Oral Microbiol Immunol 1990: 5: 155-161

Outer membrane proteins of Actinobacillus actinomycetemcomitans and Haemophilus aphropfiilus studied by SDS-PAGE and immunobiotting

Anne Isine Bolstad\ Tore Kristoffersen\ Ingar Olsen^, Hans R. Preus^ Harald B. Jensen^ Endre N. Vasstrand^ Vidar Bakken^ Departments of 'Periodontology, ^Biochemistry, University of Bergen, ^Microbiology, tjniversity of Oslo, Norway

Bolslad AI. Krisloffersen T. Olsen I. Preus HR. Jensen HB. Vassirand EN, Bakken. V. Outer membrane proteins o/ Actitiobacilliis actitioniycetetncotnitans and Haemophilus aphrophilus studied by SDS-PAGE and imnnmoblotling. Oral Microbiol Immunol 1990: 5: 155-161. This investigation characterized and compared outer membrane proteins (OMP) of the closely related Aelinobacillus aclinomveeleineomilans and Haemophilus aphrophilus by tneans of SDS-PAGE patterns atid teactions on itiimunoblots with rabbit antiserutn against A. aetinomycelemcomitans FDC Y4. Reactions with serum from a patient with Papillon Lefevre syndrome (PLS), frotn whotn periodontai wild strains of/I. actinomyeetemcomitans had been isolated, were also studied. OMP were purifted with selective solubilization from lyophilized cells of 10 wild and 4 reference strains of A. aclinomyeelemcomitans and 4 reference strains of H. aphrophilus. OMP profiles frotn wild and reference strains of A. actinomycetemeomitans were similar while those frotn A. aetittomycetemcomitans and H. aphrophilus differed. The tnost protninent difference was absence of a heat modifiable protein in H. aphrophilus strains. Itntiiunoblotting revealed strong cotnmon antigens in most strains, including a heat tnodifiable protein with mol wt 34 kDa, as well as a 29 kDa and a 16.5 kDa protein. Treattnent with pronase and sodiutn periodate confirmed the protein nature of the major OMP antigens.

Aclinohaeillus aclinomyeelemcomilatts is sotne OMP may be possible candidates a Gratn-negative, nontnotile, capnophi- for itnmunotherapy (3, 14). Although lic, coccobacillary rod often associated sotne attention has been paid to OMP of with juvenile periodontitis (33). Because some strains of A. aeiinomyeeiemeomiof close genetic relations reassigntnent ians and H. aphrophiltis (8), mostly proof ^. adinomycetemeomilans to the ge- tein profiles of whole bacterial cell exnus Haemophilus as H. actinotnycetem- tracts have been studied (29). We have comitans has been proposed (20). A. ac- previously used OMP for the taxonotny tinomycetemcotnilans and Haetnophilus of Eubaeteriuni species (1). aphrophilus show great sitnilarity in The purpose of the present study was morphology, habitat and biochetnical to see if OMP could be used for taxoreactions and taxonotnic distinction be- nomic distinction between A. aelinotntween thetn can be difficult. ycetemeomitans and H. aphrophilus by Outer tnetnbrane proteins (OMP) are cotnparing and characterizing their constituents of the surface layers of Gra- OMP with respect to protein patterns m-negative bacteria. They have many on SDS-PAGE and reactions on imtnuimportant functions, serving as phage re- noblots with rabbit antiserum against ceptors (6), diffusion pores, proteases, the strain FDC Y4 of ^, actinomveetemmitogens (7) or taking part in conju- coniilans. Reactions with serum frotn a gation (26). Due to their itntnunogenicity patient with Papillon Lefevre syndrotne

Key words: Actinobacitlus actinomycetemcomitans; Haemophilus aphrophilus; outer membrane proteins: antigens. Dr Anne Isine Bolstad, Department of Periodontology, School of Dentistry, University of Bergen, Arstadveien 17, N-5009 Bergen, Norway Accepted for publication August 22, 1989

(PLS) from whotn wild strains of A. aeiinomyeetemeomitans had been isolated (21, 23-24), were also studied. OMP of strains from the PLS patient and reference strains of A. aetinomyeeletncomitans were cotnpared to each other and with those of reference strains frotn H. aphrophilus. This was done in order to obtain tnore knowledge of the surface architecture of these organistns of which H. aphrophilus apparently does not have great pathogenic potentials in the dentogingival area (30). Material and methods Bacterial strains and growtli conditions

The different strains used, their serotypes and sources are listed in Table 1. Strains designated GI-X were wild

Bolstad et al.

