key ndecules
Molecular geneticsof antigenicvariation Piet Borst Autigenic variation is OIW of the most effective strategies developed by parasites to escape inmune destruction. It requires a large wardrobe of surface coats and mechanisms to eschange one coat for an unrelated one. The molecular principles of antigenic variation are now largely known in the bacterial species Borrelia and Neisseria and in the protozoa of the African trypanosome group and these thee examples are discussed here by Piet Borst. Faced with the formidable attack by the vertebrate immune system, parasites mainly use four evasion techniques’.‘: (1) III‘d’ m g In cells or in body sanctuaries where the immune attack is less effective, (2) mimicry, by adsorption of host protein or imitation of host surface proteins, (3) blunting the attack by immune suppression and (4) antigenic variation, that is changing the target. Antigenic variation comes in two forms, classically known in the influenza field as ‘drift’ and ‘shift’. Antigenie drift (here called antigenic variability) occurs by the gradual accumulation of mutations in genes that code for the targets of the immune response. It occurs in every parasite, but occurs in some more than in others. For instance, the sloppy copying of RNA virus genomes by RNA replicases or reverse transcriptases leads to considerable population heterogeneity and a pronounced drift in the composition of targets for the host. Antigenic variability is hardly a specific strategy, even though sloppy polymerases can be selected for; the parasite merely makes the most of random errors. In contrast, antigenic’shift’ (here called antigenic variation, sensu strict0 (s.s.)) is a specific strategy. It requires a mechanism to replace abruptly one type of surface by another. It invariably requires multiple nonallelic genes for surface proteins and usually some form of gene rearrangementI. Antigenic variation, s.s., is the topic of this review.
in a certain order is required to avoid gross population heterogeneity and rapid induction of antibody formation against all members of the surface antigen repertoire. (4) The parasite needs to eat, mate (where relevant) and adsorb to specific targets, without undue exposure of nonvariant antigens. This Achilles’ heel problem has not been analysed in detail but antigenic diversion is one way to solve it. Thus, by presenting an immunodominant antigen that can be varied, the parasite diverts its host away from the constant elements on its surface. The parasite may also hide its nonvariant antigens in a subsurface structure that is not accessible to T cells or macrophages. This clever tactic is used by trypanosomes (see below). Benefits and costs of antigenic variation The major benefit is that the parasite can remain in the bloodstream for sufficiently long periods to provide the opportunity for transmission by blood-sucking insects or by blood-blood contact. However, the cost of antigenic variation is considerable. In a chronic Trypanosoma brucei infection in mammals, each wave of parasitaemia is terminated by the killing of more than 99.9% of the parasites. All known trypanosome surface antigen variants seem to induce an effective antibody response even in athymic nude mice”. There is no evidence that variant antigens are ever selected for their ability to evade the helper T-cell response.
What is required for antigenic variation to work? Although the strategy is intuitively obvious, the re- Switching of surface antigens in K brucei quirements are in reality complex but divide into four The entire surface of an African trypanosome is categories. (1) The parasite must be able to make a large covered with a dense,protein coat that consists of a single number of immunodominant surface antigens that have protein species known as the variant-specific surface no visible determinants in common to evade the immune glycoprotein, or VSGS-Il. T. brucei contains some lo3 system for a significant period. (2) The parasite must be genes for VSGs, of which a large fraction encodes a able to switch the exposed ‘variant-specific’ antigen in a complete VSG. VSG genes can be activated in any of three subfraction of the parasite population before antibodies ways (Fig. 1). (1) They can be activated by the duplicative have wiped out the entire population. In theory the transposition of a nontelomeric gene, B, to a telomeric switch might be made in response to antibody but, in expression site, displacing the resident gene, X, which is practice, none of the parasites that rely on antigenic lost in the process r3. Indirect evidence suggests that such variation for survival does that, possibly because com- transposition is a gene conversion process using relatively plete replacement of the coat takes too long to avoid small blocks of imperfect homology situated l-2 kb in killing by an effective immune response. Instead, para- front of donor and acceptor genes and using different sites use spontaneous switching mechanisms, resulting in blocks at the 3’ end of the gene. (2) VSG genes can also be a constant low rate of switching (10-l or less per div- activated by the duplicative transposition of a silent ision)3. Hence, the parasite population always contains a telomeric VSG gene, D, into a telomeric expression site. small fraction of ‘heterotypes’ that will survive the Large blocks of sequence may be cotransposed in this specific host response that kills the predominant case as there is considerable sequence homology between ‘homotype’. (3) The expression of surface antigen genes telomeres, hence the name, telomere conversion. A 0 1991. Elrer~rr hrncr
Publ~sbers Ltd. UK.O167-4919/91/$02.00
A29
kev molecules J
Duplicative transposition of a nontelomeric gene
Telomere conversion of a silent gene
Activation, in situ, of a telomeric expreksion site
/!!
