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Dr Jonas Bunikis, Dept of Microbiology and Molecular Genetics,
University of California, Irvine, USA.
Little is known about structure and functions of the outer membrane
proteins of Borrelia spirochetes. Besides being considered as
putative pathogenic determinants, these proteins may also appear
appropriate candidates for vaccine against borrelioses. This summary
centers on topological interactions between different classes of the
borrelial outer membrane proteins, and possible implications of such
interactions for the diagnostic and preventive strategies for Lyme disease
(LD). Another focus is a novel non-genetic manipulation of the surface
make-up of borrelias, an approach to study functions of the proteins
exposed on the surface of these cells.
Like gram-negative bacteria, spirochetes have outer membrane that
surrounds the protoplasmic cylinder complex. Specifically for spirochetes,
flagellum is located in the periplasm, the space between the cytoplasmic
and outer membranes. Borrelia spirochetes do not have lipopolysaccharides
on their surface. Instead, these organisms display abundant surface
lipoproteins, which are anchored to the outer membrane by their lipid
Figure 1. A model of the outer membrane of B. burgdorferi.
(A) Depicted are two classes of the outer membrane proteins: Osp
lipoproteins anchored to the membrane by their lipid moiety, and P66, an
integral outer membrane-spanning protein. A loop region of P66 protrudes
on the cell surface. OspA and OspC are reciprocally expressed in different
hosts. Flagella (fla) of the cell are localized in the periplasm, space
between outer and cytoplasmic membranes (OM and CM, respectively). (B) Osp
proteins shield P66 from the access by specific antibody. Se below for
Figure 1b. A 3D model of the outer membrane of B. burgdorferi.
Depicted are two classes of the outer membrane proteins: more abundant Osp
lipoproteins (e.g. OspA or OspC) which are anchored to the membrane by
their lipid moiety, and scant integral outer membrane-spanning proteins (e.g.
P66 and p13). OspA and OspC are reciprocally expressed by spirochetes in
different hosts, and shield integral membrane proteins from antibody or
proteases. Flagellum of the cell is localized in the periplasm, space
between outer and cytoplasmic membranes. (Graphics by Ignas Bunikis.)
The LD borrelias alternate expression of different outer surface
proteins (Osp) in response to changes in tick versus mammalian host
environment. Outer surface protein A (OspA), and apparently OspB as well,
is expressed by spirochetes in the midgut of ticks, but is seldom if ever
expressed by organisms during early infection of mammals. OspC is
expressed by spirochetes in the tick after the blood meal and during early
infection of mammals. OspD, a protein expressed by some but not all
low-passage isolates, like OspC, is expressed in higher amounts when OspA
expression is off or reduced. The current human vaccine for LD is based on
OspA; antibodies to OspA kill borrelias in the tick's midgut, and thus
prevent transmission of the pathogen.
The relapsing fever Borrelia spp. express on their surface
variable small or large proteins (Vsp and Vlp, respectively), which are
homologous to OspC and Vls lipoproteins of LD borrelias. In Borrelia
hermsii these lipoproteins confer to these cells ability to evade
host's immune response. Vsp proteins of Borrelia turicatae appear
to determine the spirochete's tissue tropism during infection in mammals.
Another class of borrelial surface proteins is much less abundant and
includes integral outer membrane proteins (e.g. P66, p13). They span the
membrane by using hydrophobic alpha-helices, and only relatively small
loop regions of these proteins protrude onto the cell's surface (Fig. 1).
Because of scarcity of integral membrane proteins, borrelial outer
membrane is fluid and readily perturbed by fixation, antibodies, or
detergents. One integral outer membrane protein that has been identified
in all examined species of Borrelia spirochetes is P66. Although this
protein is presumably expressed in human and other mammalian hosts, the
actual function of P66 is unknown. Native P66 has porin activity in
liposomes, but P66 is unlike gram-negative bacterial porins in its
sequence, size, and predicted secondary structure. A loop at the
C-terminus of P66 protein is variable in size and sequence among different
Borrelia spp., and at least in the case of B. burgdorferi s.l.,
is antigenically polymorphic (Fig. 2), features suggesting that this loop
is subject to selective pressure by host's immune system.
Figure 2. Immune response to P66 of B. burgdorferi s. l. is
species-specific. Antibodies to P66 in the serum from Lyme disease patient
from the United States recognize P66 of B. burgdorferi, but not B. afzelii
or B. garinii, Lyme disease agents in Europe and Asia.
One consequence of a topological relationship between the two classes
of the outer membrane proteins of B. burgdorferi s.l. is steric
hindrance by abundant Osp proteins of antibody or protease access to the
integral membrane proteins (Fig. 1). When OspA or OspC is present on the surface,
higher concentrations of trypsin are required to cleave the
surface-exposed loop of P66 (Table 1).
Table 1. Trypsin-cleavage of P66 of B. burgdorferi cells with
different Osp phenotypes
In contrast, significantly lesser amount of the protease cleaves P66 of
B. burgdorferi cells that lack Osp proteins on their surface.
Similarly, a monoclonal antibody to the loop of P66 binds, agglutinates
and inhibits in vitro growth of Osp-less cells, but not borrelias that
express OspA and OspB, OspC or OspD (Fig. 3). Hindrance by Osp proteins
appears also to affect the immune response to P66. Only mice immunized
with Osp-less, but not Osp-expressing, borrelias produce antibodies to
P66, suggesting that presence of Osp proteins limits access to P66 during
antigen presentation. Furthermore, the close association of OspA and P66
is confirmed by in situ cross-linking of the two proteins by using formaldehyde.