156

Taiiie I. Bacteria investigated

Preparation of OMP by selective solubiiization of inner membrane

Species

Strain

Serotype

Souree

Site of isolation

A. actinomycetemcotnilatis

29523 29522 33384^ Y4 GI-X

a b c b

ATCC ATCC ATCC FDC" UNO'

Blood Mandibular abseess Lung abscess Juvenile periodontitis Prepubertal periodontitis

H. aphrophilus

33389^ 19415

ATCC ATCC EDC FDC

Endoearditis Endocarditis Periodontitis Periodontitis

655 654

" American Type Culture Collection, Rockville, MD, U.S.A. bEorsyth Dental Center, Boston, MA, U.S.A. c University of Oslo, Oslo, Norway. ^ Type strain of species.

strains of A. actinotnycetemcomitans isolated from subgingival plaque samples taken from a 12-year-old girl with PLS and prepubertal periodontitis (21, 23-24). Wild strains of A. actinomycetemcomitans were cultured anaerobically at 37°C for 72 h on liquid Slots medium (27). Reference strains of A. actinomycetemcomitans and H. aphr-

ophilus were cultured anaerobically in brain heart infusion (Difco Laboratories, Detroit, MI) broth. After harvesting by centrifugation, cells were washed twice with deionized water and lyophilized over (//-phosphorous pentoxide (E. Metck AG, Dannstadt, W. Gertnany). Lyophilized tnaterial was stored at — 20°C under nitrogen gas.

Am. 1a 1b 2a 2b 3a 3b 4a 4b 5a 5b 6a 6b

The procedure was in essence that described by Bakken and Jensen (2). Briefly, freeze-dried bacteria were washed in cold 0.03 M Tris/HCl, pH 8.1, resuspended in the satne buffer and broken in a French pressure cell at about 3,000 lbf in"= (20.7 MPa). After centrifugation at 2,000 X g for 5 tnin to remove unbroken cells, the eell envelopes were sedimented by centrifugation of the supernatant at 10,000 x g, 15 min, 4°C. The envelope fractions wete washed twice by resuspending thetn in 10 tnM N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES)/10 mM MgCU buffer, pH 7.4, followed by centrifugation as above. Triton X-100 was added to 2% (v/v) final concentration, and the tnixture incubated at rootn tetnperature for 20 min. After centrifugation at 10,000 x g, 15 tnin, 4°C, the pellet (OMP fraction) was washed twice by

7a 7b 8a 8b 9a 9b 10a10b11a11b

-48 -39 -34

42.7

48 43

•31.0

-28 21.5 14.4

IT

I GI

28

a Gil

-18.5 •16.5 Gill

GIV

GV

ST

GVI

GVII

Ib 2a 2b 3a 3b

4a

GIX

GX

29522

4b 5a 5b 6a 6b 7a 7b

— w w W\

I

97.4 ,^ 66.2 ..^^ Fig. 1 (A-D). SDS-PAGE analysis of OMP from A. actinomycetemcomitans wild strains Gl-X and referenee strains ATCC 29522, 33384, 29523, and EDC Y4, and from H. aphrophilus reference strains ATCC 33389, 19415, EDC 655 and 654. Samples were heated for 5 min at 50°C (lanes a) or lOOX (lanes b). Mol mass standards are indicated on the left, and the positions of main proteins on the right. Proteins with increased mobility are arrowed. Gels were stained with Coomassie billiant blue.

GVIII

42.7, »*

II

31.0-x,.,.

