Phase and Antigenic Variation in Bacteria

Colony Morphology and Opacity

Historically, phase-variable structures have been recognized by their effect on colony morphology that led to descriptions like dry versus moist, ruffled versus smooth, and opaque versus translucent. These changes in colony morphology can be attributed to phase variation of a variety of surface-exposed proteins, of the capsule, and of cell wall composition. These changes most probably lead to altered packing of cells in the colony, which determines the colony morphology. This relationship was directly shown for Vibrio parahaemolyticus with variable levels of capsule production (92).

In Haemophilus influenzae type b strains, three colony variants, opaque, intermediate, and translucent, have been found. These vary in important virulence properties like colonization in the nasospharynx and serum resistance. The colony phenotype appears to be a result of specific combinations of expression of multiple phase-variable proteins, which include variation in the level of capsule production and in the level of a cell envelope protein encoded by oapA (244, 281, 296, 388) (Table 1). A clear relation between colony phenotype and colonization or virulence also exists in Streptococcus pneumoniae (172, 387). In an animal model, opaque variants were more virulent on systemic infection whereas the translucent variants were more successful colonizers of the nasospharynx. The two variants also differed in an in vitro assay of invasion and transcytosis of endothelial cells. Opaque variants produced up to sixfold more capsule and twofold less teichoic acid compared to the transparent form (292). Similarly, Streptococcus gordonii colony morphology and virulence-associated properties, including hemolysin production, phase vary (163, 374). In contrast, in Helicobacter pylori, a variable-colony phenotype is a result of phase variation of expression of phospholipase A, which indirectly affects virulence by release of urease and VacA (347, 348) (Table 1). In the pathogen Salmonella enterica serotype Typhimurium, variable colony morphology is correlated with the coordinated control of phase variation of at least four proteins (153). However, the soil isolate Pseudomonas aeruginosa also showed variable colony morphology, which was correlated with phase variation of multiple traits including aggregation and motility (71).

Color variation in colonies grown on specific media can be caused by phase variation of proteins that interact with a dye. For example, in certain strains of Staphylococcus epidermidis, phase variation of a polysaccharide adhesin leads to variable colony color when grown on Congo red agar (419, 420) (Table 1; also see “Molecular mechanisms of phase variation” below). In summary, any reversible change in colony morphology, opacity, or color indicates that the expression of one or more proteins phase varies. Further analysis of these and related phenotypic variations may provide us with new insights into bacterial survival strategies, and the strong correlation between virulence and colony morphology in pathogens suggests that characterizing the underlying molecular basis may also provide valuable insights into bacterial pathogenesis.

Capsule

The capsule can influence interactions with the host cells and host environment, including invasion, adhesion, and serum sensitivity, and is a well-recognized virulence factor. Phase variation of capsule synthesis can involve a classic on/off switch but also has been used to describe modulations in the level of production; as mentioned above, this can change colony morphology. Capsule variation has been found to occur in both gram-positive and gram-negative bacterial species, including Campylobacter jejuni (9), Citrobacter freundii (267), S. pneumoniae (379), and specific serogroups of Neisseria meningitidis (121, 122) (Table 1). In Bacteroides fragilis, eight different capsule polysaccharides can be produced per cell. The expression of each is under the control of on/off phase variation, resulting in a very diverse clonal population (183a). In H. influenzae type b cells, the level of expression of the capsule can be modulated, and an irreversible switch to a nonexpressing phenotype can occur (see the sections on recombination [below]) (reviewed in reference 297).

Fimbriae and Pili

Fimbriae, also referred to as pili, are proteinaceous structures that extend from the cell surface. Fimbriae assembled by the chaperone/usher-dependent and the nucleator/precipitation pathways are distinct in both structure and function from type IV pili (reviewed in references 96, 227, 306, and 381). Fimbriae mediate adhesion of the bacterial cell to host tissue through interaction with receptors located on the host cell (reviewed for Escherichia coli in reference 209). These interactions, which occur either with the main structural subunit or with a fimbrial adhesin, are often specific, so that certain chemical groups of the host proteins or lipids are recognized by the fimbrial adhesin. Fimbria-mediated attachment to inorganic, solid surfaces most probably occurs by nonspecific interactions, an important feature in biofilm formation (263, 282). Phase variation of fimbriae is regulated mostly by mechanisms that affect transcription originating at the major promoter of the operon, resulting in variable (on/off) expression of most or all genes in the fimbrial operon (209).

A single species, or even a single isolate, can express multiple fimbriae that each can phase vary. The genomes of different pathogenic E. coli isolates encode different fimbriae, some of which phase vary, as well as type 1 fimbriae, which is common to all isolates and which phase varies (Table 1). The S. enterica serotype Typhimurium genome encodes at least 11 fimbrial operons, among which phase-variable expression has been identified for pef, lpf, and fim (53, 146, 251, 256) (Table 1). The biological role of phase variation of fimbriae is discussed in “Biological significance of phase variation” (below).

Fimbrial phase variation control mechanisms and the fimbrial structural genes may have evolved as separate modules. For example, the common feature of the pap-like family of fimbrial operons is the regulatory mechanism of phase variation, but the fimbrial structural genes within this family are not all related. Conversely, the subunit of the MR/P fimbriae in Proteus mirabilis resembles that of Pap fimbriae in E. coli, but the two phase variation control mechanisms are different (128, 202) (see “Molecular mechanisms of phase variation” below) (Table 1).