.. X N
p 7
Fig. 1.
Tl7e various pathways for activating a variant-specific surface glycoprotein gene in T. brucei. Boxes A, B and C are nontelomeric genes; X is a telomeric gene. Duplicative transposition of B displaces X and brings B under the expression site promoter (flag). The vertical bar marks the end of a chromosome. The other mechanisms illustrated are explained irt the text.
simplereciprocal recombination can alsooccur between telomeresi4, allowing the exchange between an active and an inactive genebut this mode of switching appears to be uncommon.(3) The other mechanismfor activation of VSG genesis the activation of an inactive telomeric expressionsite and the concomitant inactivation of the previously active site. Mechanistically, the activation of one site and the inactivation of the other appear to be independentspontaneousprocesses, but the nature of the (in)activation remainsunknown. Gene rearrangements near the promoter are not involvedi but recent unpublished evidence indicates that gene rearrangements may occur further upstream. Order of VSG geneexpressionin T. brucei With lo3 VSG genes,three switching modesand at least five, but possibly as many as twenty, telomeric expressionsites,antigenic switching in trypanosomesis clearly a complex business.One reasonfor this complexity may be the need for a temporal order in VSG gene expression.How this order isachievedisstill a matter for speculation. An essentialexperimental result that has directed thinking in this area was obtained by transferring a trypanosome population from a chronically infected animal to a freshanimal (Fig. 2). It wasfound that the order of appearanceof variants isreset,that is variant A tends to take over after the infecting variant has succumbedto host antibodies. This suggeststhat the
fly I rabbit 1 : l - nzq=E I---------------;passage rabh2: Fig. 2. A highly
A-
B-
-
D-
etc.
E -etc.
schematized representation of the ordered appearance of trypanosome variants in a chronic mammalian infection. The metacyclic coat repertoire46 is switched on in the salivary gland of the tsetse fly. Letters represent the major variants in subsequent waves of parasitaemia. The order is imprecise, for example A may go to C or D. Filled circle: metacyclic repertoire.
impreciseorder in which variants appear in a chronic infection is, to a large extent, determined by the relative frequency at which the corresponding gene is switched on. Several factors co-determine this frequency? telomeric genesin expression sitesthat are easily activated tend to come first; they can either be activated, in situ (Fig. I), or by telomeric conversion. Other telomeric genesfollow becausetheir surrounding sequencesalso provide large segmentsof homology with the active expressionsite. Theseare followed by the expressionof nontelomeric genesthat have good homology blocks at both 5’ and 3’ ends.Finally, geneswith poor homology in their flanking sequencewith the expression site will get their chance. Indeed it appears that the genesthat are expressed later are chimaeric genes assembledfrom pseudogenes,which by themselvescannot give rise to a functional VSG”-i9. Although thesegeneticfactors undoubtedly contribute to the order in which VSG genesare expressed,other factors also play a role. One is growth rate, since some variants grow faster than others”‘. A more speculative factor is the composition of mixed coats. When trypanosomesswitch from coat A to coat B, they temporarily wear a mixed, AB, coat. Mixing appearsto be complete, as suchtrypanosomesstain uniformly with either anti-A or anti-B antibodies”. Moreover, asthe turnover of coat is lowL1, VSG A is orly gradually diluted out as the trypanosomes multiply. It is therefore conceivable that the properties of mixed coats could be important in determining the order of VSG gene expression. Some mixed coats might be unstableor unprotective; in others one variant may remain highly visible to host antibodies even if it representsonly a small proportion of the total coat. Agur et a1.13have tested this in mathematical modelsof trypanosome infection. They could simulate a realistic parasitaemiaonly if some mixed coats provide better protection than others. Although the results presentedby Agur et a1.l” are striking, their model remains to be tested by experiment. With the recent development of stable trypanosome transformatiorP, it should now be relatively simpleto