[~EVIEWS

16 Pink, J.R.L. (1986) Immunol. Rev. 91, 115--128

17 Reynaud, C-A., Anquez, V., Grimal, H. and Weill, J-C. (1987) Cell 48, 379-388 18 Parvari, R. et al. (1988) EMBOJ. 7, 739-744 19 Thompson, C.B. and Neiman, P.E. (1987) Cell 48, 369-378 20 Thompson, C.B. (1989) in Mechanisms of B Cell Neoplasia, 1989 (Melchers, F. and Potter, M., eds), pp. 46-54, Editiones Roche 21 Knight, K.L. and Becker, R.S. (1990) Cell 60, 963--970 22 Allegmcci, M. et aL (1991) Eur.J. Immunol. 21,411-417 23 Knight, K.L. (1992) Annu. Rev. lmmunol. 10, 593-616 24 Parvari, R. et al. (1990) Proc. Natl Acad. Sci. USA 87, 3072-3076 25 McCormack, W.T. and Thompson, C.B. (1990) Genes Dev. 4, 548--558

Although seemingly homogeneous, microbial populations representing a single strain are almost always heterogeneous at the cellular and genetic levels. This remarkable phenomenon, which has been intensively studied in a small number of model microorganisms, particularly pathogens, is probably widely distributed in the prokaryotic kingdom. It results from spontaneous changes in the genetic information of a species, here termed genetic variation*, which occur at relatively high frequencies and thus affect even small populations. Often such changes lead to the phenotypic variation of surface components. This is particularly important in the case of pathogenic bacteria because most of the variable structures studied so far have turned out to be essential either for the colonization of the host, or for the survival of the pathogen within that host. The ability to vary the immunogenicity of important structures and to 'fine tune' the specificities of receptors or adhesins is of considerable evolutionary advantage to pathogens, which must encounter unpredictable changes in their environment. This review will cover the functional consequences and advantages of phenotypic variation, and the mechanisms that produce it in Neisseria gonorrhoeae and other pathogenic bacteria, concentrating on some of the newer discoveries in the field. Phenotypic variation includes phase and antigenic variation. For the purposes of this review we will define phase variation as the reversible loss or gain of a molecule or defined structure. Antigenic variation concerns the organization or composition of that molecule or structure, and is sometimes defined in terms of the presence or absence of epitopes; such epitopes can be referred to as 'phase variable'. Genetic variation is not limited to bacteria but is also found in a wide variety of eukaryotic organisms and viruses (discussed elsewhere in this issue). Detailed reviews on bacterial variation *Such changes have been described both as 'random' processes1, and also as 'programmed rearrangements'2. This apparent terminological contradiction is resolved by considering the temporal and spatial contexts of the terms: genetic variation is random with regard to the time of occurrence but nonrandom with regard to the genetic information involved; there appear to be genetic programs that affect loci in distinct regions of the chromosome.

Haber, J,E. (1992) Trends Genet. 8, 446-452 Kim, S. et aL (1990) Mol. Cell. Biol. 10, 3224-3231 Buerstedde, J-M. et aL (1990) F~BOJ. 9, 921-927 Carlson, L.M. et al. (1991) Cell 64, 201-208 Oettinger, M.A., Schatz, D.G., Gorka, A. and Baltimore, D. (1990) Science 248, 1517-1522 31 Takeda, S., Masteller, E.L., Thompson, C.B. and Buerstedde, J-M. (1992) Proc. NatI Acad. Sci. USA 89, 4023-4027 32 Buerstedde, J-M. and Takeda, S. (1991) Cell 67, 179-188 26 27 28 29 30

C.R THOMPSONIS IN THEHOWARDHUGHESMEDICALINSTITUTE AND DEPARTMENTS OF INTERNAL MEDICINE AND MICROBIOLOGY/IMMUNOLOGY, UNIVERSITY OF MICHIGAN MEDICALCENTEg ANNARBOI~MI 48109, USA.