Type IV pili function as adhesins and include conjugative pili. Phase variation, antigenic variation of the structural subunits, and phase-variable modification have been described. Sequence variation in the type IV conjugative pili encoded by plasmids R64, R721, and ColI-P9 occurs as a result of incorporation of only one of a set of distinct C termini in the PilV tip proteins of the pilus in an individual cell. This sequence variation is associated with different receptor specificity, thereby dictating the species that will be preferred as a DNA recipient in a conjugation reaction (154, 177, 178). N. gonorrhoeae can theoretically produce over a million different, antigenically distinct pilin subunits for its type IV pili (99, 180, 240, 319, 346; reviewed in reference 12) (see “Molecular mechanisms of phase variation” below) (Table 1). In addition, the pilus-associated protein PilC phase varies (164) (Table 1). These pili are involved in interaction with eukaryotic cells, and thus these variations are probably important for pathogenesis (28, 312). In S. enterica serotype Typhi, phase-variable expression of the PilV subunit of the type IVB pili affects the pilus-associated property of cellular autoaggregation (243) (see “Molecular mechanisms of phase variation” below). Pili can also be modified, but whether modification occurs can vary within a clone, due to phase variation in the expression of one of the enzymes involved. This is the case for glycosylation by PgtA in N. gonorrhoeae (10) (Table 1).

Flagella

Flagella mediate bacterial motility; adhesion and virulence are sometimes enhanced by flagellar expression and motility (265). Flagellin is also a pathogen-associated molecular pattern recognized by the innate immune system through Toll-like receptor 5 (204). The antigenic property of the flagellin of a bacterial isolate forms a significant part of the serological classification scheme, indicating that this is, in general, an invariant property. However, phase and antigenic variation of flagella in a clonal population does occur. As early as 1922, variation of the flagellar antigens in S. enterica serotype Typhimurium was described by Andrewes (6). This consists of variation between two forms (biphasic antigenic variation), H1 and H2, in which the flagellar subunit consists of the FliC or FljB protein, respectively (35, 326) (see “Molecular mechanisms of phase variation” below). Mutants that no longer can switch between flagellar types were altered in virulence compared to the wild-type isolate (150), and the two flagellar subunits appear to elicit different responses from eukaryotic cells (46).

In Campylobacter coli (272), Campylobacter jejuni (106, 169), and Helicobacter pylori (264), flagellar expression and motility phase vary (41, 73, 165) (Table 1). The underlying reason for this can differ. For example, C. coli expression of FlhA, which is required for the expression of flagellin, phase varies (272), whereas in H. pylori, expression of the fliP gene, encoding the flagellar basal body, phase varies (165) (Table 1). In Bordetella pertussis, phase-variable expression of the regulatory system BvgAS results in flagellar phase variation (340) (Table 1) (see “Regulatory proteins” below). In this species, flagellar synthesis is not required for virulence and may even be detrimental (2).

Other Surface-Exposed Proteins

Proteins that are integrated in the cell wall in gram-positive organisms or in the outer membrane in gram-negative organisms can have a variety of functions; these proteins include transporters, porins, receptors, colonizing factors, and enzymes. Antigenic or phase variation has been found for at least one member of each of these functional groups (Table 1).

In the M1inv⁺ clone of the gram-positive group A Streptococcus (S. pyogenes), expression of the cell wall-associated surface proteins C5a peptidase, M protein, and type IIa IgG Fc receptor phase vary, as well as expression of the capsule and pyrogenic exotoxin (52, 195). This is in part due to phase-variable expression of the DNA binding, regulatory protein Mga (see “Regulatory proteins” below) (36, 233-235, 329). Expression of the collagen-like surface protein SclB is under the control of a separate phase variation control mechanism (285) (see “Molecular mechanisms of phase variation” below) (Table 1).

In gram-negative N. gonorrhoeae and N. meningitidis strains, expression of various outer membrane proteins phase varies, including that of members of the family of outer membrane opacity proteins (opa) that facilitate adhesion (339) (see “Biological significance of phase variation” below) (Table 1) and the porin PorA (class I outer membrane protein) in serogroup B N. meningitidis (Table 1). PorA is one of the candidates for a protein-based vaccine, and phase variation, as well as the naturally occurring antigenic variation of this protein, may affect efficacy (16, 51, 355, 365). In addition, in Neisseria spp., the expression of outer membrane proteins that are involved in iron acquisition phase varies, including the siderophore receptor FetA in N. gonorrhoeae (42) and two hemoglobin receptors in N. meningitidis DNM2 (201) (Table 1). This may reflect a need to balance iron acquisition during growth in the host and to evade the immune system. Phase-varying colonizing factors include Ag43 in E. coli (64, 270) (Table 1) and Oap H. influenzae (281, 388).

In Campylobacter fetus, which is a pathogen of domestic and wild animals, a class of proteins known as surface layer proteins (SLPs) are exported to the cell surface and are noncovalently attached to the lipopolysaccharide (LPS) (80). SLPs are important virulence factors, and the absence of SLP leads to increased sensitivity to complement activity and decreased infectivity (27). These SLPs undergo extensive antigenic variation, which is achieved by so called “nested DNA inversion” (81, 83, 84, 286; reviewed in reference 82) (see “Molecular mechanisms of phase variation” below) (Table 1). Other examples of phase variation of surface proteins in gram-negative bacteria are included in Table 1.

Mycoplasma species do not have a cell wall, and lipoproteins constitute part of the surface proteins. Many of these are under the control of phase and antigenic variation (19, 20, 26, 325, 407). This includes a substrate binding component of an ABC transporter in Mycoplasma fermentans (351). Interestingly, the pMGA family of hemagglutinins phase varies in Mycoplasma gallisepticum (207, 253, 401), whereas the homologous proteins in Mycoplasma synoviae undergo antigenic variation (254). Expression of the Vlp family of lipoproteins in Mycoplasma hyorhini undergoes both phase variation and antigenic variation (47-50, 299, 300, 408). The combination of these two regulatory systems and the fact that there is a family of six related Vlp proteins that each are subject to these controls lead to a large repertoire of Vlp proteins that can be expressed (409). In Mycoplasma species that are human commensals and pathogens, phase or antigenic variation is indicated for M. hominis (190) and M. penetrans (250, 301).

