7.6 Molecular Genetics of Bordetella Pertussis Virulence

7.6 Molecular Genetics of Bordetella pertussis Virulence Vincenzo Scarlato, Dagmar Beier and Rino Rappuoli Department of Molecular Biology, IRIS, Chiron Vaccines, Italy

CONTENTS Introduction

B. pertussis phenotypes and virulence genes The bvg regulatory locus Propagation and maintenance of B. pertussis strains Genetic manipulation of B. pertussis Construction of a bvgS mutant strain Concluding remarks Acknowledgements

++++++ INTRODUCTION Bordetella pertussis is a small Gram-negative coccobacillus that infects humans causing whooping cough, an acute respiratory disease. The disease progresses in three stages. (1) An incubation period of 10-14 days is followed by the catarrhal stage characterized by cold-like symptoms. (2) After 7-10 days the illness progresses to the paroxysmal phase and symptoms then include violent coughing spasms followed by an inspiratory gasp resulting in the typical whooping sound, for which the disease is named. This stage lasts for 14 weeks. (3) Symptomsgradually become less severe and paroxysms less frequent during the convalescent period, which may last up to six months.

B. pertussis colonizes the upper respiratory tract by specificadhesion to the ciliated cells and to the alveolar macrophages (Weiss and Hewlett, 1986).Although the events that occur during the normal course of infection are still far from clear, progress has been made in idenhfymg several factors that may function as virulence determinants. These virulence factors interfere with the clearance mechanisms of the respiratory epithelium, damage host cells, and suppress the immune response. B. pertussis METHODS IN MICROBIOLOGY, VOLUME 27 ISBN 0-12-521525-8

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adheres to the cells of the respiratory tract, mostly through the action of adhesins: fimbriae (FIM) that elicit formation of agglutinating antibodies; a 69 kDa outer membrane protein (pertactin), which is a non-fimbrial agglutinogen; and filamentous hemagglutinin (FHA), a high-M, rod-like surface protein (Relman et al., 1989; Willems et al., 1990; Leininger et al., 1991). The latter two proteins contain arginine-glycine-aspartic acid (RGD) motifs, which are involved in binding to integrin receptors on mammalian cells. Pertussis toxin (PT),considered to be a major virulence factor of B. pertussis (Nicosia et al., 19861, appears also to act as an adhesin (Arici, et al., 1993). PT consists of five subunits encoded by the ptx locus, S l S 5 , found in a 1:1:1:2:1ratio (Tamura et al., 1982). Secretion of PT requires its correct assembly in the periplasmic space (Pizza et al., 1990) and the help from eight proteins A-H encoded by the ptl operon, which maps downstream from the ptx operon (Weiss et al., 1993). Subunits S2 and S3 of PT contain two different binding activities.S2 preferentially recognizes glycoconjugates on ciliated cells and S3 recognizes macrophages (Saukkonen et al., 1992). The toxic properties of PT are associated with the enzymatic activity of the S1 subunit, which has an ADP ribosylating activity and transfers ADP ribose groups to G proteins of eukaryotic cells (Pizza et al., 1989). PT has a wide range of activities and may be responsible for paroxysmal cough. It is a protective antigen and together with FHA and pertactin is included in current acellular vaccines (Rappuoli, 1996). Adenylate cyclase toxin is a bifunctional protein with adenylate cyclase and hemolysin activities. It is able to penetrate and intoxicate mammalian cells by elevation of internal cyclic AMP levels, and is thought to act primarily as an antiphagocytic factor (Glaser et al., 1988).

