Microbial Pathogenesis 1992 ; 12 : 227-235
Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection Nadia Khelef,' Hiroshi Sakamoto 2 t and Nicole Guiso'* 'Unite de Bacteriologie Moleculaire et Medicale and 2 Unite de Biochimie des Regulations Cellulaires, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France (Received October 4, 1991 ; accepted in revised form November 11, 1991)
Khelef, N . (Unite de Bacteriologie Moleculaire et Medicale, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, France), H . Sakamoto and N . Guiso . Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection . Microbial Pathogenesis 1992 ; 12 : 227235 . Among virulence factors synthesized and secreted by Bordetella pertussis, pertussis toxin (PTX) and the bifunctional adenylate cyclase-hemolysin (AC-Hly) are able to invade mammalian cells and to impair intracellular functions. Moreover, both proteins are protective antigens in murine intracerebral and respiratory models . In order to study their in vivo properties, different B . pertussis mutants, deficient in AC-Hly expression or secretion, or producing modified ACHly devoid of either adenylate cyclase or hemolytic activities, were constructed and examined . The in vivo properties of the mutants were compared to PTX deficient strains, using the murine respiratory model . We show that lack of PTX as well as adenylate cyclase or hemolytic activities results in avirulence . Furthermore, we show that mutants lacking adenylate cyclase or hemolytic activities were unable to multiply as fast as the parental strains and PTX mutants during the first 5 days following infection . Thus, both adenylate cyclase and hemolytic activities are required by B . pertussis to initiate infection . Key words : Bordetella pertussis ; pertussis toxin ; colonization .
murine respiratory model ; adenylate cyclase-hemolysin ;
Introduction Bordete/la pertussis, the agent of whooping cough, synthesizes a variety of virulence factors . Some participate in the adhesion of the bacteria to the respiratory tract of the host, such as filamentous hemagglutinin (FHA) and pertussis toxin (PTX) . Others are responsible for the local disease, such as dermonecrotic toxin (DNT) and tracheal cytotoxin (TCT), or for the systemic disease such as PTX . Bordetella pertussis also synthesizes an adenylate cyclase-hemolysin (AC-Hly), a bifunctional protein which exhibits toxic activity and is able, as PTX, to invade mammalian cells
in vitro
and to
disrupt cellular functions by increasing intracellular cAMP concentration ." Previous studies have shown that B . pertussis mutants unable to produce FHA or DNT still have the ability to cause a lethal infection and show no defects in persistence, whereas mutants that fail to produce PTX and AC-Hly are unable to cause lethality, in the infant mouse respiratory model .` Both PTX and AC-Hly are protective antigens
* Author to whom correspondence should be addressed . tAuthor to whom requests for strains should be addressed . 0882-4010/92/030227+09 $03 .00/0
© 1992 Academic Press Limited
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Table 1 Strain 18323 18323S 18HS19 18HS26 18HS17 18HS13 18HS36 18HS37 Tohama SN B P-TOX6 B P RA
Bacterial strains used in this study (B . pertussis) Genotype"
Phenotype
Reference
Wild-type Wild-type A cya ABDE A cya BDE cya A (Aaa623-779) cya A (Aaa827-886) cya A (1 88 LQ) cya (K 58 Q) Wild-type
Vir' Vir', Strep R [AC-Hly] Ac'-Hly AC' -Hly AC -Hly AC -Hly' AC-Hly' Vir', Strep R , Nal' PTX PTX
23 This study This study This study This study This study This study This study 24 25 24
Aptx Aptx
a188LQ represents two-amino acid insertion between codons 188 (D) and 189 (I) . K58 Q represents the substitution of amino acid K in position 58, by Q .
against B . pertussis lethal challenges in murine intracerebral or respiratory models, however, AC-Hly, unlike PTX, protects against bacterial colon ization . 6-8 These results, together with the findings of Goodwin and Weiss,' suggest that PTX is essential for lethality while AC-Hly plays a role in the initiation of infection . In the present study, using the murine respiratory model, we examined the in vivo properties of B . pertussis mutants (see Table 1) . Mutants carrying a deletion of the entire cya operon, comprising genes encoding AC-Hly (cya A) and proteins involved in its secretion (cya B,D,E) as well as a deletion of the cya B,D,E genes only, were constructed and compared with PTX deficient mutants . In addition, point and insertion mutants or mutants carrying internal partial in-phase deletions in the cya A gene, resulting either in inactivation of adenylate cyclase activity or hemolytic activity, were also constructed and examined . We show that both adenylate cyclase and hemolytic activities, and not PTX, are essential for B . pertussis to initiate infection . Results Characterization of B . pertussis mutants
In a first set of experiments, we checked that all AC-Hly or PTX mutants used in this study grew on BG medium or in synthetic medium as did the parental strains . The expression of AC-Hly, PTX, FHA and P .69 in the parental strains and in the different mutants was determined by using specific antibodies, or by measuring their activity . As shown in Table 2, B . pertussis strains deleted for cya A or ptx genes were not impaired in their ability to express other virulence factors (18HS19 and BPRA, BP-TOX6) . Mutants devoid, respectively, of hemolytic or adenylate cyclase activities (18HS17, 18HS13 and 18HS36, 18HS37) expressed and secreted modified or truncated ACHly proteins as well as other virulence factors . All mutations resulting in inactivation of adenylate cyclase or hemolytic activities abolished the capacity of AC-Hly to elicit cAMP synthesis in mammalian cells . It is important to note that mutants devoid of adenylate cyclase activity (18HS36 and 18HS37), and therefore devoid of toxin activity, retained full hemolytic activity . A mutant lacking cya B,D,E genes, necessary for AC-Hly secretion (18HS26) expressed all other virulence factors and, as expected, did not secrete the AC-Hly protein . It has, however, to be noted that even in the bacteria, only a very low amount of AC-Hly could be detected .
