(Received for publication, June 6, 1995; and in revised form, February 14, 1996)
From the
Heparin is a functional and structural analog of the Chlamydia trachomatis heparan sulfate-like attachment ligand
that mediates infectivity by bridging chlamydiae to eukaryotic cells.
The binding of heparin to the Chlamydia organism's
surface was characterized by a direct binding assay. Although for two C. trachomatis biovars the binding by heparin was saturable,
trachoma biovar organisms bound twice the amount of heparin than
lymphogranuloma venereum biovar organisms. To probe the structural
nature of the heparan sulfate-like ligand interactions, a range of
heparin-derived oligosaccharides and sulfation-modified species of
heparin were compared for their ability to compete with
[H]heparin for binding to chlamydial organisms
and for inhibition of chlamydial attachment and infection of eukaryotic
host cells. The assays revealed that a decasaccharide was the minimal
chain length required to effectively bind C. trachomatis organisms, compete with the host cell receptor and rescue
infectivity. In addition, a moderately sulfated adhesin analog, N-desulfated, N-acetylated heparin, was able to
compete with chlamydial organisms for host cell receptors, whereas this
derivative could not compete with [
H]heparin for
binding to chlamydial organisms. These results indicate that the
specificity of the eukaryotic cell receptor and the chlamydial surface
acceptor differ in their fine-structure requirements of ligand binding,
and that the size and sulfation density of the heparan sulfate-like
ligand each contribute to its ability to bind and bridge chlamydiae to
eukaryotic cells.
A broad range of microbial pathogens that infect humans bind
heparan sulfate moieties of eukaryotic cell surface proteoglycans.
Heparan sulfate-containing proteoglycans on eukaryotic cells have been
shown to facilitate microbial adherence and/or cellular invasion for
human immunodeficiency virus(1) , herpes simplex
virus(2, 3) , cytomegalovirus(4, 5) ,
varicella zoster virus(6) , Bordetella
pertussis(7) , Leishmania
donovani(8, 9) , Trypanosoma
cruzi(10) , and Plasmodium circumsporozoites(11) . Unlike each of these other
microbial pathogens, it has recently been shown that Chlamydia
trachomatis attachment to, and subsequent infectivity of,
eukaryotic cells is dependent upon the presence of a heparan
sulfate-like ligand on the surface of the
organism(12, 13) . Treatment of C. trachomatis organisms with a specific heparan sulfate lyase, heparitinase,
abolishes chlamydial infectivity, yet exogenous heparin or heparan
sulfate rescues chlamydial attachment and infectivity for
heparitinase-digested organisms(13) . Because exogenous heparin
or heparan sulfate can restore attachment and rescue infectivity, it is
thought that the organism binds the heparan sulfate-like
glycosaminoglycan (GAG) ()by a specific surface acceptor
molecule. Characterization of the structural requirements of heparin
binding to microbial pathogens has significant implications for
understanding the basic biology of both the pathogen and the eukaryotic
host as well as for development of antimicrobial strategies based upon
the essential adhesion step of infectivity.
C. trachomatis is phylogenetically deeply separated from other eubacteria because
chlamydiae grow only within eukaryotic host cells and have a
developmental cycle(14) . The developmental cycle is
characterized by two forms of the organism: a metabolically active
intracellular form, and a metabolically inactive extracellular form,
called the elementary body (EB), that is capable of infecting mammalian
cells. There are two biovariants of C. trachomatis that cause
a spectrum of important diseases in humans(15) . The trachoma
biovar is responsible for most genital tract and ocular infections of
mucosal surfaces, whereas the lymphogranuloma venereum (LGV) biovar is
much more invasive and primarily grows in lymphoid tissue. It has been
shown for both biovars that a heparan sulfate-like ligand on the
surface of the C. trachomatis EB is essential for infection of
mammalian cells(12, 13) . It has been proposed that
the heparan sulfate-like adhesin is synthesized by
chlamydiae(13) , but the structure of the native heparan
sulfate-like ligand that decorates the chlamydial cell surface is not
known. The ligand can be produced and labeled with
[S]sulfate in infected cells incapable of GAG
synthesis(16) , the product is susceptible to heparitinase
digestion, but it is not bound by heparan sulfate-specific monoclonal
antibodies(13) . Although the two biovars are equally dependent
upon the heparan sulfate-like ligand for infectivity(12) , the
biovars differ in surface charge (17) and in their
susceptibility to heparin inhibition of attachment to host
cells(18) . The interaction of the heparan sulfate-like GAG
with a chlamydial EB acceptor has not been established, nor has a
specific GAG acceptor on chlamydiae been identified.