Perhaps the best-studied example of multiphasic antigenic variation is that of lipoproteins in Borrelia spirochetes that are the causative agents of relapsing fever (reviewed in references 12 and 13). These lipoproteins are divided in two groups, Vlp and Vsp for large and small proteins, respectively, and can be further divided into different families, each with about 70% sequence identity (see “Biological significance of phase variation” below). One of the mechanisms involves recombination between an extensive repertoire of silent, variable vlp and vsp loci (“archival loci”) and an expression site (reviewed in reference 12). The closely related protein VlsE in B. burgdorferi, the causative agent of Lyme disease, is also under the control of antigenic variation through a similar combinatorial variation (181, 232, 411) (Table 1).

LPS and LOS Modification: Variation in Expression of Surface Epitopes

In gram-negative bacteria, LPS is the main constituent of the outer leaflet of the outer membrane. LPS consists of a lipid A moiety, a core of polysaccharide, and an O antigen. LPS variability among species and serotypes occurs mainly in the O antigen, specifically in the identity and number of sugars in the polysaccharide chain. In some species, the core lacks the multiple O-linked saccharide units and is often therefore referred to as lipooligosaccharide (LOS) (283). LPS, also referred to as endotoxin, is a powerful stimulant of the immune system due to the lipid A moiety, which is a pathogen-associated molecular pattern recognized specifically by toll-like receptor 4 (25).

The chemical identity of LPS or LOS is defined by the addition of side groups, for example as a result of the activity of glycosyltransferases or sialyltransferases, or by the addition of phosphorylcholine (ChoP). These traits can vary within a clonal population as a result of phase variation of one or more enzymes involved in the modification. An in-depth review of the chemical nature of modification of the O antigen of LPS is presented in reference 199. LPS modifications can impact antigenicity but can also affect serum sensitivity and adhesion. This is discussed in more detail below for the LOS of N. meningitidis (see “Biological significance of phase variation”). Below, additional examples are given relating to several important pathogens. Since variable LPS modification is not easily identified, it is quite possible that it occurs in other species as well.

Ganglioside mimicry of the LOS by Campylobacter jejuni is thought to be an important factor in the development of Guillain-Barré and Miller-Fisher syndromes after infection and is associated with specific modification of the LOS. Expression of the enzymes involved in the modification can phase vary. Alternate synthesis of gangliosides GM-2 and GM-1, like LOS in C. jejuni NCTC 11168, is a result of phase variation of expression of β-1,3-galactosyltransferase encoded by wlaN (206); in C. jejuni strain 81-176, reversible conversion between the GM-2 and GM-3 LOS correlates with phase-variable expression of the cgtA gene (111) (Table 1). Based on our understanding of these phase variation mechanisms, Linton et al. were able to show that a different C. jejuni strain, which had been characterized as producing only GM-2 like LOS, was able to convert to producing GM-1 like LOS (206). This illustrates how an understanding of phase variation at the molecular level may have a significant impact on the understanding of the pathogenicity and epidemiology of a pathogen.

Another well-studied example of apparent host mimicry occurs in the human gastric pathogen Helicobacter pylori, which can incorporate into its LPS variable carbohydrate modifications that resemble structures of the Lewis group of antigens of human blood groups. This variation occurs distinct from, and in addition to, LPS microheterogeneity (reviewed in reference 8, 384). Three fucosyl transferase genes (futA, futB, and futC, also referred to as the fucT genes), which are each under the control of a phase variation mechanism, we involved in the LPS modification (7, 385) (Table 1). Other genes involved in LPS modification may phase vary as well (309).

In both encapsulated and nonencapsulated H. influenzae, modification can occur by phase-variable Lic3A or LgtC, a sialyltransferase and a glycosyltransferase, respectively (136, 137, 226, 391) (Table 1). Interestingly, these enzymes compete for the same lactose disaccharide moiety on LOS. Thus, the activity of one enzyme affects the substrate availability for the other. Phase variation of lgt-mediated glycosyl modification in N. meningitidis and N. gonorrhoeae is discussed in more detail below (see “Biological significance of phase variation”) (Table 1).

The LOS of H. influenzae can also be decorated with ChoP. Phase-variable expression of the kinase encoded by licA results in phase-variable ChoP decoration, even though other genes may also contribute (315, 394) (Table 1). The presence of ChoP appears to confer sensitivity to serum-mediated killing caused by C-reactive protein, whereas in an animal model of the nasospharynx, this may confer a competitive advantage (214, 392, 395). Genetic analysis of three genes of the lic locus was performed to determine the on or off state of genes involved in LOS modification in bacteria isolated from the nasospharynx, blood, and cerebrospinal fluid in an animal model. Tissue-specific combinations were prevalent, supporting the idea that certain combinations of LOS modification may facilitate colonization or survival in different host environments (141).

Among Neisseria species, phase-variable ChoP modification of the LPS can occur, but type IV pili also contain ChoP (389, 394). Analysis of ChoP expression in a large group of isolates suggests that the commensal isolates decorate the LPS whereas pathogenic isolates decorate the pili (321, 322, 389) (Table 1).

Other phase-variable LOS modifications in H. influenzae, as well as in the related bovine pathogen H. somnus, exist but have not been characterized further (151, 152, 155, 402). Antigenic or phase variation of LPS also occurs in certain Legionella pneumophila isolates (see “Molecular mechanism of phase variation” below) (212, 213) and in S. enterica serotype Typhimurium (188). Modification of LOS or LPS in Francisella tularensis (61), Coxiella burnetii (98, 117), and Chlamydia spp. (211) varies, but the contribution of a phase variation control mechanism versus environmental regulation or mutation has not been determined.

