interaction of spirochetes with the host

(~) I~,:~TII'UT PASTEUR/ELSEVIER Res. MicrobioL Paris 1992 1992, 143, 629-639

Interaction of spirochetes with the host

P. H i n d e r s s o n (1) (*), D . T h o m a s (2) (*) (**), L. S t a m m (3), C. P e n n (4), S. Norr is (5) and L .A . Joens (6)

(0 Institute of Medical Microbiology, University of Copenhagen, Copenhagen (Denmark), (2) Bowman Gray School of Medicine, Winston-Salem, NC (USA),

¢~) The School of Public Health, Department of Epidemiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC (USA),

(4) University of Birmingham, School of Biological Sciences, Birmingham (UK), (s) Medical school, Department of Pathology and Laboratory Medicine, The University of Texas,

Health Science Center at Housto,',, Houston TX (USA), (6) Department of Veterinary Science, Universily of Arizona, Tucson, AZ (USA)

SUMMARY

The success of an invading organism must depend on several cytoplasmic, surface- associated and secreted factors. The technical difficulties in handling pathogenic spirochetes like Treponema pallidum and Borrelia burgdorferi have made it diff icult to define specific factors involved in entry and long-term survival. The problem of defining virulence factors has been attacked by several strategies: T. pallidum secretes a num- ber of immunogenic low molecular mass proteins. The most ~edominant are of molecular w6ight 15.5 and 22 k~a. Pmiiniinary data suggest that ~,ntibcdiss against these proteins induce protective immunity in rabbits experimentally infected with T. pallidum. Many 9o*cPti~lhy impo ten t st~rface-associated ar~tigen.~ o~ T. p~3~t~.dL, m h~ve now been cloned and charactezized. Two ~f these, TpD and TpE, are lipoproteins which exhibit characteristic size heterogeneity. The apparent molecular weight of TpE from T. pallidum and T. pertenue are difft, rent. The clinical symptoms in ~yphilis and yaws are very different. but sequence analys;s of TpE has shown that the TpE proteins are indeed very simi!er in th'. t'.-Jo strains. This observation make.~ it unlikely that heterogeneity of TpE can account for the different clinical symptoms of syphii~s and yaws. Sequence data for another newly sequenced surface-associated antigen of T. pallidum (molecular we:ght 41 kDa) "ndicate that this protein is involved in g~ucose transport and chemotaxis/motil ity.

Intracellular factors like the molecular chaperonin GroEL have been documented both in treponemes and borreliae. This stress protein is involved in cellular repair processes and folding/assembly of protein subunits. Indirect evidence suggests that GroEL affects the abil ity of spirochetes to survive in the stressful environment of the infected host. Several lines of evidence suggest that the Osp proteins of Borr~lia are important for host/parasite interaction. Further support for this idea has come from studie~ of ~ series of monoclonal antibodies aqainst OspA. A monoclonal antibody =.~i0~=i GspA (gB3D) is able to block at tachment of b'. burgdorferi to a cell monolayer. BorreIia loses infectivity after several passages in vitro The !oss of pathogenicity is associated wi th loss of specific plasmids and proteins. One of the low-passage-associated proteins (Lap30) has been cloned and sequenced. Lap30 is a lipoprotein encoded by a ~8-kb plasmid, not present in high passage B. burgdorferi.

(*) Chairpersons and a',:thors of the present summary of session, in collaboration wir~ i~i~o~.ti=lg p,t.ticipams at the "Molecular Biology of Spirochetes" meeting, October 1991, Annecy, France.

(**) Present affiliation: Department of Periodontics, The University of Texas Health Science Cemer at San Antonio, San Antonio. TX (USA).

630 P. HINDERSSON E T AL.

Aberrant immunological ~ocesses induced by the Iipopolysaccharide component of Treponema hyodysenteriae could explain the dramatic intestinal lesions in swine dysenteriae. But analysis by TLC reveals that the LPS Gf this treponeme is different from clas- sical Salmonella LPS. LPS-like substances from pathogenic (7". hyodysenteriae) and non-pathogenic (T. innocens) are different, giving preliminary evidence that the structure of LPS like substances is correlated to pathogenicity. Only recently have we begun to analyse and characterize spirochetal virulence factors.

Further studies of protein factors end components of the spirochetal cell wall will probably reveal how spiroch~,'.as survive in the host and which components of the spirochete are involved in tissue damage and aberrant non-productive immunological processes.

Key-woKJs: Spirochete, Virulence; Host defences, Chaperonins, LPS, lipoproteins, Pro~ective immunity.