229
Bordete/la pertussis adenylate cyclase-hemolysin
Table 2 strains
Characteristics of mutants and their parental
Strains
AC' Hly`
18323S 18HS19 18HS26 18HS17 18HS13 18HS36 18HS37 TohSN TOX6 BPRA
65 0 5910 8 >5x10 8 >5x10 8 >5910 8 >5x10 8 >5x10 8 10' >5910 8 >5x10 8
'All strains expressed FHA detected by hemagglutination and Western blotting, and P .69 detected by Western blotting . 'Adenylate cyclase activity in bacterial suspension (nmoles cAMP/min/ml) . Hemolysin activity detected on BG plates . 'Toxin activity assayed by measuring internalized adenylate cyclase activity in sheep erythrocytes . +, wild type level, -, not detectable ; NM, not measured. 'AC-Hly and PTX detected by Western blotting . 'PTX leucocytosis-promoting activity . 9 LD 50 were determined after intranasal infection of 4-week-old mice .
Virulence of B . pertussis mutants in murine respiratory model The ability of the different mutants to cause a lethal infection in 3-4-week-old mice was examined, by comparing them to the virulent parental B . pertussis 18323S or TohamaSN strains . As shown in Table 2, mutants deficient in the synthesis of AC-Hly (18HS19 and 18HS26) or PTX (BPRA or BP-TOX6), were unable to cause lethality, in agreement with the results reported by Weiss and Goodwin .' Furthermore, 18HS13 and 1 8HS1 7 impaired in hemolytic activity, and 18HS36 and 1 8HS37 lacking adenylate cyclase activity, were also unable to cause lethal infection, even at a challenge dose of 5X10 8 cfu . These results indicate that both adenylate cyclase and hemolytic activities, carried by the AC-Hly bifunctional protein, are required to cause lethal infection . Persistence of B . pertussis mutants after sublethal infection The ability to induce a persistent infection by the different mutants was compared with that of the virulent parental strains . As shown in Fig . 1, the two different PTX deficient mutants, although avirulent, were able to adhere and multiply in lungs of mice as did the parental strain, but the rate of clearance of the mutants was faster . Mutants carrying a deletion of cya A,B,D,E genes, therefore unable to synthesize ACHly (18HS19) or a deletion of secretion genes cya B,D,E (18HS26), showed decreased ability to multiply in the first 5 days of infection (Fig . 2) . 18HS36, 18HS37, 18HS13 and 18HS17, secreting modified or truncated AC-Hly protein devoid either of adenylate cyclase (Fig . 4) or hemolytic activities (Fig . 3), were also unable to multiply as fast as the virulent parental strain or ptx mutants, even though the starting number of cfu injected was 10-times higher than that of the parental strain . However, the rate of clearance of these mutants and the parental strain was the same . Induction of specific antibody synthesis after infection with live B . pertussis strains After intranasal infection of mice with 5 x 10 5 cfu of different B . pertussis strains (Tohama SN, 18323 S or BPRA), antibodies against AC-Hly, FHA, PTX and P .69
N . Khelef
23 0
et al.
8
7
0
c 0 N
° U
4
J
3 2
I I I 10 20
I
0
30
Days after challenge Fig . 1 . Bordetella pertussis Tohama, BP Tox 6 and BPRA colonization of the lungs of mice . Three-fourweek-old mice were challenged intranasally with 5x10 5 cfu of Tohama SN (0), BPRA (0) and BP-TOX6 (0) . The plots show the geometric means±SD (bars) for six mice per time point .
could be detected 2 weeks following infection and persisted more than 2 months after infection . After infection with the cya A,B,D,E genes deletion mutant (18HS19), as expected, no anti-AC-Hly antibodies could be detected . However, even anti-PTX, anti-FHA and anti-P .69 antibodies were not detected, confirming the inability of this mutant to colonize the respiratory tract of the host (Fig . 5) .
Discussion The aim of this study was to compare, in a murine respiratory model, the in vivo role of AC-Hly and PTX, two toxins produced by B . pertussis. Different AC-Hly mutants,
O'
CX
U Ot 0 J
Days after challenge Fig . 2 . Bordetella pertussis 18323S, 18HS19, 18HS26 colonization of the lungs of mice . Three-fourweek-old mice were challenged intranasally with 5x10 5 cfu of 183235 (i), 18HS19 (0) and 18HS26 (o) . The plots show the geometric means±SD (bars) for six mice per time point .
Bordete/la pertussis adenylate cyclase- hemolysin
10
231
20
30
Days after challenge Fig . 3 . Bordetella pertussis 18323S, 18HS13, 18HS17 colonization of the lungs of mice . Three-fourweek-old mice were challenged intranasally with 5 x 10 5 cfu of 18323S ( •) , 18HS17 (0) and 18HS13 (0) . The plots show the geometric means±SD (bars) for six mice per time point .
either unable to express or secrete the protein, or devoid of adenylate cyclase or hemolytic activities, were constructed and compared with two PTX deficient mutants . In a first set of experiments we characterized these strains with respect to their ability to express unrelated virulence factors, because we have observed that mutations, such as nalidixic acid resistance, could modify the synthesis of some Bordetella proteins . The different mutants used in this study were not affected in the expression of other unrelated virulence factors . It has to be noted that undetectable or very low amount of AC-Hly protein could be formed in the mutant carrying a deletion of the cya B,D,E genes necessary for AC-Hly secretion . This result suggests either a reduced expression of the protein or its instability
Days after challenge Fig . 4 . Bordetella pertussis 18323S, 18HS36, 18HS37 colonization of the lungs of mice . Three-fourweek-old mice were challenged intranasally with 5 x 10 5 cfu of 18323S (s), 18HS37 (0) and 18HS36 (0) . The plots show the geometric means±SD (bars) for six mice per time point .