++++++ B. pertussis PHENOTYPESANDVIRULENCE GENES

Like many bacterial pathogens, B. pertussis expresses virulence factors depending upon growth conditions. This phenomenon, known as phenotypic modulation, is reversible and was first observed by Lacey (1960). With the exception of tracheal cytotoxin, all other virulence factors are expressed at a temperature of 37"C, while their expression is repressed at 25°C and at 37°C in the presence of modulators such as nicotinic acid or MgSO, (Lacey, 1960; Gross and Rappuoli, 1988; Scarlato et al., 1990). A simple indicative tool to follow phenotypic modulation of virulence factors is the presence or absence of a halo of hemolysis, which colonies of B. pertussis show on agar plates containing blood (Fig. 7.4). At 37°C bacteria show hemolysis, indicating production of virulence factors. At 25"C, or at 37°C in the presence of 50 m~ MgS04or 10 m~ nicotinic acid, bacteria do not show hemolysis, indicating absence of virulence factors. Phase variation is a term indicating a genotypic change characterized by simultaneous loss of expression of virulence factors. Non-virulent phase variants arise in a population at a frequency of 103-lW. Genetic analysis has shown that both phenotypic modulation and phase variation 396

are under the control of a single genetic locus, the bug locus (Weiss et al., 1983; Aricb et al., 1989;Stibitz and Yang, 1991).Transition between the two distinct phases of B. pertussis is mediated by the products of the bvg locus. In the Bvg' phase, B. pertussis expresses most of the defined toxins and adhesins. When the bvg locus is inactivated through modulating agents or by mutations, the organism switches to the Bvg- phase. The bug locus was first reported by the pioneering work of Weiss et al. (1983). They isolated Tn5 insertion mutants defective in the expression of virulence factors including PT,adenylate cyclase, and FHA. Nucleotide sequence determination and analysis of bug revealed that the proteins encoded by this locus, BvgA and BvgS, belong to the two-component regulatory systems (Aricb et al., 1989;Stibitz and Yang, 1991).

Figure 7.4. Phenotypic differences between B. pertussis grown on agar plates containing blood and incubated at (a) 37°C and (b) 25°C. If the plates are supplemented with 5 0 m MgSO, or l o r n nicotinic acid, the phenotype obtained at 37°C is similar to that shown in (b). At 37°C (a) the bacterial colonies are surrounded by a halo of hemolysis, which is not present in colonies grown at 25°C (b): this is due to the production and secretion of the hemolysin protein, which occurs only at 37°C. Colonies of bacteria grown at 37°C are smooth, dome-shaped and translucid, whereas at 25°C they appear rough, flat, and opaque. 397

++++++ T H E bvg REGULATORY LOCUS The bvg locus occupies 5 kb of the Bordetellu genome (Fig. 7.5a), in which resides the genetic information for the two proteins:BvgA and BvgS, of 23 and 135 kDa, respectively (Aricbet ul., 1989; Stibitz and Yang, 1991). BvgA is a typical response regulator with an N-terminal receiver and a DNAbinding C-terminal helix-turn-helix motif. Three amino acid residues,

Figure 7.5. (a) Genetic organization of the bvg and fha loci. Open arrows indicate the direction of translation of the indicated genes and small arrows the direction of transcription from the various promoters. Functional domains of the BvgAS system are indicated. (b)Model showing the steps in BvgAS signal transduction in Bordetellu. Environmental signals (temperature, MgSO, and nicotinic acid) regulate the activity of BvgS. In its active form,BvgS autophosphorylates its transmitter domain. Following a cascade of phosphorylation from the transmitter to the receiver and output domains, BvgA is phosphorylated. Phosphorylation of BvgA is likely to induce a conformationalchange of the protein and bvg-regulated promoters are activated. Consequently, a Bvg- (non-virulent)phenotype is converted to a Bvg' (virulent) phenotype.

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which may play a fundamental role in the event of transactivation are strictly conserved among all two-component regulators (i.e. Asp-10, Asp54, and Lys-104 in BvgA). BvgS is an ’unorthodox’ sensor protein composed of a periplasmic input domain as well as several cytoplasmic domains termed linker, transmitter, receiver, and output domains (Fig. 7.5b). Within the linker region, located between the input and transmitter domain, several mutations have been mapped which render BvgS insensitive to modulating signals (Miller et al., 1992). While the input domain, linker, and transmitter domain, which mediates the autophosphorylation of a conserved His (729) residue, are common features of all two component sensor proteins, the presence of a receiver and output domain is limited to a subset of prokaryotic sensors (Parkinson and Kofoid, 1992). These additional domains of BvgS, harboring conserved amino acids that act both as acceptors and donors of phosphate, seem to be involved in a complex phosphorylation cascade leading to activation of the response regulator BvgA (Uhl and Miller, 1994; Beier et al., 1995; Uhl and Miller, 1996). Expression of the bvg locus is regulated by four promoters, P,, (Scarlato et al., 1990; see Fig. 7.5a). The P, and P3 promoters are autoregulated by BvgA and are repressed by the addition of MgSO, or nicotinic acid to the culture medium. The P, promoter is activated by the above-mentioned signals. The P, promoter synthesizes an RNA complementary to the 5’ untranslated region of the bvg mRNAs and is also regulated by the bug locus in response to external stimuli. Transcription from other virulence gene promoters has also been mapped and proved to be environmentally regulated.