Pathogenic spi; ochetes |ike Treponema pallidum and Borrelia burg ~:jrfer are able to survive for a long time in tile hostile environment of the ~lost. However, very little is known about the host/parasi te interaction which makes it possible for the~c spirochetes to penetrate host barriers and maintair~ residency in the hosts. The success of an invading spirochete must be due ~o the complex interaction }~ctween several cytoplasmic, surface-associated extracellular factors. Attempts to identify important key components in these compartments is ongoing in seveia! I~.borato- ties. Important new contributions in this fie~d ~i!! be reviewed here.

ExtraeeUular antigens of 7". pallidum

Lola Stamm and coworkers have idenfi;ie,:~ .,cveral low molecular mass, pathogen-specific -". ~:ullidum proteins that are localized extracellutarly, I~ut ~mder certain conditions are found in association with the treponemal cell surface. 7". pallidum cells f.-csbly extracted from infected rabbi~ testes were intrinsically labelled with 35S-methionine (Stamm and Bassford, 1985). Following removal of the treponemes by cen- trifugation, the low molecular mass proteins were recovered from the medium by precipitation with triehloroacetic acid. The low molecular mass protein~: v, cre present in very small quantities and could only be visualized on SDS-polyacrylamide gels by fluorog- raph~ (Stature and Bassford, 1985). None of the low molecular mass proteins are identical to the TritonX-114 extractable proteins described by Rudolf e: aL (1988) or the 24-kDa protein of Hsu et al. (1988).

The lgG response of experimentally infected rabbits and human patients in various stages of syphilis

to the low molecular mass proteins was examined by radioimmunoprecipitation. IgG present in the majority of the human sera taken beyond the primary stage of syphilis precipitated most or all of the low molecular mass proteins (fig. 1). Interestingly, two proteins of 22 kDa and 15.5 kDa were the first low molecular mass proteins precipitated by rabbit sera taken early during infection. Polyclonal rabbit an- tisera prepared against the low molecular mass proteins not only reacted with the 22-kDa and 15.5-kDa proteins, but did show some reactivity with the immunogenic 47-kDa (TpS) and 34-kDa (TpD) 7". pal- lidurn lipoproteins (Norris et aL, 1987) presumably due to cellular contamination.

To aid purification of the low molecular mass proteins, Stamm and co~orkers radiolabelled T. pall# dum cells with sSS-methionine in se rum-f ree medium. These cells labelled more efficiently than the cells labelled in the presence of rabbit serum, due to the lack of competing cold methionine. Cell-free supernates from treponemes labelled for 2 h in the absence or in the presence of serum were compared. Only small amounts of the low molecular mass proteins were observed in the supernate from cells labelled in the absence of serum. The addition of rabbit serum albumin to the treponemal cells resulted in the normal complement of low moiecular mass proteins in the supernate. Thus, tile majority of the low molecular mass proteins remain associated with the treponemal cell surface in the absence of serum, but are eluted in the presence of serum. Further studies confirmed that the low n~olecular mass pro- reins present on ceils incubated in the absence of serum are cell-surface-exposed based on their accessibility to proteinase K. In pulse-chase experiments with treponemes labelled in vitro for I h, Stature and coworkers demonstrated that the 22-kDa

LPSLS - itpopolys~ccharide-iike substance, i T[.C = thin-layer chromatography.

I N T E R A C T I O N OF SPIROCHETES W I T H THE H O S T 631

A N P P S S S L L T T Y Y

21 .5 - " -

14 .4- "-": ~ ~

Fig. 1. P,r.d~oimmunoprecipitation of 7". pallidum low molecular mass proteins with various human syphilitic or yaws sera.

lmmunoprecipitations were performed and analysed by SDS-PAGE and fiuorography. Lane A : 3SS-methionine low molecular mass proteins. Precipitates obtained with pooled normal human sera (lane NL primary syphilitic sera (lanes P), secondary (l~,~es $), latent (lanes L), tertiary (lanes T) or human yaws sera (lane Y). The last lane contains the precipitate obtained with serum from a rabbit that was hyperimmunized with the low molecular mass proteins. The arrows indicate the posi- tions of the 22-kDa and 15.5-kDa proteins. Molecular weight standards are in kDa. Only the relevant portion of the gel is show~_.

and 15.5-kDa proteins were slowly secreted from the cells into the medium supernate.

Finally, a preliminary vaccination experiment was conducted in rabbits using multiple injections of whole inactivated T. pallidum cells extracted in the presence of serum and then washed in phosphate- buffered saline or cells extracted in serum but not washed, Both rabbits developed a virtually identical lgG response to the T. p,41idum cellular protein antigens and to the low molecular mass protein antigens with the exception of the response to the 15.5-kDa protein. The rabbit that received the washed cells mounted a vigorous IgG response to the ;5.5-kDa protein and was completely protected against intradermal challenge with T. pallidum. In contrast, the second rabbit, ..v'.,os- igG respon.~e to the 15.5 l~Da was somewhat less pronoun:ed, was only partially protected. These results mcri: further investigation. A more detailed analysis of the role of the low molecular mas~ protein.; itl host immunity and treponemal pathogenesis will be facilitated by the cloning and sequencing of the genes encoding these proteins.

