Environmental Sensing Mechanisms in Bordetella

Environmental Sensing Mechanisms in B o r d e t e l l a J o h n G. C o o t e Division of h~/~c'tion and Immunity, Institute ~['Biomedical and Li/e Sciences, Universi O, (~ Glasgon; Joseph Black Building, Glasgow G 12 8QQ, UK

ABSTRACT The success of a bacterial pathogen may depend on its ability to sense and respond to different environments. This is particularly true of those pathogens whose survival depends on adaptation to different niches both within and outside the host. Members of the genus Bordetella cause infections in humans, other animals and birds. Two closely related species, B. pertussis and B. bronchiseptica, cause respiratory disease and express a similar range of virulence factors during infection, but exhibit different host ranges and responses to environmental change. B. pertussis has no known reservoir other than humans and is assumed to be transmitted directly via aerosol droplets between hosts. B. bronchiseptica, on the other hand, has the potential to survive and grow in the natural environment. Comparison of the manner in which these two organisms respond to external signals has provided important insights into the co-ordinate regulation of gene expression as a response to a changing environment. During infection, both species produce a range of virulence factors whose expression is co-ordinated by two members of the two-component family of signal transduction proteins, the bvg (bordetella virulence gene) and ris (regulator of intracellular stress response) loci. When active, the bvg locus directs the activity of a number of virulence determinants in both species whose products, such as adhesins and toxins, establish colonization of the host by the bacteria, although each organism has evolved a slightly different strategy during pathogenesis. B. pertussis, the causative agent of whooping cough, promotes an acute disease and tends to be more virulent than B. bronchiseptica which generally causes chronic and persistent asymptomatic colonization of the respiratory tract. The recently identified ris locus appears to control the ADVANCES IN MICROBIAL PItYSIOLOGY VOI~ 44 ISBN 0-12-027744- ]

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expression of factors important for intracellular survival of B. bronchiseptica, but a role for this regulatory locus in B. pertussis infection has not been established. Expression of the virulence determinants controlled by the bvg and ris loci is subject to modulation by different environmental signals, such as low temperature, which act through these two-component systems, Evidence indicates that, for B. bronchiseptica, bvg-controlled determinants expressed under modulating conditions, such as motility, facilitate adaptation and survival in environments outside the host. With B. pertussis, however, there is no apparent requirement for prolonged survival outside the host and this difference is reflected in the expression of different, as yet uncharacterized, determinants as a response to modulating signals. The nature of the gene products involved and their assumed role in the life cycle of B. pertussis remains to be determined. Thus, comparative analysis of these species provides an excellent model for understanding the genetic requirements for pathogenesis of respiratory infection and adaptation to changing environments, both within and outside the host. 1. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Bordetellaspecies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. P a t h o g e n e s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. V i r u l e n c e f a c t o r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The r e s p o n s e to e n v i r o n m e n t a l c h a n g e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3,1. R e g u l a t o r y d e t e r m i n a n t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. R e g u l a t i o n o f bvg l o c u s - c o n t r o l l e d g e n e s . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. The role of t h e Vag a n d Vrg g e n e p r o d u c t s in t h e Bordetella life cycle 4.1. B, pertussis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. B, bronchiseptica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. I n t e r m e d i a t e p h a s e p r o t e i n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. I n t e r a c t i o n w i t h t h e h o s t i m m u n e s y s t e m . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. T h e s i g n i f i c a n c e of t h e i n t r a c e l l u l a r stage . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. C o n c l u s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements ................................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. I N T R O D U C T I O N

Bacterial pathogens must adapt to ecological niches both within and outside the host and in both of these locations a pathogen may encounter a number of different environments. Pathogens express appropriate sets of gene products in response to the stimuli they encounter in these varied environments and their long-term survival depends on synthesis of necessary factors at appropriate times as a response to these stimuli. For example, upon entry to the host,

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different components may be required at different stages of infection. Initially, the bacteria need to adhere to epithelial tissues in sufficient numbers to establish infection. They will then need to acquire nutrients and multiply in the face of the innate defences and immune responses of the host. In some instances. they may invade host cells in order to survive and to allow dissemination to deeper tissues. Finally, they must persist for a period within the host in preparation for eventual transmission to a new susceptible host. This latter process may in some cases involve adaptation to, and survival in, external environments (Finlay and Falkow, 1997). Signals received from the environment can be physical or chemical in nature, such as alterations in temperature, osmolarity, pH, oxygen, metal ions or general nutrient availability, or may be derived from other living cells and involve diffusable molecules or signals generated by direct contact. Among the many new insights that have emerged from the study of bacterial pathogenicity is the manner in which signal transduction systems are used by bacteria to control the co-ordinated expression of different sets of genetic determinants as they transit from one environment to another (Mekalanos, 1992). Coordination is generally achieved by a regulatory locus whose expression is itself responsive to external signals and which in turn regulates the appropriate genes in a controlled manner. This feature and its relationship to pathogenicity was recognized in B. pertussis some years ago and was the subject of an earlier article (Coote, 1991 ). Bordetella provides a paradigm for investigation of the co-ordinated response to a changing environment and this review will locus in particular on two very closely related species, B. pertussis and B. bl'onchiseptica, both of which cause respiratory disease and have common regulatory mechanisms responsive to external signals, but which display clear differences in the manner in which they have adapted to host and external environments. A detailed review of the genus Bordetella and the host-pathogen interactions of those species that colonize the respiratory tract is provided by Cotter and Miller (2000).

2. B O R D E T E L L A SPECIES

Bordetella spp. are Gram-negative bacteria that cause infections in humans and other animals. Seven species have now been described: three are motile by peritrichous flagella (B. avium, B. bronchiseptica, B. hin~ii) and four are non-rnotile (B. holmesii, B. parapertussis, B. pertussis, B. trematum). B. pertussis appears to be adapted exclusively to the human respiratory tract and is the cause of whooping cough (Wardlaw and Parton, 1988). B. parapertussis generally causes a milder pertussis-like disease, but is no longer considered a strict parasite of humans as it has been isolated from sheep. The human and

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sheep strains appear to represent distinct populations and there is no evidence for transmission between man and sheep (Porter et al., 1994; Yuk et al., 1998a). B. bronchiseptica is a commensal organism of the respiratory tract of many animals, but can also cause disease, being particularly associated with atrophic rhinitis and bronchopneumonia in pigs and kennel cough in dogs, and is an opportunistic pathogen in immunocompromised humans (Goodnow, 1980; Woolfrey and Moody, 1991 ). B. avium causes upper respiratory tract disease (coryza or rhinotracheitis) in birds (Kersters et al., 1984) but has not been reported to cause disease in humans. B. holmesii and B. trematum exclusively infect humans, causing either septicaemia and endocarditis or ear and wound infections, respectively (Weyant et al., 1995; Vandamme et al., 1996). B. hinzii is the name given to a B. avium-like group of organisms from the respiratory tract of domestic poultry; human isolates have also been reported (Vandamme et al., 1995; Kattar et al., 2000).

2.1. Pathogenesis The latter three species noted above have only recently been described and their pathogenic potential has not been investigated. For the other bordetellae, apart from their different host ranges, some differences exist in the manner in which disease is manifested. Pertussis is primarily a disease of young children, but adults with waning immunity may be an important reservoir of infection. Pertussis is highly infectious with transmission being attributed to droplet infection. The clinical features of the disease tend to vary with age, general health and immune status, but typical pertussis progresses in three stages. An incubation period of 7-14 days is followed by a catarrhal stage with mild cough and mucus production. The coughing then becomes increasingly severe in the paroxysmal stage characterized by bouts of violent coughing often followed by inspiratory gasps or whoops. This stage lasts for 1-4 weeks and may be accompanied by anoxia-associated disturbances of the central nervous system. In the final convalescent stage, which may last up to 6 months, the cough gradually subsides. B. parapertussis is generally considered to cause a milder form of disease than B. pertussis, but this organism is nevertheless capable of causing typical whooping cough with paroxysmal coughing (Wirsing von Konig and Finger, 1994). B. avium infection of birds mimics pertussis infection of humans. Both organisms cause highly contagious acute diseases and Jowl and humans exhibit similar clinical symptoms including 5 to 14-day incubation periods, coughing and mucus accumulation. Bordetellosis in animals caused by B, bronchiseptica is also a highly infectious disease, but is often characterized by asymptomatic colonization of the respiratory tract. Infections are generally chronic with long-term persistence by the organisms in the upper respiratory tract. This difference is perhaps

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surprising, as B. pertussis, B. parapertussis and B. bronchiseptica are very closely related while B. avium is more distantly related (Musser et al., 1986). Interestingly, recent studies have shown substantial differences in genome organization between B. pertussis and B. parapertussis and have suggested that human isolates of B. parapertussis are representatives of a clone of B. bnmchiseptica that has adapted to the human host independently of B. pertussis (van der Zee et al., 1997; Middendorf and Gross, 1999),

