J. Mol. Microbiol. Biotechnol. (2002) 4(3): 197–203. JMMB Symposium

Regulatory Factors of Bordetella pertussis Affecting Virulence Gene Expression

Jochen Ko¨ nig, Andreas Bock, Anne-Laure Perraud, 1989). Phenotypic modulation depends on the activity Thilo M. Fuchs, Dagmar Beier, and Roy Gross* of the histidine kinase BvgS which is an environmental sensor located in the cytoplasmic membrane. Under Lehrstuhl fu¨ rMikrobiologie, Theodor-Boveri-Institut the appropriate conditions, the BvgS protein autophos- fu¨ rBiowissenschaften, Universita¨ tWu¨ rzburg, D- phorylates at a histidine residue in its transmitter 97074 Wu¨ rzburg, Germany domain and, subsequently, the phosphate is trans- ferred to an aspartic acid in the receiver domain of the BvgA response regulator. BvgA-P is then able to activate transcription from the virulence gene promo- Abstract ters leading to expression of several adhesins and toxins (Table 1) (Rappuoli, 1994). Most pathogenic bacteria encounter changing The BvgAS system differs from typical two-com- growth conditions during their infectious cycle ponent systems, because BvgS contains additional and, accordingly, have to modulate gene expres- phosphorylation sites outside of its transmitter: In fact, sion to enable the efficient colonization of different an obligate multistep His-Asp-His-Asp phosphorelay environments outside or within their host organ- occurs that involves the BvgS transmitter, receiver and isms. In Bordetella pertussis the transcription of HPt domains (Figure 1) (Arico et al., 1989; Perraud most virulence factors including several toxins et al., 1999; Uhl and Miller, 1994,1996). The high and adhesins is regulated coordinately by the energy phosphohistidine present in the C-terminal HPt BvgAS two-component system. The molecular domain is the exclusive phosphate source for BvgA characterization of the BvgAS system revealed (Perraud et al., 1998). Furthermore, transcomplemen- that it belongs to the small group of unorthodox tation of mutant sensor proteins by the separate two-component systems applying an obligate mul- expression of individual signalling domains demon- tistep phosphorelay. Moreover, despite the coordi- strated that the phosphorelay in the BvgS histidine nated control of the virulence regulon, subtle kinase involves BvgS homodimers (Beier et al., 1995, differences in the regulation of individual virulence 1996). BvgA also forms homodimers, but, in contrast to genes were observed which led to the identifica- several other response regulators such as FixJ, there is tion of sophisticated mechanisms possibly no evidence for any influence of phosphorylation on its engaged in fine tuning of virulence gene expres- oligomerisation state (Bock, Rippe and Gross, sion. unpublished; Perraud et al., 2000). This shows that activation of BvgA by phosphorylation does not Coordinate Regulation of the Bvg-Regulon involve its dimerization (Perraud et al., 2000; Bock et al., 2001). It has been known for a long time that the expression of Little is known about the mechanisms of signal per- virulence properties of B. pertussis,theetiological ception and the control of the histidine kinase activity agent of whooping cough, is unstable. In fact, avirulent present in the transmitter domain of two-component so-called phase variants may arise with high fre- sensor proteins. However, like in other sensor proteins quency. Moreover, the virulent phenotype depends on the linker region of BvgS connecting the transmitter environmental conditions and is reversibly affected by domain with the membrane spanning region and the changes in the temperature and by several chemical periplasmic domain appears to be crucial, because compounds, a phenomenon termed phenotypic mod- point mutations in this linker can either cause the ulation.For example, the virulence regulon is only inactivation of BvgS or constitutive kinase activity expressed at body but not at room temperature. Both (Beier et al., 1996; Manetti et al., 1994; Miller et al., phenomena – phase variation and phenotypic modula- 1992). Interestingly, the BvgS linker region was tion –were shown to involve the BvgAS two-component recently shown by sequence similarity to contain a system (Arico et al., 1989; Cotter and DiRita, 2000; PAS domain which should be affected by these point Gross and Rappuoli, 1988,1989; Weiss and Falkow, mutations (Taylor and Zhulin, 1999). This may indicate 1984). Phase variation leads to a spontaneous and that the BvgS protein via its PAS domain is also able to usually irreversible loss of virulence gene expression perceive oxygen and/or the energy state of the due to mutations, frequently short deletions, in the bacteria. The preception of such stimuli would be in bvgAS gene locus (Monack et al., 1989; Stibitz et al., agreement with the fact, that also housekeeping functions including the cytochrome composition of the *For correspondence. Email [email protected]; terminal oxidase of the respiratory chain are Bvg- Tel. (931) 888 4403; Fax. (931) 888 4402. regulated (Cotter and DiRita, 2000).

