The Bordetella pertussis model of exquisite gene control by the global transcription factor BvgA

Correspondence Kimberly B. Decker deckerkb@mail.nih.gov Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA


Introduction
Bordetella pertussis, the causative agent of whooping cough (pertussis), was first isolated from an infected person in 1906 (Bordet & Gengou, 1906).The small Gram-negative aerobic coccobacillus is an obligate human pathogen and thus has no known environmental reservoir.Vaccination programs have contributed to a substantial decrease in pertussis incidence, from ~260 000 cases in the USA in 1934 to ~1000 cases in 1976 (CDC, 1995).However, the increase in incidence since the early 1980s -including 27 550 cases in 2010 (CDC, 2011) -makes pertussis the most prevalent vaccine-preventable disease in industrialized countries (Mooi et al., 2009).
Whooping cough is resurging in countries with a historically low incidence attributable to high vaccine uptake (CDC, 1995;de Melker et al., 2000;Kerr & Matthews, 2000).The hallmark of this resurgence is a shift in prevalence from young children to adolescents and adults (discussed by Halperin, 2007).Multiple explanations have been offered for the reemergence of pertussis.These include an increased awareness of the disease, improved laboratory diagnostic tools, suboptimal vaccines and decreased vaccination coverage in parts of the world (Gangarosa et al., 1998).
At first glance, the persistence of pertussis despite intense vaccination efforts is unexpected because B. pertussis is highly clonal and lacks the genetic diversity of many other pathogens (Caro et al., 2006;Diavatopoulos et al., 2005;Parkhill et al., 2003;van Loo et al., 2002).Differences between B. pertussis clinical isolates are mainly due to differential expression of genes for surface-expressed proteins, mutations in genes for secreted proteins and gene reduction mediated by insertion sequence elements (Brinig et al., 2006;Caro et al., 2006;Heikkinen et al., 2007).In fact, among the major Bordetella subspecies, including Bordetella parapertussis, which causes a typically milder respiratory disease in humans, and Bordetella bronchiseptica, which infects many four-legged mammals, as well as B. pertussis, phenotypic differences have not been attributed to pathogenicity islands, plasmids, transposable elements or insertions from phage genomes.This Downloaded from www.microbiologyresearch.orgby IP: 54.191.40.80 On: Fri, 07 Jul 2017 04:19:57 finding distinguishes Bordetella from Salmonella and Vibrio species (reviewed by Cotter & DiRita, 2000).Thus, in Bordetella, the virulence regulon is differentially expressed in the different subspecies, yielding bacteria with very different niches and lifestyles (reviewed by Cotter & DiRita, 2000;Mattoo et al., 2001).
Effective pathogenesis involves tightly coordinated regulation of virulence factors in response to environmental cues (Dorman, 1995;Marteyn et al., 2010;Rhen & Dorman, 2005;Swanson & Hammer, 2000).For example, disturbing the usual pattern of virulence gene expression in Bordetella can significantly reduce colonization in a mouse model (Akerley et al., 1995;Kinnear et al., 2001).Furthermore, B. pertussis regulation employs a sophisticated repertoire to provide phenotypic diversity within highly clonal, genetically homogeneous bacterial populations.Hence, in addition to its role in global health and infectious disease, B. pertussis provides a valuable model in the laboratory to investigate regulation across all bacterial species.Here, we discuss regulation of the B. pertussis virulence genes, highlighting the promoters for fhaB, encoding filamentous haemagglutinin, and the fim genes, encoding fimbriae.We emphasize how the control of these genes differs from other well-characterized systems of bacterial activation and discuss the role of these regulatory mechanisms in the context of B. pertussis virulence.

The two-component system BvgAS regulates virulence genes in B. pertussis
To sense relevant cues in the external environment and transduce these signals into intracellular responses such as changes in gene expression, bacteria frequently utilize two-component systems.These systems typically consist of a sensor kinase (SK) and a response regulator (RR), which functions as a DNA-binding transcriptional activator (reviewed by Stock et al., 2000).The SK includes a sensing domain, situated in the bacterial periplasm, connected via a transmembrane segment to a kinase domain, located inside the cell.The SK is thought to transmit the signal to the cell interior via a conformational change within the protein, which affects the efficiency of ATPdependent autophosphorylation of one SK molecule by its homodimer partner.However, the exact molecular mechanism remains elusive.The RR activator then catalyses its own phosphorylation with the phosphate group donated by its cognate SK or by a small molecule phosphodonor (acetyl phosphate, imidazole phosphate or phosphoramidate, among others).Because these signalling mechanisms are quite rare in eukaryotes (Galperin, 2010), two-component systems are potential targets for antimicrobial therapies.