Surface-associated T. pallidum proteins

Spirochetes seem to have an abundance of lipidated protein antigens associated v, ith the cell surface which can be expected to be important for host/spirochete interaction. Although the roie of the lipids is not known at present, it can be assumed that the lipids function as an anchor which attaches the lipidate,:l protein to membranes.

Several of the lipid-containing T. pallidum proteins have been cloned and some have been se-

quenced. Two of these, TpD and TpE (van Embden et al., 1983 ; Hindersson et al., 1986) were discussed by Charles Penn. Both TpD and TpE are abundant highly immunogenic proteins recognized by syphilitic sera. They both exhibit a characteristic smear when analysed by SDS-PAGE or Western blotting (fig. 2). The reason for this apparent heterogeneity is not yet clear. The heterogeneity of TpD is also observed by crossed immunoelectrophoresis, where TpD can be separated into two fractions (Hindersson et al., 1986). Each ~f these fractions corresponds to the lipidated and nc n-lipidated form of the protein (Schouls et al., !989). TpD has a signal sequence characteristic of lipoproteins (Schouls et al., 1989) and Leo Schouls has shown experimentally that both TpD and TpE are lipidated (Schouls et al., 1991). Dick Strugnell in Melbourne has now sequenced the TpE gent, cloned in Charles Penn's laboratory, and the sequence shows that a typical signal sequence as found in TpD is also found in TpE. A preliminary sequence comparison by Leo Schouls suggests some homology in the DNA sequence, particularly in the 3' portion, between the TpD and TpE sequences. This might suggest that the two proteins have some functional homology, bu~ confirmation depends on sequence comparison yet to be performed. A 0rotein with similar electrophoretic properties to TpE is also found in other treponemes like Treponemapallidum subsp, pe;tenue (7". pertenue), the causative agent of yaws and in Treponema denticola. The apparent molecular weight of ]'pE seems to diffe; slightly between some isolates of T. pallidum subspecies (fig. 3). Since very few differences between ; . pallidum and T. pertenue are known which might account for the difference in the clinical picture between syphilis and yaws, the apparent differences in TpE prompted an examination of the T. pertenue TpE se-


Q

o

,i~ - , ~ ~ , ,~

Fig. 2. Two-dimensional PAGE of T. pallidum, silver-stained. Note, the characteristic smearing of TpD (**) and TpE (*), The acidic portion of the first dimen-

sion i: to tile right and the pH for TpD is approximately 5. extending to approximately 7.5 at the left-ilaad-side of the gel.

quence, The TpE gene of T. pertenue (Gauthier strain) was therefore isolated by PCR based on the sequence from the Nichols strain of T. pallidum, by Lesly Tag in BirmingEam. The only difference in the TpE amino acid sequence of these organisms was a deletion of three codons in T. pertenue TpE and two amino acid substitutions, one of them conservative, resulting f-orn single base changes. This cannot account for the differences in apparent molecular weight obtained by SDS-PAGE/Western blotting. The smearing of TpD is abolished by removal of the cysteine group which serves as an acylation site (Schouls et al., 1991). But electrophoretic conditions llke variation in salt concentration can also influence the degree o f smear. Other well documented lipoproteins do no! exhibit this characteristic smear and therefore other posttranslational modifications

might account for the odd electrophoretic behaviour of TpD and TpE.

The 414"Da antigen of T. pallidum is a very abundant protein cloned and expressed in E. coil in Charles Penn 's laboratory• Detergent extractions shows that the 41-kDa antigens when expressed in E. coil, is membrane-associated and sarcosyl- insoluble• Sucrose gradient fractionations of recombinant E. coil expressing this protein indicate that it is associated with the outer membrane. In 7". paili- dum, the reducing agent mercaptoethanol seems to enhance the association of the 41-kDa antigen with a TritonX-100 soluble fraction which probably contains the outer membrane, periplasmic and possibly also cytoplasmic membrane components. Preliminary sequence data show that the 41-kDa antigen has a deduced typical hydrophobic signal sequence which

I N T E R A C T I O N O F S P I R O C H E T E S W I T H T H E t t O S T 633

further suggests that the protein is surface-associated. Homology searches with the 41-kDa amino acid sequence shows an interesting homology with the so- called MglB protein of E. eoli (Scholle et al., 1987), which is a periplasmic protein involved in glucose transport and in chemotaxis/motil i ty. Presumably, chemotaxis/motility is very important for spirochetes and the possible involvement of the 41-kDa antigen in chemotaxis/motility is of potential interest for un- derstanding this aspect of the host/parasite interaction.