createa rangeof mixed coatsin the laboratory. How to changeantigenswithout starving Bloodstream trypanosomes are mastersin the art of delegation. They limit their biosynthetic efforts to the bare minimum and import most of the building blocks, even complex compounds like purines and all lipids, from their host. There is strong circumstantial evidence that uptake of someof thesebuilding blocks involves host macromoleculesbinding to parasite receptors’s. How does the trypanosome manageto open up its coat for receptor-mediated uptake of macromoleculeswithout exposing itself to the host’s immune system?This problem iselegantly solvedwith a specializedstructure, called the flagellar pocket (Fig. 3), that is found at the baseof the flagellum buried in the trypanosome body. The flagellar pocket is an area of intensive endocytosisand it is probably the only part of the trypanosome surface that can bind and take up host molecules’6.The walls of the flagellar pocket contain high-affinity binding sites for host transferrin and low-density lipoprotein (LDL), both of which are internalized at high rates. The pocket is
A30
key violecules J
readily accessible to large protein complexes, such as LDL or antibodies, but not to T cells, monocytes or macrophages. Hence, the antibodies that might eventually be made against the invariant receptors required for substrate uptake may not be very effective since they will be taken up and degraded by the parasite and will never be able to invoke the help of the cellular arm of the immune response. Nevertheless, Olenick et al.” have reported that immunization of mice with a purified flagellar pocket fraction will partially protect mice against subsequent trypanosome infection. This suggests that the therapeutic exploitation of this Achilles’ heel of the parasite might be ‘feasible. Using recently developed transformation techniques14, it should be possible to create trypanosomes that have flagellar pocket proteins all over their surface coat. Such transgenic trypanosomes might induce high antibody titers that are sufficient to saturate the substrate receptors in the flagellar pocket of normal trypanosomes and kill these by starvation or complement-mediated lysis.
Mitochondrion
Antigenic variation of Borrefia Antigenic variation in the eubacterial prokaryote Borrelia is rather similar to that in African trypanosomes28.B. hermsii causes a relapsing fever in mice resulting from its multiplication in the bloodstream. A singlecloned organismcan give rise to at least 26 different serotypes, the immunodominant antigens of which are found in a single surface protein called the variable major protein (VMP). Antigen switching is apparently spontaneousand occurs at a rate of 10-4-10-3 per cell generation. The mechanism of switching of VMPs in Borrelia is similar to VSG switching in trypanosomes, at least for the three VMPs analysed. There appear to be arrays of silent VMP geneson linear 30 kb ‘storage’ plasmids.Activation of a VMP geneis accomplishedby the duplicative transposition of the geneto an expression site29,probably by geneconversion30. As in trypanosome infections, someBorrefiu serotypes occur early in infections, whereas others are predominantly seen later. The mechanismof this ordered appearanceremainsunknown. As the infection by B. recurrentis in humansresembles B. hermsii infections in the mouse,it seemslikely that the persistenceof B. recurrentis is basedon the sametype of antigenic variation used by B. hermsii. Whether other pathogenic Borreliu specieshave a similar antigenic variation systemat their disposalis still in doubt. Although B. burgdorferi, the causative agent of Lyme disease,also contains smalllinear plasmids,the clonal polymorphisms observedin its outer membraneprotein, OspB, appearto be due to genetic drift rather than to the alternative expressionof different membersof a gene family3’. Antigenic variation of Neisseriapili Two types of surface proteins are known to vary in Neisseriu species,the pilins and the opacity protein+-34. The pilins are the protein subunits of the pili, which are long hairlike protein appendageson the bacterial surface that appear to be the prime target for host neutralizing antibodies. At first sight antigenic variation of pili resemblesantigenic variation of the surface proteins of
Fig.3. Schematic cross section of an African trypanosome showing some key structural features.