In the genus Bordetella, the primary two-component system involved in virulence gene regulation consists of the sensor kinase BvgS and the response regulator BvgA.BvgS is a 'hybrid' SK, which has three phosphorylation sites in three distinct domains that mediate a phosphorelay (Fig. 1a) (Uhl & Miller, 1996).Phosphorylated BvgA (BvgA~P) binds to different virulence gene promoters in different binding patterns, discussed below, to activate transcription.BvgAS controls the expression of over 100 virulence genes (Bootsma et al., 2002) and the BvgS and BvgA sequences are almost invariant among B. pertussis strains and clinical isolates (Herrou et al., 2009).
In 1960, Lacey reported different antigenic properties for three distinct modes of B. pertussis and chose the term 'modulation' to describe the transition between modes (Lacey, 1960).Today, we understand that the three modes described by Lacey correspond to what are now known as the Bvg + , Bvg 2 and Bvg i (intermediate) phases (Fig. 1c).The Bvg + phase, characterized by expression of all BvgAactivated adhesins and toxins, is required for virulence (Cotter & Miller, 1994).This phase is manifested under 'non-modulating' environmental conditions that are conducive to phosphorylation of BvgS, such as growth near 37 u C, the temperature in the respiratory tract of the human host.The Bvg i phase, in which the BvgAS system is not fully induced, may have a role in transmission by aerosol route or the initial stages of infection.
'Modulating' conditions [growth at lower temperatures (25 u C) or the presence of nicotinic acid or magnesium sulfate (MgSO 4 ) in the growth medium] result in a reduction of BvgS activity, leading to the Bvg 2 phase.The role of the Bvg 2 phase is not yet understood, but it is clearly not required for virulence in animal models of infection (Garcı ´a San Miguel et al., 1998).However, inappropriate expression of the Bvg 2 phase in vivo is actually detrimental to successful infection (Merkel et al., 1998).It has been hypothesized to be important for intracellular uptake and persistence, or transmission between mammalian hosts (Herrou et al., 2009;Locht et al., 2001).In B. bronchiseptica, the Bvg 2 phase is adapted for survival under conditions of extreme nutrient deprivation (Cotter & Miller, 1994).Alternatively, it may be an evolutionary remnant from an ancestor that occupied an environmental niche (Gerlach et al., 2001;von Wintzingerode et al., 2001).

Temporal gene regulation by BvgA~P coordinates the expression of proteins needed for virulence
The differential binding to the regulatory regions of different virulence genes constitutes a simple yet highly effective system by which regulation by BvgA~P can result in varied kinetics of virulence factor expression.Virulence genes whose products are presumably involved early in pathogenesis -such as surface-expressed adhesin proteins and BvgA itself -are activated almost immediately upon a shift to permissive growth conditions (37 u C) (Scarlato et al., 1991) (Fig. 1c).This rapid transcriptional response is thought to arise from the presence of high-affinity BvgA~P binding sites located upstream of the early virulence gene promoters (Fig. 1b).Virulence genes whose products are thought to play a role later in pathogenesis -such as toxins and their secretion systems -remain transcriptionally inactive until the intracellular concentration of BvgA~P increases to a level sufficient to fill the low-affinity BvgA~P sites upstream of the late virulence gene promoters (discussed by Cotter & Jones, 2003) (Fig. 1b, c).In addition, an intermediate gene has been described (bipA) in which moderate levels of BvgA~P activate expression, but high levels repress expression (Williams et al., 2005) (Fig. 1b, c).Furthermore, BvgA acts indirectly as a negative effector through its regulation of the BvgR protein, which negatively regulates a set of genes encoding outer membrane and secreted proteins (Merkel et al., 2003).