2.2. Virulence Factors

Although the events that occur during the course of infection with Bordetella spp. are far from clear, progress has been made in identifying several factors that function as virulence determinants. These have been best characterized in the human pathogen B. pertussis and have been reviewed elsewhere (Weiss and Hewlett, 1986; Patton, 1998; Cotter and Miller, 2000). Infection begins with colonization of the ciliated epithelial cells of the upper respiratory tract. Factors implicated in adhesion are filamentous haemagglutinin (FHA), a high-M r rodlike surface protein; a 69 kDa outer-membrane protein (Omp), pertactin (PRN), which is a non-fimbrial haemagglutinin, and sero-specific fimbriae. Pertussis toxin (PTX), produced only by B. pertussis and considered a major virulence factor of this organism, also appears to act as an adhesin (Sandros and Tuomanen, 1993). Two more recently characterized Omps, BrkA (Bordetella resistance to killing) and TCF (tracheal colonization./
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synergistic combination of TCT and lipopolysaccharide (LPS), is thought to be responsible for the destruction of the ciliated epithelial cells (Flak and Goldman, 1999). Dermonecrotic toxin (DNT) has a potent vasoconstrictive activity which could induce a local inflammatory reaction, but its role in human pertussis has not been defined. It has been associated with turbinate atrophy in B. bronchiseptica infection of pigs by causing bone degeneration. In target cells, it appears to render the GTP-binding protein Rho constitutively active, causing changes to the cytoskeleton (Horiguchi et aI., 1997; Lacerda et al., 1997; Masuda et al., 2000). PTX has a wide range of pathophysiological activities, including inhibition of the chemotactic and phagocytic abilities of leukocytes, most of which are due to the enzymic activity of the A subunit of the toxin that has ADP-ribosyl-transferase activity targeting the inhibitory subunit of eukaryotic adenylate cyclase (AC) (Katada et al., 1986). This has the effect of raising intracellular cyclic 3', 5' AMP (cAMP) concentrations with resulting disturbances in cell metabolism and responsiveness to external signals. Adenylate cyclase toxin (ACT) also acts to raise the intracellular concentration of cAMP in mammalian target cells and has similar effects on leukocyte chemotaxis and phagocytosis as PTX. ACT also has other modulating effects on the host immune system (Coote, 1996; Ladant and Ullmann, 1999). However, whereas PTX is an AB toxin using the cell-binding B subunit to introduce the A subunit into the target cell, ACT is a cell-invasive toxin that possesses AC activity. The Nterminal 400 amino acids of ACT have AC enzymic activity which is stimulated up to 1000-fold by host calmodulin. The remainder of the molecule has membrane-targeting and pore-forming activity. ACT invades target cells, whereupon the N-terminal adenylate cyclase enzymic moiety is activated by host calmodulin to produce high levels of cAMP, which impairs the functions such as chemotaxis and phagocytosis of immune effector cells and is assumed to assist survival of the bacterium in the initial stages of respiratory tract colonization. Interaction of B. pertussis with human monocytes impairs T cell proliferation to an exogenous antigen and this effect is also related to the action of ACT (Boschwitz et al., 1997). Other work has indicated that B. pertussis ACT is responsible for apoptosis of mouse alveolar macrophages in vivo (Gueirard et aI., 1998). With B. bronchiseptica, neutrophils are critical to the early defence against lung infection and ACT is implicated in the inhibition of this neutrophil-mediated defence mechanism (Harvill et al., 1999a). Recently, both B. pertussis and B. bronchiseptica have been shown to possess a type III secretion system (Yuk et al., 1998b; Kerr et al., 1999), a multicomponent process common to many bacterial pathogens for delivery of effector proteins directly into mammalian cells (Galan and Collmer, 1999). This system in B. bronchiseptica has been shown to play an important role in combating the host immune system (section 4.4.1 ).

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Finally, members of the bordetellae have been reported to invade a variety of cultured mammalian cells, but it is not clear if this invasive capacity is an important feature of the natural infection. The significance of a possible intracellular location is open to speculation, but it may be an explanation for the protracted course of disease. The importance of cell-mediated immunity in bordetellosis has been recognized (Redhead et al., 1993; Gueirard et al., 1996; Mills et al., 1998) and may be related to an intracellular persistent state against which humoral immunity is not effective. This aspect of the life-style of the bordetellae within the host is considered in more detail below (sections 4.4 and 4.5). Only three of the known virulence-associated factors, fimbriae, TCT and DNT, are present in all Bordetella spp. examined. In keeping with their close relatedness, B. pertussis, B. parapertussis and B. bronchiseptica express a similar set of virulence factors, with the exception of PTX and TCF, which are only produced by the human pathogen, B. pertussis. The clinical features of disease caused by B. parapertussis resemble those of pertussis, although the organism usually manifests a milder form of infection lacking the characteristic paroxysmal coughing periods. This may be due to the absence of PTX, a notion supported by the coughing rat model of pertussis. Here rats are infected intrabronchially with bacteria embedded in agarose beads to prevent rapid clearance from the lungs. Wild-type B. pertussis produces a significant number of coughing paroxysms not induced by B. parapertussis or a B. pertussis mutant unable to express PTX (Patton et al., 1994). B. avium is more distantly related within the bordetellae and has been reported to produce only DNT, TCT, fimbriae, a component similar to FHA and a distinct osteotoxin that is cytotoxic for osteogenic and tracheal cells (Gentry-Weeks et al., 1988. 1993).

3. THE R E S P O N S E T O E N V I R O N M E N T A L

CHANGE

The bordetellae exhibit antigenic modulation and phase variation, processes that allow the organisms to alternate between virulent and avirulent forms by either phenotypic (antigenic modulation) or genotypic (phase variation) means. Phenotypic modulation is a fi'eely reversible process whereby all the cells in a population can alter the expression of virulence factors in a co-ordinate manner in response to environmental change. Under laboratory conditions, low temperature (25°C) and high levels of certain salts, such as MgSO 4, or organic acids, such as nicotinic acid, will promote loss of virulence factor expression and the synthesis of other antigens in their place. This process was thoroughly investigated by Lacey ( 1960), who noted an X (xanthic) mode, which induced small, domed, haemolytic colonies able to express virulence factors on, for example, Bordet-Gengou blood agar at 37°C: a C (cyanic) mode consisting of

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large, flat, non-haemolytic colonies produced under modulating conditions such as those noted above, and an I (intermediate) mode, which was induced when the composition of ions in the medium, such as MgSO 4, was at an intermediate level. Although less well characterized, antigenic modulation has been observed in B. avium (Gentry-Weeks et al., 1991 ; Akerley et al., 1992). Phase variation is also characterized by simultaneous loss of expression of virulence factors, but here avirulent phase variants arise by spontaneous mutation in a population at a frequency of 10-3 to 10~6and in some instances this has been shown to be reversible, albeit at a lower frequency (Weiss and Falkow, 1984; Monack et al., 1989; Stibitz et al., 1989; Gentry-Weeks et al., 1991). Intermediate phase variants of B. pertussis expressing some of the known virulence factors have been isolated (Goldman et al., 1984; Carbonetti et al., 1994; Stibitz, 1994; Cotter and Miller, 1997).

3.1. Regulatory Determinants Many bacterial signal transduction systems that allow adaptation to changing environments employ two associated proteins, a sensor and a transcriptional regulator. This family of two-component sensor/regulator proteins represents a primary mechanism to allow bacteria in general to efficiently sense and adapt to their environment (Gross et al., 1989; Appleby et al., 1996; Perraud et al., 1999). Signal transduction in two-component regulatory systems is based on phosphotransfer reactions between histidine and aspartate residues in highly conserved signalling domains. The predominant type is composed of two proteins, a transmitter, which is a sensor autokinase that responds to environmental signals by autophosphorylation at a histidine-residue (H-motif), and a receiver/effector protein that is able to accept the phosphate group from the transmitter at an aspartate residue located in an acidic pocket. These two proteins, in their simplest forms, each contain two domains. The transmitter protein has a domain (generally extracytoplasmic) that responds to environmental signals and a conserved cytoplasmic domain that is autophosphorylated. Members of this histidine kinase superfamily can be identified by five conserved regions within the cytoplasmic domain where autophosphorylation takes place (Grebe and Stock, 1999). In these proteins, the two domains are most often separated by two or more hydrophobic transmembrane helices. The receiver/effector protein has a conserved receiver domain that contains the phosphorylated aspartyl residue and an output domain that is able to interact with target genes due to conformational changes induced by phosphorylation. These proteins share a conserved sequence o f - 125 amino acids containing the aspartic acid residue (Goudreau and Stock, 1998). 'Unorthodox' systems are characterized by a multistep phosphorelay between histidine and aspartate residues (His-Asp-His-Asp) that involves

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additional receiver and histidine-containing phosphotransfer (Hpt) modules. The latter have a consensus motif unrelated to the H-motif of the transmitter and, although they do not have kinase activity, they can mediate phosphotransfer reactions. The modules can exist as isolated proteins or fused with each other in various combinations. It is assumed that the extension of the signalling pathway between the sensor and the effector components provides the opportunity for additional regulatory signals to influence the phosphorelay. In the bordetellae, the phenotypic and genotypic modulation effects described above are mediated via the products of the bvg (for bordetella virulence gene) regulatory locus (originally called vir), which is a two-component signal transduction system sensitive to alterations in the external environment (Arico et al., 1989). This locus was first identified in B. pertussis by Weiss et al. (1983) as a Tn5 insertion site that created a pleiotropically negative avirulent phenotype unable to express virulence factors such as PTX, ACT, FHA and HLT. Weiss and Falkow (1984) proposed that modulating conditions influenced the expression of this locus, which in turn altered the expression of the virulence determinants in a co-ordinate manner. Recently, a second two-component regulatory system (ris) has been described in B. bronchiseptica which is also involved in the co-ordinate gain or loss of virulence-associated properties and may be required for in vivo persistence (Jungnitz et al., 1998) (section 4.5.1).