trypanosomes and Borreliu. There is an array of silent pilin genes35 and activation of a generequiresits transposition to an expression site36. However, there are three interesting Neisseriu-specificvariations on this theme. (1) All silent genes are incomplete; the missing part specifiesthe amino-terminal invariant domain of pilin and this is contributed by the expressionsite. (2) Switching can occur in two unusual ways37,38. The first method is by transformation in which competent Neisseriu cells efficiently take up exogenous homologous DNA from lysed Neisseriu and this DNA can be usedto replace the pilin expression copy at a rate of 1p3 per cell division. This appearsto be the main route for pilin switching, at leastin cultured organisms.Second,when DNA uptake is prevented, a low rate of switching still occurs. This is not due to a unidirectional genetransfer but to a reciprocal recombination betweena silent pilin geneand the genein the expressionsite. (3) Pili may be lost, usually becausea nonfunctional silent pilin gene copy is put into the expression site39. Pili are not essential for survival and under someconditions hairlessvariants may even be at an advantage. In contrast, trypanosomeswithout surface coat are instantly killed. Variation of opacity proteins in Neisseria A second surface protein of Neisseriu, encoded by a genefamily (opu), isa minor outer membraneprotein3’J3 called the opacity protein. The gene family is small in some Neisseriu speciesand it is doubtful whether the
A31
Opa domain
Signal sequence I
r
I
r
--
/-
/-
_/--
/HC
:
I
I I 1
Hydrophobic variable length
I
lO” VSG genes (some pseudo or incomplete)
>27 VMP genes?
>lO (incomplete) genes
13
Switching
(1) Mainly by duplicative transposition to one of many ESs” (2) Switching ES
By duplicative transposition to a single ES?
(1) BY gene conversion of ES using exogenous donor (2) By reciprocal recombination
Translational control by reading-frame shift
between
opa
genes
silent
geneand ES Control of switching Complex (seetext) order Probably all in flagellar pocket beyond the reach
Hiding common antigens
? (someorder present) ?
of T cells and macrophages “ES: expression site
References 1 Bloom, B.R. (1979) Nature 279, 21-26 2 Trager, W. (1986) Liviq Together: T/z 13io/o
Molecular geneticsof antigenicvariation Piet Borst Autigenic variation is OIW of the most effective strategies developed by parasites to escape inmune destruction. It requires a large wardrobe of surface coats and mechanisms to eschange one coat for an unrelated one. The molecular principles of antigenic variation are now largely known in the bacterial species Borrelia and Neisseria and in the protozoa of the African trypanosome group and these thee examples are discussed here by Piet Borst. Faced with the formidable attack by the vertebrate immune system, parasites mainly use four evasion techniques’.‘: (1) III‘d’ m g In cells or in body sanctuaries where the immune attack is less effective, (2) mimicry, by adsorption of host protein or imitation of host surface proteins, (3) blunting the attack by immune suppression and (4) antigenic variation, that is changing the target. Antigenic variation comes in two forms, classically known in the influenza field as ‘drift’ and ‘shift’. Antigenie drift (here called antigenic variability) occurs by the gradual accumulation of mutations in genes that code for the targets of the immune response. It occurs in every parasite, but occurs in some more than in others. For instance, the sloppy copying of RNA virus genomes by RNA replicases or reverse transcriptases leads to considerable population heterogeneity and a pronounced drift in the composition of targets for the host. Antigenic variability is hardly a specific strategy, even though sloppy polymerases can be selected for; the parasite merely makes the most of random errors. In contrast, antigenic’shift’ (here called antigenic variation, sensu strict0 (s.s.)) is a specific strategy. It requires a mechanism to replace abruptly one type of surface by another. It invariably requires multiple nonallelic genes for surface proteins and usually some form of gene rearrangementI. Antigenic variation, s.s., is the topic of this review.
