion, 2885 recombinant E. coli K12 clo wer isolated.

recomb1nant E. coli olones ere I scr ed ·· n c nln blood agar ptat . After two days, 15 clones

round the, colonies. le o tyse hum and

Properlies of haemolytlc E. coti K12 alones L ionella stratn are le to prod a protea and also

2 4 ·8 10 I I I I

p pEWl1>

pEWL2

1'1

PEWLUf >

cause browning of the medium and exhlbit fluorescenc · actlvity (Baine and Rasheed. 1979, Vicke and Yu. 1984; Dreyfus end tglewsJd. 1 986). ln addttion, cytotoxic effec were seen fottowfng lncubation of various ceU llnes with extn cte or StApematan ' of Legione/la (Quinn et al., 1989· seealso T able 1) .ln order to detect one o1 these propertie$ n haemotytic E. coli K12 ciones, they were inttially cultivated on L-agar plate coota ning 1.5% klm milk. Th eight clon which only exhlbited a canine.-specific hae­mo ytic aotivity showed proteolytic properties as well. Cell extracts of these clones, but no of the haemolytic clon · s with pecificit es to human and sheep erythrocytes we~ also cytotox.ic forVero cetl and CHO cells (see Table 1). ln contrast. the other seven ctones were negative, for proteo .. lytlc and cytotoxJc propartlas but were abt to confer product on of a melanin-like brawn pigment after culti· vation on MQ .. agar plate with tyrosl e (see Fig. 1). rn additlon, these cJones showed yeJlow...green fluorescenc

vity tong-wav ,u.v. ligh ( T be 1). W soggest. therefore. tha two differ . nt type of haemolytic clon were lsolated: or;e has proteolytic and cytotOJti

. activfties, and the other medi tes brownlng of the medium and also has' 1uorescenoe activity.

Subcloning of the gene codmg for the haemolyUc colour-producing, fluorescence-pos-ltive phenotype

0 · A was lsolated from the haemofrys n·positive co our producing. fluorescence-positive cosmld pBLL426. and restrio ion map w established (Fig. 2). Dlfferent 0 A fr. gment were Iigated 10to the vector pUC18 (Norrander et al~, 1983). The subclones were analysed for the three p enotypes. A 7.0kb Pstl frag n located in pEWL1 conferred thes& properties. The determinant responsible

Theleglolysln g n from Le nell pn umop . il 1137

.., Color

+

+

+

+

+

for the three phenotypes eems o tran cri r . m i own p ,omoter. since th p operties co td be de after subclon ng of the fr: gment in both orientation (dat not shown). rn order to localize the gene on the 3 4 kb BamHIIPstt Insert n plasmid pEWl11 a set of dele ion mutants was. constructed (Fig. 2). The data demon trat that the gene mus be I eated between oo-ordina es 13 and 15 in Ffg • . 2, next to the Sphl stte at pos Ion 14 A Ball-Smal fragme of 1.6kb on pJas id pEWL11 w abl to mediate the three ph notypes as the smaU Insert. Plasmid pEWL 1141, htch r ulted tr m exo nuclease m digestion o pEWL 114 confer.red ·n at v phenotypes.

Trsnsposon mapping of the gene coding for the ·th11 phenotypes

A complementary pp o ·eh to mapping of h d te , .. nant was attempted by using in ertion I inactivatio· ofth haemolysln- colour- and fluorescence-specffic en with transposon. Tn 1000 (Guy.er. 1978). For mu gene 1s th · 3.3 kb EcoAl fragment of pfasmid pEWL3 (see Fig 2) wa Iigated into vector pBR328 (Soberoo et sl .• 1980 , r ultln in plasmid ,p·EWL4. Insertion mutants in pEWL were soreened on blood agar plates as weil a M9 plate with tyrosine and the locations of · nsertions were de rmin by sultable digestion of the recomblnant ON . A set ot representativ nsertional mutants are g ven in g. 3. All3 Tn 1000 insertions obta ned that re ulted in n at v phenotypes were Jocat d between th Ball it nd h Smst site next to the Sphl ite at map position 14kb. These d ta lend further weight o the idea that the n 1

loc ted in this particular DNA region .. Muta ions located outside this region were haemolyttc and ill produced he

11 8 E. Wintermeyer et al .

..

Color: +

05

..)