3.1.1. The bvg Regulatory Locus

The BvgAS regulatory system in Bordetella is an 'unorthodox' system where three signal transduction modules (transmitter, receiver and Hpt module) are combined in a 135 kDa membrane-bound BvgS sensor protein (Fig. 1). BvgS possesses an N-terminal periplasmic region which samples the environment and this region is flanked by two transmembrane sequences that anchor the protein in the cytoplasmic membrane (Stibitz and Yang, 1991). A linker region lies between the second transmembrane region and the transmitter domain. Two proline-rich lengths of 12 and 15 amino acids join the transmitter to the receiver and C-terminal Hpt domains, respectively. BvgS autophosphorylates at His-729 in the transmitter domain followed by phosphotransfer to Asp1023 in the receiver domain and His- 1172 in the C-terminal Hpt domain before final phosphotransfer to BvgA (Uhl and Miller, 1994, 1996a). The receiver/effector BvgA is a 23 kDa protein that is a typical response regulator with an N-terminal receiver which is phosphorylated at Asp-54 and a Cterminal region containing a helix-turn-helix (HTH) DNA-binding motif (Boucher et al., 1994). There is evidence that the phosphorelay pathway involves dimerization of both BvgS and BvgA (Scarlato et al., 1990; Stibitz and Yang, 1991, Beier et al., 1995, 1996).

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Periplasm H729 Environmental signals -~

Bvg S

Bvg A

D1023 Hl172 ~ P/'-~-~" P

D54 "P I I R I HTHI

P

ATP 2¸

ADP

?~

~!i~,i!,~ !ii;i~

AAPPAAATAAT

,>

........

AALPTPPSPQAAAPA (A/P II)

Figure 1 A model for BvgAS signal transduction. Proposed steps in the phosphorelay are shown with the following domain abbreviations. BvgS (1238 amino acids, aa): P, periplasmic domain (514 aa); L, linker region (159 aa); T, transmitter (232 aa); R, receiver (131 aa); Hpt, histidine phosphotransfer domain (126 aa); A/P, alanine/proline rich sequences. BvgA (209 aa): R, receiver domain (-109 aa); HTH, helix-turn-helix molif (-100 aa). CM, cytoplasmic membrane. H729 is the site of autophosphorylation on BvgS, D1023 and H1172 are the sites of phosphoryl group transfer within BvgS, and D54 on BvgA is the final point of phosphotransfer. Data from Uhl and Miller (1994, 1996a,b).

Thus, signal transduction by the BvgAS system involves a complex phosphorelay, the significance of which is not yet clear. The natural signals that influence the activity of BvgS in vivo are unknown and whether or not the activity of BvgS on BvgA is finely tuned during the phosphorelay remains to be determined. The BvgS receiver domain contains autophosphatase activity that may modulate the phosphorelay (Uhl and Miller, 1996b). The phosphorylation status of a similar 'unorthodox' phosphorelay involved in sporulation in Bacillus subtilis (Perego, 1998) is influenced by aspartate-specific phosphatases that can dephosphorylate both an intermediate receiver domain and the final effector protein. The phosphatases themselves are regulated by various physiological conditions that allow information from several regulatory circuits to be integrated into the system before the final phosphorylated effector protein influences gene expression and sets in train the sporulation process. The sporulation process represents a major commitment on the part of the organism and the decision to embark on it must be carefully controlled. Similarly, the decision by the bordetellae to switch on a range of virulence factors needs to be regulated upon entry of the bacteria into the host and will

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presumably be influenced by the multitude of signals encountered during colonization of the host. There is clear evidence (see below) that the induction of virulence factors is temporally controlled, at least in vitro, and that this differential expression is dependent on the level of phosphorylated BvgA. The receiver and Hpt domains of BvgS are essential for signal transduction to BvgA (Uhl and Miller, 1994, 1996a; Beier et al., 1995, 1996). Thus, neither the histidine kinase transmitter portion of BvgS nor the internal receiver domain is able to pass the phosphate group to BvgA. Only the phosphohistidine of the Hpt domain can complete the phosphorelay to BvgA. This feature contrasts with other 'unorthodox' phosphorelays, such as the ArcAB system of E. toll, where either the transmitter or the Hpt domain of the redox sensor ArcB can act as the phosphodonor for the effector protein ArcA (Tsuzuki et al., 1995). Data indicate that both types of phosphorelay can operate in vivo (Matsushika and Mizuno, 1998) where the Hpt-mediated phosphorelay appears to be essential for adaptation to anaerobic conditions, but the 'short-cut' phosphorylation pathway operates under aerobic conditions. It has been suggested that the 'short-cut' phosphorelay is more promiscuous and may interconnect with other two-component systems in a process termed 'cross-talk' (Wanner, 1992), and that the extended phosphorelay is much more specific and interacts solely with its cognate effector protein (Perraud et al., 1999). This is borne out by recent data concerning the BvgAS system, which showed that the interaction of the Hpt domain of BvgS with BvgA is highly specific (Perraud et al., 1998). Thus, one role of the extended phosphorelay may be to impart specificity to the system and avoid interference by other transmitter proteins. This would be particularly important for the bordetellae, as the BvgAS system controis a large number of virulence-related genes whose expression must be carefully modulated and correlated with specific environmental signals. The predicted amino acid sequences of the BvgAS proteins of B. pertussis, B. parapertussis and B. bronchiseptica are highly conserved with an overall amino acid identity of 96% (Arico et al., 1991 ). No amino acid differences are found in BvgA and most changes are concentrated in the periplasmic sensory region of BvgS. This might suggest that this region senses different signals or responds to different environmental levels or combinations of signal for the different species. In order to investigate this, Martinez de Tejada et al. (1996) constructed a hybrid strain of B. bronchiseptica containing the bvgAS locus from B. pertussis. They found that the BvgS and BvgA proteins were functionally interchangeable in B. pertussis and B. bronchiseptica as the hybrid strain produced Bvg+ and Bvg--specific factors normally and responded to the same signals such as MgSO 4, nicotinic acid and temperature. It did, however, differ in relative sensitivity to these signals. Thus, for example, experiments in vitro indicated that a higher concentration of MgSO 4 or nicotinic acid was required to modulate the hybrid strain from the Bvg÷ to the Bvg phase. These data suggested that the by,AS locus from B. pertussis was less sensitive to

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modulating signals than that from B. bronchiseptica. The phenotype displayed by further hybrid constructions indicated that the periplasmic sensor domain of BvgS was responsible for this difference in sensitivity to the different signals, in keeping with the differences found in the amino acid sequence in this region between the two species. These subtle differences in sensitivity to signals in vitro were not reflected in differences in the ability of the hybrid strain to infect rats and cause disease, indicating that the BvgAS proteins from the two species responded in a similar manner to the in vivo environment.

3.2. Regulation of bvg Locus-controlled Genes 3.2.1. Temporal Control The bvg locus alters the expression of two types of virulence-associated genes, vir-activated (rag) genes, which encode all the well-characterized virulence factors mentioned above (except TCT), and 1,it-repressed (vrg) genes (Knapp and Mekalanos, 1988), whose products, in the case of B. pertussis, have not been characterized, but which are not implicated in the disease process (Martinez de Tejada et al., 1998; Merkel et al., 1998b) (section 4.1). In the case of B. bronchiseptica, vrg genes encode motility and other co-regulated phenotypes (section 4.2) that may be important for survival of the organism in the external environment, but again do not appear to contribute to initial infection (Cotter and Miller, 1994) (section 4.2). Thus, Bvg components act positively to activate vag genes within the regulon while at the same time repressing the expression of vrg genes (Bvg + phase). In the absence of Bvg components, rag loci are not expressed and the repression on vrg loci is lifted (Bvg- phase) (Fig. 2). Of the environmental stimuli sensed by the bordetellae, the temperature change encountered by the bacteria when entering or leaving the host is likely to be as important as any. Transcriptional analysis after a temperature switch from 25°C to 37°C in laboratory media has shown that the bvg-regulated promoters can be divided into early and late groups; the former are induced within a few minutes of exposure to the higher temperature and the latter some 2 h later (Scarlato et al., 1991 ). Included in the early group of promoters are those for the genes encoding FHA and fimbriae and also the bvg locus itself. Transcription of the bvg locus is tightly controlled by 1bur autoregulated promoters, three of which, Pl, P2 and P3, direct mRNA synthesis from the bvg operon, while P4 drives transcription of an antisense RNA of unknown function complementary to the 5" untranslated region of the bvg mRNAs (Roy et al., 1990; Scarlato et al., 1990). The activity of P2 is constitutive and is assumed to maintain synthesis of a low level of BvgAS proteins in the cell under modulating conditions. The other bvg promoters become active at an early point upon temperature shift from 25°C to 37°C and allow accumulation

ENVIRONMENTAL SENSING MECHANISMS IN BORDETELLA Bvg+ phase Active bvg locus

Environmental signals

Mutation

Activation of vag loci Repression of vrg loci

Antigenic modulation

Bvg-phase Inactive bvg locus

153

Phase variation

No activation of vag loci Repression lifted on vrg loci

Figure 2 Reciprocal expression of genes controlled by the bvg locus as a response to mutation or environmental signals, rag loci, vir or bvg-activated genes; w-g loci, vir or #vgrepressed genes. Taken from Coote (1991), with permission of the publishers.

of bvg mRNA, which in turn promotes a steady increase in the intracellular concentration of the transcriptional activator BvgA (Scarlato et al., 1991). After 2 h under these laboratory conditions, the concentration of BvgA has increased 20-fold and at this point transcription from the late group of promoters begins, inducing synthesis of, for example, PTX and ACT. The early promoters such as PFHA and P1BV~ were shown to bind BvgA, while binding to late promoters such as PPTX and PcYA could not be demonstrated (Roy and Falkow, 1991; Boucher et al., 1994). In addition, the early promoters were transcribed in Escherichia coli if the bvg locus was supplied in trans, but again this was not achieved with the late promoters except in special circumstances (see below). These observations pointed to the possibility that an additional transcriptional activator, perhaps itself induced as a response to accumulating BvgA, was required for activation of these late virulence genes. Some evidence for this has been reported (Huh and Weiss, 1991" DeShazer et al., 1995), but other data showed that in vitro phosphorylation of BvgA could enhance its capacity to bind to late promoters (Boucher et al, 1994; Boucher and Stibitz, 1995). Further in vitro transcription studies using purified RNA polymerase from B. permssis showed that phosphorylated BvgA was sufficient for activation of both early and late promoters (Steffen et al., 1996). The transcriptional capacity of the E. coil RNA polymerase was weak in comparison to that of B. pertussis, which suggested that the lack of expression of late promotets in E. coli was probably due to inappropriate formation of the transcription initiation complex. Zu et al. (1996) showed that the affinity of