The major interactions between GAG and GAG-binding proteins involve negatively charged sulfates and carboxylates on GAG and positively charged residues of proteins(19) . Such an interaction is often expected to be relatively nonspecific in nature and of low affinity; however, type IV collagen contains three separate heparin binding domains, each of which has a distinct affinity(20) . Likewise, the coagulation inhibitor antithrombin III binds specifically to a defined pentasaccharide sequence within heparin(21, 22) . Thus, it is possible that there is a ligand-specific acceptor on the chlamydial cell surface that specifically binds the functional analog, heparin. Unfortunately, the diverse nature of heparin- or heparan sulfate-binding proteins that have been identified makes it difficult to predict structural or functional motifs that are conserved among this class of molecules (19) .
The analysis of GAG-protein interactions is complicated by the inherent complexity of heparin and heparan sulfate structure(23) . Three outstanding structural features of most GAG oligosaccharides are their 1) length; 2) high negative charge, due to the presence of carboxyl and sulfate groups; and 3) constituent carbohydrate composition(24) . The unbranched carbohydrate backbone of heparin or heparan sulfate consists of disaccharide repeats of hexuronic, D-glucuronic, or L-iduronic acids, and D-glucosamine units, joined by 1,4-glycosidic linkages. The variable location of N-acetyl, N-sulfate, and O-sulfate groups on these three units can give rise to at least 10 different monosaccharide building blocks that can be combined into a large number of different oligosaccharide sequences(24) . An entire GAG chain may contain approximately 100 disaccharide repeats. The large number of possible combinations of sulfation, backbone composition, and chain length complicates the structural and functional analysis of GAGs. Nevertheless, a better understanding of the nature of the heparan sulfate-like adhesin involved in chlamydial attachment and infection would not only contribute to defining a primary chlamydial virulence determinant but also would facilitate the identification, characterization, and isolation of the active chlamydial GAG ligand, GAG acceptor, and host cell receptor that are essential for productive infection of eukaryotic cells.
In this study, heparin and heparin derivatives were used as
analogs of the chlamydial heparan sulfate-like ligand to circumscribe
the structural requirements of binding to chlamydial EBs and for
mediating infectivity of eukaryotic cells. Heparin rather than heparan
sulfate was used as the comparative standard for these studies as it
appeared to be functionally equivalent in previous studies of
chlamydial attachment and infectivity(12, 13) , and
each of the GAG derivatives used was obtained from heparin. By a direct
binding assay it was shown that binding of exogenous
[H]heparin to EBs representing both C.
trachomatis biovars was saturable. Using derivatives of heparin,
productive interaction with the EB and the eukaryotic cell differed and
was dependent upon sulfate position, sulfation density, and
oligosaccharide length.
Figure 1:
Binding of
[H]heparin to C. trachomatis organisms.
Saturability of binding to C. trachomatis EBs representing the
LGV (serovar L2) biovar (closed circles) and the
trachoma (serovar B) biovar (open circles). Various
concentrations of [
H]heparin were mixed with C. trachomatis serovar L2 (2.7
10
EBs) or
serovar B (2.1
10
EBs) at 4 °C for 1 h. After
washing, the organism-associated radioactivity (counts/min) was
determined by scintillation counting. The data are the average of three
separate experiments.
To
examine the contributions made by sulfation of heparin to bind EBs, two
modified species of heparin, CDSNS and NDSNAc, were tested for their
ability to compete with [H]heparin for binding to C. trachomatis EBs. Unlike unlabeled heparin, the modified
species of heparin did not competitively inhibit
[
H]heparin binding to C. trachomatis organisms (data not shown). The failure of these modified heparin
derivatives to competitively inhibit the binding of
[
H]heparin to chlamydial organisms suggests the
binding affinity of the sulfation-modified heparin derivatives to C. trachomatis was low.
Figure 2:
Effect of the length of heparin saccharide
chain on inhibition of C. trachomatis attachment and
infection. Attachment inhibition (A) was assessed using S-labeled EBs in the presence of 500 µg/ml indicated
heparin oligosaccharides. The amount of radioactivity that was
cell-associated was determined by scintillation counting. Infectivity
neutralization (B) was assessed in the presence of 500
µg/ml of indicated heparin oligosaccharides during cell infection
and results are expressed as IFU per coverslip. Solid bars represent C. trachomatis serovar L2. Open bars represent C. trachomatis serovar B. Vertical lines indicate S.E. The 8-mer data in panel A was based on one
triplicate and two duplicate assessments from three
experiments.