21.5%*

a

I4.4C AaY4

Ha 33389

I Ha 19415

Ha654

Ha655

Aa Aa 33384 29523

OMP 0/Actinobacillus and Haetnophilus l a 1b 2a

2b

3a

3b 4a

4b 5a

5b

6a

6b 7a

7b

8a

8b

9a

157

9b 10a10bt1a 11b

97.4 —

66.2—:; 42.7 — 31.0—

^11

34

21.5— •16.5

14.4—'

Ql

ia

GH

Gill

Ib 2a 2b

GIV

3a 3b

GV

4a

GVI

4b

48 34

GVII

GVIII

GIX

GX

Y4

5a 5b 6a 6b 7a 7b 8a 8b 9a 9b 50 48 39 34 '-.-28

39 34 -.—28 ..--.-24

#•.-24

16.5

29523

29522

33364

Ha655

Ha Ha 33389 19415

Y4

Ha654

AaY4

Fig. 2. (A-D). A-C show imtnunoblots of OMP from wild strains Gl-X and referenee strains EDC Y4, ATCC 29523, 29522, and 33384 of A. actinotnycelemcomitans probed with rabbit antiserum against whole cells of A. actinotnvcetenicomitatis strain EDC Y4. 2D shows immunoblots of the reference strains of H. aphtopiiilus ATCC 33389, 19415, EDC 655 and 654. A. aclittomycelemcomilatts EDC Y4 is ineluded for comparison. Antiserum was the same as in A-C. Samples were heated for 5 tnin at 50°C (Lane a) or 100°C (Lane b). Mol wt standards are indicated on the left in A, the position of major antigens on the right in A-D and also on the left in C.

resuspension in 10 tnM HEPES/10 tnM buffer, pH 7.4. Preparation of Triton X-100 soluble material (inner membrane)

Louis, MO). Bio-Rad (Bio-Rad Laboratories, Richmond, CA) low mol wt protein standards were used as size tnarkers.

Human and rabbif antisera

Human serutn was prepared from whole blood drawn from a 12-year old girl PLS patient with advanced periodontitis (21, 23-24). Rabbit antiserutn was prepared by itijecting rabbits subcutaneously 3 times at 2-week intervals with whole cells of A. actinomycetemcotnilans strain FDC Y4 suspended in complete Freund's adjuvant.

To 100 pX (10%v/v) Triton X-100 soluble material (intier metnbrane) 900 /d Silver staining for lipopoiysaccharide (LPS) acetone (90% v/v) was added and kept at —20°C for minimutn 24 h. After een- LPS was stained as deseribed by Tsai trifugation at 10,000 xg, 15 min, the and Frasch (32). resultant pellet was washed twice in 50% (v/v) acetone, dried at room tetn1a I b 2 a 2 b perature atid then frozen at — 20°C. Samples to be analyzed by SDS-PAGE were suspended in 10 tnM HEPES/10 mM MgCl. buffer, pH 7.4.

3a

3b

4a

4b 5a

5b

—28-29

SDS-PAGE

OMP wete separated by SDS-PAGE according to the tnethod of Laetnmli (11) using a 12.5% (w/v) polyacrylatnide gel. The proteins were stacked in a 4% polyacrylatnide gel. Preparations of OMP were heated in sample buffer at 50 or 100°C for 5 min to determine the degree of heat modiftability. The gels were stained with Cootnassie brilliant blue R 250 (CBB, Sigma Chemical, St.

E GIIGX Ha Ha 33389 19415

Ha655

Ha654

AaY4

Fig. 2 (E-F). 2E shows itnmunoblot of OMP ftom the wild strains Gil and GX of A. actinomycetemcomilans probed with human serum from the patient from whom the wild strains were eoUeeted. Samples were heated lor 5 min at lOO'C. This figure was representative for all the wild strains. Eig. 2F shows immunoblot of OMP Irom H. aphrophilus strains ATCC 33389, 19415, EDC 655, 654, and from EDC Y4 o f / i . aetinoniycetemconiitans probed with the same human serum as in E. Samples were heated at 50°C (Lane a) or 100°C (Lane b). The positions of major antigens are indicated.

Bolstad et al.