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BvgA-P for PFHAwas higher than that of BvgA and that recognition of PPTX was not possible unless BvgA was phosphorylated and present in at least a 10told higher concentration than that required for PFHA"Similarly, only BvgA-P and not BvgA could bind PCYA(Karimova et al., 1996; Steffen et al., 1996). Gel retardation and DNAase 1 footprinting studies have shown that BvgA-P interacts directly with inverted repeat regions containing the consensus sequence TTTC (C/T)TA upstream of the RNA polymerase binding sites in virulence gene promoters (Roy and Falkow, 1991; Karimova et al., 1996: Karimova and Ullmann, 1997; Marques and Carbonetti, 1997; Kinnear et al., 1999). Currently, it is thought that there is direct protein-protein interaction between BvgA-P acting as a dimer and the RNA polymerase on the same face of the DNA helix (Boucher et al., 1997: Stibitz, 1998). However, BvgA~P interacts with smaller upstream regions for PVHA(--67 to --101) and PlBv G (-52 to -84) (Roy and Falkow, 1991) than for PPTX (-60 to -168) (Zu et al., 1996) and PcY/, (-51 to -137) (Karimova et al., 1996), which suggests that BvgA-P acts with high affinity to stimulate transcription on early gene promoters, but that co-operative binding at higher concentrations of the effector is required at several low affinity binding sites for efficient transcription at late promoters (Boucher and Stibitz, 1995; Karimova and Ullmann, 1997; Marques and Carbonetti, 1997). Functional differences between the two types of promoter are further emphasized by characterization of mutations causing specific loss of expression of PTX and ACT, but not FHA, which were found to map to either the t p o A gene (encoding the c¢ subunit of RNA polymerase) or the Cterminal portion of BvgA, downstream of the putative helix-turn-helix DNA binding motif (Carbonetti et al., 1994; Stibitz, 1994). These results indicate that BvgA interaction with RNA polymerase may differ, depending on the promoter context. A recent report examined the kinetics of transcription from the locus encoding PRN (Kinnear et al., 1999). In this case, an intermediate pattern was obtained with transcription from PPRNinitiating at about 60 rain, between that of the early PFHA and late PPTX' Transcription from this promoter required BvgA-P bound to an inverted repeat with 9 of 14 bp of the consensus sequence within the region from -52 to -94, with BvgA being inactive and unable to bind to the PPRNregion. What happens if the bacteria are 'down-shifted' from .37 °C to 25°C or exposed to other modulating conditions, such as the presence of MgSO4? The amount of P 1BVC,'PrHA and PcYAmRNA was shown to decrease with estimated half-lives of 45, 40 and 9 rain, respectively, on 'down-shift' from 37°C to 25°C (Prugnola et al., 1995). Similarly, exposure of cells growing exponentially at 37°C to 50 mM MgSO4 promoted little change for the first 6 min, but after this time the PI BVG'PFHA'PPTXand PCYAmRNAs decayed with half-lives of 12, 4, 2 and 2.5 min, respectively (Scarlato and Rappuoli, 1991 ). However, the P 1BW~ mRNA persisted at a low level lbr 2 h and only became undetectable alter 8 h.

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Thus, the external modulating signal required only 6 min to block RNA transcription of the genes, although transcription of the bvg locus continued at a much reduced level for longer. The effect of a 'down-shift' in temperature apparently took longer to repress the genes. In keeping with this, the amount of BvgA did not obviously decrease over the first 2 h when cells were shifted from 37°C to 25°C, although the degree of phosphorylation of the effector protein was not determined (Prugnola et al., 1995). Thus, the expression of rag genes appears to be temporally programmed, at least in vitro, which would coincide with an early requirement for adhesins during infection and a later need tBr toxins as the infection becomes established and promotes a response from the host immune system. However, what role this differential regulation may play in vivo is unknown and it may simply reflect the capacity of the organism to respond to small changes in the environment in different ways, Thus, for example, Melton and Weiss (1993) showed that a TnS-lacZ fusion to a gene involved in PTX secretion responded to a threshold level of MgSO 4 with an all-or-none response whereas induction of a JhaB-lacZ fusion varied in parallel with MgSO 4 concentration. These observations would fit with a model where BvgA-P has a high affinity for PrnA at low concentrations, but where expression t?om PPTX requires a higher concentration of BvgA-P acting in a co-operative manner. It also explains the intermediate phase of antigen expression, which has been noted under semimodulating conditions (Lacey, 1960; Cotter and Miller, 1997), whereby a subset of Bvg-activated factors such as FHA and fimbriae are expressed together with antigens specific to this intermediate phase (section 4.3).

3.2.2. Signal Transmission What is not clear from these experiments is the manner in which modulating signals, in particular temperature shift, are able to regulate expression from the bvg-associated loci. The activity of some bacterial promoters that respond to temperature and other environmental changes is known to correlate with the degree of DNA supercoiling, which has emerged as an important link between environmental change and gene transcription (Dorman. 1995). A clue as to how alteration in temperature may control the bvg-associated loci in bordetellae was provided by Scarlato et al. (1993), who showed that transcription of the PPTX promoter in E. coil depended on the presence of the bvg operon, but the cloning configuration of the two loci was critical for activity,. Thus, environmentally regulated transcription of PPTX occurred if it was cloned in cis with the bvg operon on the same plasmid. Co-transformation of E. coil with the bvg locus on a low-copy-number plasmid, with PPTX on a high-copy-number plasmid, only allowed transcription from PPTX if this region was bounded by tnmscriptional terminators that prevented any readthrough of transcription

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from cryptic plasmid promoters and, even then, no environmental regulation was achieved. These data suggested that an elongating RNA polymerase complex, intruding into the region of the Pvrx promoter, might alter the structure of the DNA template and prevent transcription. It is established that transcription can alter the topological state of DNA (Dorman, 1995). In keeping with this, addition of novobiocin, an inhibitor of DNA gyrase, the enzyme responsible for implementation of negative DNA supercoiling, promoted increased transcription from Pwrx in E. coli, indicating that DNA relaxation had stimulated transcription. These experiments, therefore, provided evidence that transcriptional regulation can be influenced by topological changes in the promotet region. Investigation of the effect of novobiocin on vag and vrg gene expression in B. pertussis also showed that these loci were very sensitive to the drug. All of those examined exhibited a marked decrease in expression (including Pvrx), except for PPRN, which was up-regulated (Graeff-Wohlleben et al., 1995). The reason for the significant differences in PPTX behaviour in the two bacterial hosts is unclear. Aside from the equilibrium afforded by the reciprocal action of DNA gyrase (DNA topoisomerase II) and DNA topoisomerase I, which function to promote DNA supercoiling and relaxation of DNA supercoils, respectively, other protein elements are known to influence DNA topology. In E. coli, one of the most important of these from a regulatory viewpoint is H-NS, a small histone-like DNA-binding protein involved in maintaining the architecture of the condensed bacterial chromosome. The protein is implicated in a variety of enviromnental effects on gene transcription in E. coli, including repression of virulence-associated loci at low temperatures, and it is suggested that increased temperature compromises the ability of H-NS to interact with DNA to repress these loci (Atlung and Ingmer, 1997; Hurme and Rhen, 1998). Proteins with similar properties to H-NS have been implicated in temperature regulation of virulence loci in several pathogens (Dorman, 1995; Hurme and Rhen, 1998; Nye et al., 2000). A DNA-binding protein, BpH3, with significant homology to H-NS, has been described in B. pertussis (Goyard and Bertin, 1997), but defining a precise role for this protein in temperature-regulated transcription has been hampered by the inability to disrupt the bph3 gene, suggesting that it is essential for cell viability. Taken together, the data discussed above suggest that the promoters of the bvg-associated loci may be directly influenced by changes in temperature via alteration in the conformation of the DNA through supercoiling changes and/or interaction with H-NS-like proteins. It is unlikely that other modulating signals such as the presence of MgSO 4 or nicotinic acid influence promoter activity in this way. Other possibilities tbr differential regulation that have to be considered are environmental influences on mRNA stability and a direct influence on the BvgS sensor protein itself. Graeff-Wohlleben et al. (1995) noted that the half-life of a vrg transcript increased fi'om 5 min at 37°C to 26 rain at 25°C, in

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keeping with greater expression of vrg-encoded proteins at the lower temperature. Interestingly, other modulating signals affected the half-life of the vrg transcript in different ways. Addition of nicotinic acid only increased the halflife at 37°C to 12 rain, and MgSO 4 had no effect at all. The authors suggested that the cellular responses to changing growth conditions may be fine-tuned by differential action of the various modulating signals on mRNA stability of some bvg-associated genes. Thus, modulating signals such as MgSO 4 and nicotinic acid, particularly the former, must presumably exert their effect on increased vrg gene transcription by means other than DNA topology and mRNA stability. A direct effect on the BvgS protein, which prevents the phosphorelay to BvgA, is the obvious possibility. The SO42- anion is important for this as MgC12 has no modulating effect, but other cations such as Na +, K + or NH4 + can replace Mg 2+ (Brownlie et al., 1985; Melton and Weiss, 1989). The necessary features of an efficient modulating compound were investigated by Melton and Weiss (1993). Modulators must be negatively charged and induction of modulation by analogues of nicotinic acid showed that the carboxyl group was important, as nicotinamide was inactive, but the pyridine ring nitrogen was not necessary because benzoic acid was active. Binding of modulators to the bacterial cell surface could not be demonstrated. How external signals influence the BvgS protein is not known. Perhaps the negatively charged modulator interacts with positively charged amino acids in the periplasmic domain of BvgS which may induce a conformational change that prevents dimerization of BvgS and consequent phosphorelay to BvgA. Although the data of Melton and Weiss (1993) produced a clear picture of the structural requirements for a modulating compound, attempts to identify a natural modulator possessing the required features, such as retinoic acid or a derivative of aromatic amino acids, were unsuccessful.