Genetic variation in pathogenic bacteria BRIAN D. ROBERTSONAND THOMAS F. MEYER In contrast to textbook ideas o f pure cultures and defined strains, genetic variation is a fact o f life in the microbial world It not only allows pathogens to establish themselves in their chosen host, but also allows them to resist that host's subsequent attempts to evict thent Here we review some o f the mechanisms that bring about this variation, and some o f the functional consequences that result f r o m it.

emphasizing different aspects of this subject, have been published1, 2.

Functional consequences of variation Genetic variation as a means o f escaping the i m m u n e response

The function of the immune system in higher organisms depends on extensive genetic rearrangements, which are crucial for maintaining the diversity of antibodies and the T cell receptor (see review by Gellert, this issue). One of the first examples of molecular variation described, for the African trypanosome (see Van der Ploeg et aL, this issue), is a response to this enormously diverse immune response. As far as is known, a single variable protein covers the entire surface of this pathogen, allowing it to avoid the host immune response. The spirochaete Borrelia hermsii, which causes relapsing fever, may also use antigenic variation primarily to escape the immune response. This bacterium produces large quantities of a variable lipoprotein, which could be sufficient for a complete surface coat. In contrast, the surface of N. gonorrhoeae is composed of both variable and conserved surface components, and the major invariant surface antigens are also targets for the host immune response. Therefore surface protein variation in the case of N. gonorrhoeae is unlikely to be a way of avoiding the immune response in the conventional sense. The fact that gonorrhoea is a persistent infection probably results

TIG DECEMBER1992 VOL. 8 NO. 12 ©1992 Elsevier S¢-ience Publishers Ltd (ILK)

I]EVIEWS from a number of pathogenic mechanisms, including antigenic variation.

Haemophilus influenzae lipopolysaccharide 1° and Bacillus thuringiensis toxinlL

Variation as a means of microenvironmental adaptation

Mechanisms of variation General homologous recombination

During the establishment and course of an infection, pathogens encounter a wide range of microenvironments. Each of these presents special problems in terms of the available receptors, the need for particular bacterial structures and the immune response mounted by the host. In N. gonorrhoeae, four factors involved in microenvironmental adaptation have Been described. The first indications that variation took place in gonococci came from observations of differences in colony morphology 3. A variety of different colonies were described that were either transparent or opaque to varying degrees, and that differed in shape. These morphological characteristics were correlated with changes in outer membrane proteins and the presence or absence of pili, proteinaceous filaments protruding from the cell surface. Pili are important for the establishment of an infection in vivo, initially anchoring bacteria to cell surfaces. Variation in pilin (PilE), the major subunit of pili, probably allows optimization of the bacteria-host cell interaction and certainly influences adhesion to epithelial cells 4, although the specific role of pilin in adhesion is unclear. However, pili seem to inhibit the invasion of epithelial cells by adherent gonococci, at least in vitro, and loss of pili by phase variation facilitates the invasion process 5. PilC is another phase variable gonococcal protein which plays a role both in the assembly of pili and in adhesion 6. The PilC protein that is expressed determines the type of pili assembled and whether or not the bacteria adhere to epithelial cells4. Proteins of a third type, those responsible for the opaque colony phenotype (opacity proteins, Opa), are also involved in the invasion of epithelial cells 5,7 and interaction with human leukocytes (Ref. 8; E-M. Kupsch, B. Knepper, T. Kuroki, I. Heuer and T.F. Meyer, submitted). Opa proteins undergo both phase and antigenic variation and are determinants of cellular tropisms. Variation of PilE, PilC and Opa proteins thus allows the fine tuning of intermolecular interactions that are important at various stages of the infection. Another type of variable surface component found in gonococci, lipopolysaccharide (LPS), has been shown to play a role in the ability of the bacteria to invade epithelial cells in vitro (J.P.M. van Putten and C.T.P. Hopman, submitted). Gonococcal LPS can be sialylated in vivo and in vitro using cytidine 5'monophospho-N-acetyl neuraminic acid (CMP-NANA) as substrate, conferring serum resistance on the bacteria9. When this is done in vitro not only are the organisms resistant to killing by normal human serum, but otherwise invasive bacteria are rendered noninvasive. However, spontaneous LPS variants arise that bind decreased amounts of sialic acid and that can still invade epithelial cells, even in the presence of CMPNANA. Thus LPS variation aids the gonococcus in adapting to its environment. Variation in a number of other pathogens also has direct functional consequences; examples include