(-)

pigment ... Jike colour and showed fluorescence ctivlty. With the exception of one mutant, which was non-haemo­lytic but showed very weal< brown ng and fluorescence, all the haemolysin-negative mutants a so ·failed in their abil ty to mediate cofour productlon and to produce uoresce ce ac ivity

Comparison of leglolysJn and protease determinants

DNA wa isolated from p asmids pEWL 113 (see Fig. 2) and pBLL4133 (Table 1) ln additio , chro osomal DNA was isolated from L pneumophila stratn Philadelphia I. The e DNAs were c·leaved wtth EaoRI-Smal Bgiii-Sall. and with Hindlll, respectively, and were hybridized wtth the radioactively Iabeiied 2.4 kb EcoRI-Sma I fragment of pEWL1 3. As indicated 1n Fig. 4 no homology wa found between the specific ONA probe and the protease-encod­ing ptasmid DNA, pBLL4133, bu a strong signaJ rep­resenting a HlndiU fragment of 10 kb was observed atter hybridization with the Leglonella-specJfic DNA. These data confirm that the gene responsible for haemoly.sis, colour production and fluorescence is not identical to the protea speoific determinant of L. pneumophila descrlbed recently (Quinn and Tompkins~ 1989} and is o inated from the Legionella genome. The correspond­lng e1e . minant was termed 1egiolysin (1/y).

Detectlon of the leglotysin protein in minicel/s

ln order o analyse he protein encoded by the legiolysin· specific gene. mimoells were isofated from E. coli strain P678·54 carrying plasmids pEWL 114 which conferred the three phenotypes. and pEWL 114 , which represent a defetion mutant ot pEWL 114 (see F1g. 2). As mdicated in Fig. 5 (lane C), minicefls harbouring plasmld pEWL114 expressed a protein of about 39 kDa. The vector plasmid pUC18 and the legiotysin-negative deletion mutant clone P678-54 {pEWL 1141) (lanes Band 0) did not produce this protetn. We therefore concluded. following sodium dod -cyl sulf)hate/polyacrylamlde get etectrophoresis (SOs­PAGE) analysts, that the leglotystn gene lly codes for a protein w1th a molecular ma of 39k0a.

+ +

+

+

Westarn blot analysis with anriserum dlrected agalnst a LacZ -Liy fusion protein

Vectors pSEM1-3 (Knapp et al 1990) ere used to construct fusions between the proximal (5 ') part of the lacZ gene coding for the enzym ß-galac osidase and /ly-speclfic sequences. Consequently, a Sphl-Sph I frag­ment of plasmid pEWL114. with one Sph l site located in the /ly-speci tc insert (see Fig. 2) and the othe Sphl ite being part of the polylinker of vector pUC18 was I ga ed into the polylinker of plasmid pSEM-2 (Fig. 6). A fusjon protein: was ncoded by the resulting clone W311 O­(pAAD114) wtth a molecular mass of 90kDa follow~ng induction with isopropyl-ß-o-thiogalac opyr nos· (IPTG). Owing to the large amount of fuslon prot in, inctusion bodies were formed whtch facil tat th i ol -tion of the protein from SOs-PAGE {dat not hown;

f'~g. 4. Southem hybndlzar•on of Bg/11-58/l·cleaved pla mid D of p8LL4133 (prot production, lane • ol EcoRI-Smsl ctea of pEWL 113 (Jegiotysin produc1 n. Ia b) a.nd of Hindill-e ved chromosomal DNA of L pneuf'I10Phi PhU. I ne c). The 0NAs w hybndized with t 2.4kb ScoRJ-Smal frag nt of p 3 whiCh w Iabeiied by the randorn plim 1'19 proeedur . . sil marker P..~·ONA

V with Hindill w s u • DNA ' 10 •

kd A B c D

97

4

Lly

29

Knapp et 8/. 1990). These protein preparation ere u for immunization of rabbits. Antisera gen rated against the LacZ-Uy fusion protein were used in Westem blotting experiment .