158

Immunoblotting

Electrophoretic transfer onto Nitrocellulose membrane (NC) of pore size 0.45 pm (Schleicher & Sehuell, W Germany) was performed using buffers recommended by Towbin et al. (31). After SDS-PAGE of OMP, transfer of gel proteins was carried out by employing a Nova Blot Electrophoretic Transfer kit together with the Multiphor II base (LKB, Bromma, Sweden). Successful transfer was controlled by staining the NC niter containing the mol wt standards with fast green. The staining solution was made of 0.1% fast green, 50% methanol and 10% acetic acid in distilled water (dHjO). Prior to overnight incubation with the first antibody the NC filter was blocked with 0.05% Tween 20 in Trisbuffered saline (TTBS) (20 mM Tris, 500 mM NaCl, pH 7.5) for 1 h at room temperature. The immune sera were used at 1:200 (v/v) dilution (rabbit antiserum to FDC Y4) or 1:100 (v/v) (serutn from the PLS patient) with 1% gelatinTBS. Non-specifically bound primary antibody was retnoved by a short rinse with dHiO, followed by washing in TTBS for 2 X 10 min under gentle agitation. The tnembranes were then incubated with the second antibody solution for 1 h. Goat anti-rabbit IgG, conjugated with horseradish peroxidase (HRP) (Bio-Rad) or goat anti-human IgG HRP (DAKO-immunoglobulins a/s, Denmark), were used as a seeond

antibody. Free and non-specifically bound antibodies were removed by washing the NC tnembrane in TTBS for 2x10 min with gentle agitation followed by a short rinse with TBS to retnove Tween. HRP color development solution (Bio-Rad) was prepared immediately prior to use and consisted per blot (9x6 em-) of 10 tng 4-chloro-lnaphthol dissolved in 3.3 ml ice cold tnethanol mixed with iee cold 30% H2O2 {W pf) in 16.7 tnl TBS. Treatment with pronase

Pronase (Proteinase K, BoehringerMannheim, W Gertnany) was added to OMP samples in SDS-PAGE sarnple buffer at concentrations of 10 pg per sample and kept at 60°C for 60 tnin. The mixtures were then heated at 50 °C or 100°C for 5 min before they were loaded onto gels. Eleetrophoretic transfer of these gels was performed as previously. Treatment with sodium periodate

Sodium periodate (Sigma), 50 mM final concentration, was added to OMP in SDS-PAGE sample buffer and kept at 37°C for 15 min. The satnples were then heated at 50°C or 100°C for 5 tnin atid subjected to SDS-PAGE. Sotne of these gels were stained with CBB, with or without subsequent silver staining for LPS. Other such gels were used for immunoblotting with rabbit antiserum.

1a 1b 2a 2b 3a 3b 4a 4b 5a 5b

6a 6b

-37 34-

-30

28-

16.5-*

B GI

Gl-P

Y4

Y4-P

Y4IM Y4 IM-P

Results Outer membrane protein profiles

The 10 wild strains of A. actinomycetemcomilans displayed great sitnilarity in the main patterns of OMP (Fig. 1AB). Four dominant protein bands were visible when Triton X insoluble material was heated with sample buffer at 100°C for 5 tnin. The apparent tnol wts of these proteins corresponded to the 48, 39, 34 and 28 kDa zones of the standards included for comparison. Less prominent were bands corresponding to 43, 32.5, 29, 18.5 and 16.5 kDa. When OMP from the wild strains of A. aeiinomyeetemeomitans were heated at 50°C for 5 tnin, only the 34 kDa protein detnonstrated greater electrophoretic tnobility than it did when heated at 100°C (Fig. lA-D). This heat tnodifiable ability was seen also in the reference strains or A. aclinomyeelenieomitans (Fig. 1 A-D). The protein profiles of reference strains of A. actinomveetemcomitans ATCC 29522 (Fig. IB),'FDC Y4 (Fig. IC), ATCC 29523 and ATCC 33384 (Fig. ID) were very tnuch like those of the wild type strains of this organistn. A. actinomycelemcotnilans ATCC 29522 exhibited weak staining of band 39 kDa. The differences in protein profiles between reference strains of A. aclinomyeelemeotnitans were quantitative, rather than qualitative. The OMP composition of H. aphrophilus could be distinguished frotn that of A. aelinotnvcetemeotnitans in SDSPAGE, despite tnany sitnilar bands (Fig. 1 C-D). The most promitient difference was the apparent absence in H. aphrophilus strains of bands with greater electrophoretic tnobility in satnples heated at 50°C compared to 100°C, although a tnore distinet staining of the band 34 kDa could be observed. The profiles of H. aphrophilus strains were very sitnilar to each other. Mol wts of main OMP in H. aphrophilus strains were 48, 39, 34 and 16.5 kDa (Fig. 1 C, D). Besides, //. aphrophilus strains ATCC 33389 and ATCC 19415 showed marked staining of the proteins with tnol wt 43 and 37 kDa.

