Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6521-6525, July 1992 Medical Sciences Modulation of adenylate cyclase production as pertussis enters human (bacterial invasion/gene expression) H. ROBERT MASURE Laboratory of Molecular Infectious Diseases, The Rockefeller University, 1230 York Avenue, New York, NY 10021 Communicated by Maclyn McCarty, April 20, 1992

ABSTRACT During the course of human , Bor- distinct phenotypes (Vir+ and Vir-) in response to defined detella pertussis colonizes sequential niches in the respiratory medium components. An increased concentration of MgSO4 tract that include intracellular and extracellular environments. or nicotinic acid or a decreased temperature produces a Vir- In vitro the expression ofvirulence factors such as the adenylate phenotype (8-11). Genes expressed in the Vir+ state have cyclase toxin is coordinately regulated by the bvg locus, which been termed virulence-activated genes (i.e., those encoding is an example ofa two-component sensory transduction system. Ptx, Fha, and AC) and those expressed in the Vir- state have With this toxin as a reporter, activities were compared been termed virulence-repressed genes (9). Little is known between a wild-type and an altered strain to determine whether about the in vivo role of phenotypic modulation because of bacterial entry into human macrophages affected gene expres- the inability to measure the expression of the regulated genes sion. BPRU140, a strain containing an inducible expression such as Ptx, Fha, and Fim with sufficient sensitivity and vector, produced enzyme activity independent of bvg. Samples specificity in vivo in response to a changing environment. In of the parent, the induced, and the uninduced BPRU140 were contrast, the bvg-regulated AC, with its high specific activity, incubated individually with macrophages for 30 min. Extra- was chosen as a reporter for the bacterial response to the cellular were then killed by gentamicin. The number natural transition of B. pertussis into human macrophages. of viable intracellular bacteria and the internalized bacterial The cya operon, responsible for the AC (Cya) and hemo- enzyme activity were measured over time. By 2.5 hr all samples lytic (Hly) phenotypes, contains five genes, cyaA, -B, -C, -D, reached a steady-state concentration of 105 bacteria per 106 and -E (12, 13) with the genetic organization depicted in Fig. macrophages. Following an initial peak of enzyme activity, 1. Transcription of the structural gene, cyaA, is driven by a adenylate cyclase values for the parent and the uninduced single promoter that is dependent on bvg for expression. An BPRU140 decreased to a basal level, while the values for the additional promoter is present between cyaA and cyaB that is constitutive for the transcription of cyaB, -D, and -E (15). induced strain remained at least 3-fold greater. Therefore, The gene product of cyaA is a -stimulated, extra- compared with the persistence of enzyme in the induced strain cellular AC that also confers the hemolytic phenotype and the BPRU140, the decrease in enzyme production by the parent ability to increase cAMP in mammalian cells (16-18). The and the uninduced BPRU140 upon entry into macrophages products of the other contiguous genes cyaB, -D, and -E are indicates in vivo down-modulation of gene expression. These required for of AC (12). In the opposite orientation observations support the hypothesis that sensory transduction to this polycistron and separated by a short, 260-base-pair contributes to adaptations for bacterial survival in the infected intergenic region is another gene, cyaC, whose product host. conveys the hemolytic and invasive properties to the toxin, presumably through covalent modification of AC (19-21). Several genera of pathogenic bacteria, such as the gastroin- testinal pathogens Salmonella, Yersinia, Shigella, and List- eria, invade and survive within host cells. Recent reports MATERIALS AND METHODS describing the entry and intracellular survival ofBordetellae Bacterial Strains and Media. strains used from rabbit alveolar macrophages (1) and cultured mamma- were DH5a, which is F-080dlacZA(lacZYA-argF)U169 lian cells (2,3) suggest at least two different niches in the host: recAl endAl hsdRJ7 (rK-mK+) supE44 A- thy-i gyrA relAl one extracellular on the ciliated epithelium (4) and another (Bethesda Research Laboratories), and SM10, which is thi, within leukocytes (1,5). The recovery ofbacteria from within thr, leu, su111, RP4-2-Tc::Mu (22). B. pertussis strains used rabbit alveolar macrophages in vivo indicates an intracellular were BP348, a cyaA::TnS derivative of BP338 (12, 14); state during infection (1). It is assumed that bacteria mount BP536, a spontaneous naladixic acid-resistant derivative of an adaptive response to a changing environment by the BP338 (23); BP537, a spontaneous bvg- derivative of BP536 coordinate regulation ofthose genes which convey a selective (23); and BPRU44, a cya::TnStacl derivative of BP338 (20). advantage for survival. BPRU176, -178, and -182 are derivatives of BP348 that The virulence factors of , such as contain plasmids pAMN2, -10, and -13, respectively (Fig. 1). pertussis toxin (Ptx), filamentous hemaglutinin (Fha), fim- The conjugal transfer of plasmids from E. coli to B. pertussis briae (Fim), and the adenylate cyclase (AC) toxin, are was performed as described (23, 24). organized into a regulon. Activation of expression of the E. coli were grown in Luria-Bertani (LB) liquid or solid genes in this regulon is controlled by the genetic locus bvg, medium (25). B. pertussis were grown in Stainer Scholte (SS) which encodes two , BvgA and BvgS, that are medium (26) or on Bordet-Gengeou (BG) blood agar plates. members of the two-component family of bacterial sensory To phenotypically modulate B. pertussis in vitro, bacteria transduction proteins (6, 7). In vitro coordinate regulation by were grown in medium supplemented with 40mM MgSO4. To bvg, a process termed phenotypic modulation, produces two cultivate B. pertussis under growth-limiting conditions, bac- terial suspensions were incubated in SS medium lacking a The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: AC, adenylate cyclase; IPTG, isopropyl .6-D- in accordance with 18 U.S.C. §1734 solely to indicate this fact. thiogalactopyranoside; cfu, colony-forming unit(s).

6521 Downloaded by guest on October 1, 2021 6522 Medical Sciences: Masure Proc. Natl. Acad. Sci. USA 89 (1992)

Th5 oi BP348 cyaoperon 1640 and resuspended in a fresh 2 ml of RPMI-1640 plus gentamicin (100 ,g/ml) to kill extracellular bacteria. Inter- A I B D I E I nalized bacteria were determined at timed intervals by plating Plasnid consbcons serial dilutions of a lysate on BG medium. The B B K lysate was prepared by pelleting and resuspension ofa 100-,lI PAMN13 cyaA sample ofthe bacterial/macrophage mixture with phosphate- B buffered saline containing 0.1% Triton X-100. K Determination of the Internalized Bacterial AC Activity. pAMN2O cyaA Trypsin treatment has been shown to completely destroy any bacteria-associated AC activity (31). Thus to distinguish AC K activity derived from internalized bacteria from that of ex- cyaA tracellular bacteria, samples of the bacteria/macrophage suspension were treated with trypsin to destroy any enzyme FIG. 1. Structural organization of the cya locus and plasmid activity outside the macrophage. Samples from the bacteria/ constructions. The cya operon consists ofa polycistron that contains macrophage mixture were removed at timed intervals, cyaA, -B, -D, and -E. cyaC is in the opposite orientation and is adjusted to a final trypsin concentration of 20 ,ug/ml, and separated by a 260-base-pair intergenic region. The TnS chromoso- incubated for 10 min at 37°C. After a 5-min incubation with mal insertion in the mutant strain BP348 (14) resides in cyaA at the a 20 molar excess of soybean trypsin inhibitor, the macro- position indicated (12). Restriction sites are indicated by vertical phages were lysed as described above, and the samples were lines (B, BamHI; K, Kpn I) and triangles represent promoters. Ptac, frozen and subsequently assayed for AC activity. AC activity tac promoter. measured in the macrophage lysates was derived from the supplement ofessential components required for growth (26). bacteria and not the endogenous eukaryotic enzyme since Antibiotics when required were used at the following con- enzyme activity in a lysate of macrophages challenged with centrations: ampicillin, 100 ,ug/ml; chloramphenicol, 34 a cya- strain, BPRU44, was three log units less than with the ,ug/ml for E. coli or 10 pAg/ml for B. pertussis; and strepto- Cya+ strains (data not shown). mycin, 