N . Khelef eta[
232 A
B
C
I
2
D
3
4
I
2
3
4
Fig . 5 . Antibodies detection in the sera of infected mice by PTX and AC-Hly deletion mutants and parental strains . Groups of 3-4-week-old mice were infected intranasally with 5 x 10 5 cfu of (A) B . pertussis Tohama SN, (B) BPRA, (C) B . pertussis 18323S and (D) 18HS19 . Mice were bled every week following the infection during 2 months and antibodies against AC-Hly (lane 1), PTX (lane 2), FHA (lane 3) and P .69 (lane 4) were detected by Western blotting . The results presented in the figure were obtained with sera collected 5 weeks after infection .
in the absence of the secretion genes . Similar results were also obtained with the Escherichia coli a-hemolysin . 19 The murine respiratory model we used to study Bordete/la pathogenesis reproduces some parameters of the human disease, such as attachment to ciliated cells of the respiratory tract, growth and multiplication of the bacteria, systemic disease such as hyperleucocytosis, and local disease such as destruction of the ciliated cells of the respiratory tract . In general, in the murine respiratory model, suckling mice are used because of the age-dependent severity of the disease, as also observed in the human disease . However, we have previously shown that B . pertussis freshly re-isolated from lung homogenates of infected mice are not only virulent for infant mice but also for adult mice ." Consequently, all mutants were freshly re-isolated from infected mice and their virulence tested . We show that mutants which did not express PTX or ACHly, or were impaired in adenylate cyclase or hemolytic activities, were unable to cause lethal infection, in the adult mice model, in agreement with previous data reported by Weiss with infant mice .' Our results show, for the first time, that both adenylate cyclase and hemolytic activities carried by the AC-Hly protein are required for virulence and initiation of infection . The main difference we observed, between ptx and cya mutants, was that ptx mutants, like the parental strains, attach, grow and multiply, whereas cya mutants did multiply much more slowly, in the first 5 days of infection, even at high challenge dose . This latter result is at variance with the finding of Weiss, who has shown that a high challenge dose, an AC-Hly deficient mutant was still able to colonize the lungs of suckling mice .' However, cya mutants were cleared at the same rate as the parental strain, suggesting a role for AC-Hly in the early steps
Bordetella pertussis adenylate cyclase-hemolysin
233
of infection . ptx mutants behaved like the parental strain at the beginning of infection, but are cleared faster, suggesting a role for PTX in colonization . This result is in agreement with the findings of Finn et al ., who showed that 14 days after respiratory infection, ptx mutants colonized 100-fold less than the parental strain . 5 It has to be noted that mutants without adenylate cyclase activity retained full hemolytic activity . This demonstrates for the first time that hemolytic activity does not require adenylate cyclase activity . These mutants lacked the ability of rapid multiplication, suggesting that hemolysin, necessary for the toxic activity of the ACHly protein, 14 by itself does not play a role in bacterial colonization . The role of ACHly in initial growth of the bacteria in the respiratory tract has been corroborated by the finding that the cya A,B,D,E mutant, unlike the ptx mutant, did not elicit antibodies directed against other virulence factors . It has been recently shown that AC-Hly, as PTX, is a protective antigen against B . pertussis lethal intranasal and intracerebral challenges . 9 - 20 However, AC-Hly is able to protect against brain or lung colonization by bacteria,' contrary to PTX . 9 Taken together, all these results suggest that AC-Hly is required for initial growth of the bacteria and is a protective antigen against bacterial colonization, whereas PTX is required for lethality and is able to protect against the disease . These results are in agreement with the Swedish clinical trials which showed that PTX is an immunogen against the disease but not against the infection, 21 Using a murine respiratory model, it has been recently shown that strains deficient in production of P .69 and FHA, two other virulence factors of B. pertussis, were able to colonize the lungs of mice as the parental strains ." The authors proposed that additional vir-regulated factor is necessary for colonization, proliferation and surv ;val of the bacteria in the murine respiratory tract . Our present results suggest that AC-Hly could be this factor .
Materials and methods
Bacterial strains and growth conditions . Bordetella pertussis strains used in this study are listed in Table 1 . All strains were lung-passaged derivatives isolated as described previously." Bacteria were grown on Bordet Gengou agar supplemented with 15% defibrinated sheep blood (BG) at 36°C for 48 h . Subcultures in liquid medium were performed on Stainer Scholte medium" for 20 h until absorbance measured at 650 nm reached 1 .0 . DNA manipulation and construction of B . pertussis mutants . All B. pertussis mutants were constructed by chromosome-borne double allelic exchanges, using pRTP1 derivative suicide plasmids and the selection method described previously . 12 The suicide plasmids were introduced into the B . pertussis 18323S strain by electroporation .' 3 The suicide plasmids used to construct 18HS13, 18HS17, 18HS19 were described by Bellalou et al." The 18HS26 mutant was constructed using a suicide plasmid carrying the 5' end of the cya B gene ligated to the 3' end of the cya E gene . As a result, the cya B gene was deleted from amino acid 191 to the end, the cya D gene was totally deleted, and the cya E gene lacked its translation initiation signals and amino acids 1-355 . The 18HS36 and 18HS37 strains were constructed using suicide plasmids carrying the 5' end of the cya A gene, mutated either by insertion of the oligonucleotide sequence CTGCAG between codons 188 and 189, 15 or by substituting AAA codon to CAA, at position 58 . 16 All mutants, except 18HS36 and 18HS37, were checked both by Southern hybridization and complementation analysis . Adenylate cyclase assay. Adenylate cyclase activity was measured on bacterial suspensions as described previously ." One unit corresponds to 1 nmol of cAMP formed per min at 30°C at pH 8 .
Determination of adenylate cyclase toxin capacity . Adenylate cyclase toxin activity was
234
N . Khelef et al.
assayed by measuring the internalized adenylate cyclase activity as described ." Wild-type activity was taken as 100% .
Pertussis toxin assay. PTX leucocytosis-promoting activity was determined by peripheral leucocyte counts, 6 days after infection of mice with the various Bordetella challenge strains ." Filamentous hemagglutinin assay . The ability of Bordetella strains to agglutinate sheep erythrocytes was assayed as described previously ." Electrophoresis and immunoblotting . Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using ready to use 4-15% or 8-25% gels and a PhastSystem (Pharmacia) . For immunoblotting experiments, proteins were transferred from polyacrylamide gels to Hybond C-Super membranes (Amersham) and incubated with monoclonal anti-AC, polyclonal anti-FHA, polyclonal anti-PTx or polyclonal anti-P .69 (gift of C . Capiau, Smith-Kline Laboratories) . The immunochemical detection was performed with peroxydase-labelled anti-mouse immunoglobulins using the Amersham ECL detection reagents . Mouse intranasal infection . Bordetella pertussis strains were grown for 24 h on BG plates at 36°C . Bacteria were resuspended in saline and then serially diluted to provide challenge inoculum dilutions to evaluate the LD 50 . For the respiratory infection, 50 yl of bacterial suspension were injected intranasally, to groups of 10, 3-4-week-old female Swiss mice (CERJ, St . Berthevin, France) . The LD 50 for the challenging inocula were determined by recording daily the number of dead mice during 30 days . Sublethal challenges were performed by intranasal injection as described above . Infected mice were sacrificed by cervical dislocation 1 h after exposure (at time designated day 0) and at various days thereafter (six mice per time point) . The lungs were removed and homogenized in saline with tissue grinders . Dilutions of lung homogenates were sampled on BG plates and cfu were counted after 3-4 days of incubation at 36°C . Statistical analysis. Data were tested for statistical significance by Student's t-test . We are grateful to Agnes Ullmann for her constant interest in this work, stimulating discussions and critical reading of the manuscript . We thank C . Locht and R . Rappuoli for the gift of PTX deficient mutants, J . Bellalou for the gift of 18323 S strain and advice for the electroporations experiments, D . Ladant for the pRAC 3 plasmid, M . J . Quentin-Millet for purified FHA and PTX, and C . Capiau for the anti-P .69 antibodies . This work was supported by funds from Institut Pasteur, Centre National de la Recherche Scientifique (URA1129), Pasteur Merieux Serum Vaccins and from DRET to Agnes Ullmann, in whose laboratory part of the work was carried out .