The B. pertussis s factor is an essential component of the transcription machinery B. pertussis RNA polymerase (RNAP), like that of other bacteria, consists of an enzymic core (a 1 a 2 bb9v) plus a s factor required to direct core to the promoter region at the start of a gene.Bacteria typically have multiple s factors, which direct RNAP to different classes of genes based on the cell's needs (reviewed by Gruber & Gross, 2003).
In primary s factors, which are responsible for most transcription during exponential growth, four conserved regions (regions 1-4) mediate interactions between s and the core and/or promoter specificity (reviewed by Hook- Barnard & Hinton, 2007).Regions 2, 3 and 4 bind to specific recognition sites in the promoter DNA: the 210 element ( -12 TATAAT -7 ), an extended -10 element ( -15 TG -14 ) and the 235 element ( -35 TTGACA -30 ), respectively.Typically, two out of three such sequence elements are sufficient for recognition.In addition, the a subunit C-terminal domains (aCTDs) may also directly contact the DNA through AT-rich 'UP' elements, usually located between -40 and -60.
In B. pertussis, the primary s is termed s 80 .Its counterpart in Escherichia coli is the well-studied s 70 .s 80 is 71 % similar and 55 % identical to E. coli s 70 , and it has been shown that several known promoters are active using RNAP reconstituted with either s factor (Baxter et al., 2006;Boucher et al., 1997;Decker et al., 2011;Steffen & Ullmann, 1998).Furthermore, within s region 4, a portion of s known to play a major role in gene regulation, s 70 and s 80 are 84 % similar and 73 % identical.Thus, it is reasonable to extrapolate much of the detailed characterization of s 70 to s 80 .
One discrepancy between E. coli s 70 and B. pertussis s 80 resides in the N-terminal region where s 80 has a positively charged 'region P' (residues 1 to ~150).Such a domain is present in many pathogenic bacteria but absent in E. coli s 70 (Yang et al., 2010).In Helicobacter pylori, region P binds polyphosphate under conditions of limited nutrients, and mutations that eliminate this interaction lead to accelerated cell death during starvation.However, how and if this binding regulates gene expression is not yet known.

Bacterial transcription activation occurs through a variety of mechanisms
A promoter with 'perfect' elements does not equal a perfect promoter; such a construct cannot be regulated and, therefore, could be detrimental to an adaptive organism.Consequently, regulated promoters, such as those that drive expression of B. pertussis virulence genes, contain a complement of core promoter sequence elements that is suboptimal and then use DNA-binding factors to activate RNAP under the appropriate conditions.
Transcription initiation proceeds through multiple steps (Kontur et al., 2008;Saecker et al., 2002; reviewed by Hook- Barnard & Hinton, 2007), from an initial closed complex (RP C ), in which the DNA is fully double-stranded, to an open complex (RP O ), in which polymerase has isomerized and the DNA has bent, opened and descended into the active site (Gries et al., 2010).Consequently, there are multiple points at which an activator might function.
Several classes of activators, distinguished by their varying mechanisms, have been described.
Bacterial class I activators bind to promoter DNA upstream of the -35 element (typically near -60) and directly interact with the RNAP aCTDs (Fig. 2a).It is thought that this interaction, by stimulating initial binding of RNAP, helps to recruit the enzyme to the promoter (reviewed by Gourse et al., 2000).In contrast, class II activators bind to promoter DNA adjacent to or overlapping the -35 element (Fig. 2b).At this location, they are positioned to interact with the DNA-recognition helix within s 70 region 4 and/or a subunits CTDs or NTDs (Dove et al., 2003).These activators can also help recruitment and/or accelerate ratelimiting steps in the formation of RP O (Barnard et al., 2004;Browning & Busby, 2004;Lawson et al., 2004).Interestingly, among class II activators, there is variation in the precise binding site and orientation of the activator, discussed below.In addition, some promoters use a combination of class I and class II (Fig. 2c).
Mechanisms of some other transcription factors bear little resemblance to class I or class II activation.For example, MerR activates expression of the mercuric ion operons by binding to the unusually long spacer region between the -10 and -35 core promoter elements (Fig. 2d).Its binding contorts the DNA, effectively shortening the spacer and creating a functional promoter for RNAP (Hobman et al., 2005;Watanabe et al., 2008).