Figure 3:
Effect of the length of heparin saccharide
chain on restoration of attachment and infectivity for
heparitinase-digested C. trachomatis. Attachment restoration (A) was assessed using S-labeled EBs and
infectivity restoration (B) was assessed using unlabeled C. trachomatis EBs. Solid bars represent C.
trachomatis serovar L2. Open bars represent C.
trachomatis serovar B. 25 µg/ml indicated heparin
oligosaccharide or heparin was added to aliquots of
heparitinase-digested EBs, incubated at 4 °C for 1 h, and washed
prior to inoculation of HeLa 229 cell monolayers. The amount of
radioactivity that was cell-associated was determined by scintillation
counting. Results from attachment assays are expressed as percentage of
untreated samples from the average of triplicate assessments; results
from infectivity assays are expressed as IFU/coverslip from the average
of duplicate assessments.
Figure 4:
Effect of modified sulfation of heparin on
chlamydial attachment and infection. Attachment (A) was
assessed using S-labeled EBs in the presence of 500
µg/ml of modified or unmodified heparin. Infectivity neutralization (B) is expressed as IFU/coverslip. Vertical lines indicate S.E.
Figure 5: Effect of modified sulfation of heparin on restoration of attachment and infectivity for heparitinase-digested C. trachomatis. Attachment restoration (A) and infectivity restoration (B) were assessed using heparitinase-digested EBs as described for Fig. 3. Results of attachment assays are expressed as percentage of untreated samples from the average of triplicate assessments. Results from infectivity assays are expressed as IFU/coverslip from the average of duplicate assessments. Solid bars represent C. trachomatis serovar L2. Open bars represent C. trachomatis serovar B.
Figure 6: Dextran sulfate restoration of infectivity for heparitinase-digested C. trachomatis. Infectivity inhibition was determined by incubating aliquots of C. trachomatis serovar L2 alone (Control) or in the presence of 500 µg/ml heparin or dextran sulfate. For the same experiment aliquots of C. trachomatis serovar L2 were digested with heparitinase (Heptn'ase) for 1 h, and 25 µg/ml heparin or 25 µg/ml dextran sulfate was then added to aliquots of heparitinase digested EBs and washed prior to inoculation of HeLa 229 cell monolayers. The results were expressed as IFU/coverslip from the average of duplicate assessments.
A trimolecular working model of chlamydial attachment has been proposed in which a Chlamydia-synthesized heparan sulfate-like ligand is bound by chlamydial acceptors and host cell receptors, thereby mediating chlamydial attachment and infectivity (13) . This model was tested by investigating the role of analogs of the heparan sulfate-like ligand for binding chlamydial EBs and mediating interactions with mammalian host cells. Heparin and heparin derivatives were used in direct binding assays to chlamydial organisms and in indirect competition assays for organism binding to host cells to independently probe the structural and functional requirements for binding the chlamydial GAG acceptor and the host cell receptor, respectively. In the direct binding assays, for EBs representing both C. trachomatis biovars, binding of heparin was saturable suggesting that heparin binding to EBs is specific. Although the kinetics of heparin binding to EBs was similar for the two biovars, trachoma biovar EBs bound twice the amount of heparin than LGV biovar EBs. These estimates could be confounded by the amount and avidity of bound natural ligand, and by estimates of the number of EBs used in these experiments. Nevertheless, these data suggest a significant difference in the native surface architecture for these biovariants. This conclusion is consistent with ion-exchange chromatography of native EBs demonstrating that the LGV biovar has a higher negative surface charge than trachoma biovar EBs(17) . If the difference in surface charge is attributable to the presence of the native heparan sulfate-like ligand, then the LGV biovar may have quantitatively more or more highly sulfated native ligand on its surface than the trachoma biovar.