3.2.3. The Reciprocal Expression ofvag and vrg Loci The expression of the bvg regulon is complex because the BvgAS products promote transcription of the vag genes and repression of the vrg genes; this scenario is reversed by modulating conditions or mutation in the bvg locus. Because of the requirement for an intact bvg locus to repress the vrg genes, it was assumed that either the BvgA effector protein itself acted as a repressor at vt~4 gene promoters or it activated a gene that encoded the vrg gene repressor. By examining transposon insertion mutants of B. pertussis that demonstrated constitutive expression of a bvg-repressed gene under conditions where the vag genes were expressed, Merkel and Stibitz (1995) were able to identify a putative open reading frame (ORF), close to the bvgAS operon, with homology to sequences of unknown function in other species. This locus, termed bvgR, is

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transcribed in the opposite orientation to bvgAS and is dependent on BvgA for expression (Merkel et al., 1998a). It encodes a predicted 32 kDa protein, which may act to repress the vrg genes, Evidence in support of this was provided earlier by Beattie et al. (1990, 1993), who identified a conserved 32 bp sequence at the 5' end of four of five vrg genes within the coding region, which was shown by Southwestern analysis to bind a -34 kDa protein present in nonmodulated cells but absent in modulated cells. Thus, the BvgR protein may bind this conserved sequence within the coding region of a vrg gene and repress transcription. The sequence was not identified in one vrg gene and an additional mode of regulation, perhaps involving an activator itself repressed by BvgR, would need to be invoked in this case. A model for the control of the bvg regulon is shown in Fig. 3.

3.2.4. Consequences of Alteration in Signal Transduction

Several mutations that render the expression of the bvg operon constitutive and insensitive to environmental signals have been described (Knapp and Mekalanos, 1988; Miller et al., 1992; Cotter and Miller, 1994; Goyard et al., 1994; Manetti et al., 1994). All result from point mutations in the linker region of BvgS (Fig. 1), which emphasizes the importance of this region for signal transduction. Somewhat surprisingly, no constitutive mutants of this type have been characterized with alterations in the periplasmic domain, where environment sensing is assumed to occur. Linker insertion in this region prevents bvgAS activation of rag genes (Miller et al., 1992), perhaps by preventing oligomerization of BvgS. It has been suggested that the mutations in the linker region act to stabilize BvgS in an oligomeric conformation that is unable to respond to a modulating signal, and is, therefore, continually primed to autophosphorylate. A point mutation in the transmitter domain of BvgS (Fig. 1) at position 733 close to His-729, the proposed site of phosphorylation of the transmitter domain, creates a constitutive phenotype that is intermediate (Bvg i) between the Bvg+ and Bvg- phases (Cotter and Miller, 1997).

4. THE ROLE OF THE Vag A N D Vrg GENE P R O D U C T S IN THE B O R D E T E L L A LIFE CYCLE Bordetella is related phylogenetically to Alcaligenes and other free-living bacteria, which suggests that the ancestral bordetellae evolved to infect warm-blooded hosts. B. bronchiseptica is able to survive and grow under nutrient-limiting conditions (Porter et al., 1991; Cotter and Miller, 1994) and in natural waters (Porter and Wardlaw, 1993). Transmission of B. avium is

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reported to occur via contact with contaminated litter and water rather than aerosol droplets (Cimiotti et al., 1982), which indicates an ability to survive in the external environment. These properties may still reflect the ancestral origin of the bordetellae. B. pertussis and B. parapertussis appear to have lost the facility for prolonged survival in the external environment; they thus represent pathogens highly adapted to the mammalian host. The maintenance of the BvgAS system in the bordetellae would indicate that it plays an important role in the life-style of the organisms, allowing them to adapt to marked changes in their environment. Thus, it can be assumed that the two types of reciprocally expressed gene products act at different stages of the life cycle. Under one set of conditions, the expression of the vag genes would be important, while under a different set of conditions, the expression of the vrg genes would be advantageous. These responses might conceivably occur within the host, at different stages of infection, or outside the host during transmission to another host or in the environment.

4.1. B. pertussis The human respiratory tract is the only known habitat for B. pertussis. There is little evidence to suggest that vrg gene expression is required during the disease process, at least in the mouse model of infection. Martinez de Tejada et al. (1998) and Merkel et al. (1998b) constructed isogenic mutants of B. pertussis 18323 that had constitutive Bvg + or Bvg- phenotypes and compared their ability to cause disease in mice. The Bvg c mutants had point mutations in the linker region of BvgS (Miller et al., 1992), and exhibited a constitutive phenotype for Vag products under all environmental conditions. The Bvg- mutants had truncated bvgS genes which rendered the bvgAS operon inactive and were therefore phenotypically constitutive for expression of the Vrg products. The Bvg R mutants contained in-frame deletions in the bvgR gene and, therefore, constitutively expressed Vrg products even in the Bvg + phase. There was no significant difference up to day 35 between the numbers of bacteria recovered from the nasal cavity, trachea and lungs of infant mice inoculated intranasally with -5 x 104 CFU of the parent 18323 or the Bvg c mutant strains. A Bvg mutant was not recovered from any location at day 6, confirming that lack of expression of Vag products prevented colonization of the mouse respiratory tract. The numbers of Bvg R bacteria recovered from the respiratory tract were decreased compared with the wild-type, particularly when the infection with the parent strain was at its peak, and the survival rate of mice infected with the Bvg R strain was much higher than that of mice infected with the wild-type strain. These data showed that the Bvg + phase is necessary and sufficient tbr establishment of mouse respiratory tract colonization and, because the Bvg c strain was as virulent as the wild-type, the expression of the Vrg products is not

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required. In other words, constitutive expression of the Bvg + antigens had no effect on infection, which indicated that alternate expression of Vag and Vrg antigens at different stages of the infection was not apparently required. Moreover, ectopic expression of Vrg antigens during infection had a detrimental effect on the process, emphasizing the importance of bvgAS-mediated repression, via BvgR, of the vrg genes in vivo. Martinez de Tejada et al. (1998) did, however, examine more closely the expression of one vrg gene, vrg6, during infection. This gene was originally associated with virulence in B. pertussis (Beattie et al., 1992), but Martinez de Tejada et al. (1998) showed that the colonization defect was caused by an unlinked mutation in the original transposon insertion mutant, and construction of a new vrg6 deletion derivative had no effect on the ability of the strain to colonize the respiratory tract of mice. Nevertheless, the expression of vrg6 was examined in vivo using a promoterless cassette containing resolvase functions flanking a tetracycline-resistance (Tc R) gene placed downstream of the vrg6 promoter. Transcription of vrg6 would then drive expression of the cassette and the resolvase gene products would elicit loss of the Tc R gene by site-specific recombination. In vitro on agar, 7% of B. pertussis colonies were Tc-sensitive in the Bvg + phase and 90% were Tc-sensitive in the Bvg- phase, indicating quite clearly the expression of vrg6 under modulating conditions. In vivo after colonization of the nasal cavity, -17% of colonies recovered 20 days postinfection were Tc-sensitive, suggesting some activity of vrg6 in cells in vivo. Martinez de Tejada et al. (1998) interpreted these data with caution due to inherent experimental variation and possible differences in growth rate, but this approach may well be informative. It suggests that there may either have been increased activity of vrg6 in vivo in some of the infecting population of bacteria or the low basal level of vrg6 expression in the inoculating population encouraged a better overall growth and/or survival of those cells expressing the Vrg6 antigen. Thus, overall it is difficult with B. pertussis to visualize when the alternative expression of bvg-dependent genes would be needed. If there is such a requirement, the point(s) at which it is called upon, whether inside or outside the host, remains to be determined. If it is required at some point in the host, it must be at a late stage. It should be remembered, though, that the data to support these conclusions have been obtained from a mouse model of infection and should, therefore, be interpreted with some degree of caution as B. pertussis normally infects only humans. Lacey (1960) noted that in human sera from 50 convalescent patients, a small minority possessed antibodies to avirulent phase antigens while the remainder had antigens specific to the virulent phase. The former patients had only a mild cough, but were culture-positive for 2-5 months. It is conceivable, therefore, that the avirulent phase represents a pathologically dormant state associated with the later stages of infection and may provide a strategy for immune evasion and persistence. Lacey (1960) also

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showed that avirulent phase antigens expressed under modulating conditions were less immunogenic than those from the virulent phase. These may be the products of the vrg genes, which may contribute to persistence within the host. An alternative explanation tbr the appearance of antibodies to Vrg products is the appearance of spontaneous avirulent phase variants due to mutation in the bvgAS locus. Such variants were noted in an early report (Kasuga et al., 1954) and may explain the presence of Vrg antigens at late stages of infection. For example, Stibitz et al. (1989) noted that in one strain of B. pertussis the genotypic change to the avirulent form (phase variation) was reversible and caused by insertion or deletion of a cytosine residue in a run of six such residues within the bvgS gene. It is conceivable that reversible frame-shift mutations in this region of bvgS may represent a useful genotypic change that has been maintained against evolutionary elimination because it offers some advantage to this highly adapted pathogen. Supplementary to this, is the possibility that expression of avirulent phase antigens may be coupled to development of a transmission-competent state for aerosol transit between hosts. Here, lbr example, lack of expression of antigens acting as adhesins would aid expulsion of the bacteria. As yet, no function has been assigned to any of the vrg gene products produced by B. pertussis and any knowledge of their contribution to the life-style of the organism may have to await a model system that is able to examine the entire life cycle.