++++++ PROPAGATIONAND MAINTENANCE OF B. pertussis STRAINS

Under laboratory conditions, B. pertussis is more difficult to grow than other bacteria such as Escherichia coli and Salmonella. Growth requires special media and a long incubation time (bacterial duplication time is approximately 4-6 hours at 35°C). Other members of the Bordetella genus such as B. bronchiseptica and B. parapertussis are easier to grow. B. bronchiseptica grows in E. coli media at the same rate as E. coli, while B. parapertussis has an intermediate generation time. Colonies of most B. pertussis strains can be maintained for a period of 7-10 days on agar plates stored inverted at room temperature or at 4°C. Bacteria can be stored for many years in media containing glycerol at low temperature without significant loss of viability. To establish colonies for further use, cells should be streaked on Bordet-Gengou (BG) agar plates containing 25% defibrinated sheep blood (Scarlato et al., 1996), and incubated for three days at 35°C (see Fig. 7.4). A large inoculum, such as a loop of bacteria from a plate grown to confluence is necessary to start the primary liquid culture in Stainer-Scholte ( S S ) medium (Stainer and Scholte, 1971; Scarlato et al., 1996). The rate of growth is dependent upon the strain, the temperature, 399

and the degree of aeration. Culture aeration is best achieved on a water rotary bath at about 300 rpm. A rough estimate of the number of B. pertussis cells can be obtained by considering that 1 OD, is approximately 8 x 10"cells ml-I. During genetic manipulations, it may be useful to use strains that can be easily selected on antibiotic containing plates. The most useful antibiotics for B. pertussis are streptomycin and nalidixic acid. In order to obtain naturally resistant strains to these antibiotics, 10 ml of mid log cultured bacteria are centrifuged and resuspended in 0.1 ml of fresh SS medium. Aliquots of this bacterial suspension (89 pl, 10pl, 1pl, respectively) are then plated on BG agar plates containing 1mgml-' of streptomycin, or 50pg ml-' of nalidixic acid, and incubated at 35°Cfor 5-8 days to allow the growth of the spontaneous resistant strains.

++++++ GENETIC MANIPULATION OF 6. pertussis The virulence factors and their regulators have been studied in detail using techniques for genetic manipulation of B. pertussis which were developed during 1980-1990. These techniques made possible the identification, alteration, and manipulation of specific genes in the chromosome and, therefore, the construction of isogenic B. pertussis mutants. The first attempt at B. pertussis transformation was made by Weiss and Falkow (1982)who described that B. pertussis cells cannot be transformed by classical methods such as calcium heat shock treatment because this procedure reduces the viability of the cells more than 1000-fold. However, a cold shock or freezing of the cells results in transformation by plasmids of the P and W incompatibility groups, which replicate in B. pertussis. The transformation frequency obtained was between 16and 103pg-'of DNA. The same authors have also reported that DNA isolated from E. coli could not be introduced into B. pertussis by transformation if this DNA contains Hind111 recognition sequences. A major factor in the efficiency of transformation is the availability of a restriction defective recipient. Unfortunately, the Bordetellu restriction system has not been investigated and such mutants have not been isolated as yet. Obviously, the problem of poor transformation efficiency makes it, for most practical purposes, unrealistic to attempt direct transformation of Bordetellu with DNA from a ligation. Recombinant plasmids obtained in E. coli can be used to transform the desired Bordetellu strain. Although this method of transformation of Bordetellu has been developed, the low efficiency renders the system not very easy to work with. In more recent years, it has been reported that electroporation of B. pertussis has been successfully used to introduce either plasmid DNAs or linear DNA fragments (Zealey et ul., 1988, 1991). A number of parameters can influence the frequency of B. pertussis transformation by electroporation; however, this technique can yield a frequency of transformation in the magnitude of 106pg' of DNA. In the range of 10"-109cells ml-I, the efficiency of transformation is independent of the cell concentration (Zealey 400