In order to begin elucidation of the structural requirements of the heparan sulfate-like ligand as an adhesin, chemically modified species of heparin and homogeneously sized oligosaccharides obtained from depolymerized heparin were tested. To examine the length requirements of the heparan sulfate-like ligand for mediation of cell adhesion, oligosaccharides of defined length were used to compete with chlamydial organisms for binding host cells. Inhibition of attachment and neutralization of chlamydial infectivity were measured to estimate the ability of competing oligosaccharides to bind to the host cell receptor. As the size of oligosaccharide decreased, the ability to inhibit chlamydial attachment or infection also decreased, with heparin fragments smaller than octasaccharide having no effect on inhibition of attachment or infectivity. These results suggest the heparin molecules longer than decasaccharides were able to compete for binding to host cell receptors with the native chlamydial adhesin. The ability of these molecules to rescue chlamydial attachment and infectivity that was abolished by pretreatment of EBs with heparitinase, revealed that the decasaccharide or dodecasaccharide heparin oligosaccharides fall short of fully rescuing chlamydial infectivity despite increased molar concentrations of oligosaccharides. The lack of full restoration may indicate 1) the relatively poor binding of heparin oligosaccharides to the EB surface, 2) longer oligosaccharides are required to optimally bridge and interact simultaneously with two different proteins, or 3) other structural differences between the natural C. trachomatis heparan-sulfate-like ligand and heparin oligosaccharides.
Structurally heparin and heparan sulfate molecules consist of repeated disaccharide units that are sulfated differently. Each of these molecules can be subdivided into domains based on a combination of sulfation density and saccharide units. In general, heparin contains a higher proportion of sulfation and a higher L-iduronic acid/D-glucuronic acid ratio than heparan sulfate; however, heparan sulfate shares some highly sulfated regions that are similar to heparin(28) . It is possible that the different GAG-sulfation requirements observed for the host receptor and chlamydial acceptor derive from the polymeric nature of the adhesin ligand to which they bind. The adhesin bridge model (13) predicts that both the host cell receptor and chlamydial acceptor bind the GAG adhesin, but the receptor and acceptor may contact different domains of the adhesin molecule that could be substantially different with respect to sulfation location and density. In the experiments designed to investigate whether the sulfation of the chlamydial adhesin affected chlamydial infectivity, differentially sulfated forms of heparin were used in inhibition of attachment or infectivity neutralization assays. Although the N-sulfated heparin derivative (CDSNS) was unable to inhibit chlamydial attachment or infection, the O-sulfated heparin derivative (NDSNAc) showed nearly the same strong inhibition ability as native heparin for chlamydial neutralization. The limited restoration of chlamydial attachment and infection observed by incubating the sulfation-modified derivatives of heparin with heparitinase-digested EBs was due to the poor binding of sulfation-modified heparin derivatives to chlamydial organisms as these derivatives were weak competitors of heparin binding to EBs. These results indicate that the chlamydial adhesin acceptor has a sulfation requirement that differs from that required by the host cell receptor.
The effect of the O-sulfation or N-sulfation of the heparin molecule on the ability of heparin to bind to host cells or chlamydial organisms cannot be determined unequivocally because heparin molecules carrying the same high sulfate content with either O- or N-sulfate groups are not available. Nevertheless, the NDSNAc heparin derivative and dextran sulfate (both O-sulfated) inhibited chlamydial attachment and infectivity to the host cell but did not effectively rescue chlamydial infectivity. These data implicate a requirement for both O- and N-sulfation of GAG for binding the chlamydial acceptor and only a requirement for O-sulfation to effectively interact with the host cell receptor. It has been shown previously that chondroitin sulfate does not strongly inhibit chlamydial infection(12, 13) . Chondroitin sulfate is moderately sulfated and contains only O-sulfated saccharide residues (23) suggesting that other structural features defined by the carbohydrate backbone contribute to the specificity of binding. Thus, while a relatively high sulfation density of GAG appears essential, it is evident that the position of sulfate groups and the composition and length of the carbohydrate backbone also play crucial roles in the interaction of heparan-sulfate-like ligand to its chlamydial acceptor and eukaryotic cell receptor.
The findings that the chlamydial acceptor and eukaryotic cell receptor have different requirements for binding heparin derivatives have significant implications for the design and use of sulfated compounds for prophylactic and therapeutic applications. While the use of a compound like heparan sulfate might be considered as an antimicrobial antagonist for a variety of pathogens that bind heparan sulfate proteoglycans, this may be contraindicated for chlamydiae as such compounds can enhance attachment and rescue infectivity over a broad range of concentrations. However, the NDSNAc derivative of heparin did not bind chlamydiae, could not rescue attachment or infectivity, yet was nearly as potent as heparin in its ability to competitively inhibit chlamydial interactions with eukaryotic cells. Thus understanding the structural requirements of sulfated compounds in relation to their functional interactions with both the eukaryotic host cell and microbial pathogen promises to permit the design of infectivity antagonists with targeted specificity and defined activity.