Figure 3. A monoclonal antibody to the loop of P66 (aloop)
binds, agglutinates and inhibits growth of Osp-less B. burgdorferi, but
not the cells that express OspA. A monoclonal antibody to a non
surface-exposed region of P66 (non-aloop) does not affect Osp-less
cells and is used as negative control.
One implication of steric interaction between proteins on the surface
of borrelias may be lack of the effectiveness of antibodies to what
otherwise would be an appropriate vaccine target (Table 2) or diagnostic
antigen. Polyclonal and monoclonal antibodies to P66 or p13 integral
membrane proteins kill only B. burgdorferi cells that lack surface
lipoproteins. However, due to apparent irregularity of Osp expression by
borrelias during advancement of infection (OspC is expressed only during
early infection and OspA may or may not be expressed during persistent
infection), there may be time-points when spirochetes are effectively
Osp-less. Antibodies to the loop of P66 are found in patients with late
Lyme disease and in field mice, an indication of unmasking of this protein
during natural infection. Antibodies to the "late" antigens
(e.g. P66) of B. burgdorferi seem to last longer after infection or be
more specific than antibodies against "early" antigens (e.g.
OspC or flagellin). This may explain the absence of re-infection in
patients with disseminated Lyme disease, as opposed to those with early
localized infection, and suggests also that the "late" antigens
could be protective.
Table 2. Anti-borrelial effect of antibodies to integral
outer membrane proteins of B. burgdorferi.
Polyclonal anti-native P66*
* Exner et al., 2000; ** Sadziene et al., 1995
A more comprehensive understanding of the functions of borrelial outer
membrane proteins is hampered by the incomplete development of genetic
systems. Genetic transformation of Borrelia spp. has been limited
to insertional inactivation of a few B. burgdorferi genes.
Expressing recombinant genes for outer membrane proteins has yet to be
achieved. Moreover, there has not been successful transformation of any of
the relapsing fever Borrelia spp. to date. Alternatively, phenotype of
borrelias can be transiently modified by a non-heritable decoration of
their surface with exogenous lipoproteins. Purified recombinant Osp
lipoproteins stably insert in the outer membrane of viable cells of both
homologous and heterologous Borrelia spp. lacking surface
lipoproteins of their own (Fig. 4A). The findings suggest that exogenous
lipoprotein inserts into the outer membrane by its lipid moiety rather
through a more specific ligand-ligand interaction. In situ proteolysis and
formaldehyde cross-linking of decorated B. burgdorferi cells
indicate that the topology of the inserted recombinant protein is similar
to that of its native counterpart. It appears, therefore, that insertion
of lipoproteins into a membrane may occur without the assistance of
transport or chaperone-type proteins.
Figure 4. Phenotype of viable Borrelia cells lacking
endogenous surface lipoproteins of their own is modified by decorating
cell surface with exogenous lipoprotein. (A) Osp-less or Vsp-less B.
burgdorferi and B. hermsii cells, respectively, are decorated with
recombinant OspA lipoprotein, which is subsequently detected in IFA using
OspA-specific monoclonal antibody. (B) A polyclonal antiserum to OspA
kills B. hermsii cells decorated with exogenous OspA, as well as OspA+ B.
burgdorferi cells used as a control.
Importantly, treatment of B. burgdorferi and B. hermsii cells
with recombinant OspA lipoprotein changes their phenotype, at least with
respect to susceptibility to OspA-specific antibody. The morphological
effects of antibody on B. burgdorferi cells expressing OspA and B.
hermsii cells displaying exogenous OspA are indistinguishable (Fig.
4B), a suggestion that the phenotype manipulations can be evaluated in
vivo as well as in vitro.
In conclusion, the outer membrane of Borrelia spp. contains
abundant surface lipoproteins and relatively scant integral membrane
proteins, which functions are not well understood. In B. burgdorferi,
the proximity and possible contact between these two classes of proteins
results in steric hindrance by Osp lipoproteins of antibody or protease
access to the integral membrane proteins, candidates for a new-generation
vaccine and diagnostics. A technique of non-genetic modification of the
phenotype of viable borrelias by decorating their surface with exogenous
lipoproteins may be used to study functions and properties of lipoproteins
Holt, S. C. 1978. Anatomy and chemistry of spirochetes. Microbiol
Barbour, A. G., and S. F. Hayes. 1986. Biology of Borrelia
species. Microbiol Rev. 50:381–400.
Bunikis, J., A. G.Barbour. 1999. Access of antibody or trypsin to an
integral outer membrane protein (P66) of Borrelia burgdorferi
is hindered by Osp lipoproteins. Infect. Immun., 67:2874–2883.
Bunikis, J., H. Mirian, E. Bunikiene, A. G. Barbour. 2001.
Non-heritable change of a spirochete's phenotype by decoration of the
cell surface with exogenous lipoproteins. Mol Microbiol., 40:387–396.
Part of the proceedings of the symposium Current Research on
Tick-Borne Infections, Kalmar, Sweden, March 28–30, 2001. © 2001, Jonas Bunikis,
Dept of Microbiology and Molecular Genetics, University of California, Irvine,
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CRTBI - Extended Abstracts
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Bunikis J et al.: Structure and Function of the Surface Proteins of Borrelia Spirochetes
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