et al., 1988). On the contrary, the efficiency of transformation depends upon the strength of the electric field. The main tool used to transform B. pertussis is the conjugation of mobilizable plasmids from E. coli. Two classes of mobilizing plasmids can be used: the broad host range plasmids, which replicate in B. pertussis, and the so-called suicide plasmids, which do not. Replicating plasmids can be used for complementation purposes of mutated genes. Suicide plasmids can be used for random transposon mutagenesis as well as for gene replacement into the chromosome to create defined mutants. No special considerations are required when using these plasmids, except that as they are generally larger than the E. coli vectors, it is necessary to use a higher concentration of linear vector DNA in the ligation cocktail to achieve the desired molar concentration of DNA ends (Sambrook et al., 1989). The use of transposon-mediated mutagenesis has greatly contributed to the identification and characterization of specific virulence genes. Whereas in spontaneous or chemically induced mutants it is difficult to determine the site and the number of mutations resulting in an altered phenotype, transposon mutants can be easily mapped by genetic (antibiotic resistance) or physical (probe hybridization) techniques. Transposon mutagenesis has played an important role in the understanding of pertussis disease. The use of this technique in Bordetella does not require special consideration and it is easily achieved following plasmid conjugation from E. coli (Weiss et al., 1982,1983). Since conjungation between E. coli and B. pertussis has been the most common way to introduce new DNA into the B. pertussis chromosome, thus allowing genetic manipulation and characterization of specific genes, here we describe the use of this technique to construct a specific mutant. The structure of the most used conjugative plasmid in Bordetella, pRTP1, has been previously described (Stibitz et al., 1986). A derivative of this plasmid, pSS1129 (Stibitz and Yang, 1991) carrying a gentamicin resistance cassette has also been successfully used to create suitable B. pertussis mutants. These plasmids contain the oriT for conjugative transfer from the broad host range plasmid RK2, a vegetative origin of replication from ColE1, an ampicillin resistance gene, and the gene encoding the E. coli ribosomal protein S12. Expression of the ribosomal protein S12 is dominant over streptomycin resistance, therefore converting a streptomycin-resistantstrain to a streptomycin-sensitive strain. Plasmids pRTPl and pSS1129 are unable to replicate in B. pertussis and thus if the recipient strain is subject to antibiotic selection carried by the plasmid, only bacteria which have the plasmid integrated into the chromosome will grow. Consequently, integration of the plasmid occurs through a single homologous recombination event, the exconjugants acquire the genetic markers of the plasmid, and a streptomycin-resistant strain becomes streptomycin sensitive. A second event of homologous (intrachromosomal) recombination could be selected on streptomycin-containing plates. This recombination allows the selection of strains that have lost the S12 gene and all the genetic markers carried on the plasmid, allowing substitution of target sequences. These plasmids are the most useful tools available today.

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The procedure used to manipulate a B. pertussis strain to create a mutant with a kanamycin resistance cassette interrupting the bvgS gene is an example. A step-by-step protocol of conjugation is reported in Table 7.16. All cloning manipulations are performed in an E. coli recA mutant using a small size and high copy number vector such as a pBR322 derivative. Once the desired DNA fragments are cloned in the correct order, the fragment is recovered and inserted into the pSS1129 conjugative vector and then used to transform the conjugative strain E. coli SMlO (Simon et al., 1983). This strain carries mobilizing genes of the broad host range IncP-type plasmid RP4 integrated into the chromosome. A freshly transformed colony of SMlO is then used for bacterial conjugation with B. pertussis. Since the efficiency of conjugation may vary with unpredictable parameters, it is advisable to perform at least three or four conjugations at a time by using single well isolated colonies of freshly transformed E. coli.