GX-P

Fig. 3 (A-B). Treatment with pronase. A: Imtnunoblot of A. actinotvycelenicomiians GI and EDC Y4 Triton X-100 insoluble fraction (OMP) and EDC Y4 Triton X-100 soluble fraetion (inner membrane = IM). Samples were heated for 5 min at 50"C (Lane a) or lOO'C (Lane b). Those treated with pronase, designated P, were heated at 60"C for 60 min. Positions of major antigens are indieated. B; Silver staining for LPS of a gel previously stained with Coomassie. OMP from A. aeiinomyeeiemeomiians strain GX had been treated with pronase (P). Sample was heated for 5 min at 100"C.

Immunoblotting with rabbit anti A. actinomycetemcomitans FDC Y4 serum

The wild type strains of A. aclinomyceletncotnitans gave strong reactions at the bands corresponding to 34, 28-29 and 16.5 kDa when the satnples were heated

OMP 0/Aetinobaeillus and Haemophilus

1a

Ib

2a

2b 3a 3b

4a^

4b

5 a 51

la

ib 2a 2b 3a 3b 4a,^ _4b.

5a

159

5b

•«»•

3934« * mm '»*'«

2818.5--r 5-^

Ql

Gil

AaY4 Ha33389Ha655

61

Gil

AaY4

Ha33389 Ha655

Fig. 4 (A-B). Coomassie stained gel of OMP from A. aclitiomycetetncomitans GI-II, EDC Y4 and from H. aphrophilus ATCC 33389 and EDC 655. Samples were heated for 5 min at 50"C (Lane a) or 100°C (Lane b). Proteins with increased mobility are arrowed. B: The Coomassie stained gel in A is silver stained for LPS.

at 100°C (Fig. 2A-B). When the preparations were heated to either 100"C or 50°C, a change in the electrophoretic mobility of the heat tnodifiable protein from 34 kDa to 28 kDa occurred (Fig. 2A-B). Strains ATCC 29522 and FDC Y4 of A. actinomycelemeotniians showed well-defined staining of band 24 kDa (Fig. 2B) while strains ATCC 29523 and 33384 did not (Fig. 2C). Strain ATCC 29523 of ^. aetinotnycelemeomitans exhibited heavy staining of band 48 kDa (Fig. 2C), whereas the other strains showed tnoderate staining of this band. Reference strain FDC Y4 and the wild strains of A. actinomyeetemcomitans also reacted at the 39 kDa band (Fig. 2 B-C). The main difference between A. actinomycetemcomilans and H. aphrophilus strains was the absence in the latter of heat modifiability of the protein at 34 kDa (Fig. 2D). The 34 kDa band, howevet; was tnore heavily stained when the satnple was exposed to 100°C than to 50°C. This may be due either to a higher solubility of the protein at 100°C, or, more unlikely, to a conformational change which infiuenced the binding of the antiserutn. As with the A. aetinomycetemcomitans strains, the H. aphrophilus strains possessed a heavily stained 16.5 kDa band. In addition, H. aphrophilus ATCC 33389 revealed a strong reaction of a band of approximately 50 kDa (Fig. 2D). On some blots H. aphrophilus ATCC 19415 showed a similar, but less prominent reaction. When heated at 100°C, the H. aphrophilus strains ATCC 33389, ATCC 19415, FDC 655 and FDC 654 (Fig. 2D) showed strong reactions at 34,