G H N

m - - N

w m

m

Fig. 3. SDS-PAGE analysis of starins Gauthier (G) and Haiti B (H) of T. pallidum subsp, pertenue, and Starin Nichols (N) of subsp, pallidum. Note the apparent molecular ~eight difference in the TpE pioteir, (bu~) bet~ecn

strains Gauthier and Niche,Is.

S ~ i r o c h e t a l c h a p e r o n i n s

Also, intraceilular proteins can be expected to be important for long-term survival of pathogenic spirochetes in the host. Stress proteins are induced in some bacteria in response to heat shock and other forms of cellular stress like reactive oxygen radicals (Christman et al., 1985; vanBogelen et al., 1987; Buchmeier and Heffron, 1990). Stress proteins pro- tect the bacteria against the damage caused by such bactericidal components. One of the stress proteins which has attracted a lot of interest recently is the GroEL protein. The GroEL protein is structurally conserved and the expression of this protein has been documented in many different bacteria (Hindersson et al., 1984). A eukaryotic analogue has been found in mitochondria and chloroplasts (Jindal et aL, 1989; Hemmingsen et al., 1988) and it is now believed that GroEL is present in all living ceils. Although GroEL expression is upreguiated in response to stress, it has an important function under non-stress conditions as well (Fayet et aL, 1989). The biological function of GroEL in the celi is to facilitate protein folding and assembly of macromolecular protein complexes (Hemmingsen et al., 1988). The term "molecular chaperonins" has been used by Ellis to describe this new class of cellular enzymes (Ellis et al., i989). Another, smaller protein, "GroES" , collaborates and interacts with GroEL (Tilly and Georgopoulos, i982). The GroEL protein of B. burgdorferi, T. pallidum and Treponema phagedenis has been known for many years as the common antigen (Hindersson et eL, !984; Hansen et al., !988). The protein was designated common antigen because of its extreme cross-reactivity with corresponding proteins in other microorganism: (H¢iby, 1975). Immunochemical data as well as sequence data have now firmly estab- lished that common antigen is equivalent to GroEL (H!ndersson et al., 1987; Thole et al., 1988; Sl~,nafc~t .7 al., 1991). Alignment of GroEL sequences from different organisms shows that the highly conserved regions are placed in lumps with less cc, nserved regions in between (Shanafelt et al., 1991 ; Hindersson et al., 1990). Both T. pallidum and B. burgdorferi GroEL terminate in a characteristic Gly Gly Met rep:at. This structure is found in most GroEL except m the GroEL of chloroplasts~

The native GroEL protein is assembled into a macromolecular complex of fourteen 60-kDa subunits (fig. 4). Two rings of seven 60-kDa subunits are stacked and contribute to the characteristic donut- like appearance of the complex observed by electron microscopy. Spirochetal GroEL shows the same macromolecular organization. This has been inlt:rred from gel filtration studies (Petersen et al., 1982; Houston et aL, 1990) and from recent elec'ron microscopy studies performed by Houston et al. (Houston et al., 1990) (figs. 4 and 5).

634 P. H I N D E R S S O N E T AL.

Fig. 4. Model of the T. pallidum GroEL complex consist- ing of two stacked 7-mers.

The model is based on interpretation of the electron micrographs shown in figure 5.

It can be expected that the spirochetal GroEL is important for survival of spirochetes in the human host and for B. burgdorferi during transition from the vector at low temperature to higher temperature in the warm-blooded animal, but this has not yet been documented experimentally. Despite intensive efforts (Stamm et aL, 1991 ; Hindersson and Norris, unpub- lished), it has not been possible to demonstrate heat induction of GroEL in B. burgdorferi and T. pallidum. Spirochetes-like Leptospira interrogans and T. phagedenis show normal heat !nduction of GroEL and DnaK. B. burgdorferi and T. pallidum probably express stress proteins at high levels under a wide range of conditions. In many bacteria, GroES and GroEL genes are organized as a coregulated operon controlled by heat shock promoters (Hinder:;son et al., 1990). The two sequenced GroEL genes of T. pallidum and B. burgdorferi are not preceded by a GroES gene and no typical heat shock expression signals can be identified immediately upstream (within 1000 bases) of the GroEL genes, it may be that GroEL expression is regulated quite differently in pathogenic spirochetes. Another possibility is that the GroEL gene is part of a larger operon. But the DNA sequences upstream of B. burgdorferi and 7". pall# dum GroEL share no homology. A detailed analysis of the transcribed messenger RNA will be needed to make a proper identification of the spirochetal expression signals.