DNA Restriction-Modification Systems

DNA restriction-modification (R/M) systems allow a bacterium to recognize and restrict foreign DNA that is not appropriately modified. Expression of some of these systems phase varies, but protein sequence variation analogous to antigenic variation also occurs (also see “Biological significance of phase variation”) This was first identified in Mycoplasma pulmonis, a rodent pathogen. Recombination-dependent rearrangement occurs within the hsdS genes at two loci, each containing two copies of hsdS (85). The variable HsdS proteins that are synthesized from these recombinant genes each determine a different DNA sequence specificity for the DNA R/M system (84). In addition, inversion can result in incorrect orientation of the hsdMR genes relative to the promoter, and therefore on/off phase variation also occurs (86, 87, 331).

Sequence analysis of other bacterial genomes suggests that phase variation of (putative) DNA R/M systems occurs in a variety of species (66, 68, 307, 309, 354) (see “Genomics and phase variation” below). Confirmation of phase variation has been obtained for expression of the mod gene in H. influenzae (66), for a type III modification system in Pasteurella haemolytica (304), for both the modification and restriction enzymes of a type III R/M system in H. pylori (70), and for a modification system in S. pneumoniae (275, 349) (Table 1).

In the gram-positive soil bacterium Streptomyces coelicolor A3(2), the phage growth limitation system determines reversible sensitivity and resistance to φC31 phage. The complete molecular basis is not clear, but it is associated with phase variation of a DNA methyltransferase, PgIX (192, 344). Thus, some phase-variable DNA modification systems may have evolved as a protection mechanism against phage infection.

Regulatory Proteins

DNA binding proteins that function as activators or repressors can be categorized as “global” regulators with genome-wide target sequences or as “operon-specific” or “local” regulators. The expression of multiple regulatory proteins is now known to phase vary and includes representatives of both groups. The expression state of all genes that dependent on the regulator, under both positive or negative control, will depend on the expression state of the phase-varying regulator itself. Examples are the global, virulence-associated regulatory protein in S. pyogenes, Mga (formerly Mry or VirR) (36, 329), and the BvgS protein of the global, two-component BvgAS regulatory system in Bordetella pertussis (340; reviewed in reference 228) (Table 1). The Bvg⁺, Bvg⁻, or Bvgⁱ phase variants of B. pertussis of later studies are not related to this phase variation mechanism. Rather, these phase variants result from the environmental modulation of BvgAS-dependent regulation (60). Expression of conjugation-related genes phase varies in Enterococcus faecalis, also as a result of phase-variable expression of a regulator, TraE (124, 280).

Phase-variable expression of operons is often the result of a mechanism that regulates the initiation of transcription at the main promoter of the operon (see “Molecular mechanism of phase variation” below). As a result, expression of local regulators encoded by genes in the operon also phase varies. For example, in E. coli, expression of the local regulator PapB, encoded by the pap operon, phase varies (Table 1) (33). This not only affects pap expression through an autoregulatory loop, but also affects type 1 fimbrial expression (97, 403) (see “Cross regulation” below) (Table 1). Together, these examples demonstrate that phase variation of a single regulatory protein can lead to coordinated, phase-variable expression of multiple cellular proteins and can establish an interdependent network of phase-variable gene expression.

Metabolism-Associated Genes

Recently, phase variation of metabolism-associated proteins was identified in the human pathogen Streptococcus pneumoniae. A comparison of protein expression patterns between two colony variants showed that at least three proteins were differentially expressed, including pyruvate oxidase (SpxB), a putative elongation factor, and a proteinase maturation protein (268). The significance of SpxB phase variation appears to be related to the hydrogen peroxide that is produced in the pyruvate oxidase-mediated conversion of pyruvate to acetyl-phosphate. The level is sufficiently high to be lethal to other species and may provide a SpxB⁺ isolate with a competitive advantage in a mixed-species environment (275, 276). This example shows that identification of differentially expressed proteins between colony phase variants can also yield insight into bacterial virulence strategies.

Phage Genes

The composition of the tail fiber of E. coli phage Mu determines the bacterial host range of the phage, presumably in conjunction with strain- or species-specific bacterial LPS. The composition alternates between two forms as a result of a DNA inversion event mediated by the site-specific recombinase Gin (110, 279, 363). Phage Mu is not associated with virulence of E. coli, but some virulence factors are phage encoded, for example cholera toxin (reviewed in reference 39). It is therefore conceivable that phase variation will be identified in phage proteins that (indirectly) affect the spread of virulence traits among natural bacterial populations and therefore will be an important feature from an epidemiological standpoint.

Concluding Remarks

Most of the genes that are known to undergo phase-variable expression encode surface-exposed proteins or proteins that modify or regulate surface proteins, and most of the putative phase-varying genes identified in genome-wide screens also belong to these classes (Table 1). However, in this section we have presented numerous examples showing that phase variation is not limited to proteins with a specific function or specific cellular location. Most phase-varying proteins were identified in bacterial pathogens of mammalian hosts, and many are proteins that affect virulence. This bias may reflect the significant focus of the research community on these organisms and does not rule out the occurrence of phase variation in commensal species or species that do not reside in or on a host. It is thus tempting to speculate that the occurrence of phase variation is more prevalent. Novel phase-varying proteins may be identified as a result of our increasing understanding of the role(s) of phase variation and through the facilitated identification of some of the underlying mechanisms using genomics. Conversely, as more phase-varying genes are identified, we will be challenged to determine their biological significance.