region (15), and the reaction with anti Y4 serutn here is probably due to the presence of such LPS. Two proteins iti the inner tnembrane of FDC Y4 reacted strongly with the anti Y4 serum (Fig. 3A, lanes 5a-b). Apparent tnol wts of these proteins were Immunoblotting with human antiserum 37 and 30 kDa. Pronase treattnent elimProbing with serutn frotn the PLS pa- inated all reactions on blots of the inner tient on blots of wild strain OMP from tnetnbrane fraction (Fig. 3A, lanes A. aetitwmyceteincotnitans heated to 6a-b), pointing to a pure proteinaceous 100°C (Fig. 2E), detnonstrated reac- nature of the antigenic substance in this tions with proteins 39, 34, 29 and 16.5 fraction. All CBB stainable bands disappeared kDa. The lanes of OMP frotn A. aetinom- as a result of pronase treatment. When yceietneomilans FDC Y4 showed batids the same gel was silver stained for LPS, at 34, 28-29 and 16.5 kDa (Fig. 2F). only the region of the gel below 14.5 OMP (Vom H. aphrophilus strains react- kDa reaeted (Fig. 3B). Also with several ed at 16.5 kDa and with antigen prep- other A. aelinomyeetemeomilans strains, arations heated at 100°C, also proteins CBB staining for protein (Fig. 4A) and with mol wt 34 kDa became visible (Fig. subsequent silver staining for LPS (Fig. 4B) revealed LPS reactions only in the 2F). same low mol wt region of the gel. These observations suggested that LPS of the Treatment with pronase and sodium A. aeiinomyeetemeomitans strains were periodate of the short chain type, and that the To verify that the antigenic substances reactions on the blots of bands with mol which reacted with sera on blots were wts above approximately 14.5 kDa were proteins, various preparations of outer all due to reaetions with proteins. A and inner tnembranes were subjected to possible reaction with stnall atnounts of treattnent with pronase prior to analysis LPS associated with the proteins, could by SDS-PAGE and immunoblotting. not be conclusively exeluded. However, The results frotn using A. aetinomyee- this possibility was weakened by the temeomitans strains GI and FDC Y4 finding that sodium periodate treattnent outer tnembrane material and Y4 inner of OMP frotn A. aclinomyeelemcomitnembrane tnaterial are shown in Fig. tans strain Gil did not negatively affect 3A. After pronase treattnent of outer the antigen-antibody reaction of the tnetnbrane samples, anti Y4 serutn re- protein bands. acted only with tnaterial located in the low tnol wt region of the gel, i.e. below Discussion about 14.5 kDa (Fig. 3A). One would The extensive interstrain hotnology exexpect to find short chain LPS in this hibited by A. actinomycetemcomitans 28-29 atid 16.5 kDa. Strain FDC 655 and 654 did not yield the strong staining of the 50 kDa protein, but their other profiles were sitnilar to those of H. aphrophilus ATCC 19415 and 33389.

160

Bolstad el al.