• ,{

: . . . - . ' ~ .'.:-'~

Fig. 5. Electron micrograph of purified T. pallidum GroEL comglexes (Houston et al., 1990). (a) and (b) shows the Grot-k seen from the top. (c) and (d) are side views (origi-

nal pictures kindly provided by S. Norris).

Outer membrane antigens of B. burgdorferi

The outer membrane of spirochetes can be expected to contain antigens of importance for the host/parasite interaction. Denee Thomas and cowor- kels developed a panel of monoclonal antibodies against outer membrane enriched fractions of B. burgdorferi. OspA and OspB are well documented, abundant , highly immunogenic proteins present in this cell fraction (Barbmtr and Schrumpf, 1986). Not quite unexpectedly, many of the hybridoma cell lines derived by using outer-membrane-enriched material for immunization were directed against OspA and OspB. In competition experiments using peroxidase-labelled monoclonal antibodies, the panel of antibodies (table l) could be divided into five different competition groups for each Osp protein. Some of the monoclonal antibodies against OspA strowed a broad reactivity with a var.;.ety of different isolates derived from a wide range of locations world- wide. Other antibodies were very specific for Ameri- can isolates. One of the OspA monoclonal antibodies (9B3D) inhibited adherence of B. burgdorferi to en- dothelial cell monolayers. A dose-response curve for the inhibition o f adherence of B. b,trgdorferi to cell monolayers by Fab fragments of monoclonal antibody 9B3D is shown in figure 6. Following the cell association assay, cell-surface bound bacteria could

I N T E R A C T I O N OF SPIROCHETES WITH THE H O S T 635

Table !. Isotype and campetition grouping of B. burgdorferi HBI9 monoclonals.

Monoclonal Competition antibody lsotype group

BPIE10 IgG! BPIEIO 6A4B (*) IgG2B 72B lgGl OspA I 9A6C igG 1 9B3A IgG 1 9BIC lgGl OspA 2 9B2B lgGl OspA 3 9B3D lgGl OspA 4 6B2C IgG2b OspA 5 82C lgG 1 83C (') IgG2b OspB 1 84B lgG l 84C lgG2b OspB 2 6B2B IgG3 OspB 3 3A3 igGl OspB 4 3A5 I'l IgGl OspB 5 3D3 (*) IgGl 3A2 lgGl 35 kDa 1 3B2 IgGl 35 kDa 2 3C2 IgG2b 35 kDa 3 3D2 lgGl 35 kDa 4 9A5D IgG2b 35 kDa 5

(*) Hybridoma selected to generate ascitic fluid from groups containing more than one hybridoma.

be observed by immunofluorescence. Bacterial attachment was demonstrated to be greatly reduced by Fab fragments of 9B3D. The anti-OspA antibody 9B2B is of the same isotype as 9B3D, but does not inhibit cell association of the bacteria. To more con- chtsively show that 9B3D was inhibiting initial adherence of spirochetes to the cell monolayer, the attachment experiments were also performed at 4°C. At this temperature, invasion and penetration of the monolayers is inhibited and the bacteria can only at- tach. Cell association at 4°C was inhibited 84 % by antibody 9B3D compared to untreated spirochetes at the same temperature. Together with the encourag- ing immunization studies of Fikrig and eoworkers (1990) using purified OspA, these result indicate a central role of the " O s p " proteins, especially OspA, in pathogenesis.

L o w passage-associated proteins o f 11. burgdorferi

B. burgdorferi, like Neisserta gonorr~oeae, loses its infectivity and virulence following prolonged in

Oo,

| ~ 6o.

z~ ~ 40 - ¢jr~

J CONCENTRATION OF 9B3D (p.g/ml)

Fig. 6. lnhibiiion of binding of B. burgdorferi to endotheli- al cell monolayer by murine anti-OspA monoclonal anti-

body 9B3D.

vitro culture. For example, Scbwan el al. (1988) showed that the Sh-2-82 strain was infectious to white-footed mice up to 10 in vitro passages, but was not infectious in subsequent cultures. Loss of virulence coincides with changes in the plasmid content and protein profile~ of B. burgdorferi isolates. By comparing low-passage, virulent strains with their isogenic high-passage, non-infectious counterparts, it may be possible to identify proteins (and, at least in some cases, their encoding plasmids) which are re- quired for infectivity and virulence. These virulence factors are most likely a small subset of the proteins expressed by low-passage strains, but are absent or underexpressed in high-passage strains; this larger group could be called low-passage-associated proteins (Laps).