Homologous recombination is widely used to generate molecular variation in bacteria and depends on the RecA function. RecA protein promotes the annealing of single-stranded DNA to any complementary sequences in double-stranded DNA, displacing one of the original strands. Preliminary evidence that phase and antigenic variation in the pili of N. gonorrhoeae (reviewed in Ref. 12) might result from intragenic recombination came from sequencing pilin transcripts from variants expressing different types of pilin 13. It was then demonstrated that recombinational exchange occurred within the pilin expression locus pilE, and involved the transfer of variable sequences from a repertoire of non-expressed or silent loci (pilS) to the expression locus TM. This finding was subsequently extended by other investigations15,16. The exact mechanism by which this recombination occurs is still a matter of contention (see below), since gonococci can naturally take up species-related DNA released by spontaneous cell lysis, and could therefore make use of exogenous pilin sequences. Not only are there more than a million possible combinations of pilin sequences leading to the production of antigenically variant pili, but the production of variant pilins also causes pilus phase variation. Revertible nonpiliated phase variants commonly fall into two groups. When propilin is cleaved at amino acid 40 rather than position 1 after the leader peptide, the resulting pilin molecule is incompatible with pilus assembly and is secreted into the medium as soluble or S-pilin 17. This alternative cleavage seems to depend on the PilC protein produced 6. The second group of nonpiliated variants results from the production of very long or L-pilin molecules, which do not form pili but instead are found in the periplasm or outer membrane of the cell. Such L-variants are the product of a recombination between pilS and pilE that leads to a tandem multiplication of the number of copies of pilS in the expression site. When the intervening pilS sequences are in frame this leads to long, often unstable, pilin molecules. L-variants can revert to the piliated phenotype if the tandem copies of pilS within the pilE gene are deleted or replaced. However, similar deletions can lead to the irreversible loss of the pilE gene. Borrelia hermsii uses a mechanism similar to that of gonococci and trypanosomes to alter its variable major proteins (Vmp). There are at least 27 variant vmp genes, mostly promoterless silent copies on linear plasmids. They are expressed after a recombination event that transfers a copy of the silent gene to a single telomeric expression site on another linear plasmid TM. The gene that was originally expressed is lost during this process, but how this occurs remains unresolved. In Bacillus thuringiensis, new toxin specificities are produced by recombination between and within regions of the toxin genes n, while in gonococci, recombination events between opa genes

TIG DECEMBER1992 VOL.8 NO. 12

[]~EVIEWS

Case A

Case B

before

after

I

before I

,i~iiiiiiiiiiiiiii~=

I

>

> I

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reciprocal recombination 2 or 3

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I

=

probeX

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FIGI! Possible pathways of recombination between pilS and pilE loci. In the upper panel are schematic representations of Southern blots of chromosomal DNA isolated from an isogenic strain before and after two p/l-associated recombinational events (A and B). These have been probed for sequences X and Y, which are contained in pilS and pilE loci respectively. The lower panel shows the possible recombination pathways that could produce the observed hybridization patterns. Case A has the typical hybridization pattern associated with a reciprocal recombination, as could result from pathway 1. On first inspection Case B appears to be the result of a nonreciprocal recombination or gene-conversion event. However, such a pattern could also result from 'hidden' reciprocal recombinations. In pathway 2 there is reciprocal recombination between opposite strands of a replicating chromosome, resulting in two different chromosomes within a single cell. In pathway 3 the reciprocal recombination occurs between chromosomal DNA entering the cell by transformation and the resident chromosome. This leaves the resident pilS copy containing sequence X intact, while exogenous DNA carrying X recombines with pilE. Since variants are selected for changes in pilE, changes in p/lS are not detectable if they involve only the opposite strand. Nothing is known about which of these pathways is preferred at any one time, and the relative positions of the participating pilS a n d pilE loci and the origin of replication add further complications. are a source of variation leading to the reassortment of the hypervariable regions19, 2°.