References 1 . Glaser P, Sakamoto H, Bellalou J, Ullmann A, Danchin A . Secretion of cyclolysin, the calmodulinsensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis . EMBO J 1988 ; 7 : 3997-4004 . 2 . Hanski E . Invasive adenylate cyclase toxin of Bordete/la pertussis . Trends Biochem Sci 1989 ; 14 : 45963 . 3 . Ui M . The multiple biological activities of pertussis toxin . In : Wardlaw AC, Parton R, eds . J Wiley, 1988 ;121-45 . 4 . Weiss AA, Goodwin MM . Lethal infection by B. pertussis mutants in the infant mouse model . Infect Immun 1989; 57 : 3757-64 . 5 . Finn TM, Shahin R, Mekalanos JJ . Characterization of TnphoA gene fusions in Bordetella pertussis . Infect Immun 1991 ; 59 : 3273-9 . 6 . Sato Y, Sato H . Animal models of pertussis. In : Wardlaw AC, Parton R, eds. J Wiley 1988; 309-25 . 7 . Guiso N, Szatanik M, Rocancourt M . Protective activity of Bordete//a adenylate cyclase-hemolysin against bacterial colonization . Microb Pathogen 1991 ; 11 : 423-31 . 8 . Guiso N, Khelef N . Murine models to study Bordetella pathogenesis and to characterize protective antigens. In : Proceeding of the Fifth European Workshop on Bacterial protein toxins . 1991 (in press) . 9 . Goodwin M St M, Weiss AA . Adenylate cyclase toxin is critical for colonization and pertussis toxin for lethal infection by Bordetella pertussis in infant mice . Infect Immun 1990; 58 : 3445-7 .
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10 . Guiso N, Rocancourt M, Szatanik M, Alonso JM . Bordetella adenylate cyclase is a virulence associated factor and an immunoprotective antigen . Microb Pathogen 1989; 7 : 373-80 . 11 . Stainer DW, Scholte MJ . A simple chemically defined medium for the production of Phase I Bordetella pertussis . J Gen Microb 1971 ; 63 : 211-20 . 12 . Stibitz S, Black W, Falkow S . The construction of a cloning vector designed for gene replacement in Bordetella pertussis. Gene 1986; 50 : 133-40 . 13 . Zealey G, Dion M, Loosmore S, Yacoob R, Klein M . High frequency transformation of Bordetella by electroporation . FEMS Microbiol Lett 1988 ; 56 : 123-6 . 14 . Bellalou J, Sakamoto H, Ladant D, Geoffroy C, Ullmann A . Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase . Infect Immun 1990; 58 : 3242-7 . 15 . Ladant D, Glaser P, Ullmann A . Insertional mutagenesis of Bordete//a pertussis adenylate cyclase . J Biol Chem 1992 ; 267 : 2244-50 . 16 . Glaser P, Elmaoglou-Zaridou A, Krin E, Ladant D, Barzu 0, Danchin A . Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by sitedirected mutagenesis . EM BO J 1989 ; 3 : 967-72 . 17 . Ladant D, Brezin C, Alonso JM, Crenon I, Guiso N . Bordete/la pertussis adenylate cyclase : purification, characterization and radioimmunoassay . J Biol Chem 1987 ; 261 : 16264-9 . 18 . Munoz JJ, Arai H, Cole RL. Mouse-protecting and histamine-sensitizing activities of pertussinogen and fimbrial hemagglutinin from Bordetella pertussis . Infect Immun 1981 ; 32 : 243-50 . 19 . Oropeza-Wekerle RL, Speth W, Imhof B, Genstschev, Goebel W . Translocation and compartimentalization of Escherichia coli hemolysin (HIyA) . J Bacteriol 1990 ; 172 : 3711-7 . 20 . Guiso N, Szatanik M, Rocancourt M . Bordetella adenylate cyclase : a protective antigen against lethality and bacterial colonization in murine intranasal and intracerebral models . In : Proceedings of the Sixth International Symposium on Pertussis . Bethesda : Department of Health and Human Services, 1990 ; 207-11 . 21 . Olin P . New conclusions and lessons learned from the vaccine trial in Sweden . In : Proceedings of the Sixth International Symposium on Pertussis . Bethesda : Department of Health and Human Services, 1990 ;299-302 . 22 . Roberts M, Fairweather NF, Leininger E et al. Construction and characterization of Bordetella pertussis mutants lacking the Vir-regulated P .69 outer membrane protein . Mol Microbiol 1991 ; 5 : 1393-404 . 23 . Pittman M . Genus Bordetella. In : Krieg NR, Holt JG, eds . Bergey's manual of systemic bacteriology, vol . 1 . Baltimore : Williams & Wilkins Co ., 1984 ; 388-93 . 24 . Antoine R, Locht C . Roles of the disulfide bond and the carboxy-terminal region of the S1 subunit in the assembly and biosynthesis of pertussis toxin . Infect Immun 1990; 58 : 1518-26 . 25 . Relman DA, Domenighini M, Tuomanen EU, Rappuoli R, Falkow S . Filamentous hemagglutinin of Bordetella pertussis : nucleotide sequence and crucial role in adherence . Proc Natl Acad Sci USA 1989 ; 86 :2637-41 .
Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection Nadia Khelef,' Hiroshi Sakamoto 2 t and Nicole Guiso'* 'Unite de Bacteriologie Moleculaire et Medicale and 2 Unite de Biochimie des Regulations Cellulaires, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France (Received October 4, 1991 ; accepted in revised form November 11, 1991)
Khelef, N . (Unite de Bacteriologie Moleculaire et Medicale, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, France), H . Sakamoto and N . Guiso . Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection . Microbial Pathogenesis 1992 ; 12 : 227235 . Among virulence factors synthesized and secreted by Bordetella pertussis, pertussis toxin (PTX) and the bifunctional adenylate cyclase-hemolysin (AC-Hly) are able to invade mammalian cells and to impair intracellular functions. Moreover, both proteins are protective antigens in murine intracerebral and respiratory models . In order to study their in vivo properties, different B . pertussis mutants, deficient in AC-Hly expression or secretion, or producing modified ACHly devoid of either adenylate cyclase or hemolytic activities, were constructed and examined . The in vivo properties of the mutants were compared to PTX deficient strains, using the murine respiratory model . We show that lack of PTX as well as adenylate cyclase or hemolytic activities results in avirulence . Furthermore, we show that mutants lacking adenylate cyclase or hemolytic activities were unable to multiply as fast as the parental strains and PTX mutants during the first 5 days following infection . Thus, both adenylate cyclase and hemolytic activities are required by B . pertussis to initiate infection . Key words : Bordetella pertussis ; pertussis toxin ; colonization .
murine respiratory model ; adenylate cyclase-hemolysin ;
Introduction Bordete/la pertussis, the agent of whooping cough, synthesizes a variety of virulence factors . Some participate in the adhesion of the bacteria to the respiratory tract of the host, such as filamentous hemagglutinin (FHA) and pertussis toxin (PTX) . Others are responsible for the local disease, such as dermonecrotic toxin (DNT) and tracheal cytotoxin (TCT), or for the systemic disease such as PTX . Bordetella pertussis also synthesizes an adenylate cyclase-hemolysin (AC-Hly), a bifunctional protein which exhibits toxic activity and is able, as PTX, to invade mammalian cells
in vitro
and to
disrupt cellular functions by increasing intracellular cAMP concentration ." Previous studies have shown that B . pertussis mutants unable to produce FHA or DNT still have the ability to cause a lethal infection and show no defects in persistence, whereas mutants that fail to produce PTX and AC-Hly are unable to cause lethality, in the infant mouse respiratory model .` Both PTX and AC-Hly are protective antigens
* Author to whom correspondence should be addressed . tAuthor to whom requests for strains should be addressed . 0882-4010/92/030227+09 $03 .00/0
© 1992 Academic Press Limited
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Table 1 Strain 18323 18323S 18HS19 18HS26 18HS17 18HS13 18HS36 18HS37 Tohama SN B P-TOX6 B P RA
Bacterial strains used in this study (B . pertussis) Genotype"
Phenotype
Reference
Wild-type Wild-type A cya ABDE A cya BDE cya A (Aaa623-779) cya A (Aaa827-886) cya A (1 88 LQ) cya (K 58 Q) Wild-type
Vir' Vir', Strep R [AC-Hly] Ac'-Hly AC' -Hly AC -Hly AC -Hly' AC-Hly' Vir', Strep R , Nal' PTX PTX
23 This study This study This study This study This study This study This study 24 25 24
Aptx Aptx
a188LQ represents two-amino acid insertion between codons 188 (D) and 189 (I) . K58 Q represents the substitution of amino acid K in position 58, by Q .
against B . pertussis lethal challenges in murine intracerebral or respiratory models, however, AC-Hly, unlike PTX, protects against bacterial colon ization . 6-8 These results, together with the findings of Goodwin and Weiss,' suggest that PTX is essential for lethality while AC-Hly plays a role in the initiation of infection . In the present study, using the murine respiratory model, we examined the in vivo properties of B . pertussis mutants (see Table 1) . Mutants carrying a deletion of the entire cya operon, comprising genes encoding AC-Hly (cya A) and proteins involved in its secretion (cya B,D,E) as well as a deletion of the cya B,D,E genes only, were constructed and compared with PTX deficient mutants . In addition, point and insertion mutants or mutants carrying internal partial in-phase deletions in the cya A gene, resulting either in inactivation of adenylate cyclase activity or hemolytic activity, were also constructed and examined . We show that both adenylate cyclase and hemolytic activities, and not PTX, are essential for B . pertussis to initiate infection . Results Characterization of B . pertussis mutants
In a first set of experiments, we checked that all AC-Hly or PTX mutants used in this study grew on BG medium or in synthetic medium as did the parental strains . The expression of AC-Hly, PTX, FHA and P .69 in the parental strains and in the different mutants was determined by using specific antibodies, or by measuring their activity . As shown in Table 2, B . pertussis strains deleted for cya A or ptx genes were not impaired in their ability to express other virulence factors (18HS19 and BPRA, BP-TOX6) . Mutants devoid, respectively, of hemolytic or adenylate cyclase activities (18HS17, 18HS13 and 18HS36, 18HS37) expressed and secreted modified or truncated ACHly proteins as well as other virulence factors . All mutations resulting in inactivation of adenylate cyclase or hemolytic activities abolished the capacity of AC-Hly to elicit cAMP synthesis in mammalian cells . It is important to note that mutants devoid of adenylate cyclase activity (18HS36 and 18HS37), and therefore devoid of toxin activity, retained full hemolytic activity . A mutant lacking cya B,D,E genes, necessary for AC-Hly secretion (18HS26) expressed all other virulence factors and, as expected, did not secrete the AC-Hly protein . It has, however, to be noted that even in the bacteria, only a very low amount of AC-Hly could be detected .