To identify Laps, and hence, possible B. burgdorferi virulence factt~:s, Norris and coworkers compared the two-dimensional gel electrophoresis (2DGE) profiles of virulent, low-passage B31 strain (< 10 in vitro passages) and avirulent, high passage B31 strain (cultured continuously in vitro for several years) (S.J. Norris, C.J. Carter, J.K. Arnett, and A.G. Barbour, manuscript in preparation). Non- equilibrium pH gradient electrophoresis (O'Farrel et al., 1977) was used in the first dimension because it allows the resolution of basic pro~,eins (such as OspA and OspB) as well as acidic proteins. Polypeptides with apparent molecular weights (Mr) of 35, 30, 24, and 20 kDa were present in low-passage B31 but were


absent or underexpressed in high-passage B31. The 30,000 Mr polypeptlde (Lap30) is an abundant protein in low-passage B31 and other B. burgdorferi low- passage strains and was thus selected for further characterization.

Lap30 was found to be a lipoprotein encoded by a plasmid with an apparent size of 38 kb; this plasmid is not present in the high-passage B31 strain. The gene was identified and cloned using an oligonucleo- tide sequence derl,ed from amino acid sequences of Lap30 CNBr cleavage fragments. The nucleotidc sequence revealed a typical prokaryotic gene structure, except that 7 direct repeats of an 18-base sequence were located immediately upstream of the open read- ing frame; this repeating sequence included both - 35 and - 10 consensus transcription start signals. The deduced amino acid sequence possessed a 19-amino acid hydrophobic leader sequence followed by a consensus signal peptidase II cleavage site. The lipoprotein nature of Lap30 was confirmed by 3H- palmitate labelling of the low-passage B31 strain; specific incorporation of labelled palmitate into Lap30 was revealed by 2DGE and autofluorography. Beyond the leader sequence, the deduced amino acid sequence is hydrophilic; like OspA, OspB and other lipoproteins, : ap30 appears to be anchored by its lipid tail into cellular membranes. The Lap30 sequence lacks significant homology with the B. burgdorferi protein geo:=s sequenced to date or with other GcnBank entries. Future studies will include deter- mination of the structural location, immunogenici- ty and possible pathogenetic roles of Lap30 and other low-passage-associated proteins.

Lipooligosaccharide-like substances o f 7". hyodvsen- teriae

Treponema hyodysenteriae, another member o f the order spirochetales, is a porcine pathogen that was isolated in 1971 from pigs afflicted with swine dysentery. Acute stages of the disease are characterized by mucohaemorrhagic diarrhoea followed by progressive dehydration. The causative organism is highly infectious and morbidity rates of 70 % with subsequent morta!ity rates of 30 % are commonly seen in herds. Pigs which recover often become asymptomatic carriers, al though symptoms may reoccur if the animal becomes stressed. The treponemes causing the symptoms are considered non- invasive, since they colonize the intestinal crypts without penetration of the lamina propria. The specific factors involved in pathogenesis are poorly characterized. Toxins secreted by T. hyodysenteriae could play a role in pathogenesis, but supernatants from cultures of the organism do not show the presence of toxins when assayed in live animals or in tissue cultures. T. hyodysenteriae produces an ac-

tive haemolys in which appears to be non- immunogenic. Convalescent sera does not contain antibodies against this haemolysin and rabbits and mice immunized with either crude or purified haemolysin fail to develop antibodies against it. It is assumed that this haemolysin plays a key role in the pathogenicity of T. hyodysenteriae. Research on the role of haemolysin in the pathogenicity of T. hyodysenteriae is an active research area ill several laboratories.

The action of haemolysin can explain many of the observed pathological findings. However, other cel lular constituents could prove to be equally important causes of the dramatic symptoms observed in porcine swine dysenteriae. These components could be part o f the cell wall. Like other Gram-negative bacteriae, 7". hyodysenteriae possesses a lipopolysaccharide component in its outer membrane. In 1982 and 1983, Nue~sen et al. demonstrated that purified extracts o f T. hyodysenteriae had a significant biological activity when aseayed by cell culture on live animal inoculation (Nucssen et al., 1982, 1983). The material was shown to increase macrophage receptor- mediated phagocytosis in addition to demonstrat ing cytotoxicity, mitogenicity, chemotaxic attraction for porcine leukocytes and, when combined with ac- tinomycin D, mut ine lethality. In spite o f its similar biological activity toward LPS derived from En- terobacteriaceae, questions about its nature arose when the LPS were compared by SDS-PAGE. The spirochetal LPS did not produce the "stepladder ef- fect" and did not show an electrophoretic band that comigrated with the evolutionary conserved core region of LPS from other Gram-negative bacteria. The biologically active component o f LPS resides in the core region, like lipid A.

Joens and coworkers in Tucson, Arizona ad- dressed the question as to whether T. hyodysenteriae LPS contains an Enterobacteriaceae-like lipid A molecule and whether this component is capable of inducing immune response (la) molecules on the surface of macrophages.

