Regulatory Factors of Bordetella pertussis Affecting Virulence Gene Expression

J. Mol. Microbiol. Biotechnol. (2002) 4(3): 197 203. JMMB Symposium Regulatory Factors of Bordetella pertussis Affecting Virulence Gene Expression Jochen König, Andreas Bock, Anne-Laure Perraud, Thilo M. Fuchs, Dagmar Beier, and Roy Gross* Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften, Universität Würzburg, D- 97074 Würzburg, Germany Abstract Most pathogenic bacteria encounter changing growth conditions during their infectious cycle and, accordingly, have to modulate gene expression to enable the efficient colonization of different environments outside or within their host organisms. In Bordetella pertussis the transcription of most virulence factors including several toxins and adhesins is regulated coordinately by the BvgAS two-component system. The molecular characterization of the BvgAS system revealed that it belongs to the small group of unorthodox two-component systems applying an obligate multistep phosphorelay. Moreover, despite the coordinated control of the virulence regulon, subtle differences in the regulation of individual virulence genes were observed which led to the identification of sophisticated mechanisms possibly engaged in fine tuning of virulence gene expression. Coordinate Regulation of the Bvg-Regulon It has been known for a long time that the expression of virulence properties of B. pertussis, the etiological agent of whooping cough, is unstable. In fact, avirulent so-called phase variants may arise with high frequency. Moreover, the virulent phenotype depends on environmental conditions and is reversibly affected by changes in the temperature and by several chemical compounds, a phenomenon termed phenotypic modulation. For example, the virulence regulon is only expressed at body but not at room temperature. Both phenomena phase variation and phenotypic modulation were shown to involve the BvgAS two-component system (Arico et al., 1989; Cotter and DiRita, 2000; Gross and Rappuoli, 1988,1989; Weiss and Falkow, 1984). Phase variation leads to a spontaneous and usually irreversible loss of virulence gene expression due to mutations, frequently short deletions, in the bvgas gene locus (Monack et al., 1989; Stibitz et al., *For correspondence. Email roy@biozentrum.uni-wuerzburg.de; Tel. (931) 888 4403; Fax. (931) 888 4402. 1989). Phenotypic modulation depends on the activity of the histidine kinase BvgS which is an environmental sensor located in the cytoplasmic membrane. Under the appropriate conditions, the BvgS protein autophosphorylates at a histidine residue in its transmitter domain and, subsequently, the phosphate is transferred 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 promoters leading to expression of several adhesins and toxins (Table 1) (Rappuoli, 1994). The BvgAS system differs from typical two-component systems, because BvgS contains additional phosphorylation sites outside of its transmitter: In fact, an obligate multistep His-Asp-His-Asp phosphorelay occurs that involves the BvgS transmitter, receiver and HPt domains (Figure 1) (Arico et al., 1989; Perraud et al., 1999; Uhl and Miller, 1994,1996). The high energy phosphohistidine present in the C-terminal HPt domain is the exclusive phosphate source for BvgA (Perraud et al., 1998). Furthermore, transcomplementation of mutant sensor proteins by the separate expression of individual signalling domains demonstrated that the phosphorelay in the BvgS histidine kinase involves BvgS homodimers (Beier et al., 1995, 1996). BvgA also forms homodimers, but, in contrast to several other response regulators such as FixJ, there is no evidence for any influence of phosphorylation on its oligomerisation state (Bock, Rippe and Gross, unpublished; Perraud et al., 2000). This shows that activation of BvgA by phosphorylation does not involve its dimerization (Perraud et al., 2000; Bock et al., 2001). Little is known about the mechanisms of signal perception and the control of the histidine kinase activity present in the transmitter domain of two-component sensor proteins. However, like in other sensor proteins the linker region of BvgS connecting the transmitter domain with the membrane spanning region and the periplasmic domain appears to be crucial, because point mutations in this linker can either cause the inactivation of BvgS or constitutive kinase activity (Beier et al., 1996; Manetti et al., 1994; Miller et al., 1992). Interestingly, the BvgS linker region was recently shown by sequence similarity to contain a PAS domain which should be affected by these point mutations (Taylor and Zhulin, 1999). This may indicate that the BvgS protein via its PAS domain is also able to perceive oxygen and/or the energy state of the bacteria. The preception of such stimuli would be in agreement with the fact, that also housekeeping functions including the cytochrome composition of the terminal oxidase of the respiratory chain are Bvgregulated (Cotter and DiRita, 2000). # 2002 Horizon Scientific Press