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Table 2 strains
Characteristics of mutants and their parental
Strains
AC' Hly`
18323S 18HS19 18HS26 18HS17 18HS13 18HS36 18HS37 TohSN TOX6 BPRA
65 0 5910 8 >5x10 8 >5x10 8 >5910 8 >5x10 8 >5x10 8 10' >5910 8 >5x10 8
'All strains expressed FHA detected by hemagglutination and Western blotting, and P .69 detected by Western blotting . 'Adenylate cyclase activity in bacterial suspension (nmoles cAMP/min/ml) . Hemolysin activity detected on BG plates . 'Toxin activity assayed by measuring internalized adenylate cyclase activity in sheep erythrocytes . +, wild type level, -, not detectable ; NM, not measured. 'AC-Hly and PTX detected by Western blotting . 'PTX leucocytosis-promoting activity . 9 LD 50 were determined after intranasal infection of 4-week-old mice .
Virulence of B . pertussis mutants in murine respiratory model The ability of the different mutants to cause a lethal infection in 3-4-week-old mice was examined, by comparing them to the virulent parental B . pertussis 18323S or TohamaSN strains . As shown in Table 2, mutants deficient in the synthesis of AC-Hly (18HS19 and 18HS26) or PTX (BPRA or BP-TOX6), were unable to cause lethality, in agreement with the results reported by Weiss and Goodwin .' Furthermore, 18HS13 and 1 8HS1 7 impaired in hemolytic activity, and 18HS36 and 1 8HS37 lacking adenylate cyclase activity, were also unable to cause lethal infection, even at a challenge dose of 5X10 8 cfu . These results indicate that both adenylate cyclase and hemolytic activities, carried by the AC-Hly bifunctional protein, are required to cause lethal infection . Persistence of B . pertussis mutants after sublethal infection The ability to induce a persistent infection by the different mutants was compared with that of the virulent parental strains . As shown in Fig . 1, the two different PTX deficient mutants, although avirulent, were able to adhere and multiply in lungs of mice as did the parental strain, but the rate of clearance of the mutants was faster . Mutants carrying a deletion of cya A,B,D,E genes, therefore unable to synthesize ACHly (18HS19) or a deletion of secretion genes cya B,D,E (18HS26), showed decreased ability to multiply in the first 5 days of infection (Fig . 2) . 18HS36, 18HS37, 18HS13 and 18HS17, secreting modified or truncated AC-Hly protein devoid either of adenylate cyclase (Fig . 4) or hemolytic activities (Fig . 3), were also unable to multiply as fast as the virulent parental strain or ptx mutants, even though the starting number of cfu injected was 10-times higher than that of the parental strain . However, the rate of clearance of these mutants and the parental strain was the same . Induction of specific antibody synthesis after infection with live B . pertussis strains After intranasal infection of mice with 5 x 10 5 cfu of different B . pertussis strains (Tohama SN, 18323 S or BPRA), antibodies against AC-Hly, FHA, PTX and P .69
N . Khelef
23 0
et al.
8
7
0
c 0 N
° U
4
J
3 2
I I I 10 20
I
0
30
Days after challenge Fig . 1 . Bordetella pertussis Tohama, BP Tox 6 and BPRA colonization of the lungs of mice . Three-fourweek-old mice were challenged intranasally with 5x10 5 cfu of Tohama SN (0), BPRA (0) and BP-TOX6 (0) . The plots show the geometric means±SD (bars) for six mice per time point .
could be detected 2 weeks following infection and persisted more than 2 months after infection . After infection with the cya A,B,D,E genes deletion mutant (18HS19), as expected, no anti-AC-Hly antibodies could be detected . However, even anti-PTX, anti-FHA and anti-P .69 antibodies were not detected, confirming the inability of this mutant to colonize the respiratory tract of the host (Fig . 5) .
Discussion The aim of this study was to compare, in a murine respiratory model, the in vivo role of AC-Hly and PTX, two toxins produced by B . pertussis. Different AC-Hly mutants,
O'
CX
U Ot 0 J
Days after challenge Fig . 2 . Bordetella pertussis 18323S, 18HS19, 18HS26 colonization of the lungs of mice . Three-fourweek-old mice were challenged intranasally with 5x10 5 cfu of 183235 (i), 18HS19 (0) and 18HS26 (o) . The plots show the geometric means±SD (bars) for six mice per time point .
Bordete/la pertussis adenylate cyclase- hemolysin
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20
30
Days after challenge Fig . 3 . Bordetella pertussis 18323S, 18HS13, 18HS17 colonization of the lungs of mice . Three-fourweek-old mice were challenged intranasally with 5 x 10 5 cfu of 18323S ( •) , 18HS17 (0) and 18HS13 (0) . The plots show the geometric means±SD (bars) for six mice per time point .
either unable to express or secrete the protein, or devoid of adenylate cyclase or hemolytic activities, were constructed and compared with two PTX deficient mutants . In a first set of experiments we characterized these strains with respect to their ability to express unrelated virulence factors, because we have observed that mutations, such as nalidixic acid resistance, could modify the synthesis of some Bordetella proteins . The different mutants used in this study were not affected in the expression of other unrelated virulence factors . It has to be noted that undetectable or very low amount of AC-Hly protein could be formed in the mutant carrying a deletion of the cya B,D,E genes necessary for AC-Hly secretion . This result suggests either a reduced expression of the protein or its instability
Days after challenge Fig . 4 . Bordetella pertussis 18323S, 18HS36, 18HS37 colonization of the lungs of mice . Three-fourweek-old mice were challenged intranasally with 5 x 10 5 cfu of 18323S (s), 18HS37 (0) and 18HS36 (0) . The plots show the geometric means±SD (bars) for six mice per time point .
N . Khelef eta[
232 A
B
C
I
2
D
3
4
I
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3
4
Fig . 5 . Antibodies detection in the sera of infected mice by PTX and AC-Hly deletion mutants and parental strains . Groups of 3-4-week-old mice were infected intranasally with 5 x 10 5 cfu of (A) B . pertussis Tohama SN, (B) BPRA, (C) B . pertussis 18323S and (D) 18HS19 . Mice were bled every week following the infection during 2 months and antibodies against AC-Hly (lane 1), PTX (lane 2), FHA (lane 3) and P .69 (lane 4) were detected by Western blotting . The results presented in the figure were obtained with sera collected 5 weeks after infection .