and H. aphrophilus reflected minor plei- ophilus (9, 16). The eriteria presently es- itntnunoblots. However, since A. aetiotrophy and preeluded use of the envel- tablished add to those already suggested nomyeetemeomitans and H. aphrophilus ope protein profiles of these organisms for taxonomic distinction between these differ in their pathogenic potential in for typing clinical isolates at the strain organistns (4, 9, 16-18, 28). It should be the dentogingival area, it is unlikely that level. This was in eontrast to high-resol- realized though that differenees between P6 is a critical antigen in this context. ution two-dimensional electrophoresis bacteria on the species level do not ex- Whether other differences in OMP surof cellular proteins where strains from elude relatedness at the genus level. We face arehitecture of these organisms the tested organisms could be dis- have recently suggested frotn studies on eontribute to the difference in virulence tinguished (9). However, OMP profiles oligotiucleotide sequences of ribosotnal will have to be elucidated. could apparently be useful to group RNA (18) that A. actinomyeetemcomistrains of these organisms into species, tans should be transferred to genus HaAcknowledgements although it would have been desirable emophilus. to test more than these 2 species to claim Our present results were also close This work was supported by the this as a tnethod generally useful for to those achieved with SDS-PAGE and Norwegian Researeh Couticil for distinguishing among species. This find- itnmunoblotting of OMP frotn Haem- Science and the Humanities. ing was in accordance with those of Di- ophilus pleuropneumoniae (12, 25). Pohl Rietizo & Spieler (8) although the mol et al. (19) and Maclnnes & Rosendahl wts reported in their study did not fit (12) found that H. pleuropneumoniae References exactly with ours. Also Moore etal. (13) shared greater homology with members 1. Baardsen R, Bakken V, .lensen HB, Hofstad T. Outer membrane pattern of Euand Tanner (29) have differentiated oral of genus Aetinobaeillus based on phenobaeteriurn plaiitii. .1 Gen Mierobiol 1988; bacteria by PAGE of cellular proteins. typic and deoxyribonucleie acid related44: 1561-1564. The proteins obtained from a bac- ness than with metnbers of genus Haem2. Bakken V, Jensen HB. Outer membrane terial cell extract is believed to refiect ophilus. proteins of Eusobacterium nucieatum the genetic background of that strain The present study did not detnonEev 1. J Gen Microbiol 1986: 132: and can be cotnpared with protein strate any elear correlation between 1069-1078. fingerprints from other strains as a OMP and serotype distribution of A. 3. Blaser MJ, Hopkins JA, Vasil Ml. Catnmeasure of relatedtiess (10). This was aetinomyeetemeomitans. This was in pytoixwtcr jejuni outer membrane proteins are antigens for humans. Infeet Imsupported by the sitnilarity in OMP agreetnent with Zatnbon et al. (34) who mun 1984: 43: 986-993. demonstrated between wild strains and found that the serotype c antigen of A. reference strains of ^. actinomycetemeo- aeiinomyeetemeomitans was composed 4. Brondz I, Olsen I. Review. Chemotaxonomy of selected species of the Actinobamitans grown in different media whieh of 95% carbohydrate, 2% protein and ciiitis-Haemopiulus-Pasleureiia group by may imply that the isolated OMP were 3% phosphate. means of gas ehrotnatography, gasconstitutionally expressed and therefore Imtnunoblotting with rabbit antichromatography-mass spectrometry and particularly suited for taxonomic pur- serum or hutnan serum showed a strong bioenzymatic methods. J Chromatogr poses. Previously, restriction endo- reaction with the heat modifiable pro1986: 380: 1-17. nuclease mapping of chromosomal tein and with a protein of approxitnate 5. Calhoon DA, Mayberry WR, Slots J. DNA has suggested that wild strain mol wt 16.5 kDa. Some baeteria also Cellular fatty aeid and soluble protein composition of ActinobaeiUus actitiomGIX-X differ in genotype from that of showed strong reaction with the 24 kDa ycetemcomitans and related organisms. J GI-GVIII, while the reference strains protein. While the heat modifiable proClin Microbiol 1981: 14: 376-382. referred to showed only minor differ- tein 34(28) kDa was protninent both in 6. Datta DB, Arden B, Henning U. Major ences in their DNA fingerprints (22). gels and on blots, the 16.5 kDa protein proteins of the Escherichia coli outer cell Apparently, genotypic differences were exhibited weak reactions in gels, but envelope metnbrane as baeteriophage renot expressed in recognizable protein strong reactions on blots. The protein ceptors. J Bacteriol 1977: 131: 821-829. profiles of the strains either by SDS- with mol wt 24 kDa was visible only 7. DiRienzo JM, Nakamura K, Inoye M. PAGE or on itntnunoblots (Figs. lA-B, on blots. This indicated that even stnall The outer membrane proteins of Gram2A-B). negative bacteria: biosynthesis, assembly amounts of these proteins (24 and 16.5 and functions. Annu Rev Biochem 1978: In our SDS-PAGE study A. actinom- kDa) were sufficient to produce a strong 47: 481-532. yeelemcomitatrs and H. aphrophilus immunologie reaction, and that the 24 8. DiRienzo JM, Spieler EL. Identifieation could be distinguished. First, some kDa protein seemed to be a particularly and characterization of the major cell OMP from these organisms differed in potent antigen. It is interesting that a envelope proteins of oral strains of Actimol wts, and secondly, OMP from H. 16.6 kDa OMP, referred to as P6, has nobaeiUus aciitiotnycftetncomitatis. Infect aphrophilus strains did not exhibit the been identified so far in all examined Immun 19B3: 39: 253-261. heat modifiable protein. The tnain dif- strains of Haemophilus itrflttenzae (14), 9. Jcllum E, Tingelstad V, Olsen I. Differenference when cotnparing A. actinomyce- an important pathogen in both adults tiation between Aelinobacillus actinointemcomitans strains and H. aphrophilus and ehildren. P6, being a target for baeycetemcomitans and Haemophilus aphrophiius by high-resolution, two-dimenstrains on blots was the absence of a terieidal antibodies, may be an itnportsional protein electrophoresis. Int J heat modifiable protein in H. aphrophi- ant surfaee antigen with regard to host System Bacteriol 1984: 34: 478^83. lus. These findings are in agreement immunity and vaccine development 10. Jones D, Krieg NR. Bacterial classiftwith studies on soluble eellular proteins towards this organistn. It was notecation V. Serology and chemotaxonomy. with one-dimensional PAGE (5) and worthy that both A. aetinomyeetetneoIn: Krieg NR, Holt JG, eds. Bergey's high resolution, two-ditnensional elee- milans and H. aphrophilus possessed a manual of systematic bacteriology. Vol. trophoresis of whole cell proteins frotn protein in the same region which dem1. Baltimore: Williams & Wilkins, 1984: A. actinotnycelemcomitans and H. aphr- onstrated strong antigenic reaction on 15-18.