++++++ CONSTRUCTION OF A bvgS MUTANT STRAIN In the proposed experiment (see Table 7.161, a plasmid carrying the 2749 bp EcoRI fragment from the bvgS gene (Aricb et al., 1989) is digested with enzyme NcoI and end-repaired with Klenow enzyme (Fig. 7.6). This digestion removes the coding information for the BvgS receiver and output domain leaving 1096 bp at the 5’-end of the fragment and 610 bp at the 3’end of the fragment. A blunt-ended kanamycin cassette is cloned between the 5’- and 3’-flanking regions of bvgS. This new EcoRI recombinant DNA fragment (bvgS::kan) is cloned into the conjugation suicide vector pSSll29 and transformed into SM10. Colonies of SMlO(pSS1129/bvgS::kan) are used for conjugation with a suitable B. pertussis strain. For mutagenesis

Table 7.16. Conjugation between E. coli and 6. pertussis

1. Collect B. pertussis cells from a fresh plate and spread uniformly on a BG plate supplemented with 10 rn MgCl,. 2. Spread one fresh transformed colony of SMlO(pSS1129/bvgS::kan) on top of the B. pertussis cells and incubate for 5 h at 35°C. 3. Collect bacteria and plate onto a BG plate supplemented with nalidixic acid, kanamycin, and gentamicin and incubate at 35°C for 6-7 days. 4. To select for the loss of the plasmid and the second recombination event, plate the exconjugants on BG plates supplemented with streptomycin and incubate at 35°C for 4-5 days. 5. Analyze bacteria grown on BGstreptomycin plates for the loss of the plasmid and the acquisition of the kanamycin resistance by streaking single colonies on a BG plate containing kanamycin. Incubate for three days at 35°C. 6. Confirm the insertion of the kanamycin cassette into the desired gene by Southern blot analysis on chromosomal DNA extracted from kanamycin-resistant isolates. 402

Figure 7.6. Schematic representation of the events of recombination following conjugation to construct a bugs mutant. The dark black arrows indicate the wildtype bvg locus; the dotted box, a kanamycin cassette. Important restrictionsites are indicated. Key: E, EcoRI; N, NcoI; Gm, gentamicin;Kan, kanamycin; Nal, nalidixic acid; Sm, streptomycin.'Resistant; 'sensitive.

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purposes, a clinical isolate of B. pertussis should be made resistant to nalidixic acid and streptomycin. Then, exconjugants are selected on plates containing nalidixic acid, gentamicin, and kanamycin. Nalidixic acid is used to counterselect E. coli, whereas gentamicin and kanamycin are used to select for acquisition of the plasmid by B. pertussis. Since the vector cannot replicate in B. pertussis, growing colonies are exconjugants that have the plasmid integrated into the chromosome by homologous recombination. The schematic representation of a conjugation event to generate the h g S mutant is shown in Fig. 7.6.Propagation of an exconjugant strain on plates containing streptomycin will select those strains that had a second event of intrachromosomal homologous recombination of the h g S gene, resulting in the loss of plasmid DNA. Additional selection for kanamycin resistance will discriminate between strains with a definitive replacement of the bvgS gene with the kanamycin cassette and revertants to wild-type.

++++++ CONCLUDING REMARKS Although €3. pertussis is a fastidious organism which is difficult to grow and manipulate, the technology currently available enables us to construct isogenic mutants, mutagenize or delete natural genes, or introduce recombinant genes in Bordetella. Mutations in the bvg, fha,ptx, pertactinencoding gene and other virulence-encoding genes have been obtained using these technologies. Genetic, molecular, and biochemical studies have allowed dissection of the molecular mechanisms of B. pertussis pathogenicity, elucidation of the role of virulence factors, and manipulation of toxin genes to obtain strains that produce safe vaccine molecules. B. pertussis is a beautiful example of how genetic tools have been successfully used to advance science and our understanding of bacterial pathogenesis.

++++++ ACKNOWLEDGEMENTS We thank G. Corsi for the figures and Catherine Mallia for editing. Studies in our laboratory were partially supported by the Human Frontier Science Program Organization.

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