in a certain order is required to avoid gross population heterogeneity and rapid induction of antibody formation against all members of the surface antigen repertoire. (4) The parasite needs to eat, mate (where relevant) and adsorb to specific targets, without undue exposure of nonvariant antigens. This Achilles’ heel problem has not been analysed in detail but antigenic diversion is one way to solve it. Thus, by presenting an immunodominant antigen that can be varied, the parasite diverts its host away from the constant elements on its surface. The parasite may also hide its nonvariant antigens in a subsurface structure that is not accessible to T cells or macrophages. This clever tactic is used by trypanosomes (see below). Benefits and costs of antigenic variation The major benefit is that the parasite can remain in the bloodstream for sufficiently long periods to provide the opportunity for transmission by blood-sucking insects or by blood-blood contact. However, the cost of antigenic variation is considerable. In a chronic Trypanosoma brucei infection in mammals, each wave of parasitaemia is terminated by the killing of more than 99.9% of the parasites. All known trypanosome surface antigen variants seem to induce an effective antibody response even in athymic nude mice”. There is no evidence that variant antigens are ever selected for their ability to evade the helper T-cell response.
What is required for antigenic variation to work? Although the strategy is intuitively obvious, the re- Switching of surface antigens in K brucei quirements are in reality complex but divide into four The entire surface of an African trypanosome is categories. (1) The parasite must be able to make a large covered with a dense,protein coat that consists of a single number of immunodominant surface antigens that have protein species known as the variant-specific surface no visible determinants in common to evade the immune glycoprotein, or VSGS-Il. T. brucei contains some lo3 system for a significant period. (2) The parasite must be genes for VSGs, of which a large fraction encodes a able to switch the exposed ‘variant-specific’ antigen in a complete VSG. VSG genes can be activated in any of three subfraction of the parasite population before antibodies ways (Fig. 1). (1) They can be activated by the duplicative have wiped out the entire population. In theory the transposition of a nontelomeric gene, B, to a telomeric switch might be made in response to antibody but, in expression site, displacing the resident gene, X, which is practice, none of the parasites that rely on antigenic lost in the process r3. Indirect evidence suggests that such variation for survival does that, possibly because com- transposition is a gene conversion process using relatively plete replacement of the coat takes too long to avoid small blocks of imperfect homology situated l-2 kb in killing by an effective immune response. Instead, para- front of donor and acceptor genes and using different sites use spontaneous switching mechanisms, resulting in blocks at the 3’ end of the gene. (2) VSG genes can also be a constant low rate of switching (10-l or less per div- activated by the duplicative transposition of a silent ision)3. Hence, the parasite population always contains a telomeric VSG gene, D, into a telomeric expression site. small fraction of ‘heterotypes’ that will survive the Large blocks of sequence may be cotransposed in this specific host response that kills the predominant case as there is considerable sequence homology between ‘homotype’. (3) The expression of surface antigen genes telomeres, hence the name, telomere conversion. A 0 1991. Elrer~rr hrncr
Publ~sbers Ltd. UK.O167-4919/91/$02.00
A29
kev molecules J
Duplicative transposition of a nontelomeric gene
Telomere conversion of a silent gene
Activation, in situ, of a telomeric expreksion site
/!!
.. X N
p 7
Fig. 1.
Tl7e various pathways for activating a variant-specific surface glycoprotein gene in T. brucei. Boxes A, B and C are nontelomeric genes; X is a telomeric gene. Duplicative transposition of B displaces X and brings B under the expression site promoter (flag). The vertical bar marks the end of a chromosome. The other mechanisms illustrated are explained irt the text.
simplereciprocal recombination can alsooccur between telomeresi4, allowing the exchange between an active and an inactive genebut this mode of switching appears to be uncommon.(3) The other mechanismfor activation of VSG genesis the activation of an inactive telomeric expressionsite and the concomitant inactivation of the previously active site. Mechanistically, the activation of one site and the inactivation of the other appear to be independentspontaneousprocesses, but the nature of the (in)activation remainsunknown. Gene rearrangements near the promoter are not involvedi but recent unpublished evidence indicates that gene rearrangements may occur further upstream. Order of VSG geneexpressionin T. brucei With lo3 VSG genes,three switching modesand at least five, but possibly as many as twenty, telomeric expressionsites,antigenic switching in trypanosomesis clearly a complex business.One reasonfor this complexity may be the need for a temporal order in VSG gene expression.How this order isachievedisstill a matter for speculation. An essentialexperimental result that has directed thinking in this area was obtained by transferring a trypanosome population from a chronically infected animal to a freshanimal (Fig. 2). It wasfound that the order of appearanceof variants isreset,that is variant A tends to take over after the infecting variant has succumbedto host antibodies. This suggeststhat the
fly I rabbit 1 : l - nzq=E I---------------;passage rabh2: Fig. 2. A highly
A-
B-
-
D-
etc.