4 . 2 . B. bronchiseptica

In contrast to B. pertussis, a reservoir of B. bronchiseptica outside the host is a real possibility as this organism, unlike B. pertussis, is able to survive and grow in natural waters and in buffered saline without added nutrients (Porter et al., 1991; Porter and Wardlaw, 1993). By contrast with the distinct lack of knowledge of the repertoire of Vrg factors in B. pertussis, features of this phase in B. bronchiseptica have been characterized. They include motility (Akerley and Miller, 1993), LPS variation (van den Akker, 1998) and synthesis of an adhesin (Register and Ackermann, 1997), urease (McMillan et al., 1996), acid phosphatase (Chhatwal et al., 1997) and, in some strains, siderophore production (Giardina et al., 1995). To date, no Bvg phase-specific factor common to both species has been noted. Animal infection experiments similar to those described above with B. pertussis were done with constitutive Bvg + or Bvg-strains of B. bronchiseptica (Cotter and Miller, 1994). In this case, however, there is the significant advantage of studying infection in a natural host. The wild-type strain used was originally isolated from the rabbit and was able to induce infection with as few as 200 CFU administered intranasally. The same constitutive mutation in the linker region of BvgS as was used in B. pertussis was introduced to create the Bvg c B. brom:hiseptica

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strain and an in-frame deletion in bvgS was used for the Bvg phase-locked strain. Experimental infection of the rabbit with these strains again showed that the Bvg + phase is necessary and sufficient for respiratory infection, as a Bvg+-phase-locked mutant infected rabbits in a manner indistinguishable from the wild-type (Cotter and Miller, 1994). The Bvg strain was unable to establish infection and no antibodies to flagellin (which would be expected to be expressed in the Bvg- phase) were detected in the sera of rabbits, indicating that this organism was cleared very rapidly from the animals. In separate experiments, i! was again shown that bvg-mediated repression of Vrg factors was crucial to infection as ectopic expression of a Bvg -phase phenotype (motility) in the Bvg + phase significantly reduced colonization in the rat (Akerley et ell.. 1995). However, outside the host, the Bvg--phase-locked mutant grew and survived in phosphate-buffered saline (PBS) medium to a greater extent than the wildtype, indicating a clear advantage for the Bvg- phase under nutrient-limiting conditions (Cotter and Miller, 1994, 1997). The Bvg c strain was unable to survive at all, which indicated that adaptation to the nutrient-limited PBS medium was dependent on Vrg factors expressed in the Bvg phase. Thus, for B. blvnchiseptica, these data suggest that the Bvg + phase is required for infection and that Vrg factors expressed during the Bvg phase aid survival and perhaps transmission of the organism outside the host. The ability to survive and multiply in an external reservoir may, therefore, be a crucial aspect of the B. bronchiseptica life cycle, but may not be as important for B. pertussis, which has evolved to rely on direct host-to-host transmission. It may explain why B. pertussis possesses non-expressed flagellin gene sequences (Akerley and Miller, 1993; Leigh et al., 1993) and has a bvg regulatory locus less sensitive to environmental signals (section 3.1. l ). It is noteworthy that an adhesin is produced by B. bronchiseptica in the Bvg phase. It is clear that adhesins produced in the Bvg + phase, such as FHA and fimbriae (Cotter et al., 1998; Mattoo et al., 2000), play an important role in colonization of the respiratory tract in vivo. However, other data have shown that greater numbers of temperature-modulated Bvg phase bacteria adhered to individual nasal epithelial or macrophage cells in vitro than non-modulated Bvg + phase bacteria (Register and Ackermann, 1997: Brockmeier, 1999) and, in experimental infection of the rat, greater numbers of Bvg phase bacteria adhered initially to the nasal cavity than non-modulated Bvg + phase organisms (Brockmeier, 1999). These data are interpreted to indicate that when bacteria first enter the host from the environment they will be in the Bvg- phase and the adhesin expressed in this phase is required to ensure that high numbers of bacteria can initially adhere, so as to prevent clearance mechanisms from removing the organisms, until the shift to the Bvg + phase is completed at the higher temperature and Bvg+-phase adhesins, such as the fimbriae and FHA, can take over.

164 4.3. Intermediate

J.G. COOTE Phase Proteins

The points discussed above raise the question as to what role, if any, intermediate or I mode antigens might play in the life cycle of the bordetellae. These are induced in vitro when the composition of ions in the medium, such as MgSO 4, is at an intermediate level (Lacey, 1960; Cotter and Miller, 1997). The I mode is characterized by expression of a unique subset of antigens. An understanding of this intermediate phase has been facilitated by the identification of a mutant strain of B. bronchiseptica with a point mutation in the transmitter domain of BvgS (Fig. l ). This creates a constitutive phenotype that is phase-locked in this intermediate (Bvg i) condition between the Bvg + and Bvg- phases (Cotter and Miller, 1997). The Bvg i mutation conferred an intermediate ability to survive under nutrient-limiting conditions compared with a phase-locked Bvg + strain that was unable to survive and a phase-locked Bvgstrain that exhibited prolonged survival in these conditions. Similarly, an intermediate capacity to establish infection in rats was demonstrated, with the Bvg i strain showing reduced ability to colonize the nasal septum and inability to colonize the trachea compared with the wild-type strain. Analysis of rag and vrg gene transcription showed that bvgAS, ,[haB and prn transcription in the Bvg i strain was somewhat reduced and cya transcription was markedly reduced. Conversely, transcription of a vrg gene associated with motility was slightly increased, but the Vrg products were not expressed to an extent that motility was exhibited by the Bvg i strain. This type of pattern was similar to that seen during temperature shift studies from 25°C to 37°C (Scarlato et al., 1991 ; section 3,2. l ), where temporal expression of rag genes was assumed to coincide with increased accumulation of the transcriptional activator BvgA-E If it is assumed that many, if not all, of the unique proteins produced by the Bvg i strain represent the products of bvg-regulated genes, it follows that varying environmental conditions may be precisely reflected in the intracellular concentration of BvgA-P which could, in turn, control the expression of different subsets of genes. The mutation in the transmitter domain of BvgS in the Bvg i strain may therefore have an adverse effect on autophosphorylation of the sensor protein or subsequent phosphorelay to BvgA, which results in a constitutive intermediate level of BvgA-P. The nature and function of the I mode gene products represent important areas for future investigation. One such product has recently been identified (K. E. Stockbauer, B. Fuchslocher, J. E Miller and P. A. Cotter, submitted for publication). BipA (Bvg-intermediate phase protein) is a surface-exposed outer membrane protein in B, bronchiseptica, orientated such that its C-terminus extends from the cell surface. The gene sequence of bipA predicts a 165kDa protein with significant similarity at the N-terminal end with the membrane localization regions of intimin and invasin, proteins important for interaction of enteropathogenic and enterohaemorrhagic E. coil and YeJwiniaspp., respectively,

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with mammalian cells. The protein is characterized by a series of highly conserved 90 amino acid repeats whose role is unknown, but which may serve to separate the membrane localization region from the C-terminal, region which mediates cell/cell interaction. The bipA gene sequence from B. pertussis predicts a very similar protein of 137 kDa protein, which is smaller only by virtue of having five of these repeat regions compared with eight for BipA of B. bronchiseptica. In B. bronchiseptica, BipA was not expressed in the Bvg- phase (created by addition of 20 mM nicotinic acid to the growth medium) and was maximally expressed under intermediate-phase (Bvg i) modulating conditions (addition of 0.5 mM nicotinic acid), although some production was manifest in the Bvg + phase. This contrasts with B. pertussis, where BipA was not produced in the Bvg + phase. Stockbauer et al. and Cotter and Miller (2000) propose a model in which BvgAS mediates activation of bipA under Bvg i- phase conditions and repression of bipA transcription in the Bvg + phase. The model would predict that the Bvg i gene promoters contain both high- and low-affinity binding sites for BvgA-R the former acting to stimulate transcription of early vag genes and Bvg i genes and the latter acting to repress Bvg i gene transcription (and activate late vag gene transcription) as BvgA-P accumulates. If this is correct, it identifies the first gene under BvgAS control that can be differentially activated or repressed in response to changes in environmental conditions, a feature that may turn out to be true for all of the Bvg i- phase genes. Cotter and Miller (1997) showed that at least some of the Bvg i- phase proteins were immunogenic in the rat after infection with the Bvg i mutant. Stockbauer et al. noted anti-BipA antibodies in sera of rabbits infected with wild-type B. bronchisel)tica, although antibodies to the Bvg i- phase polypeptides were not detected in sera from rats infected with the wild-type strain (Cotter and Miller, 1997). Sera from children recovering from pertussis were reported to contain antibodies that recognize some of the Bvg i antigens (Martinez de Tejada et al., 1998). Examination of a bipA null mutant of B. bronchiseptica in a rabbit model of infection showed that lack of the ability to produce BipA did not compromise respiratory tract colonization up to 5 weeks post-intranasal inoculation. It remains unclear what role the I phase antigens have in the life cycle of the organisms, but certainly the transition periods between environment and the host would seem likely points, although transient expression during the life cycle, for example at the earliest points of establishment of infection in the host or at a point prior to aerosol transmission, cannot be ruled out, In conclusion, the Bvg + phases of B. pertussis and B. bronchiseptica are represented by a similar set of Vag products, but the Bvg phases would seem to have radically diverged as the species evolved to produce different survival strategies. Both species produce 1 phase antigens: their role in the life cycle remains to be clarified, but they have been suggested to play an important role in aerosol transmission (Cotter and Miller, 1997, 2000; Martinez de Tejada et al., 1998).