LPS from pathogenic strains (B234, B204, B169, A1, 6933, 8044 and Ack 300/8, serotypes 1, 2, 3, 4, 5, 6 and 7, respectively) of T. hyodysenteriae, attenuated T. hyodysenteriae (T-22 serotype 1, > 131 passages in culture) and Treponema innocens (strain B1555a) was isolated by standard techniques (West- phal et al., 1952; Mclutirc et al., 1967) and analy~cd by TLC. Rather vigorous conditions (1 M I--ICI at 100 C) were necessary to obtain complete hydroly- sis of the LPS. Strain B204 LPS produced three spots of Rf 0.5, 0.56 and 0.61. Lipophilic material from serotypes 1 and 2 seemed te be identical. Serotypes 1, 2, 3, 4, 6, 7 and T. innocens B1555a all shared spots with R f = 0 . 5 and 0.56. Serotypes 3 and 5 possessed a spot of 0.66 instead of the Rf=0.61 spot. Serotype 5 lacked a spot with Rf= 0.5, but had two

INTERACTION OF SPIROCHETES WITH THE HOST 637

spots at 0.56 and 0.66. Attenuated serotype 1 (T-22), serotype 2 ( > 13 ! passages), serotypes 6 and 7 (non- pathogens) and T. innocens (B1555a) all had a common spot with an Rf=0 .18 . T. hyodysenteriae and T. innocens did not share common spf~ts with lipid A.

These data indicate that the above spirochetes have a related LPS structure, but gross differences exist between lipid A of the spirochetes and Salmonel- la. Very limited comparative data are available for other spirochetes. Beck et el. (1985) described biologically active lipopolysaccharide from B. burgdorferi. When this component was scrutmized, another group of authors concluded that, based on chemical and structural criteria, the molecule was not a true lipopolTsaccharide (Takayama et al., 1987). In addition, the Borrelia preparation lacked some of the biological properties ~ssociated with both lipid A and LPS. However, variations in certain biological ac- tivities of lipid A extracted from various sources is commonly observed. The occurrence of a spot (Rf = 0.18) coinciding with avirulent spirochetes is an interesting finding which might reflect the biological activity of the native LPS.

Joens and ccworkers concluded that the chemical and structural nature of the T. hyodysenteriae LPS is so different from Enterobacteriaceae LPS that it seems more appropriate to call these components "lipopolysaccharide-like sub!,tances" (LPSLS).

In addition, Joens and eoworkers found that the expression of la k molecules on murine macrophages was significantly induced by Salmonella LPS and T. hyodysenteriae strains LPSLS (fig. 7). However,

°I= 8 r~ C3H/HeJ

5~ 6 ~

~,e + 3 2 1

S.typhimur. S.minn. 204 1555a Mclntlm Saline LPS LPS LPSLS LPSLS LPSLS Fig. 7. Induction of la molecules on murine peritoneal macrophages (quantitated by the method of Ziegler (1984).

The designations 204 and 1555a, correspond to the T. hyodysenteriae strains described in the text. LPSLS prepared by the method of Mclntire is marked Mclntire. The variation of each value is indicated by bars. S. typhimur. and S. ntinn, designate Sahnonella typhimurium and Sahnonella minnesota, respectively.

the technique of LPS preparation influenced the biological activity. Only LPS prepared by the West- phal method (Westphal et al., 1952) proved biologically active.

The intestinal lesions of swine dysentery are es- sentially inflammatory in nature, with signs of coagulative necrosis. An innapropiate/overactive im- munologially mediated event involving spirochetal LPSLS could explain the clinical and pathological findings. As powerful modulators of the immune sys- tem, lipopolysaccharides or lipopolysaccharide-like substances may play an integral role in me disease process.

Hopefully, comparative data for other important pathogenic spirochetes like T. p~!lidum and a closer characterization of the LPSLS constituents will soon become available.

Concluding remarks

Although many groups attempt to understand the host/spirochete interaction at the molecular level, the technical problems associated with handling pathogenic spirochetes have not yet made it possible to at- tain this goal. Only recently have we begun to understand the response of the host to spirochete infection and identify spirochete molecules of potential importance for host /paras i te interaction. Sequence charactexization of important candidate proteins and putative virulence-associated plasmid- encoded proteins will be a valuable tool for ex- trapolating functional details and making a rational design for future experiments. Antigens which can induce protective immunity like B. burgdorferi OspA and possibly also the low molecular mass 7". palli- dun+ proteins described by L. Stature are prime can- didates for a more detailed investigation of their role in pathogenesis.