in the absence of the secretion genes . Similar results were also obtained with the Escherichia coli a-hemolysin . 19 The murine respiratory model we used to study Bordete/la pathogenesis reproduces some parameters of the human disease, such as attachment to ciliated cells of the respiratory tract, growth and multiplication of the bacteria, systemic disease such as hyperleucocytosis, and local disease such as destruction of the ciliated cells of the respiratory tract . In general, in the murine respiratory model, suckling mice are used because of the age-dependent severity of the disease, as also observed in the human disease . However, we have previously shown that B . pertussis freshly re-isolated from lung homogenates of infected mice are not only virulent for infant mice but also for adult mice ." Consequently, all mutants were freshly re-isolated from infected mice and their virulence tested . We show that mutants which did not express PTX or ACHly, or were impaired in adenylate cyclase or hemolytic activities, were unable to cause lethal infection, in the adult mice model, in agreement with previous data reported by Weiss with infant mice .' Our results show, for the first time, that both adenylate cyclase and hemolytic activities carried by the AC-Hly protein are required for virulence and initiation of infection . The main difference we observed, between ptx and cya mutants, was that ptx mutants, like the parental strains, attach, grow and multiply, whereas cya mutants did multiply much more slowly, in the first 5 days of infection, even at high challenge dose . This latter result is at variance with the finding of Weiss, who has shown that a high challenge dose, an AC-Hly deficient mutant was still able to colonize the lungs of suckling mice .' However, cya mutants were cleared at the same rate as the parental strain, suggesting a role for AC-Hly in the early steps
Bordetella pertussis adenylate cyclase-hemolysin
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of infection . ptx mutants behaved like the parental strain at the beginning of infection, but are cleared faster, suggesting a role for PTX in colonization . This result is in agreement with the findings of Finn et al ., who showed that 14 days after respiratory infection, ptx mutants colonized 100-fold less than the parental strain . 5 It has to be noted that mutants without adenylate cyclase activity retained full hemolytic activity . This demonstrates for the first time that hemolytic activity does not require adenylate cyclase activity . These mutants lacked the ability of rapid multiplication, suggesting that hemolysin, necessary for the toxic activity of the ACHly protein, 14 by itself does not play a role in bacterial colonization . The role of ACHly in initial growth of the bacteria in the respiratory tract has been corroborated by the finding that the cya A,B,D,E mutant, unlike the ptx mutant, did not elicit antibodies directed against other virulence factors . It has been recently shown that AC-Hly, as PTX, is a protective antigen against B . pertussis lethal intranasal and intracerebral challenges . 9 - 20 However, AC-Hly is able to protect against brain or lung colonization by bacteria,' contrary to PTX . 9 Taken together, all these results suggest that AC-Hly is required for initial growth of the bacteria and is a protective antigen against bacterial colonization, whereas PTX is required for lethality and is able to protect against the disease . These results are in agreement with the Swedish clinical trials which showed that PTX is an immunogen against the disease but not against the infection, 21 Using a murine respiratory model, it has been recently shown that strains deficient in production of P .69 and FHA, two other virulence factors of B. pertussis, were able to colonize the lungs of mice as the parental strains ." The authors proposed that additional vir-regulated factor is necessary for colonization, proliferation and surv ;val of the bacteria in the murine respiratory tract . Our present results suggest that AC-Hly could be this factor .
Materials and methods
Bacterial strains and growth conditions . Bordetella pertussis strains used in this study are listed in Table 1 . All strains were lung-passaged derivatives isolated as described previously." Bacteria were grown on Bordet Gengou agar supplemented with 15% defibrinated sheep blood (BG) at 36°C for 48 h . Subcultures in liquid medium were performed on Stainer Scholte medium" for 20 h until absorbance measured at 650 nm reached 1 .0 . DNA manipulation and construction of B . pertussis mutants . All B. pertussis mutants were constructed by chromosome-borne double allelic exchanges, using pRTP1 derivative suicide plasmids and the selection method described previously . 12 The suicide plasmids were introduced into the B . pertussis 18323S strain by electroporation .' 3 The suicide plasmids used to construct 18HS13, 18HS17, 18HS19 were described by Bellalou et al." The 18HS26 mutant was constructed using a suicide plasmid carrying the 5' end of the cya B gene ligated to the 3' end of the cya E gene . As a result, the cya B gene was deleted from amino acid 191 to the end, the cya D gene was totally deleted, and the cya E gene lacked its translation initiation signals and amino acids 1-355 . The 18HS36 and 18HS37 strains were constructed using suicide plasmids carrying the 5' end of the cya A gene, mutated either by insertion of the oligonucleotide sequence CTGCAG between codons 188 and 189, 15 or by substituting AAA codon to CAA, at position 58 . 16 All mutants, except 18HS36 and 18HS37, were checked both by Southern hybridization and complementation analysis . Adenylate cyclase assay. Adenylate cyclase activity was measured on bacterial suspensions as described previously ." One unit corresponds to 1 nmol of cAMP formed per min at 30°C at pH 8 .
Determination of adenylate cyclase toxin capacity . Adenylate cyclase toxin activity was
234
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assayed by measuring the internalized adenylate cyclase activity as described ." Wild-type activity was taken as 100% .
Pertussis toxin assay. PTX leucocytosis-promoting activity was determined by peripheral leucocyte counts, 6 days after infection of mice with the various Bordetella challenge strains ." Filamentous hemagglutinin assay . The ability of Bordetella strains to agglutinate sheep erythrocytes was assayed as described previously ." Electrophoresis and immunoblotting . Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using ready to use 4-15% or 8-25% gels and a PhastSystem (Pharmacia) . For immunoblotting experiments, proteins were transferred from polyacrylamide gels to Hybond C-Super membranes (Amersham) and incubated with monoclonal anti-AC, polyclonal anti-FHA, polyclonal anti-PTx or polyclonal anti-P .69 (gift of C . Capiau, Smith-Kline Laboratories) . The immunochemical detection was performed with peroxydase-labelled anti-mouse immunoglobulins using the Amersham ECL detection reagents . Mouse intranasal infection . Bordetella pertussis strains were grown for 24 h on BG plates at 36°C . Bacteria were resuspended in saline and then serially diluted to provide challenge inoculum dilutions to evaluate the LD 50 . For the respiratory infection, 50 yl of bacterial suspension were injected intranasally, to groups of 10, 3-4-week-old female Swiss mice (CERJ, St . Berthevin, France) . The LD 50 for the challenging inocula were determined by recording daily the number of dead mice during 30 days . Sublethal challenges were performed by intranasal injection as described above . Infected mice were sacrificed by cervical dislocation 1 h after exposure (at time designated day 0) and at various days thereafter (six mice per time point) . The lungs were removed and homogenized in saline with tissue grinders . Dilutions of lung homogenates were sampled on BG plates and cfu were counted after 3-4 days of incubation at 36°C . Statistical analysis. Data were tested for statistical significance by Student's t-test . We are grateful to Agnes Ullmann for her constant interest in this work, stimulating discussions and critical reading of the manuscript . We thank C . Locht and R . Rappuoli for the gift of PTX deficient mutants, J . Bellalou for the gift of 18323 S strain and advice for the electroporations experiments, D . Ladant for the pRAC 3 plasmid, M . J . Quentin-Millet for purified FHA and PTX, and C . Capiau for the anti-P .69 antibodies . This work was supported by funds from Institut Pasteur, Centre National de la Recherche Scientifique (URA1129), Pasteur Merieux Serum Vaccins and from DRET to Agnes Ullmann, in whose laboratory part of the work was carried out .