As indlcated in Fig. 7, the antiserumwas spectf1c for a proteln of about 50 kDa which represents the N·termlnal h lfo LacZinstratnW3110(pSEM 2)andforthel.acZ-l1y fusion protein of 90kDa produced by W3110(pAAD114) (1anes A and B). ln addition, a 39kDa protein in strrun P678-54(pEWL 114) was recognized by th antiserum. The molecula weight of this protein · ident cal to that of e plasmid .. encoded protein detected tn mlnlcells (see above). No signals were observed in a Western bio wUh ttle proteolytie clone HB101{p·BLL4133) ane 0). ln L pneumophila wild-type strains a protein of the same size can be de ected in Western b ots usmg the LacZ-Uy- pe­cific anti rum (Fig. 7, lanes E and F). I is concluded, 1herefore. that the legiolysin responstble for haemolysis, cotour prod ction and fluorescence aoUvity repre ents a protein of 39 kOa, produced either by recombinant E. coli K12 clones or by L. pneumophila witd-ty strains.

Di cu Ion

Jn recent years. cytotoxfc, haemolytlc and proteolyttc ac vities as weil as phospholipase C production hav been ascrtbed to L pneumophila, the causative agent of ~ionnaires' disesse (B in et al., 1979; Friedman et al.,

The legio/ys n gene from Legioneil 1139

1 80; Thorpe and Miller. 198 ; Berdal t al., 1982· me. 1 84; Bornstein tal. 1988· Belyi etal., 1989).1n addltion. L. pneumophi/. eKhlb1ts several properties such as oo our production, fluorescence or the· secret1on of v r ous nzym (Pine et al .• 198 · ci< rs and Yu, 1 84· 1nn ,

1988). Of thes f ctors, only a z.inc-metallopro aase which also co fer haemolysis was cloned nd u ied tntensively (Dreyfus and Jgfewski, 1986; Quinn nd Tomp­kins. 1989: Quinn et a/., 1989; Blaok et al. 1 990). lt as demonstrated reoently that this prolease s not req ired for intracellular growth in cell cultures or for in vivo v rule c of L pneumophila in a guin -pig m el (Sze o and Shuman, 990; Blander et al .• 1990).

By screentng a L pneumophila-spec fic genomic Ii ary in suitabl phenotypic t s nd cytotoxicity as became evident that in addltion to the prote , no h r type ot Lf!l9iOn . U pec1~· c haemolysin exists wn eh wa ermed f.egiolysin (Liy} .. Clones coding for legiolysin di ..

ated tysis of canine, human. and sheep erythrocyte . Th new ha molysin also conferred cotour productton of recombinant E. coli strain . Th1s p enomenon. J o termed browning' or prod ction of melanin-li e p' en·

t1on' was prevlou ly describ d by several au hors (B lne end Rasheed, 1979· Pine et al.t 1984; Berget sJ., 1985) tor L. pneumophl7a and other species of he Legionellaceae following cultrvatlon on plates contalnlng tyro in . Colour producttoo wa used, therefore, a 1axonomlc ma in differen ia ·an tests. A a thlrd phenotype, the legto ys1n· spec· c clon produced yellow .. gree fluorescence ctl .. v ty which was al o described for Leglonella s r ins , everaryearsago(Vioker andYu, 1984).1tisinterest gto ote h t one ONA oence, coding fo prot in of

39 kDa is re · ponslble fo the three different ph no pic properties.

Diffe en methods have b n u o show th th

pAAD 1 4

Flg. e. c map of the pl m d pM0114 produciog cZ-Uy fu ion prot ns. Clon ng sltes are ndicated.

14 E. Wintermeyer et al.

G

z· 1.1,-

Z'

ly in s not identical to e h emofytic prot . Th r striction map of the correspo ding ONA p ovtd lfi g arg ment for non-ldenlity of the lly to the

dy known prot , determin n , rm pro or msp Fig. 2). ln ad t on, the phenotyp c tes st South .rn

n izatJon data (Fig 4), and Weste blot u ing peclfic pofyclonal antibod dtrected g inst th , LacZ-uy hybrid protein c fir th t I iolys n

gen 1s different from t . z1nc metafloprote de mi-n nt However. dat concerning the isol tion ot mu nt

rytng an Insertion n th chromosome of a L. pneumo-phila stra1n which result in protease-n a iv on .. h

lytie phenotype ( ugg ttng tha the metalloprotease

·r t the only haemotytfc substance prOduc by pneumophila (Keen and Hoffmann 1989)). contr st with t resul presented her . One reason or th ncy may be tha , legioly in is. not d tectabf · with t '