Genetic Regulation

In this section the mechanisms are discussed in which the change in expression phase in an “on” cell and an “off” cell can be attribute to a DNA sequence change at a specific locus. All antigenic variation is a result of DNA sequence change and is due to one of several genetic mechanisms; this is also briefly discussed in this section. The change can be minor, with a single nucleotide change in the case of slipped-strand mispairing (SSM), or extensive, involving DNA rearrangements of fragments up to several kilobases. Phenotypic variation as a result of DNA rearrangement, irrespective of molecular mechanism involved, has been referred to as a shufflon (177).

Epigenetic Regulation

In contrast to the mechanisms described above, epigenetic regulation of phase variation occurs in the absence of a change in DNA sequence. Instead, it involves differentially methylated sequences in the regulatory regions of the phase-varying gene or operon. Since the expressed state is heritable but reversible and since regulation is not caused by a DNA sequence change, it is by definition epigenetic. The essential feature of the DNA methylation-dependent phase variation systems is that the methylation state of a target sequence at a specific site in the chromosome affects the DNA binding of a regulatory protein that directly regulates transcription. Expression of the DNA methylase itself does not undergo phase variation. Methylation-dependent phase variation has been identified in E. coli and S. enterica serotype Typhimurium. An epigenetic mechanism was first elucidated for phase variation of expression of the pap operon encoding pyelonephritis-associated pili in E. coli and is now known to regulate phase variation of a family of pap-like fimbrial operons in these species and of the outer membrane protein Ag43. The essential features of these two epigenetic phase variation systems are outlined below.

Phase Variation as Part of the Cell's Regulatory Network

Concluding Remarks

Regardless of the mechanism, per cell the switching event of phase variation is a stochastic event and thus is random. However, our increased understanding of the molecular mechanisms underlying phase variation is emphasizing that phase variation and the regulatory systems that determine the general physiological state of the bacterium are not separate processes and that the switch frequency can be modified by specific growth conditions or physiological states. Furthermore, most systems appear to require DNA synthesis, either for DNA repair or for DNA replication, and this also establishes a link between a change in expression phase and the physiological state of the bacterium. Thus, perceiving phase variation as a random, nonregulated process may lead to incorrect conclusions when addressing the significance of phase variation. Furthermore, some phase variation systems can affect the expression of other, nonrelated cellular proteins. Finally, it is clear that the expression of certain genes that undergo phase variation can enhance survival. How the different mechanisms, environmental regulation, and the gene products each contribute to the success of the bacterial population largely remains to be determined. However, this, together with ongoing efforts to identify environmental signals and regulatory networks that influence phase variation, will probably contribute to our understanding of the global changes that allow bacteria to adapt and survive in a changing environment.

Persistence through Surface Variation

Variation of all major surface antigens is a hallmark of pathogens persistently colonizing mucosal surfaces, such as N. gonorrhoeae (gonococcus), N. meningitidis (meningococcus), and H. influenzae. Here, expression of surface structures is altered by a combination of antigenic variation and phase variation mechanisms. The resulting surface variation has been proposed to allow immune evasion during chronic or recurrent infection and/or the generation of variants with altered ability to colonize niches in the host (67, 245, 346). As discussed below, these conclusions are based mainly on in vitro models while studies using the natural host provide little support for preferential colonization of niches by antigenic variants and are often not designed to monitor the evasion of adaptive immune responses over time.

One prototypical example of an organism persistently colonizing mucosal surfaces is the gonococcus, a strictly human-adapted pathogen. Gonococci can persist in the human population because they are able to cause chronic infections of the mucosa in the genitourinary tract, thereby ensuring continued transmission within the population even if the opportunity to encounter a new susceptible host may be relatively rare (as is the case for sexual transmission). Since gonococci do not survive in the environment, a large part of their genome is devoted to encoding factors necessary for survival in their niche, the genitourinary tract (249). Colonization of the mucosa of the genitourinary tract leads to a local inflammatory response that is rarely followed by bacteremia. One mechanism used by gonococci to persist in the genitourinary tract is thought to be the evasion of adaptive immune responses directed against antigens displayed on their surface. The variation of surface structures is mediated by a combination of antigenic variation and phase variation mechanisms to alter the composition or expression of prominent surface antigens, including type IV fimbriae (10, 118, 318), LOS (11, 63), colony opacity proteins (Opa) (23, 246) and other outer membrane proteins (42, 43, 58).

One challenge of constantly altering the antigenicity of the bacterial surface for the purpose of immune evasion is to maintain functionality of surface structures required for the host-pathogen interaction. It is easy to envision how this problem can be solved through antigenic variation because this mechanism can generate variants with altered immunodominant epitopes while conserving functionally important parts of the structure. Antigenic variation of the major subunit of gonococcus type IV pili is thought to be a good example of this strategy. Type IV pili are expressed during infection, and antigenic variation is observed in volunteers, although this experimental model is not well suited to studying the contribution of an adaptive immune response because antibiotic treatment is initiated within 4 days of infection (320). Antigenic variation of the major fimbrial subunit generates variation of epitopes exposed at the filament surface while leaving sequences required for its assembly unaltered (96, 271). This mechanism has little effect on the biological function of pili because adherence is mediated by a minor pilus subunit encoded by the pilC gene (303). Antigenic variation is thus expected to enable gonococci to evade an immune response directed against the major pilus subunit while maintaining its ability to interact with host cells through type IV pili.