References 1 . Glaser P, Sakamoto H, Bellalou J, Ullmann A, Danchin A . Secretion of cyclolysin, the calmodulinsensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis . EMBO J 1988 ; 7 : 3997-4004 . 2 . Hanski E . Invasive adenylate cyclase toxin of Bordete/la pertussis . Trends Biochem Sci 1989 ; 14 : 45963 . 3 . Ui M . The multiple biological activities of pertussis toxin . In : Wardlaw AC, Parton R, eds . J Wiley, 1988 ;121-45 . 4 . Weiss AA, Goodwin MM . Lethal infection by B. pertussis mutants in the infant mouse model . Infect Immun 1989; 57 : 3757-64 . 5 . Finn TM, Shahin R, Mekalanos JJ . Characterization of TnphoA gene fusions in Bordetella pertussis . Infect Immun 1991 ; 59 : 3273-9 . 6 . Sato Y, Sato H . Animal models of pertussis. In : Wardlaw AC, Parton R, eds. J Wiley 1988; 309-25 . 7 . Guiso N, Szatanik M, Rocancourt M . Protective activity of Bordete//a adenylate cyclase-hemolysin against bacterial colonization . Microb Pathogen 1991 ; 11 : 423-31 . 8 . Guiso N, Khelef N . Murine models to study Bordetella pathogenesis and to characterize protective antigens. In : Proceeding of the Fifth European Workshop on Bacterial protein toxins . 1991 (in press) . 9 . Goodwin M St M, Weiss AA . Adenylate cyclase toxin is critical for colonization and pertussis toxin for lethal infection by Bordetella pertussis in infant mice . Infect Immun 1990; 58 : 3445-7 .
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10 . Guiso N, Rocancourt M, Szatanik M, Alonso JM . Bordetella adenylate cyclase is a virulence associated factor and an immunoprotective antigen . Microb Pathogen 1989; 7 : 373-80 . 11 . Stainer DW, Scholte MJ . A simple chemically defined medium for the production of Phase I Bordetella pertussis . J Gen Microb 1971 ; 63 : 211-20 . 12 . Stibitz S, Black W, Falkow S . The construction of a cloning vector designed for gene replacement in Bordetella pertussis. Gene 1986; 50 : 133-40 . 13 . Zealey G, Dion M, Loosmore S, Yacoob R, Klein M . High frequency transformation of Bordetella by electroporation . FEMS Microbiol Lett 1988 ; 56 : 123-6 . 14 . Bellalou J, Sakamoto H, Ladant D, Geoffroy C, Ullmann A . Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase . Infect Immun 1990; 58 : 3242-7 . 15 . Ladant D, Glaser P, Ullmann A . Insertional mutagenesis of Bordete//a pertussis adenylate cyclase . J Biol Chem 1992 ; 267 : 2244-50 . 16 . Glaser P, Elmaoglou-Zaridou A, Krin E, Ladant D, Barzu 0, Danchin A . Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by sitedirected mutagenesis . EM BO J 1989 ; 3 : 967-72 . 17 . Ladant D, Brezin C, Alonso JM, Crenon I, Guiso N . Bordete/la pertussis adenylate cyclase : purification, characterization and radioimmunoassay . J Biol Chem 1987 ; 261 : 16264-9 . 18 . Munoz JJ, Arai H, Cole RL. Mouse-protecting and histamine-sensitizing activities of pertussinogen and fimbrial hemagglutinin from Bordetella pertussis . Infect Immun 1981 ; 32 : 243-50 . 19 . Oropeza-Wekerle RL, Speth W, Imhof B, Genstschev, Goebel W . Translocation and compartimentalization of Escherichia coli hemolysin (HIyA) . J Bacteriol 1990 ; 172 : 3711-7 . 20 . Guiso N, Szatanik M, Rocancourt M . Bordetella adenylate cyclase : a protective antigen against lethality and bacterial colonization in murine intranasal and intracerebral models . In : Proceedings of the Sixth International Symposium on Pertussis . Bethesda : Department of Health and Human Services, 1990 ; 207-11 . 21 . Olin P . New conclusions and lessons learned from the vaccine trial in Sweden . In : Proceedings of the Sixth International Symposium on Pertussis . Bethesda : Department of Health and Human Services, 1990 ;299-302 . 22 . Roberts M, Fairweather NF, Leininger E et al. Construction and characterization of Bordetella pertussis mutants lacking the Vir-regulated P .69 outer membrane protein . Mol Microbiol 1991 ; 5 : 1393-404 . 23 . Pittman M . Genus Bordetella. In : Krieg NR, Holt JG, eds . Bergey's manual of systemic bacteriology, vol . 1 . Baltimore : Williams & Wilkins Co ., 1984 ; 388-93 . 24 . Antoine R, Locht C . Roles of the disulfide bond and the carboxy-terminal region of the S1 subunit in the assembly and biosynthesis of pertussis toxin . Infect Immun 1990; 58 : 1518-26 . 25 . Relman DA, Domenighini M, Tuomanen EU, Rappuoli R, Falkow S . Filamentous hemagglutinin of Bordetella pertussis : nucleotide sequence and crucial role in adherence . Proc Natl Acad Sci USA 1989 ; 86 :2637-41 .