Interactions des spirochetes avec I'h6te

Le pouvoir invasif d 'un organisme d6pend de plusieurs facteurs cytoplasmiques, associ~s ~t la surface et secr6t6s. Les difficult6s techniques li6es ~. la mani- pulation de spirochetes pathog;enes comme Z'epe- nema pallidum et Borrelia burgdorferi ont rendu difficile la reconnaissance des facteurs sp6cifiques associ6s h l'entrde et /t la survie ~ long terme dans l 'h6te de ces bact6ries. Plusieurs strat6gies ont per- mis d'aborder !e probl~me de la raise en 6vidence des facteurs de virulence : T. pallidum s6cr~te un grand nombre de prot~ines immunog6nes de petit poids mol6culaire. Les plus abondantes sont de poids mol6- culaire 15,5 et 22 kDa. Des r6sultats pr61iminaires

638 P. H I N D E R S S O N E T AL.

sugg~rent que les anticorps dirig6s contre ces prot6i- nes induisent une immunit6 protective chez des lapins infect6s cxp6rimentalement avec T. pallidum. Les g6nes sp6cifiant plusieurs antig~nes potentiellement importants associ6s/~ la surface de T. pallidum ont 6t6 clon6s et caract6ris6s. Deux de ces antig6nes, TpD et TpE, sont des lipoprot6ines, de taille h6t6rog6ne caractgristique. Les .;ympt6mes de la syphilis et du pain sont diff6rents f/ , si l 'analyse de la s6quence de TpE a montr6 que les prot6ines TpE 6taient vraiment tr~s similaires chez 7 pallidum et T. pertenue, leurs poids mol6culaires respectifs sont diff6rents. Cette observation montre qu'il est peu probable que l'hgt6- rog6neit6 des protgines TpE puisse &re responsable de la diffgrence observ6e entre les sympt6mes clini- ques de la syphilig et du pain. Les s6quences r6cem- ment obtenues pour un autre antig6ne associ6 & la surface (poids mol6culaire 41 kDa) indiquent que cette protgine est impliqu6e dans le transport du glucose et dans la chimiotaxie et la mobilit6.

Des facteurs intracellulaires comme les chapero- nines, en particulier GroEL, ont 6t6 &udi6s chez T. pallidum et B. burgdorferi. Cette prot6ine de stress est impliqu~e clans les processus de r6paration cellulaire et l 'assemblage des sous-unit6s prot6iques. Des arguments indirects sugg~rent que GroEL modi- fie la capacit6 des spiroch6tes/l survivre dans I'envi- ronnement hostile de l 'h6te infect6. Plusieurs arguments, tels les r6sultats d'6tudes effectu6es avec une s6rie d 'anticorps monoclonaux contre O~pA, sugg~rent que !es prot6ines Osp de Borrelia sont importantes pour les interactions h6te/parasite. Un anticorps monoclonal (973D) dirig6 contre OspA est capable de bloquer l 'at tachement de B. burgdorferi ~t une monocouche de cellules. Borrelia perd son caract6re infectieux apr~s quelques passages in vitro. La perte de ce caract/~re est associ6e h la perte de pro- t6ines et de plasmides sp6cifiques. Le g~ne sp6cifiant une des prot6ines perdue apr~s passage in vitro a 6t6 clon6 et sa s6quence d6termin6e. I1 s 'agit d 'une lipo- prot6ine (Lap30) sp6cifi6e par un plasmide de 38 kb, qui n 'est plus retrouv6 chez B. burgdorferi apr~s de nombreuses subcultures.

Des processus immunologiquement aberrants induits par le lipopolyoside (LPS) de Treponema hyodysenteriae pourraient expliquer les 16sions intes- tinales dramatiques de la dysenteric du porc. Cepen- dam I'analyse par chromatographic sur couche mince r6v/~le que le LPS de ce tr6pon~me e~t diff'.rent du LPS classique de Salmonella. Les substances de type LPS de 7: hyodysenteriae (pathog~ne) et de Trepo- nema innocens (non-pathog~ne) sont diff&er.tes. Ces r6sultats sont en faveur de l 'hypoth~se que ces substances de type LPS sont corrd6es ~ la pathog~nicit6. L'analyse et la caract6risation des facteurs de virulence des spiroch6tes a commenc6 tr6s r6cemment. Des &udes plus d&aill~es des constituants de la paroi des spirochetes permettront probablement de r6v~-

ler comment ces bact6ries survivent chez l 'h6te, et lesquels parmi ces constituants sont impliqu6s darts le dommage tissulaire et les processus immunologi- ques aberrants.

Mots-clds: Spiroch6te, Virulence; D6fenses de l 'h6te, Lipoprot6ine, LPS, Chaperonine, lmmunit6 protective.

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interaction of spirochetes with the host

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