E -etc.
schematized representation of the ordered appearance of trypanosome variants in a chronic mammalian infection. The metacyclic coat repertoire46 is switched on in the salivary gland of the tsetse fly. Letters represent the major variants in subsequent waves of parasitaemia. The order is imprecise, for example A may go to C or D. Filled circle: metacyclic repertoire.
impreciseorder in which variants appear in a chronic infection is, to a large extent, determined by the relative frequency at which the corresponding gene is switched on. Several factors co-determine this frequency? telomeric genesin expression sitesthat are easily activated tend to come first; they can either be activated, in situ (Fig. I), or by telomeric conversion. Other telomeric genesfollow becausetheir surrounding sequencesalso provide large segmentsof homology with the active expressionsite. Theseare followed by the expressionof nontelomeric genesthat have good homology blocks at both 5’ and 3’ ends.Finally, geneswith poor homology in their flanking sequencewith the expression site will get their chance. Indeed it appears that the genesthat are expressed later are chimaeric genes assembledfrom pseudogenes,which by themselvescannot give rise to a functional VSG”-i9. Although thesegeneticfactors undoubtedly contribute to the order in which VSG genesare expressed,other factors also play a role. One is growth rate, since some variants grow faster than others”‘. A more speculative factor is the composition of mixed coats. When trypanosomesswitch from coat A to coat B, they temporarily wear a mixed, AB, coat. Mixing appearsto be complete, as suchtrypanosomesstain uniformly with either anti-A or anti-B antibodies”. Moreover, asthe turnover of coat is lowL1, VSG A is orly gradually diluted out as the trypanosomes multiply. It is therefore conceivable that the properties of mixed coats could be important in determining the order of VSG gene expression. Some mixed coats might be unstableor unprotective; in others one variant may remain highly visible to host antibodies even if it representsonly a small proportion of the total coat. Agur et a1.13have tested this in mathematical modelsof trypanosome infection. They could simulate a realistic parasitaemiaonly if some mixed coats provide better protection than others. Although the results presentedby Agur et a1.l” are striking, their model remains to be tested by experiment. With the recent development of stable trypanosome transformatiorP, it should now be relatively simpleto createa rangeof mixed coatsin the laboratory. How to changeantigenswithout starving Bloodstream trypanosomes are mastersin the art of delegation. They limit their biosynthetic efforts to the bare minimum and import most of the building blocks, even complex compounds like purines and all lipids, from their host. There is strong circumstantial evidence that uptake of someof thesebuilding blocks involves host macromoleculesbinding to parasite receptors’s. How does the trypanosome manageto open up its coat for receptor-mediated uptake of macromoleculeswithout exposing itself to the host’s immune system?This problem iselegantly solvedwith a specializedstructure, called the flagellar pocket (Fig. 3), that is found at the baseof the flagellum buried in the trypanosome body. The flagellar pocket is an area of intensive endocytosisand it is probably the only part of the trypanosome surface that can bind and take up host molecules’6.The walls of the flagellar pocket contain high-affinity binding sites for host transferrin and low-density lipoprotein (LDL), both of which are internalized at high rates. The pocket is
A30
key violecules J
readily accessible to large protein complexes, such as LDL or antibodies, but not to T cells, monocytes or macrophages. Hence, the antibodies that might eventually be made against the invariant receptors required for substrate uptake may not be very effective since they will be taken up and degraded by the parasite and will never be able to invoke the help of the cellular arm of the immune response. Nevertheless, Olenick et al.” have reported that immunization of mice with a purified flagellar pocket fraction will partially protect mice against subsequent trypanosome infection. This suggests that the therapeutic exploitation of this Achilles’ heel of the parasite might be ‘feasible. Using recently developed transformation techniques14, it should be possible to create trypanosomes that have flagellar pocket proteins all over their surface coat. Such transgenic trypanosomes might induce high antibody titers that are sufficient to saturate the substrate receptors in the flagellar pocket of normal trypanosomes and kill these by starvation or complement-mediated lysis.