Another differing mechanism is s appropriation, by which a class of bacteriophage T4 promoters are activated and host promoters are silenced (reviewed by Hinton, 2010).In this system, a small T4 protein, AsiA, binds tightly to E. coli s 70 region 4 and structurally remodels it to preclude binding to the -35 element of E. coli promoters (Fig. 2e) (Lambert et al., 2004).The remodelled s factor is correctly positioned to interact with another T4 protein, MotA, which redirects transcription activity to T4's own middle promoters via recognition of a specific site at -30.Thus, this system works by replacing s region 4 specificity for one sequence with the activator's specificity for a different sequence.

BvgA~P-regulated promoters have a characteristic architecture
At typical B. pertussis early promoters, such as those for the genes fha and bipA, a head-to-head dimer of BvgA~P binds to a primary binding site [inverted heptads with consensus sequence ( T / A )TTC( C / T )TA typically located ¢60 base pairs upstream of a virulence gene promoter; Fig. 1b; Boucher et al., 1997;Roy & Falkow, 1991], and additional dimers of BvgA~P bind to adjacent, secondary binding sites in a cooperative manner that can be relatively independent of the DNA sequence (Boucher & Stibitz, 1995;Boucher et al., 2001;Marques & Carbonetti, 1997).
Other promoters, such as the late promoters driving ptx and cya expression, utilize a consortium of binding sites with poorer matches to the consensus sequence.These sites, acting cooperatively, are filled and stimulate transcription only at higher intracellular concentrations of BvgA~P.
A structure of BvgA has not yet been obtained.Consequently, the DNA-binding domain of BvgA has been conventionally modelled on the response regulator NarL because of the sequence similarity between the two proteins (Boucher et al., 2003).The binding behaviour of BvgA has been revealed by studies using BvgA~P modified at single residues with the cleavage reagent Fe-BABE (Boucher et al., 2003).The details of this binding are entirely consistent with the X-ray crystal structure of NarL bound to its DNA site (Proulx et al., 2002).However, the activity of BvgA seems to differ from that of NarL.For example, NarL does not detectably bind DNA unless it is phosphorylated (Proulx et al., 2002).In contrast, unphosphorylated BvgA has been shown to bind DNA (Boucher et al., 1994(Boucher et al., , 1997;;Karimova et al., 1996;Zu et al., 1996;K. B. Decker, unpublished data).In addition, BvgA~P binds to virulence gene promoters with greater affinity and in at least one case, in a different binding pattern than does BvgA (Boucher et al., 1994(Boucher et al., , 1997;;Boucher & Stibitz, 1995;Karimova et al., 1996;Steffen et al., 1996;Zu et al., 1996;K. B. Decker, unpublished data).
For most of the B. pertussis virulence gene promoters, the binding site for the downstream-most BvgA~P dimer is located near the -35 region of the promoter, in a position to interact with s and/or aCTD, as in class II activation, while the upstream binding sites could interact with the other aCTD, as in class I (Boucher & Stibitz, 1995;Boucher et al., 1997Boucher et al., , 2001;;Karimova et al., 1996;Karimova & Ullmann, 1997;Kinnear et al., 1999;Merkel et al., 2003;Zu et al., 1996).Consistent with a combination class I/II  mechanism (Fig. 2c), BvgA~P activation at P fhaB requires residues within aCTD (Boucher et al., 1997) and s region 4 (Decker et al., 2011).
Despite these similarities with class I/II promoters, the architecture of RNAP/BvgA~P at P fhaB is not like other characterized class II activators.The molecular structures and models in Fig. 3 illustrate this point.The left panels in Fig. 3(b-f) depict activator/aCTD/s region 4 complexes along the promoter DNA (upstream to downstream) whereas the right panels depict an end-on view, in which s region 4 (when shown) is kept in the same orientation.In Fig. 3(a), s region 4 can be seen at the 235 element of host promoter DNA.For the promoters at which region 4 is shown, this position remains generally the same, positioned at the 235 region of the promoter (compare the purple region 4 in the right panels).