blood-agar plares used by he utnors ln add t1on. o e c not excrude th po sib l'1ty th t th mutatlon whic abolished protease .. nduced haemotys s atso ffec ed t pr uction of tegiQiystn in L pneumophila. s ulated ha a r ufatory e e nt I p ot locus may influenee expression of th lly Str tin age of vt I nce-associated genes and r ul -tory locf has been descn recently for variou · p I Ii Bordeteils pertu s. Staphy/ococcu ureus, Escherichla coll nd othe (Refman stal., 1989; Roy et al. 1989; Janzon and Arvidson, 1990; Caron et /., 1989). Ye another. ibility is that produ ton of th pr 1

p equisite for expression o legiolysin in L. pneumophila. ChnicaJ obsetvat· d histological udie su

tox•n in t pathog nes· (Friedman et s/. 1980; Winn, 988).

oxic ub c I k fegio y tn m y contribu e to tl ue darnage or y att ck potymorphonuclear leucocytes Furth rmo , toxin seem to be nece ry for productio of a necrot1c re ction in the lung tlssue of inf ted atl ts. Mark exch n experiment wi the clon J ioly in­spectfic gene will be useful for determtni g putative ole

i lys n Legionnaires' isease.

ExJ""tmtantal lprocedure

Media, enzymes and chemieals

BacterlaJ stralns nd plasm a:

Cosmld loni procedure·

Recom nant DNA t hniques

alysis, DNA wa reated w h pprop e n resutting tragmeots wer ted by t Jec roph es on 0.8-1.0% agarose gels (Knapp et al., 1984) ONA fragments w re lsolated from the garose gel by the freeze·squeeze method (Thuring et al .• 1975). For clon ng, ONA frag nts were Iigated nto suitable vector molecules after at-in vat1on of the r Ion endonucleases at 6500 for 10 m•n (Sambrook et a/., 1989). Digestion of DNAs with exonuclease UJ w done accord­ingto HenJkoff(1987) E. col/t<12 strainswere r nsformed byth CaCI2 method (Lederberg and Cohen, 1974).

Tn1 000 (y8) transposon mutagenesls

Tn 1000 InsertiOn in the legiotys n (//}1 gen w · sol ted followlng mobilization of /Jy-spectffc plasmld pEW 4 by tha F-factor which occurs by Tn TOOD-mediated cointeg tton of the two plasmlds. Resotvation ot the colntegrate in recipient cell resutts ln tly::TnUXJO InsertiOn derivatives whlch w e analy by suftable dlgestlon (Guyer. 1978).

Gene p obes and radlosctiVe labelllng

As a leg ·n (lly) .. speciflc pr the 2.4 EcoRVS l fragment of pEWL 113 (see Ftg. 2) was used After lut1on fro a ose s 1h DNA rt gt]\ent w Iabeiied y the method of

. Feinberg nd Vogelsteirt (1983) wlth random p . ml 't purohased from Boehrlnger.

Southem hybridlzation

The transter of DNA from agarose get to n trocel u paper, and the w sh g and autoradlography of the fllters were tormed as desctlbed eart (Sotrthem. 197 5). The fllters w hybridized iA 50o/o formamlde for 24h a 4211C. S ringen con-

tt oos w used for the washing Pf'()Cedure ( broo e 1~,

1989).

Detection of haemolysis, protease activity, colour production and Ruorescence

H molys n productlon of L pnfiUmophll w ld-type and of , · ombinant E. coll K12 clon was ected on r p contalning 5% rythrocytes trom el. •her humans, dogs or eep fter cutt vation fOC' 2d t 3-r'C. Prote actv ty was detect

using L .. broth contalnlng 1.5% im ml Cotour p oduction brown ng} of strains and clone was a yed fter cult vation on a ar plates with 4mM tyro ·ne. A long-wave u.v.-tam w n the darK to detect yellow-green ftuor scence excreted l to the medi m.

Analysis of proteln synthesis in mln cell

Analysi of plasmld encoded pro eins ln min cells w performed as described previousJy (Noegel et al., 1979).

Construction of LaCZ-Uy fuslon proteins and preparatlon of Uy-speclfio ntibodles

A suitabl IJyospeeific DNA fr toontaln dstalp o

The /egioly. in g n from ,

We tern blots

Western blot cc rd1ng tot

Cytotoxiclty a ~

CHO· and Vet"o cell wefe cult r

1141

1 142 E. Wintermeyer e,t al.

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