It is less obvious how functionality can be conserved when a phase-variable on/off switch is used to alter the expression of surface antigens. Phase-variable expression of a surface structure that is important for host-pathogen interactions, such as an adhesin, presents the pathogen with a problem. That is, once the host has mounted a specific adaptive response to the adhesin, those cells expressing it (i.e., phase “on” variants) will be cleared by immune mechanisms. Those cells not expressing the adhesin (phase “off” variants) will also be cleared because the adhesin is required for colonization. At a first glance, phase-variable expression of surface structures essential for the host-pathogen interaction therefore appears not to be suited for immune evasion during chronic infection. One possible solution of the problem of using phase variation for immune evasion without losing the ability to adhere to or invade tissue is revealed by the analysis of Opa expression in gonococci. Here, phase variation appears to be paired with functional redundancy of a family of outer membrane proteins to allow the organism to persist in the mucosa of the genitourinary tract. Gonococci can express up to 12 different Opa proteins, each encoded by a gene whose expression shows phase variation as a result of an SSM mechanism (67). Opa phase variation results in a heterogenous bacterial population containing cells expressing no, one, or multiple Opa proteins. Most Opa proteins bind to host cell receptors of the CD66 family (CD66a, CD66c, CD66d, and/or CD66e) (38, 44, 45, 108, 109, 375), while one variant binds heparan sulfate (373). Although the receptor specificities of most Opa proteins are similar, phenotypic differences resulting from Opa phase variation can be detected in vitro during the interaction of gonococci with epithelial cell lines or with professional phagocytes (67). It has therefore been speculated that the role of Opa phase variation may be the generation of variants that differ in their ability to colonize a particular niche in vivo. A correlate of the postulate that a particular combination of Opa proteins may be required for colonization of a particular niche in the host is that this Opa combination would be selected for and would be isolated preferentially from this niche during infection. Volunteer studies show that while the majority of isolates from infected subjects express one or more Opa proteins, no Opa protein is preferentially expressed during early infection in the male urethra (159, 345). These in vivo results do not support the view that Opa phase variation functions in the generation of variants with different fitness for colonizing a niche. Instead, these studies support the view that Opa proteins mediate overlapping or redundant functions in vivo and that these functions are required during initial colonization of the urethra (159, 345). The overlapping or redundant function mediated by different Opa proteins is likely to be a tight adherence to epithelial cells via CD66 followed by transcytosis of gonococci into the lamina propria (249). The available evidence does not exclude the possibility that the principal role of Opa phase variation is the generation of a repertoire of strains, each expressing different combinations of surface antigens with similar function, thereby allowing newly arising phase variants to evade adaptive immunity while maintaining their ability to colonize the urethra. However, this hypothesis has not yet been tested experimentally in the natural host, because treatment of volunteers needs to be initiated before adaptive immunity fully develops. Thus, it remains to be seen whether the finite combination of gonococcal Opa proteins generated by phase variation is sufficient for immune evasion during chronic infection of mucosal surfaces.

Several other human-adapted pathogens appear to use strategies for persistent colonization of mucosal surfaces that are similar to those employed by the gonococcus. For example, N. meningitidis (meningococcus) and H. influenzae infect only humans and do not survive in the environment. The vast majority of individuals infected with these organisms are carriers in whom the nasopharynx is colonized asymptomatically. Similar to the gonococcus, the meningococcus and H. influenzae are both capable of altering all major surface antigens by phase or antigenic variation. These variable antigens include the capsule (122), LOS (167), type IV pili (158, 248, 352), opacity proteins (Opa and Opc) (170, 305, 339), and other outer membrane proteins (201, 290, 366) of meningococcus as well as LOS (296, 391, 392), fimbriae (372), opacity proteins (Oap) (388), and outer membrane proteins involved in iron acquisition (57) of H. influenzae. A well-studied example is the meningococcus LOS, which is required for colonization of the nasopharynx, as suggested by animal experiments (406). Analysis of meningococcal LOS biosynthesis provides an intriguing example of how phase variation (i.e., a heritable on/off switch of gene expression) can be used to generate antigenic variation (i.e., changes in the chemical composition) of a surface structure. In this case, expression of several genes (lgt genes) encoding glycosyltransferases shows phase variation (11, 156, 157). These glycosyltransferases are required for the expression of terminal LOS structures knows as α-chain and β-chain extensions. Variation of α-chain and β-chain extensions dramatically changes the antigenic properties of LOS, thereby forming the basis for its classification into 14 immunotypes, L1 to L14 (167, 220, 314, 421). In many meningococcal isolates, individual genes are missing from the lgt-1 locus (lgtABCDE) and/or the lgt-3 locus (lgtG), thus representing meningococci capable of expressing only a small subset (often 2 or 3) of the 14 known LOS immunotypes (157, 417, 418). It is difficult to envision how switching between a limited repertoire of LOS structures can be sufficient to allow continued immune evasion during chronic infection of the nasopharynx. A mechanism that may contribute to immune evasion is the concealment of antigens by LOS sialylation. The sialylation of α-chain extensions carrying terminal galactose residues (present in the LOS of immunotypes L2, L3, L4, L7, and L9) gives rise to LOS carbohydrate moieties mimicking carbohydrates present in glycosphingolipids of human cells (218, 219). The LOS sialylation is also thought to result in a general down-regulation of antibody and complement binding to the bacterial surface (287). In addition to a possible role in immune evasion, LOS variation has been postulated to be a mechanism that facilitates the adaptation of the meningococcus to environmental changes during its transition from the mucosa of the nasopharynx into the blood. This view is supported by in vitro studies showing that the sialylated LOS of immunotypes L3, L7, and L9 confers increased serum resistance but impairs invasion of nasopharyngeal epithelial cells compared to LOS of immunotype L8 (69, 242). Furthermore, an investigation of an outbreak caused by a meningococcus clone (ET-5 complex) capable of expressing six immunotypes (L1, L3, L7, L8, L9, and L10) showed that 97% isolates from patients but only 43% of isolates from healthy carriers expressed immunotypes L3, L7, and L9 (162). It has thus been postulated that colonization of the nasopharyngeal mucosa would select for meningococcus variants expressing a nonsialylated LOS (e.g., immunotype L8) while introduction into the bloodstream would result in selection for sialylated LOS phase variants (e.g., immunotype L3) (167, 236). This hypothesis would predict that isolates from multiple sites within an individual patient (i.e., from the blood and throat) are likely to differ in their immunotype if the strain possesses a repertoire of lgt genes that enables it to potentially express immunotype L8 (preferentially isolated from the throat) as well as immunotype L3 (preferentially isolated from the blood). However, a recent analysis of paired isolates (all capable of expressing L3 and L8) from the blood and the throat of individual patients showed no evidence of phase variation within individual pairs, thereby apparently refuting this hypothesis (24). In summary, compelling experimental evidence for any of the proposed in vivo roles of LOS phase variation in meningococci is still missing.