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Structurally, the BvgAS system is highly related Table 1. Selected virulence factors of B. pertussis encoded by genes activated by the BvgAS two-component system. to several other bacterial phosphorelay systems including the ArcAB and EvgAS systems of E. coli. Factors Activity/Function The ArcAB system controls the aerobic modulation regulon and the EvgAS system controls expression of Pertussis Toxin (PTX) ADP-ribosylation of G-proteins adrugeffluxpump(Katoet al., 2000; Sawers 1999). In Adenylate cyclase toxin (CYA) Invasive adenylate cyclase general, the interaction of the histidine kinase and its and hemolysin cognate response regulator occurs with high specifi- Dermonecrotic toxin (DNT) Transglutaminase city, although in some particular two-component Filamentous hemagglutinin (FHA) Adhesion and colonization systems or under artificial conditions a cross-talk Pertactin (PRN) Adhesion and colonization Tracheal colonizing factor (TCF) Adhesion and colonization between non-cognate proteins could be observed BrkA Serum resistance (Wanner, 1992). However, for the ArcB histidine kinase it was shown that its HPt domain is quite promiscuous being able to act as a quite efficient phosphodonor even for non-cognate response regula- tors such as CheY or OmpR (Yaku et al., 1997).

Figure 1. The BvgAS two-component system and its regulon. Virulence Regulation in Bordetella pertussis 199

Accordingly, it was proposed that HPt domains may be and are switched off again when BvgA-P concentration signalling devices which may link different two-compo- reaches levels required for the activation of the ‘‘late’’ nent systems thus creating a regulatory network based genes (Cotter and DiRita, 2000; Deora et al., 2001). on cross-talk phenomena. To investigate signalling Among these intermediate factors is the bipA gene specificity mediated by the BvgS HPt domain we encoding a high molecular weight protein that shares performed domain swapping experiments and con- amino acid sequence similarity at its N-terminus with structed chimaeric histidine kinase proteins containing the proposed outer membrane localization domains of the highly related BvgS and EvgS receiver and HPt intimin of enteropathogenic and enterohaemorrhagic domains fused to the BvgS transmitter. Interestingly, Escherichia coli and invasin of Yersinia spp. (Stock- the chimaeric proteins harbouring either BvgS or EvgS bauer et al., 2001). The promoter regions of the receiver and HPt domains, respectively, were as active different classes of virulence genes show interesting as the wild type proteins regarding the intramolecular structural differences implying that additional regula- phosphorelay of the sensor protein. However, these tory elements may be involved in their control. In fact, experiments demonstrated that, despite their high there is evidence that DNA supercoiling affects level of sequence conservation, BvgA and EvgA could virulence gene expression in B. pertussis as well as only be phosphorylated by their respective BvgS and aprotein termed Baf which under certain conditions EvgS HPt domains (Perraud et al., 1998). Therefore, contributes to ptx expression (DeShazer et al., 1994; these data show that signal transduction via HPt Graeff-Wohlleben et al., 1995; Scarlato et al., 1993). domains does occur in a highly specific manner and Interestingly, spontaneous phase variants were does not support a general concept of HPt mediated identified which showed a very peculiar phenotype: In cross-talk phenomena (Perraud et al., 1999). contrast to typical phase variants which harbour The BvgAS system does not only control expres- inactivating mutations in the bvgAS gene locus and do sion of the BvgA activated virulence genes (also not express the entire virulence regulon (Monack et al., termed vag = virulence activated genes), but also 1989; Stibitz et al.,1989), in these variants only negatively controls expression of an additional set of theexpression of the ‘‘late’’ virulence genes encoding genes (also termed vrg = virulence repressed genes) PTX and CYA was abolished (Carbonetti et al., 1993; (Figure 1) (Akerley and Miller, 1996; Beattie et al., Cookson et al., 1988). The subsequent characterization 1993; Knapp and Mekalanos, 1988; Martinez de of these ‘‘partial’’ phase variants revealed a novel Tejada et al., 1998). Neither the functions of the vrg regulatory mechanism termed phenotypic variation.In genes nor their regulation are yet well characterized. these variants, single point mutations in the transla- However, a Bvg-activated repressor protein, BvgR, tional control region of the rpoA gene encoding the RNA was identified, which very likely controls expression of polymerase a subunit caused an up to three-fold over- at least several of the vrg genes (Merkel et al., 1998). production of the a subunit (Carbonetti et al., 1994). Overproduction of a most likely caused the lack of expression of the toxin promoters by a direct interaction Differential Regulation within the Bvg-Regulon of excess a with BvgA, thereby reducing the amount of available BvgA below the threshold concentration The bvgAS gene locus is autoregulated and a weak required for expression of the ‘‘late’’ toxin promoters constitutive promoter as well as two strong BvgA (Boucher et al., 1997; Carbonetti et al., 2000). So far, dependent promoters control its transcription (Scarlato only two of these phenotypic variants have been et al.,1990).Therefore, a shift of the bacteria from identified, which may indicate that phenotypic variation non-permissive to permissive growth conditions is is a relatively rare event. However, no extensive survey followed by a strong and long-lasting increase in BvgA has been carried out yet to characterize the molecular concentration. This autoregulation is a prerequisite for basis of phase variation in a sufficient number of differential gene activation phenomena observed with- different phase variants and phenotypic variation may in the Bvg-regulon: There are different subsets of vag well contribute to the gradual disappearance of viru- genes which are characterized by fast or very slow lence traits in B. pertussis and the closely related kinetics of transcriptional induction that were classified organism B. bronchiseptica observed previously as ‘‘early’’ or ‘‘late’’ genes, respectively (Gross and (Goldman et al., 1984; Gueirard et al., 1995). Rappuoli, 1989; Scarlato et al., 1991). Whereas the As explained above the phenotypic variation is major adhesin FHA is an early factor, the toxins PTX caused by a disequilibrium of factors building up the and CYA will be transcribed only several hours after transcription machinery resulting in the lack of expres- the switch in the environmental conditions. These sion of PTX and CYA. These non-hemolytic variants differences in gene expression mainly depend on the were the basis for a novel strategy to identify regulatory concentration of phosphorylated BvgA (BvgA-P) as the genes possibly involved in toxin expression (Fuchs promoters have different affinities for the transcrip- et al., 1996). It was assumed that the phenotypic tional regulator and their maximal expression may variants were more sensitive to further perturbations in either be achieved with low amount of BvgA-P (FHA) or their transcription apparatus than the wild type strains. may require high concentrations of the activator (PTX Mutations affecting toxin gene expression were gener- and CYA) (Steffen et al.,1996; Zu et al., 1996). ated by chemical mutagenesis of the phenotypic Recently, a third class of vag genes was identified, variants and screening was performed for colonies with which show an ‘‘intermediate’’ kinetics of expression areconstituted hemolytic phenotype. In most cases the 200 Ko¨nig et al.