In contrast, the relative positions of aCTD and the activator are not constant at these various promoters.At P fhaB (Fig. 3b), each a contacts the same region of DNA as a BvgA dimer, but on a different helical face (Boucher et al., 2003).This is unlike previously characterized class I/II systems (reviewed by Barnard et al., 2004), such as the CRP dimer/aCTD/galP1 structure (Fig. 3d) (Benoff et al., 2002) or the modelled structure of l cII dimer/s region 4/aCTD/P RE (Fig. 3e) (Jain et al., 2005), in which the activator and aCTD are adjacent to one another (compare the different locations of the yellow aCTD at P fhaB in the left panel of Fig. 3b versus its locations in the left panels of Fig. 3d and e).Thus, the RNAP/BvgA~P complex at P fhaB represents a new twist on class I/II activation.Furthermore, as discussed in detail below, recent evidence indicates that BvgA~P activation at the promoters for the fim genes (P fim2 and P fim3 ) involves an even more radical departure from the typical class II architecture.
The requirements for BvgA~P activation seem to involve more than just proximity between activator and polymerase.The positions of the proteins around the faces of the DNA double-helix also appear to be important.DNA mutations that disrupt the pattern of multiple sites along one face of the DNA double-helix are deleterious to promoter activity.However, mutations which remove an entire binding site yet maintain the dimer positions still allow promoter activity (Boucher et al., 2001; Marques & Carbonetti, 1997).The orientation of BvgA~P dimers along the same face of the double-helix may be important for cooperative binding of activators to the promoter or for the correct BvgA~P-RNAP interaction.Consistent with this idea is the effect of different lengths of homopolymeric tracts in the promoter regions of the fim genes, described below.Changing the lengths of these promoter regions, which alters the spacing between promoter elements as well as relative orientation of one bound protein to another around the double-helix, has dramatic effects on promoter activity (Chen et al., 2010;Riboli et al., 1991;Willems et al., 1990).

Phase variation in the fim genes generates phenotypic diversity within a population
The fim promoters are emerging as instructive models for multiple levels of gene regulation that together create a fine-tuned response to the environment and ensure the success of a multicellular population.fim2 and fim3 encode subunits of the long serrated fimbriae, serotypes 2 and 3, respectively (Heck et al., 1996;Mooi et al., 1987).Fimbriae (also called pili) allow B. pertussis to adhere to host cells and are required for efficient establishment of tracheal colonization and persistence in mouse and rat models (Geuijen et al., 1997;Mooi et al., 1992).
Expression of the fim genes is regulated at multiple levelsas part of the BvgAS regulon and at the level of the individual gene through phase variation (Heikkinen et al., 2008;Willems et al., 1990).Phase variation is a phenomenon that allows expression of a given factor to switch between 'on' or 'off' states at a rate greater than that of random mutation, frequently affecting fimbriae, flagella, outer-membrane proteins and lipopolysaccharide components in Gram-negative bacteria (reviewed by Dybvig, 1993;Henderson et al., 1999).Phase variation can occur by DNA inversion, recombination, differential methylation or, as in the case of the fim genes, by slipped-strand mispairing resulting in alteration of the length of a repetitive sequence in the regulatory or coding regions of a gene (Seifert & So, 1988;Streisinger & Owen, 1985).
The fim2 and fim3 promoters each contain a homopolymeric tract of cytosines ('C-tract') overlapping the 235 region, and each promoter can be activated by BvgA~P only when the C-tract is of a permissive length (Fig. 4) (Chen et al., 2010;Willems et al., 1990).This tract does not contain sequence-specific information, but instead appears to function as a spacer between transcription activation machinery bound to different elements of the promoter (Chen et al., 2010).Because C-tract regulation of one fim promoter operates independently from the other, cells can express any combination of fim proteins on their surface, contributing to phenotypic diversity within a population.Interestingly, a B. pertussis gene with sequence homology to fim3 and fim2 has an extremely truncated promoter Ctract: 7 Cs compared with 15 Cs in the active form of fim3.This gene, called fimX, appears to be transcriptionally silent due to the deletion in the C-tract (Chen et al., 2010;Willems et al., 1990).Moreover, the shorter homopolymeric tract limits the amount of slipped-strand mispairing that is likely to occur, making a reversion to activity by addition of Cs highly improbable.Why B. pertussis has maintained an intact copy of this supposedly 'silent' gene remains unclear.