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

200 König et al. Table 2. Distribution of Tex protein homologs in Eubacteria. The names of those species that according to their complete genome sequences do not contain the tex gene are underlined. Bacteria Aquificales Chlamydiales Cyanobacteria Firmicutes Proteobacteria Spirochaetales Thermotogales Aquificaceae Aquifex aeolicus Chlamydiaceae Chlamydia trachomatis, C. pneumoniae Chroococcales Synechocystis PCC6803 Bacillus/Clostridium Gruppe (low G/C gram + ) Bacillus/Lactobacillus/Streptococcus Gruppe Bacillus anthracis, B. halodurans, B. subtilis, B. stearothermophilus Staphylococcus aureus Streptococcus pneumoniae, S. pyogenes, S. mutans, S. equii Mycoplasmataceae Mycoplasma genitalium, M. pneumoniae Ureaplasma urealyticum Clostridiaceae Clostridium acetobotylicum, C. difficile Enterococcaceae Enterococcus faecalis Actinobacteria (high G/C gram + ) Corynebacterineae Corynebacterium diphteriae Mycobacterium tuberculosis Streptomycineae Streptomyces coeliclor a Gruppe Rickettsiaceae Rickettsia prowazekii b Gruppe Alcaligenacea Bordetella bronchiseptica, B. pertussis Neisseriaceae Neisseria gonorrhoeae, N. meningitidis g Gruppe Enterobacteriaceae Escherichia coli Salmonella typhi, S. typhimurium, S. paratyphi A Yersinia pestis Buchnera aphidicola Pasteurellaceae Pasteurella multocida Haemophilus influenzae, H. ducreyi Actinobacillus actinomycetemcomitans Pseudomonaceae Pseudomonas aeruginosa, P. putida Alteromonadaceae Shewanella putrefaciens Vibrionaceae Gruppe Vibrio cholerae Xanthomonas Gruppe Xylella fastidiosa Legionellaceae e Legionella pneumophila d Gruppe Geobacter sulfurreducens Desulfuromonas Gruppe Desulfovibrio vulgaris e Gruppe Helicobacter pylori Campylobacter jejuni Spirochaetaceae Treponema pallidum, T. denticola Thermotoga maritima resulting mutants carried either reversions or suppressor mutations in the rpoa gene itself or in the bvga gene (Fuchs and Gross, unpublished). However, several mutants carried suppressor mutations in unknown gene loci. These mutants were used for the search of antisuppressor loci by the introduction of a genomic library and allowed the identification of a gene termed tex (= toxin expression) which, when slightly overexpressed, exerted a negative effect on transcription of PTX and CYA in the mutant background. The deduced aminoacid sequence of the Tex protein revealed that it is strongly conserved in most of the eubacteria sequenced so far with amino acid similarities ranging from 50 to 80% (Table 2). Such a degree of sequence conservation is also observed with essential factors including RpoA and GyrB. However, in some phylogenetic lineages no Tex homologue is found, e.g. in the e group of the Proteobacteria, or in several obligate parasites or symbionts with extremely reduced genomes such as Mycoplasma spp. or the endosymbiont of aphids Buchnera aphidicola. Nevertheless, the high degree of sequence conservation of the Tex protein in most other eubacteria indicates a basic role of this factor. In fact, the tex gene could not be deleted from B. pertussis, but, apparently, it is not essential for other bacteria including E. coli and Neisseria gonorrhoea (Fuchs et al., 1996; König and Gross, unpublished; Petering et al., 1996). The protein shows interesting sequence similarities with the mannitol repressor (MtlR) of E. coli in its N-terminal domain and harbours as1domain at its C-terminus (Figure 2) (Bycroft et al., 1997; Fuchs et al., 1996). The presence of the S1 domain suggested that the protein may be a nucleic acid binding protein, because in most cases other proteins carrying a S1 domain were reported to interact with RNA (Bycroft et al., 1997). In fact, in solid phase binding assays using the purified Tex protein of E. coli linked to magnetic beads, RNA but not DNA could be found as a specific ligand (Figure 3). Apparently, the specific binding of Tex to RNA requires its N-terminus, because the deletion of several N-terminal amino acids abolished any preference for RNA and resulted in highly efficient binding of DNA as well as RNA (König and Gross, unpublished). Several proteins harbouring the S1 domain are involved in stress response pathways. For example, under coldshock conditions polynucleotide phosphorylase (PNPase), containing a S1 domain at its C-terminus, proved to be essential and is one of the key enzymes for mrna turnover at low temperatures (Jones et al., 1987; Luttinger et al., 1996). However, as shown by Western-blot analysis and 2-D gel electrophoresis, in E. coli expression of Tex occurs at a very low level (Fountoulakis et al., 1999; König and Gross, unpublished), and no growth conditions could be identified yet leading to a significant increase in its expression. Attempts to identify a specific RNA target for the purified E. coli Tex protein were carried out once more 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 3 H-labelled RNA (open symbols) and DNA (closed symbols) to the purified E. coli Tex protein. findings remains to be investigated. CsrB is a regulatory RNA, which by interaction with the CsrA protein controls message turnover in E. coli. Interestingly, the homologous regulator pairs of Salmonella typhimurium and Erwinia carotovora were found to be involved in virulence gene expression (Altier et al., 2000; Cui et al., 1999). Acknowledgements We thank Verena Weiss, Kirsten Jung, Karsten Rippe, Marcus Bantscheff, Michael Glocker, Nick Carbonetti and Vincenzo Scarlato for advice and fruitful collaboration throughout this project. This work was supported by grants from the Human Frontier Science Program Organization, the Priority Program Regulatory Networks in Bacteria of the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. References 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. 2000. Characterization of two novel regulatory genes affecting Salmonella invasion gene expression. Mol. Microbiol. 35: 635 646. Aricó, B.,Miller,J.,Roy, C., Stibitz, S., Monack, D., Falkow, S., Gross, R., Rappuoli, R. 1989. Sequences required for the expression of Bordetella pertussis virulence factors share homology with

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