Gene conversion and the role of transformation in pilin variation: unresolved questions As mentioned above, pathogenic Neisseriae c a n take up species-related DNA and integrate it into their chromosomes by homologous recombination. The horizontal exchange of chromosomal markers and

virulence determinants can be demonstrated by cocultivating bacteria 21. Similar studies in vitro suggest that transformation plays a role in the exchange of pil sequences 22,23. When transformation is blocked, the pathway for certain pil gene recombinations shifts from being apparently asymmetric, to an intracellular reciprocal recombination process between silent and expressed copies 23. Therefore pilin variation occurs by asymmetrical exchanges in the presence of transformation 23, and by intracellular recombination in the absence of transformation 25-25. There are a number of indications (summarized in Ref. 21) that horizontal exchange of virulence factors has taken place in vivo, although this cannot be demonstrated directly and its importance remains controversial. The term 'gene conversion' has been used to describe the apparently asymmetric distribution of variable sequences in both silent and expressed loci u p o n pilin phase transition, as shown by Southern blot hybridizations 14-16. In our view, an asymmetric recombination mechanism for pilin variation is plausible but has not been proven. In contrast, there is evidence in gonococci for reciprocal recombination involving pil genes 23, and this could also account for what has been designated gene conversion on the basis of Southern blots: if reciprocal recombination involved, for example, separate chromosomes within a cell or two branches of replication intermediates, it would probably appear to be an asymmetrical event (Fig. 1).

Variation via repetitive protein domains

There are a number of examples of variation resulting from the gain or loss of repeated domains within a molecule. Streptococcal M protein (reviewed in Ref. 26) is highly diverse in size, with over 80 antigenically different serotypes. In serotype M6, the protein consists of amino acid repeats grouped into four regions. Size variation probably occurs when repeats are deleted by recombination between the tandemly repeated elements within the coding sequence. Mycoplasma hyorhinis expresses variable combinations of multiple, size-variant surface lipoproteins (Vlp) 27. Vlp size varies when repetitive sequences coding for the divergent extracellular domain are lost or gained. The immunodominant surface antigen of Anaplasma marginale is also polymorphic in size, and this correlates with variations in the number of tandemly repeated sequences near the 5' end of the gene 2s. In none of these examples is it k n o w n whether the changes occur by homologous recombination or a RecA-independent mechanism.

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VOL. 8 NO. 1 2

[~EVIEWS TAI3LE1. S u m m a r y o f m e c h a n i s m s u s e d to generate genetic variation in pathogenic bacteria

Component

Function or element involved

Ref.

N. gonorrhoeae B. berms,i B. thuringiensis

pilE vmp c~lA

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14-16 18 11

Variation via repetitive domains

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a ~ ?

28 27 26

Site-specific recombination (inversion)

S. typhimurium

Mechanism of variation

Organism

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h2

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N. gonorrhoeae N. meningitidis H. influenzae H. influenzae M byorhinis Y pestis B-pertussis N. meningitidis B. pertussis N. gonorrhoeae

Phase variation by DNA modification

E. coli

aSee van de Putte and Goosen, this issue. bS.M. van Ham. submitted. cAlso, M Achtman. pets. commun.