resulting mutants carried either reversions or suppres- Table 2. Distribution of Tex protein homologs in Eubacteria.The names of those species that according to their complete genome sor mutations in the rpoA gene itself or in the bvgA gene sequences do not contain the tex gene are underlined. (Fuchs and Gross, unpublished). However, several mutants carried suppressor mutations in unknown gene Bacteria loci. These mutants were used for the search of Aquificales Aquificaceae antisuppressor loci by the introduction of a genomic Aquifex aeolicus Chlamydiales Chlamydiaceae library and allowed the identification of a gene termed Chlamydia trachomatis, C. pneumoniae tex (= toxin expression) which, when slightly over- Cyanobacteria Chroococcales expressed, exerted a negative effect on transcription of Synechocystis PCC6803 PTX and CYA in the mutant background. Firmicutes Bacillus/Clostridium Gruppe The deduced aminoacid sequence of the Tex (low G/C gram+) Bacillus/Lactobacillus/Streptococcus Gruppe protein revealed that it is strongly conserved in most of Bacillus anthracis, B. halodurans, the eubacteria sequenced so far with amino acid B. subtilis, B. stearothermophilus similarities ranging from 50 to 80% (Table 2). Such a Staphylococcus aureus degree of sequence conservation is also observed Streptococcus pneumoniae, S. pyogenes, with essential factors including RpoA and GyrB. S. mutans, S. equii Mycoplasmataceae However, in some phylogenetic lineages no Tex Mycoplasma genitalium, M. pneumoniae homologue is found, e.g. in the e group of the Ureaplasma urealyticum Proteobacteria,orinseveral obligate parasites or Clostridiaceae symbionts with extremely reduced genomes such as Clostridium acetobotylicum, C. difficile Mycoplasma spp. or the endosymbiont of aphids Enterococcaceae Enterococcus faecalis Buchnera aphidicola.Nevertheless, the high degree Actinobacteria (high G/C gram+) of sequence conservation of the Tex protein in most Corynebacterineae other eubacteria indicates a basic role of this factor. In Corynebacterium diphteriae fact, the tex gene could not be deleted from B. Mycobacterium tuberculosis pertussis, but, apparently, it is not essential for other Streptomycineae bacteria including E. coli and Neisseria gonorrhoea Streptomyces coeliclor Proteobacteria a Gruppe (Fuchs et al., 1996; Ko¨nig and Gross, unpublished; Rickettsiaceae Petering et al., 1996). The protein shows interesting Rickettsia prowazekii sequence similarities with the mannitol repressor b Gruppe (MtlR) of E. coli in its N-terminal domain and harbours Alcaligenacea aS1domain at its C-terminus (Figure 2) (Bycroft et al., Bordetella bronchiseptica, B. pertussis Neisseriaceae 1997; Fuchs et al., 1996). The presence of the S1 Neisseria gonorrhoeae, N. meningitidis domain suggested that the protein may be a nucleic g Gruppe acid binding protein, because in most cases other Enterobacteriaceae proteins carrying a S1 domain were reported to Escherichia coli interact with RNA (Bycroft et al., 1997). In fact, in Salmonella typhi, S. typhimurium, S. paratyphi A solid phase binding assays using the purified Tex Yersinia pestis protein of E. coli linked to magnetic beads, RNA but Buchnera aphidicola not DNA could be found as a specific ligand (Figure 3). Pasteurellaceae Apparently, the specific binding of Tex to RNA requires Pasteurella multocida its N-terminus, because the deletion of several Haemophilus influenzae, H. ducreyi Actinobacillus actinomycetemcomitans N-terminal amino acids abolished any preference for Pseudomonaceae RNA and resulted in highly efficient binding of DNA as Pseudomonas aeruginosa, P. putida well as RNA (Ko¨ nig and Gross, unpublished). Several Alteromonadaceae proteins harbouring the S1 domain are involved in Shewanella putrefaciens stress response pathways. For example, under cold- Vibrionaceae Gruppe Vibrio cholerae shock conditions polynucleotide phosphorylase Xanthomonas Gruppe (PNPase), containing a S1 domain at its C-terminus, Xylella fastidiosa proved to be essential and is one of the key enzymes Legionellaceae e formRNA turnover at low temperatures (Jones et al., Legionella pneumophila 1987; Luttinger et al., 1996). However, as shown by d Gruppe Geobacter sulfurreducens Western-blot analysis and 2-D gel electrophoresis, in Desulfuromonas Gruppe E. coli expression of Tex occurs at a very low level Desulfovibrio vulgaris (Fountoulakis et al., 1999; Ko¨ nig and Gross, unpub- e Gruppe lished), and no growth conditions could be identified Helicobacter pylori yet leading to a significant increase in its expression. Campylobacter jejuni Spirochaetales Spirochaetaceae Attempts to identify a specific RNA target for the Treponema pallidum, T. denticola purified E. coli Tex protein were carried out once more Thermotogales Thermotoga maritima using a solid phase binding assay. This approach identified 16S rRNA and CsrB as preferential binding partners. However, the functional relevance of these Virulence Regulation in Bordetella pertussis 201