Evasion of Cross-Immunity

There are also several well-studied examples of phase-variable surface antigens in members of the family Enterobacteriaceae. In these organisms, the potential of phase-variable expression of surface appendages (flagella and fimbriae) for immune evasion is offset by the constitutive expression of LPS, a major surface antigen eliciting protective immune responses. Unlike the LOS of gonococci, meningococci, or H. influenzae, the LPS of the members of the Enterobacteriaceae carries terminal structures formed by repeating oligosaccharide units (O antigen) that are highly immunogenic. Constitutive expression of these invariant antigens on their surface illustrates that members of the Enterobacteriaceae show little potential for evasion of adaptive immune responses during an infection. As a result, these organisms are eventually cleared from host tissues by adaptive immune responses directed against invariant surface antigens and therefore generally cause self-limited infections that leave the host immune to reinfection. The strategies underlying host-pathogen interactions for the Enterobacteriaceae are thus fundamentally different from those employed by pathogens causing chronic or recurrent infections by evading adaptive immune responses through variation and concealment of all major surface antigens.

A prototypical example of a pathogen belonging to the family Enterobacteriaceae is Salmonella enterica, the organism in which the phenomenon of phase variation was first described (6). Based on polymorphisms in their O antigens and their flagellin proteins, S. enterica is subdivided into more than 2,000 serotypes. However, unlike meningococcal strains, which are capable of switching between different LOS immunotypes at high frequency, each S. enterica serotype expresses an invariant O-antigen type. As a result, after infection with a particular S. enterica serotype, a mammalian host acquires immunity against infection with S. enterica serotypes expressing the same or similar O antigens. This immunity is based largely on a response to immunodominant O-antigen epitopes that are traditionally used to classify S. enterica serotypes into serogroups. For example, the O-antigen repeat units of members of serogroups B and D1 possess an identical trisaccharide backbone (the O12 antigen) but have abequose (the O4 antigen) and tyvelose (the O9 antigen), respectively, as the immunodominant sugar branch (173). The immunodominance of the O4 and O9 antigens in LPS of serogroups B and D1, respectively, is illustrated by analyzing the serological response generated during infection with S. enterica serotypes Typhimurium (serogroup B) and Enteritidis (serogroup D1). Exposure of animals to S. enterica serotype Enteritidis elicits higher antibody titers against the O9 antigen than against the O12 antigen (14). Similarly, immunization with S. enterica serotype Typhimurium results in higher titers against the O4 antigen than against the O12 antigen (333). The importance of immunodominant O-antigen epitopes in eliciting immunity is convincingly demonstrated by challenge experiments performed with mice. Immunization of mice with a S. enterica serotype Enteritidis aroA mutant (expressing the O9 antigen) elicits protection against challenge with a virulent S. enterica serotype Enteritidis strain (expressing the O9 antigen) but not against a virulent strain of S. enterica serotype Typhimurium (expressing the O4 antigen). However, cross-protection between the two serotypes is observed when mice immunized with the S. enterica serotype Enteritidis aroA mutant are challenged with a virulent S. enterica serotype Typhimurium mutant that is genetically engineered to express the O9 antigen instead of the O4 antigen (139). O-antigen polymorphism is thus a mechanism that allows S. enterica serotypes to evade cross-immunity between members of different serogroups (i.e., between S. enterica serotypes expressing different immunodominant O-antigen epitopes) (173).

While S. enterica serotype Enteritidis and S. enterica serotype Typhimurium evade cross-immunity against LPS by O-antigen polymorphism, other surface structures are well conserved between the two organisms. For instance, fimbriae encoded by the lpf operon are composed of a major fimbrial subunit, LpfA, whose primary sequence differs by only one amino acid between the two serotypes (255). Recent evidence suggests that phase-variable expression of the lpf operon is required for evasion of cross-immunity between S. enterica serotypes expressing different immunodominant O-antigen epitopes. As outlined above, mice immunized with S. enterica serotype Typhimurium (serogroup B) do not possess cross-immunity against challenge with S. enterica serotype Enteritidis (serogroup D1) (139, 255). However, cross-immunity between S. enterica serotype Typhimurium and S. enterica serotype Enteritidis is observed if the challenge strain expresses the lpf operon constitutively (i.e., once lpf fimbrial phase variation has been eliminated by replacing the lpf promoter region with the lac promoter) (252). The finding that lpf phase “off” variants of S. enterica serotype Enteritidis can evade cross-immunity against S. enterica serotype Typhimurium suggests that they retain considerable virulence because they express alternate attachment factors (255). The genome of S. enterica serotype Typhimurium contains 13 fimbrial operons (231), and the synergistic effect of inactivating a subset of these operons provides evidence for redundant functions of the encoded adhesins during infection of mice (367). These data suggest that partial functional redundancy of the 13 fimbrial operons present in S. enterica serotype Typhimurium is a prerequisite for effectively using phase variation as a mechanism for immune evasion. The genetic makeup of fimbrial gene sequences in S. enterica serotypes is thus reminiscent of the phase-variable expression of functionally related Opa proteins in the gonococcus. However, while it is difficult to envision how the finite combination of Opa proteins generated by phase variation can enable the gonococcus to continuously stay ahead of an adaptive immune response during a chronic infection, evasion of cross-immunity poses a less complicated problem. For example, S. enterica serotype Enteritidis (expressing the O9 antigen) has to overcome cross-immunity against fimbrial proteins of S. enterica serotype Typhimurium (expressing the O4 antigen) only once (because the host subsequently mounts an adaptive response to its O9 antigen), a task that appears manageable by a phase-variable on/off switch for the individual fimbrial operons. In this context, the recent finding that each S. enterica serotype possesses a distinct repertoire of fimbrial operons appears relevant, because this genetic design probably facilitates evasion of cross-immunity against fimbrial proteins between different serotypes (357). Their role in evasion of cross-immunity provides an attractive explanation of why genomes of S. enterica serotypes contain large but distinct repertoires of fimbrial operons that are regulated by phase variation and exhibit partial functional redundancy. To understand the importance of fimbrial phase variation, it is thus necessary to review the biological significance of evading cross-immunity.