Site-specific recombination Many bacteria have surface appendages used for motility or attachment to surfaces, and these are often phase variable. In contrast to the general recombination mechanism used by gonococci for pilin variation, other organisms use site-specific inversion of a genetic element to switch genes encoding fimbriae (filamentous appendages) and flagella on and off (see review by van de Putte and Goosen, this issue). Examples include the flagellum of Salmonella typhimurium, mannose-sensitive fimbriae of E. coli 29 and pill of Moraxella bovis3O. Inversion in these systems generally requires both element-specific and common bacterial functions, such as integration host factor (IHF), factor for inversion stimulation (Fis) and the histone-like protein HU (see Table 1). The pill of M. bovis undergo a phase switch between two antigenically different pilins, I and Q (previously cz and J3), which is mediated by the inversion of a sequence whose endpoints lie within the coding region of the pilin gene. In one orientation the I sequences are adjacent to the promoter and contribute I-specific leader and amino-terminal sequences30. In the other orientation, the Q-specific sequences are in the expression locus next to the promoter and provide the leader and amino-terminal amino acids. In at least one strain of Moraxella lacunata, the nonpiliated state resulted from an imperfect inversion leading to a frameshift mutation31; but in M. bovis, switches from piliated to nonpiliated cells can occur without inversions (Carl Marrs, pers. commun.) and the means by which the nonpiliated phenotype has been generated remains unknown.

Phase variation via short repeats Short repeated motifs are used in some bacteria to alter the expression level of a protein. These repeats occur upstream or at the 5' end of genes, and affect either transcription (via the promoter) or translation (by altering the reading frame). Loss or gain of repeats is thought to occur by a process involving DNA replication, termed slipped-strand mispairing32 (Fig. 2). Genes encoding gonococcal Opa proteins exist in multiple copies scattered around the genome. Each copy includes 5' repeats of CTCTI', which encode the hydrophobic core of the leader peptide. Tile translational frame of the gene is determined by the number of repeats, in turn determining the phase status of the protein 19. Slipped-strand mispairing was proposed to be responsible for altering the number of repeats, and is RecA independen#3. In vitro experiments by Belland34 indicate that the opa repeat region can form triple-stranded H-DNA, where a stretch of repeats forms an antiparallel alignment with the opposite strand, exposing a stretch of single-stranded DNA, which is a likely target for single-strand-specific nucleases. Single-strand breaks may initiate a repair process during which the number of repeats could vary (Fig. 2). DNA structures such as H-DNA, inverted repeats or AT-rich regions, which favour singlestranded conformations under high negative superhelical tension, may support this process and also explain the probable influence of superhelicity on the switching frequency. The LPS of H. influenzae is antigenically variable. This can be described in terms of the phase variation of monoclonal antibody-defined epitopes, some of

TIG DECEMBER1992 VOt. 8 NO. 12

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tains, in addition to phase variable Opa proteins35, another variable outer membrane protein (Opc) whose expression is controlled differently36. New evidence in addition to the published sequence indicates that the opc gene has a tract of 10-14 cytosines between the -35 and -10 boxes. The number of cytosines correlates with the level of expression of the protein, presumably by an effect on the efficiency of transcription (M. Achtman, pers. commun.). The proteins encoded by the bvg locus of Bordetella pertussis are required for the expression of virulence-associated genes. Bordetella pertussis also varies between virulent and avirulent phases, and for at least one strain this correlates with a reversible frameshift mutation in a poly C tract in the bvgS gene37. Fimbrial transcription is additionally controlled by the insertion or deletion of nucleotides in a run of 15 cytosine residues within the promoter region of the tim gene ~s. The genus Yersinia provides a special example of changes in a polynucleotide region associated with increased virulence. The I7. pestis yopA sequence only differs from that of the less virulent Y. pseudotuberculosis by 15 nucleotides, including a single base deletion in a poly A tract, causing a frameshift mutation39. If Y. pestis is made YopA +, then its virulence is reduced. Other examples of RecAindependent recombination via short repeats are listed in Table 127,37-39.