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4.4. Interaction w i t h the Host I m m u n e System In both humans and mice, immunization with the whole-cell pertussis vaccine has been shown to induce antibodies to Vag products, such as PTX, FHA, PRN, ACT and the fimbrial agglutinogens. Several of these are important protective antigens and are included in the recently developed acellular pertussis vaccines (Corbel and Xing, 1997; Hewlett and Halperin, 1998). Humoral immunity is assumed to have a central role in immunity to pertussis, but recent studies have indicated that cell-mediated immunity also plays an important part in the development of protective immunity to both B. pertussis and B. bronchiseptica infection (Gueirard et al., 1996; Mills et al., 1998). In the mouse model, intranasal infection with B. pertussis or B. bronchiseptica induces an influx of neutrophils and macrophages into the lung compartments. The bacteria respond to this hostile environment; many of the immune effector cells become progressively apoptotic during the course of the infection, indicating a vigorous cytotoxic response by the bacteria to these innate immune responses (Gueirard et al., 1998; Harvill et al., 1999b). Until recently, the bordetellae were generally considered to be extracellular pathogens associated predominantly with the respiratory tract of humans, animals and birds. However, a number of studies have shown that Bordetella spp. can both adhere to and invade, or be taken up by, a variety of eukaryotic cells in culture, including epithelial cells, dendritic cells, macrophages and neutrophils or polymorphonuclear leukocytes (PMN). These in vitro studies have shown that B. bronchiseptica is able to persist intracellularly tbr extended time periods, but B. pertussis survives less well and for shorter periods (Savelkoul et al., 1993; Guzman et al., 1994; Schipper et al., 1994; Banemann and Gross, 1997; Forde et al., 1998; Bassinet et al., 2000; Lenz et al., 2000). Uptake of B. pertussis has been clearly shown to occur in a bvg-dependent manner, as bvg mutants are found intracellularly in lower numbers than the wild-type and are cleared more rapidly (Ewanowich et al., 1989; Lee et al., 1990; Roberts et al., 1991 ; Friedman et al., 1992; Bassinet et al., 2000). In contrast, there exist conflicting reports on the role of the bvg locus in the invasive phenotype of B. bronchiseptica. It has been reported that Bvg- phase or bvg mutants of B. bronchiseptica are as able or more able than their wildtype parent to invade and survive intracellularly in Caco-2, A549, dendritic and HeLa cells and macrophages (Guzman et al., 1994; Schipper et al., 1994; McMillan et al., 1996; Banemann and Gross, 1997; West et al., 1997). However, in two other reports, Bvg + phase B. bronchiseptica was shown to invade HeLa cells or PMN 10-fold more efficiently than Bvg B. bronchiseptica (Savelkoul et al., 1993; Register et al., 1994). The reason for this discrepancy may be related to differences between strains of B. bronchiseptica in cytotoxicity to mammalian cells. A cytotoxic effect on epithelial cells, only expressed in the Bvg + phase of B. bronchiseptica and not by B. pertussis or B.

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parapertussis, was described by van den Akker (1997). The same toxicity towards macrophages has also been shown (Forde et al., 1999; Harvill et al., 1999b), but this phenotype appears to be strain-dependent. With a non-toxic strain, intracellular bacteria originally in the Bvg- phase were recovered in numbers 20-fold less than those in the Bvg + phase from cultured macrophages, and similar results were obtained with a more toxic strain if the multiplicity of infection was reduced and the infection period was limited to 1 h (C. B. Forde, J. G. Coote, R. Patton, S. Campbell and M. Roberts, submitted for publication). it appears, therefore, that uptake of B. bronchiseptica into mammalian cells is bvg-dependent, but this may be masked by the potential toxicity of some strains in the Bvg + phase. Although B. pertussis does not show toxicity towards epithelial cells, it is toxic towards macrophages and this effect is mediated via ACT, both in vitro in cultured macrophages and in vivo in mouse alveolar macrophages, in a process that resembles apoptosis (Khelef et al., 1993; Khelef and Guiso, 1995: Gueirard et al., 1998). The toxicity exhibited by B. bronchiseptica is more rapid in its action and seems to involve a mainly necrotic pathway with a small proportion of cells undergoing apoptosis (Forde et al., 1999), although it was noted in these experiments that high levels of toxicity were only exhibited if the bacteria were centrifuged onto the macrophages to bring the cell types into close proximity. Without this treatment, the major proportion of mammalian cells in the population remained viable, which agreed with the original report that the factor responsible for toxicity was not secreted by the bacterium (van den Akker, 1997). The bvg-dependent type 111 secretion system possessed by B. bronchiseptica and only expressed in the Bvg + phase (Yuk et al., 1998b) appears to be involved in this toxicity. A mutant strain lacking a functional type IIl secretion system was considerably less toxic towards macrophages than the parent strain and a further mutation that rendered the strain ACT abolished cytotoxicity altogether (Harvill et al., 1999b). It seems that, with B. bronchiseptica, macrophage cell death is only partly dependent on ACT and can be caused by more than one means. There is also extensive diversity within the macrophage population (Gordon et al., 1992) and this may influence the manner of interaction between the two cell types.

4.4.1. The lmportatwe o['the Type III Secretion System The type lIl secretion system in B. bronchiseptica has been shown to export several proteins when the bacterium is in the Bvg + phase, some of which are detected by antibodies present in serum from animals previously infected with the pathogen (Yuk et al., 1998b, 2000). The role of these proteins in the infection process is not clear as yet, although a strain deficient in the type 111

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secretion system was unable to establish long-term colonization of rat trachea. Upon infection, similar numbers of wild-type and mutant bacteria were retrieved from the nasal septum at 14 and 35 days, and also from the trachea at 14 days, but at 35 days the mutant bacteria had been cleared from the trachea while the wild-type strain had a firmly established infection. These results indicated that the proteins delivered by the type IIl secretion system were specifically aiding persistence in the tracheal region of the lower respiratory tract, but were not important for initial colonization. Yuk et al. (1998b) suggested that this may be a reflection of the requirement to combat induced immune responses in the normally sterile region of the trachea. Type III system-defective mutants induced higher titres of anti-Bordetella antibodies than the wild-type organism, suggesting that the proteins of the type III system normally interfere with adaptive immunity. How this occurs is not clear, but may be due to inactivation of antigen-presenting cells such as macrophages, as Yuk et al. (1998b, 2000) showed that type lII system-defective mutants were less toxic towards macrophages and neutrophils and did not induce apoptotic features characteristic of the wild-type organism. Other data showed that the type Ill proteins were responsible for dephosphorylation of tyrosine-phosphorylated proteins in cultured rat lung epithelial cells (Yuk et al., 1998b) and prevented translocation of the important transcriptional regulator NF-nB to the nucleus (Yuk et al., 2000). Many host immune responses, including cytokine production, are regulated by this family of transcriptional regulators (Ghosh et al., 1998). The proteins of the type IIl secretion system, therefore, seem important for modulating the adaptive host immune response to B. bronchiseptica infection, but their precise mode of action awaits further investigation. Only certain strains of B. pertussis and B. parapertussis were shown to express the type III secretion system under laboratory conditions (Yuk et al., 1998b) and the relevance of this sytem to B. pertussis infection has not been established.

4 . 5 . T h e S i g n i f i c a n c e o f an I n t r a c e l l u l a r S t a g e

The relevance to natural infection of in vitro observations on mammalian cell cytotoxicity and uptake and survival remains to be confirmed, but intracellular persistence may represent a chronic or quiescent stage in vivo. Bacteria would be protected from antibody and complement and, in a quiescent state, would represent an asymptomatic carrier condition. It is worth noting that certain invasive pathogens, such as Shigella and Salmonella, have well-characterized type IlI secretion systems that are involved in entry into mammalian cells (Galan, 1996; Menard et al., 1996) or are necessary for intracellular survival (Cirillo et al., 1998; Hensel et al., 1998). The bordetellae are among a growing number of pathogenic bacteria, including Helicobacter, Neisseria, Staphylococcus and Campylobacter, which were considered to be exclusively

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extracellular but for which there now exists some evidence of an intracellular phase. Pathogens such as these can be considered to be weakly invasive, in terms of the numbers of bacteria internalized, when compared with facultative intracellular bacteria such as Salmonella and Shigella, and the role, if any, that this facility has in vivo is difficult to assess. Certainly, B. pertussis has been reported within rabbit pulmonary macrophages, and within cells in mouse bronchoalveolar lavage fluid and rat lung tissue after experimental infection (Woods et al., 1989; Saukkonen et al., 1991; Hellwig et al., 1999). It has also been reported within pulmonary alveolar macrophages from children with HIV (Bromberg et al., 1991). However, intracellular survival and cytotoxicity towards the host cell are mutually contradictory. The organisms may have the capacity to kill mature, potentially more bactericidal, macrophages preferentially, and some evidence for this has been provided by the observation that freshly harvested pulmonary alveolar macrophages were more susceptible to killing by B. bronchiseptica than a murine macrophagelike cell line (Forde et al., 1999). Other cells may present a more favourable niche for bacterial survival, which may require down-regulation of toxic factors and expression of other components needed for such a survival mechanism. It was clear, however, as discussed above, that in experimental infection models, the Bvg + phase was necessary and sufficient for establishment of animal respiratory tract colonization and Bvg -phase antigens were apparently not expressed or required. These experiments do not rule out the possibility that bordetellae switch to the Bvg phase at a location within the host where an antibody response would not be generated, or that certain factors needed for longer term survival assume greater importance at late stages of infection.