OMP 0/Actinobacillus and Haemophilus 11. Laemmli, UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 1970: 227: 680-685. 12. Maelnnes JI, Rosendal, S. Analysis of major antigens of Hactnophiius (Actinobacillus) pleiiropneutnoniae and related organisms. Infect Immun 1987: 55: 1626-1634. 13. Moore WEC, Holdeman LV, Cato EP. Polyaerylamide slab gel eleetrophoresis of soluble proteins for studies of baeterial floras. Appl Environ Mierobiol 1980: 39: 900-907. 14. Murphy TE, Bartos LC, Compagnari AA, Nelson MB, Apieella MA. Antigenic characterization of the P6 protein of non-typable Haemophilus influenzae. Infeet Immun 1986: 54: 774-779. 15. Okuda K, Kato T. Hemagglutinating activity of lipopolysaccharides from subgingival plaque baeteria. Infect Immun 1987: 55: 3192-3196. 16. Olsen I. Rosseland SK. Thorsrud AK, Jellum E. Differentiation between Haemophiius paraphrophilus. H. aphrophiius. H. influctizae. ActinobaeiUus actinotnycetetneotnitans. Pasleweila multocida. P. haetnolytica and P. ureae by high resolution, two-dimensional protein eleetrophoresis. Electrophoresis 1987: 8: 532-535. 17. Olsen I, Caugant DA. Genetic diversity and relationships among strains of A. actinotnycetetneomilans, H. aphrophiius and H. paraphrophiius. J Dent Res 1988: 67: 395. 18. Paster B, Dewhurst E, Olsen I. The phylogeny of Aetinobaeillus. Haetnophilus

19.

20.

21.

22.

23.

24.

25.

and Pasteurella by 16S rRNA sequeneing. J Dent Res 1988: 67: 395. Pohl S, Bertsehinger HU, Erederiksen 'W, Mannheim W. Transfer of Haemophilus pleuropnewnoniae and the Pasteurella i\aemoiytica-\\k

Outer membrane proteins of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus studied by SDS-PAGE and immunoblotting.

This investigation characterized and compared outer membrane proteins (OMP) of the closely related Actinobacillus actinomycetemcomitans and Haemophilu...
7MB Sizes 0 Downloads 0 Views