Genetic variation and gene regulation: the interplay between two fundamentallydifferent processes

FIGE Superhelicity-dependent single-strand breaks as initiators of repeat unit variation. AT-richregions that are able to form cmcfformsor triple-stranded H-DNAmay expose single-stranded DNA under conditions of negative supercoiling. Nicking of this single-stranded DNA would allow its degradation by exonudeases. Following degradation the gap would be repaired by DNAsynthesis, during which mispairing could occur, leading eventuallyto the deletion (or addition) of one or more repeats. which lead to variations in LPS size. Two loci have been identified (licl and lic2) that are involved in this variation 1°. The licl locus contains four genes and is responsible for expression of two structurally related monoclonal antibody-defined epitopes. The first gene in the locus, licA, has 5' tandem repeats of CAAT, and is essential for the phase variation of the epitopes but not their synthesis. Alterations in the number of repeats is RecA independen0 ° and correlates with three levels of expression of the epitopes. Thus the translational frame of licA determines the level of expression of the epitopes and therefore their phase variation. Homopolymeric repeats are used by a number of organisms to control the transcription and translation of genes. Deletion or addition of single nucleotides in these sequences probably occurs during DNA replication. The gonococcus also provides us with an example of this phenomenon, which controls phase variation of PilC 6. There are two variant copies of this gene on the chromosome of strain MS11. The part of the pilC gene encoding the signal peptide contains a poly G tract, and deletions or additions in this region cause frameshifts with respect to the start codon, determining translation 6. Neisseria meningitidis con-

Two principles underlie the mechanisms that control gene expression in bacteria. Sensing, by which organisms respond to signals such as pH, temperature and the presence or absence of particular nutrients or chemicals, forms the basis of gene regulation. This allows bacteria to optimize gene expression for any given environment or stage of development, and tends to lead to a phenotypically homogeneous population. This review has concentrated on the other principal mechanism, genetic variation (summarized in Table 1). However, in the real world these principles do not operate in isolation, since both the frequency and direction of switching can be regulated, for example by DNA supercoiling 4°, methylation 41 or environmental changes 42. In turn, phase variable proteins may regulate other genes. For example, the phase variable products of the B. pertussis bvg locus regulate a number of virulence factors including the filamentous haemagglutinin, pertussis toxin and fimbriae. BvgA and BvgS are thought to form a two-component sensor-regulator system that modulates virulence in response to environmental stimuli. Therefore, nonrandom regulatory control mechanisms are often superimposed on random genetic variation, allowing the organism to coordinate its survival strategy.

Acknowledgements We thank our colleagues for communicating data prior to publication and for critical comments.

References 1 DiRita, v.J. and Mekalanos, JJ. (1989) Annu. Rev. Genet. 23, 455-482

TIG DECEMBER1992 VOL.8 NO. 12

tm

]REVIEWS 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

17 18 19 20 21

22 23

24

(1992) Proc. Natl Acad. Sci. USA 89, 5366-5370 25 Swanson, J., Morrison, S., Barrera, O. and Hill, S. (1990) J. Exp. Med. 171, 2131-2139 2 6 Scott, J.R. (1990) The Bacteria XI, 177-203 2 7 Yogev, D., Rosengarten, R., Watson-McKown, R. and Wise, K. (1991) EMBOJ. 10, 4069-4079 28 Alh-ed, D.R. el al. (1990) Prec. Nall Acad. 3c~. &3A b7, 3220-3224 29 Abraham, J.M., Freitag, C.S., Clements, J.R. and Eisenstein, B.I. (1985) Proc. Natl Acad. Sci. USA 82, 5724-5727 3 0 Marts, C.F. et al. (1988) J. Bacteriol. 170, 3032-3039 31 Rozsa, F.W. and Marrs, C.F. (1991)J. Bacteriol. 173, 40O0-4006 3 2 Levinson, G. and Gutman, G.A. (1987) Mol. Biol. Evol. 4, 203-221 33 Murphy, G.L. et al. (1989) Cell 56, 53%547 34 BeUand, R.J. (1991) Mol. Microbiol. 5, 2351-2360 35 Stern, A. and Meyer, T.F. (1987) Mol. Microbiol. 1, 5-12 36 Olyhock, AJM. el al (1991).lltcr~/~ l'