Figure 2. Schematic presentation of several members of the S1 protein family.Theblack boxes represent S1 domains, KH indicates the presence of KH-domains which are independent nucleic-acid-binding units. The figure has been adapted from Bycroft et al., 1997 and Sugita et al., 2000.

Figure 3. Binding of 3H-labelled RNA (open symbols) and DNA(closed symbols) to the purified E. coli Tex protein.

findings remains to be investigated. CsrB is a was supported by grants from the Human Frontier Science Program regulatory RNA, which by interaction with the CsrA Organization, the Priority Program ‘‘Regulatory Networks in Bacteria’’ of the Deutsche Forschungsgemeinschaft and by the Fonds der protein controls message turnover in E. coli.Interest- Chemischen Industrie. ingly, the homologous regulator pairs of Salmonella typhimurium and Erwiniacarotovora were found to be References involved in virulence gene expression (Altier et al., 2000; Cui et al., 1999). Akerley, B.J., and Miller, J.F. 1996. Understanding signal transduction during bacterial infection. Trends Microbiol. 4: 141–146. Altier, C., Suyemoto, M., Ruiz, A.I., Burnham, K.D., and Maurer, R. Acknowledgements 2000. Characterization of two novel regulatory genes affecting Salmonella invasion gene expression. Mol. Microbiol. 35: 635–646. We thank Verena Weiss, Kirsten Jung, Karsten Rippe, Marcus Arico´ ,B.,Miller,J.,Roy, C., Stibitz, S., Monack, D., Falkow, S., Gross, Bantscheff, Michael Glocker, Nick Carbonetti and Vincenzo Scarlato R., Rappuoli, R. 1989. Sequences required for the expression for advice and fruitful collaboration throughout this project. This work of Bordetella pertussis virulence factors share homology with 202 Ko¨nig et al.

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