4.5. I. The Role of the ris Locus On the assumption that an intracellular step may represent an important aspect of at least the B. bronchiseptica life cycle, Jungnitz et al. (1998) examined transposon insertion mutants for a decreased survival capacity in cultured macrophage cells. One such mutant was chosen for study and shown to have a transposon insertion in a locus with homology to other bacterial two-component regulatory systems. The locus, termed ris (regulator of intracellular stress response), was composed of a sensor kinase, RisS, and a response regulator, RisA, which showed 25% and 23% similarity to BvgS and BvgA, respectively. Analysis of the effects of various environmental conditions on a ris mutant, created by insertion of an antibiotic resistance locus into the chromosomal ris locus, indicated that expression of RisAS was required for resistance to oxidative stress, production of the bvg-repressed acid phosphatase and persistence in the mouse. Previous data had indicated

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that the acid phosphatase produced by B. bronchiseptica, but not by other bordetellae, had a role in intracellular survival (Chhatwal et al., 1997). RisAS expression, like that of BvgAS, was modulated by temperature, although not to the same extent. Expression of RisAS was reduced by 42% after growth at 25°C compared with 37°C. A similar down-regulation was observed in the presence of MgSO 4 or MgCI 2, but not nicotinic acid, which indicated that Mg 2+ ion concentration had an effect on ris expression. This is reminiscent of the regulation of the PhoP/PhoQ two-component system required for virulence in Salmonella, which is thought to be expressed as a response to low intracellular Mg 2+ levels (Vescovi et al., 1996). The bvg locus appeared to have no direct influence on ris expression, hut resistance to oxidative stress and acid phosphatase production were repressed by BvgAS expression. These data imply that these important factors regulated by RisAS and involved in intracellular survival depend on down-regulation of BvgAS expression (Fig. 3). Interestingly, the ris mutant was cleared rapidly after intranasal infection of mice and no bacteria were detected in the lungs after 14 days. The implications of this are that ris expression is an important regulator of factors required for successful colonization of the host. This might suggest that the locus has a role in combating the innate immune response of the host, by responding to signals in the intracellular environment that allow the bacteria to persist within immune effector cells. However, the factors controlled by ris may not be solely concerned with intracellular survival as Jungnitz et al. (1998) showed by 2D electrophoresis that ris controlled the expression of at least 18 proteins. As their expression appears to depend on down-regulation of BvgAS expression, some of these proteins may be represented among the factors normally referred to as Vrg products. The sequence of the ris locus is highly conserved in the bordetellae (Jungnitz et al.. 1998), but its significance to the life cycle of species other than B. bronchiseptica remains to be clarified. The role of the ris locus and indeed the bvg locus in the strategies employed by the bordetellae during infection demand further study. The data of Jungnitz et al. (1998) would suggest that these regulatory loci are expressed in a reciprocal manner, which might suggest, because expression of the ris locus is down-regulated by Bvg products, that Vag products would be switched off in the intracellular environment where the ris-controlled gene products are required. However, it is not altogether clear at present how the bvg locus responds to the intracellular environment, although certain rag gene products of B. pertussis, including ACT, have been reported not to be expressed within alveolar macrophages (Saukkonen et al., 1991 ; Masure, 1992). Relevant data for intracellular bvg-regulated gene expression by B. bronchiseptica is not available and a clearer picture needs to be gained of the role of these regulatory loci in the life cycle of the bordetellae.

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5. C O N C L U S I O N S A good overview is now available about the way in which the bordetellae control the expression of different factors as a response to a changing environment, but much of this information has been gained from laboratory experiments. Molecular genetic analysis has revealed the importance of differential transcription to in vivo infection or survival in the external environment and has also revealed that these alterations in gene transcription are controlled to a great extent by the central bvg and ris regulatory loci. However, the natural signals that influence the activity of the bvg and ris regulons are unknown, and how these signals are 'sensed' by the bacterium is not clear. There is still a need to know when and where in the host, or outside the host, virulence or survival genes are regulated; this depends on techniques that can monitor the expression of genes both in vivo and in the environment. The use of reporter-based assays has proved a reliable means to monitor the fate of bordetellae within mammalian cells. Investigations of low-level internalization and survival are enhanced by real-time methods for monitoring the activities of intracellular bacteria within eukaryotic cells, and are helpful in isolating non-invasive mutants and monitoring the level of transcription of a gene (Valdivia and Falkow, 1997). Luciferase and green fluorescent protein (GFP) reporters have both been employed with the bordetellae. Internalization and persistence of B. bronchiseptica was examined in a murine macrophage-like cell line and in murine peritoneal phagocytes using bioluminescence (Lux) as a reporter of bacterial viability (Forde et al., 1998). A mini-Tn5-based suicide vector carrying the htx genes from Photorhabdus Ittmittescens acted as a promoter probe in B. bronchiseptica and allowed the generation of a random pool of stable bioluminescent fusion strains. In addition to its potential for studying the regulation of gene expression, this system offers a number of other advantages for monitoring intracellular survival. The intact htx operon is carried within the mini-transposon and there is no requirement for addition of exogenous substrates, which are often toxic to cell lines, to render the bacteria bioluminescent. The P. luminescens luciferase is stable at 37°C and, as bioluminescence is directly related to cellular viability, monitoring of light output from internalized bacteria allows a real-time semiquantitative estimate of the numbers of intracellular bacteria and of the length of time for which they are able to persist. Cytoplasmic expression of GFP has also been used to study the interaction of B. pertussis with human neutrophils (Weingart el al., 1999: Lenz et al., 2000). Here, g,¢/~genes were expressed from a plasmid or incorporated into the chromosome and fluorescence microscopy in the presence of ethidium bromide used to distinguish attached and internalized bacteria. Intracellular bacteria resist staining with ethidium bromide and appear green, but

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extracellular bacteria take up the stain and appear orange. It will be informative to adapt these techniques to follow the fate of the bordetellae within the mammalian host and to investigate the in vivo regulation of important virulence determinants. The genomes of B. pertussis, B. parapertussis and B. bronchiseptica are currently being sequenced (http://www.sanger.ac.uk/Projects/B_pertussis/). These will allow comparative studies to be done on these closely related organisms, which nevertheless manifest differences in the diseases they cause, their host range and interactions with their environment. The genome-based approach will reveal genes unique to a particular species or genes differentially expressed in the different organisms, which can then be tested for their roles in disease. Gene sequence analysis will also reveal more gene candidates for a role in pathogenesis. For example, 10 ORFs are present in the B. pertussis genome sequence that have similarity with the autotransporter family of outer membrane proteins (Henderson et al., 1998). They include the sequences for PRN, BrkA and TCF (E Blackburn and M. Roberts, personal communication). These sequences may represent genes whose products play a role in disease. Besides bvg and ris, 9 other ORFs with consensus motifs associated with histidine-kinase sensor proteins have been identified in the B. bronchiseptica genome sequence, three of which have adjacent ORFs with conserved residues indicative of response regulator proteins (M. Lynch, personal communication). Again, these genes with similarity to other two-component systems may play an as yet unidentified role in the life cycle of the organism. Knowledge of the genome sequence will allow microarrays spanning all the known ORFs to be hybridized with mRNA or cDNA samples prepared from bacterial cells harvested from different locations or subjected to different environmental signals, for example, growth in laboratory conditions and in a disease-related environment. This will allow a 'global' picture of gene transcription to be obtained, pinpointing which genes are expressed or repressed in the different conditions. A time-course analysis upon transition from one environment to another will identify co-ordinately regulated genes. This type of analysis also permits some indication of the function of particular genes under the different circumstances, because genes exclusively expressed under a given condition should be important for the existence of the organism in that environment. Advances and refinements in protein separation techniques will allow the full impact of proteome analysis to be applied to both bacteria and host cells for the detection and characterization of proteins specifically expressed as a response to the types of environmental changes and cell-cell interactions discussed here. The members of the bordetellae represent highly adapted microorganisms that are exposed to three entirely different environments - the external environment, the surface of the respiratory tract and a possible intracellular condition - during their life cycle. They are able to sense and respond to the

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different stresses of low nutrients (starvation) and host antimicrobial factors associated with transition from one state to another. Despite the wealth of information already accumulated, much still needs to be investigated: the reasons for differences in host range between the B o r d e t e l l a species; characterization of the Vrg and intermediate-phase proteins produced by B. p e r t u s s i s and B. b r o n c h i s e p t i c a and their role in the life cycle of the two species; the manner in which the rag and vrg genes are regulated throughout the life cycle; the significance of a possible intracellular stage related to persistence in the host; possible effects on gene expression dependent on quorum-sensing mechanisms as cell densities increase during infection; and the contribution that the properties of different subsets of bacterial or host cells might play in the interplay between host and pathogen.

ACKNOWLEDGEMENTS I thank Roger Parton for valuable discussion and Jeff Miller and Peggy Cotter for making material available prior to publication. Work in the authors' laboratory was supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust.

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NOTE ADDED

AT PROOF:

1. A recent study (Antoine, R. el al. J Bacteriol. 182, 3902-3905, 2000) has shown the potential that the B. pertussis genome sequence provides for the identification of potential new virulence factors. A report by Merkel, T.J. and Keith, J.M. (Int. J. Med. Microbiol. 290, A3, 2000) provides the first evidence for quorum sensing in B. pertussis and suggests a role for vrg genes in sensing bacterial density. A paper by Belcher. C.E. et al. ( Proc. Natl. Acad. Sci. USA 97, 13847-13852, 2000) used high-density DNA microarrays to measure the transcriptional responses of host respiratory epithelial cells to B. pertusxis infection.