Evasion of cross-immunity appears to provide a significant selective advantage for S. enterica serotypes during coexistence in their animal reservoirs. A major source of direct competition between S. enterica serotypes occupying the same ecological niche (i.e., the intestine of a mammalian host species) is the host immune response, which may effectively limit the available resources (i.e., susceptible individuals suitable for transmission) (115). Mathematical models predict that cross-immunity between S. enterica serotypes which share immunodominant O antigen epitopes will result in competition, with the likely outcome being that the most successful (in terms of transmission success) serotype will outcompete the others in a particular host population (5, 173). However, S. enterica serotypes can coexist within a host population by evading cross-immunity through O-antigen polymorphism. Thus, the host immune response is predicted to reduce populations of different S. enterica serotypes sharing an immunodominant O antigen to a single strain, while coexistence of two or more serotypes in a host population is possible, if they evade cross-immunity by O-antigen polymorphism (i.e., by expressing different immunodominant O-antigen epitopes) (173). These theoretical predictions are in good agreement with the results of epidemiological studies showing that S. enterica serotypes causing the majority of cases of disease in a mammalian reservoir possess different immunodominant O-antigen epitopes (i.e., belong to different serogroups). For example, S. enterica serotype Dublin (serogroup D1) and S. enterica serotype Typhimurium (serogroup B) are associated with the majority of reported disease incidents is cattle (198, 336-338). S. enterica serotype Choleraesuis (serogroup C1), S. enterica serotype Typhimurium (serogroup B), and S. enterica serotype Dublin (serogroup D1) cause the majority of illnesses in pigs (88, 198, 336, 337). Furthermore, strictly host-adapted S. enterica serotypes causing typhoid fever coexist in the human population, and each expresses a different immunodominant O-antigen epitope. These include S. enterica serotype Typhi (serogroup D1), S. enterica serotype Paratyphi A (serogroup A), S. enterica serotype Paratyphi B (serogroup B), and S. enterica serotype Paratyphi C (serogroup C1) (173). These epidemiological data thus support the view that the host immune response to immunodominant O-antigen epitopes has shaped the population structure of S. enterica serotypes. The value of evading cross-immunity with a well-adapted competitor becomes clear when considering the alternative outcome. For example, S. enterica serotype Paratyphi A is human adapted and does not have an animal reservoir. If S. enterica serotype Paratyphi A expressed the same immunodominant O antigen as S. enterica serotype Typhi, the serotype with the greater transmission success (probably S. enterica serotype Typhi) would over time outcompete the other serotype in the human population. This would result in the elimination of S. enterica serotype Paratyphi A from its only reservoir and therefore would lead to its eradication. By expressing a different immunodominant O antigen (that of serogroup A), S. enterica serotype Paratyphi A is able to evade competition with the highly specialized S. enterica serotype Typhi (serogroup D1) and avoid eradication. The evolutionary cost of this strategy is that S. enterica serotype Paratyphi A has to compete with other S. enterica serotypes of serogroup A that circulate in the human population. The evolutionary gain (avoiding eradication) clearly outweighs the costs, since there is no other human-adapted Salmonella serotype of serogroup A that could provide stiff competition with S. enterica serotype Paratyphi A. In conclusion, evasion of cross-immunity may represent one of the driving forces for the regulation of fimbrial expression by phase variation and for the maintenance of O-antigen polymorphism among S. enterica serotypes (147, 173).

DNA Restriction-Modification Systems

Consistent with a role of phase variation in host-pathogen interactions, most phase-variable genes are predicted to be involved in the biosynthesis of surface structures. Notable exceptions are genes encoding R/M enzymes. Phase-variable expression of R/M enzymes has been found in a variety of bacterial pathogens, including S. pneumoniae (349), M. pulmonis (86), H. pylori (309), P. haemolytica (304), and H. influenzae (66). However, the significance of R/M enzyme phase variation is currently unclear. Phenotypic switching of putative R/M enzymes in H. pylori is induced on contact with human gastric cells (76). In M. pulmonis a high level of R/M enzyme (HsdS) variation is induced in vivo during infection of the rat trachea (112). These observations appear to support a role for R/M enzyme variation during host pathogen interaction. This interpretation appears less convincing in light of recent data demonstrating that HsdS variation and phase variation of the M. pulmonis variable surface protein (VsaA) are mediated by the same recombinase (HvsR) (330). The link between HsdS and VsaA phase variation in M. pulmonis suggests that isolation of variants in vivo may result from selection for surface antigen variants, not R/M enzyme variants. Thus, the significance of R/M enzyme variation for host-pathogen interactions, if any, remains to be determined.