Mitochondrion
Antigenic variation of Borrefia Antigenic variation in the eubacterial prokaryote Borrelia is rather similar to that in African trypanosomes28.B. hermsii causes a relapsing fever in mice resulting from its multiplication in the bloodstream. A singlecloned organismcan give rise to at least 26 different serotypes, the immunodominant antigens of which are found in a single surface protein called the variable major protein (VMP). Antigen switching is apparently spontaneousand occurs at a rate of 10-4-10-3 per cell generation. The mechanism of switching of VMPs in Borrelia is similar to VSG switching in trypanosomes, at least for the three VMPs analysed. There appear to be arrays of silent VMP geneson linear 30 kb ‘storage’ plasmids.Activation of a VMP geneis accomplishedby the duplicative transposition of the geneto an expression site29,probably by geneconversion30. As in trypanosome infections, someBorrefiu serotypes occur early in infections, whereas others are predominantly seen later. The mechanismof this ordered appearanceremainsunknown. As the infection by B. recurrentis in humansresembles B. hermsii infections in the mouse,it seemslikely that the persistenceof B. recurrentis is basedon the sametype of antigenic variation used by B. hermsii. Whether other pathogenic Borreliu specieshave a similar antigenic variation systemat their disposalis still in doubt. Although B. burgdorferi, the causative agent of Lyme disease,also contains smalllinear plasmids,the clonal polymorphisms observedin its outer membraneprotein, OspB, appearto be due to genetic drift rather than to the alternative expressionof different membersof a gene family3’. Antigenic variation of Neisseriapili Two types of surface proteins are known to vary in Neisseriu species,the pilins and the opacity protein+-34. The pilins are the protein subunits of the pili, which are long hairlike protein appendageson the bacterial surface that appear to be the prime target for host neutralizing antibodies. At first sight antigenic variation of pili resemblesantigenic variation of the surface proteins of
Fig.3. Schematic cross section of an African trypanosome showing some key structural features.
trypanosomes and Borreliu. There is an array of silent pilin genes35 and activation of a generequiresits transposition to an expression site36. However, there are three interesting Neisseriu-specificvariations on this theme. (1) All silent genes are incomplete; the missing part specifiesthe amino-terminal invariant domain of pilin and this is contributed by the expressionsite. (2) Switching can occur in two unusual ways37,38. The first method is by transformation in which competent Neisseriu cells efficiently take up exogenous homologous DNA from lysed Neisseriu and this DNA can be usedto replace the pilin expression copy at a rate of 1p3 per cell division. This appearsto be the main route for pilin switching, at leastin cultured organisms.Second,when DNA uptake is prevented, a low rate of switching still occurs. This is not due to a unidirectional genetransfer but to a reciprocal recombination betweena silent pilin geneand the genein the expressionsite. (3) Pili may be lost, usually becausea nonfunctional silent pilin gene copy is put into the expression site39. Pili are not essential for survival and under someconditions hairlessvariants may even be at an advantage. In contrast, trypanosomeswithout surface coat are instantly killed. Variation of opacity proteins in Neisseria A second surface protein of Neisseriu, encoded by a genefamily (opu), isa minor outer membraneprotein3’J3 called the opacity protein. The gene family is small in some Neisseriu speciesand it is doubtful whether the
A31
Opa domain
Signal sequence I
r
I
r
--
/-
/-
_/--
/HC
:
I
I I 1
Hydrophobic variable length
I
lO” VSG genes (some pseudo or incomplete)
>27 VMP genes?
>lO (incomplete) genes
13
Switching
(1) Mainly by duplicative transposition to one of many ESs” (2) Switching ES
By duplicative transposition to a single ES?
(1) BY gene conversion of ES using exogenous donor (2) By reciprocal recombination
Translational control by reading-frame shift
between
opa
genes
silent
geneand ES Control of switching Complex (seetext) order Probably all in flagellar pocket beyond the reach
Hiding common antigens
? (someorder present) ?
of T cells and macrophages “ES: expression site
References 1 Bloom, B.R. (1979) Nature 279, 21-26 2 Trager, W. (1986) Liviq Together: T/z 13io/o