Phase variation by slipped-strand mispairing can be considered a 'programmed' random event: the insertion or deletion of nucleotides is stochastic, but the frequency with which it occurs increases for homopolymeric tracts of increasing length (Streisinger & Owen, 1985).One benefit of phase variation is that it allows an organism to create diversity in an otherwise clonal population -a valuable trait for B. pertussis which has unusually poor genomic diversity for a pathogen (Gogol et al., 2007).Phenotypic diversity among surface-exposed adhesins is no doubt important for evading the host surveillance system and may be important to ensure some bacteria are poised to move to a new environment through detachment and shedding (Dybvig, 1993;Henderson et al., 1999).
Notably, the bvgS open reading frame also contains a Ctract, which is susceptible in certain strain backgrounds to slipped-strand mispairing during replication (Levinson & Gutman, 1987); insertion or deletion of a C yields a Fig. 4. Promoter architecture of P fim3 and P fim2 .The -35 region, extended -10 (TGn) element, -10 element and +1 transcription start site are outlined in boxes.BvgA~P binding sites are marked with inverted arrows: thick line, higher-affinity primary site; thin line, lower-affinity secondary site (Chen et al., 2010).The positions at which the aCTD subunits are observed to bind at P fim3 are marked (Decker et al., 2011).The C-tract, which can vary in length in each promoter, is marked.Sequence identity between fim paralogues is marked with two dots.IP: 54.191.40.80 On: Fri, 07 Jul 2017 04:19:57 truncated, nonfunctional BvgS protein (Stibitz et al., 1989).As a result, virulence genes that are normally activated by the BvgAS system are not expressed, rendering them avirulent (Stibitz et al., 1989).However, the biological relevance of this system during an infection is not known.
The molecular mechanism of fim activation is complex and elegant Besides the ability to undergo phase variation, the fim promoters are unusual because the position of the downstream BvgA~P binding site surrounds the 235 region of the promoter DNA (Chen et al., 2010) (Fig. 4).Recent work has sought to define the interaction between the activators and RNAP within this unusual architecture (Decker et al., 2011).Surprisingly, although s region 4 is not required for BvgA~P activation of P fim3 , it is still located on or near its usual position at the -35 region of P fim3 , despite the fact that this region consists of the monotonic C-tract.In addition, the aCTD subunits of RNAP bind to the same regions of the DNA as the BvgA~P dimers, and in the same configuration relative to BvgA as shown for P fhaB .This arrangement places s region 4, one aCTD and a BvgA~P dimer at the 235 region of P fim3 , suggesting that the proteins bind to different faces of the same stretch of promoter DNA.
A speculative model of the protein arrangement at P fim3 consists of the three subunits arranged around the DNA double helix in a conformation that likely depends on specific protein-protein interactions, since the DNA lacks specific sequence information to direct the protein position (Decker et al., 2011) (Fig. 3c).This model explains the exquisite control over regulation by the length of the Ctract.The insertion or deletion of one C would alter the orientation of the protein subunits around the double helix and thus should disrupt the correct positioning needed for activation.
The protein arrangement in BvgA~P activation of the fim3 promoter offers an architecture that differs even more dramatically from that seen at typical class II promoters, or even at P fhaB .This is because the aCTD within the 235 region of P fim3 is positioned nearly a helical turn farther downstream than has been seen previously.Furthermore, for class II activators like CRP at galP1 (Fig. 3d) (Benoff et al., 2002) or PhoB at pho box DNA (Fig. 3f) (Blanco et al., 2011), the activator is poised to interact with a common set of region 4 residues (discussed by Bonocora et al., 2008).However, the particular positioning of BvgA~P at P fim3 means that these residues are not available for a region 4/activator interaction.Finally, despite the fact that the BvgA~P site includes the 235 region for the promoter, just as the binding site of the T4 MotA activator includes this portion of the DNA, the fim promoter activation complex is completely different from that formed by s region 4 with the bacteriophage T4 proteins AsiA and MotA (reviewed by Hinton, 2010).Thus, activation at the fim promoters provides another example of how s region 4 can be utilized in an activation system.

