Structure and Function of the Surface Proteins of Borrelia Spirochetes

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 moiety (Fig1).

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 3D model.

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.

* 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 in situ.

Recommended literature:

1. Holt, S. C. 1978. Anatomy and chemistry of spirochetes. Microbiol Rev. 38:114–160.

2. Barbour, A. G., and S. F. Hayes. 1986. Biology of Borrelia species. Microbiol Rev. 50:381–400.

3. 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.

4. 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, USA.

Bunikis J et al.: Structure and Function of the Surface Proteins of Borrelia Spirochetes

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Gray J S et al.: The biology of Ixodes ticks, with special reference to Ixodes ricinus

Guillaume B et al.: Human Granulocytic Ehrlichia Infection in Belgium

Haglund M et al.: Tick-borne encephalitis (TBE) - an overview

Larsen K et al.: Tick species and arthropod-transmitted infections from Danish cats and dogs

Lundkvist Å et al.: Characterization of Tick-borne enchephalitis virus from Latvia – evidence for co-circulation of three distinct subtypes

Malmgren L et al.: A field trial of the effectiveness of 65% permetrin spot-on and 9.7% fipronil spot-on against ticks (Ixodes ricinus) on dogs

Massung R F et al.: Genetic Variants of Ehrlichia phagocytophila in the United States

Nilsson I et al.: Serological evidence of Lyme arthritis in Egypt

Nyman D et al.: Ticks have preferences in choosing human hosts

Ornstein K et al.: Quantification of spirochete burden in Borrelia burgdorferi infected ticks fed on OspA immunized mice by 16S rRNA RT real-time PCR

Randolph S et al.: Epidemiological consequences of tick ecology

Skarpaas T et al.: Tick-borne encephalitis in Norway

Soutschek E et al.: A defined mixture of recombinant antigens from several Borrelia genospecies improves serodiagnosis of Lyme disease

von Stedingk L V et al.: Recent research on human babesiosis – the Scandinavian perspective

Stuen S et al.: Granulocytic Ehrlichia infection in domestic and wild ruminants in Norway

Widhe M et al.: Cytokines in Lyme Borreliosis: in vivo levels of TGF-b1, TNF-a and IL-6 in serum and cerebrospinal fluid from patients with neuroborreliosis or erythema migrans in relation to clinical outcome