Conclusions
A mechanistic understanding of the activation of B. pertussis virulence genes is vital given the reemergence of the pathogen and the fact that some acellular vaccines directly and exclusively target virulence factors of the bacterium, including Fim2 and Fim3 (Bouchez et al., 2008(Bouchez et al., , 2009;;Geier & Geier, 2002).In addition, evidence from clinical studies may suggest that the Fim antigens are perhaps being subjected to immune selection due to vaccine-induced and natural-antibody-driven adaptation (Gogol et al., 2007;Tsang et al., 2004).The sophisticated controls used by B. pertussis to regulate virulence genes (differential binding of the BvgA~P activator to the promoters, different RNAP/activator architectures and phase variation by programmed mutation) demonstrate how a pathogen that is highly clonal and lacks the genetic diversity of many other pathogens can be quite successful as an obligate human pathogen (Fig. 5).A more complete understanding of the virulence factors and their regulation, and the host immune response is essential to develop the next generation of pertussis vaccines and treatments.

Fig. 1 .
Fig. 1.The BvgAS system and temporal gene regulation.(a) The BvgS sensor kinase is anchored in the inner membrane.Activating signals trigger BvgS autophosphorylation, initiating a phosphorelay that results in phosphorylation of the BvgA response regulator.(b) Phosphorylated BvgA (BvgA~P) binds to virulence gene promoters to regulate activity.BvgA binding sites are marked with inverted arrows (thick lines, high-affinity sites; thin lines, low-affinity sites).At those promoters where the aCTD subunit positions have been identified, aCTD binds to the same region of DNA as the promoter-proximal BvgA~P dimers(Boucher et al., 2003;Decker et al., 2011).(c) Schematic illustrating temporal gene regulation by BvgAS.BvgA~P activates its own expression, so intracellular BvgA~P concentration increases with time at 37 6C (x-axis labels).The Bvg " phase is characterized by expression of the vir-repressed genes; Bvg i by expression of the early and intermediate genes; Bvg + by expression of the early and late genes, whose products are required for virulence.

Fig. 2 .
Fig. 2. Characterized mechanisms of prokaryotic gene activation.Purple, s; yellow, aCTD; green, specified activator.(a) Class I activators bind upstream of the core promoter elements and interact with the RNAP aCTD subunit(s).(b) Class II activators bind closer to the core promoter elements, adjacent to or overlapping the "35 element, and interact with s region 4 and/or aCTD.(c) Combination class I/II activation relies on independent interactions between an upstream activator and aCTD, and a downstream activator and s region 4 and/or aCTD.(d) The MerR activator causes a conformational change in the alignment of the "10 and "35 elements that allows promoter recognition and gene activation.(e) During activation by s appropriation, bacteriophage T4 proteins AsiA (red) and MotA (blue) interact with s 70 region 4 to redirect RNAP from host E. coli promoters to T4 middle promoters, which contain a MotA box sequence centred at "30.
Fig. 3.The protein conformation at BvgA~P-activated P fim3 differs from that at characterized class II promoters.Purple, s region 4; yellow, aCTD; green, specified activator.For (a)-(c) and (e)-(f), the "35 nontemplate strand nucleotide (nt) is shaded cyan and marked with a carat (left panel) and oriented in the 12 o'clock position (as viewed in the right panel).For (d), the "56 nontemplate strand nt is shaded cyan and oriented in the 12 o'clock position; because the "56 nt is ~two helical turns upstream from the "35 nt in B-form DNA, the complex in (d) is similarly oriented to (a)-(c) and (e)-(f).The red carat in (d) marks nt position "41.5.(a) s Region 4 bound to "35 region DNA, as a reference for (b)-(f) (Campbell et al., 2002); PDB no.1KU7.(b) BvgA~P activation at P fhaB : s region 4, aCTD, BvgA~P dimer bound to its promoter-proximal site; modelled on the work of Boucher et al. (2003).(c) BvgA~P activation at P fim3 : s region 4, aCTD, BvgA~P dimer bound to its promoter proximal site, overlapping the "35 region DNA; modelled on work of Decker et al. (2011).(d) Class II activation by CRP at galP1: a dimer of

Fig. 5 .
Fig. 5. Multiple independent regulatory mechanisms ensure appropriate virulence gene expression and create phenotypic diversity in a Bordetella population.The bvgS sequence, environmental temperature and the length of the fim promoter C-tract together determine fim activity.Modulating conditions include lower temperature (25 6C) or the presence of MgSO 4 or nicotinic acid; non-modulating conditions include growth at 37 6C.How frameshifting within bvgS contributes to pertussis infection is not clear.