300 ,g/ml. When indicated, isopropyl f3-D- thiogalactopyranoside (IPTG) was added at 250 ,ug/ml. RESULTS AND DISCUSSION Plasmid Construction and DNA Manipulation. To identify Identification of a Regulatory Region for the Expression of the bvg-dependent regulatory region for cyaA and create tac AC and Construction of an Inducible Gene (cyaA). In vitro promoter fusions, a 6.1-kilobase (kb) BamHI-Kpn I fragment expression of the AC in a derivative of a wild-type strain, that contains the gene and its promoter but lacks any up- BP536, is mediated by the bvg locus and can be down- stream sequence was subcloned from pRM003 (27) and modulated by the addition of MgSO4 (40 mM) to the culture inserted into the corresponding sites ofthe broad-host-range medium of growing B. pertussis (Table 1). BP536 expresses cloning vectors pMMB207 and pMMB208 (28) to generate 30-fold less enzyme activity in the presence of MgSO4. In pAMN10 and pAMN2, respectively (Fig. 1). These plasmids contrast, the bvg- strain BP537 expresses only a low level constitute a pair in which cyaA is in opposite orientations to enzyme activity regardless of the presence or absence of the tac promoter of the expression vector, creating inducible MgSO4. B. pertussis may down-modulate the expression of (pAMN2) and noninducible (pAMN10) vectors. Plasmid the AC as the bacteria make the transition from outside to pAMN13, which contains cyaA, its promoter, and 2.4 kb of inside the macrophage in a manner similar to that observed upstream sequence spanning the intergenic region and cyaC, in vitro with MgSO4. To test this hypothesis it was necessary was created by subcloning a 2.4-kb BamHI fragment (20) into to create a strain in which expression of the structural gene pAMN10. Plasmids derived from pUC derivatives were cyaA was inducible and independent of bvg regulation. The isolated by the alkaline hydrolysis method (29), whereas expression of enzyme activity in this strain would serve as a plasmids derived from pMMB207 and pMMB208 were iso- basis for comparison (positive control) to the predicted lated by a boiling method (30). Recombinant DNA manipu- decreased expression in the parent strain. Plasmid-borne lations were performed by standard protocols. DNA- copies ofthe cyaA gene were constructed first to identify the modifying were obtained from United States Bio- bvg-dependent regulatory region and second to place the chemical. cyaA gene under the control of the inducible tac promoter. AC Assay. Extracellular cell-associated AC was isolated (Plasmid constructions are depicted in Fig. 1.) To test plas- from whole bacteria as a detergent extract (31). Production of cAMP was measured as described (32). Units of specific Table 1. The bvg-dependent and IPTG-induced expression of enzyme activity are presented as nmol ofcAMP produced per AC from B. pertussis min per mg of total . Samples were assayed in the Specific activity, nmol of cAMP presence of 2.5 ,uM calmodulin, and protein concentrations min of were determined by the Coomassie blue G250 method (33). per per mg protein Bacteria--Macrophage Invasion Assay. Human No MgSO4 were isolated from buffy coats and harvested from Teflon Strain Description addition MgSO4 IPTG + IPTG beakers after 5-10 days and suspended at 2 x 106 per ml in BP536 bvg+ 6.15 0.22 6.44 0.12 RPMI-1640 medium (GIBCO) (34). Frozen stocks ofisogenic BP537 bvg- 0.15 0.19 ND ND strains of B. pertussis were grown for 1-2 days on selective BP348 cyaA::Tn5 0 0 ND ND BG medium. Bacterial cells were harvested from the plates BPRU182 BP348/pAMN13 4.2 0.66 ND ND and resuspended in RPMI-1640 and adjusted to an OD620 of BPRU178 BP348/pAMN10 0.61 0.51 0.17 0.61 0.5 [109 colony-forming units (cfu)/ml]. At zero time, 1 ml of BPRU176 BP348/pAMN2 0.43 0.17 6.08 5.19 the bacterial cell suspension was mixed with 1 ml of macro- (2 x and on a tumbler at Bacteria were grown for 48 hr on selective BG agar in the presence phages 106 per ml) placed 3TC for or absence of 40 mM MgSO4 and 250 AuM IPTG as indicated. 30 min. Macrophages and bound bacteria were separated Extracellular AC was solubilized from whole bacteria in 0.1% Triton from unbound bacteria by centrifugation for 10 min at 800 X-100 and assayed for the production ofcAMP (25, 32). Mean values rpm in a Beckman Accuspin centrifuge. The bacteria/ of two independent determinations are presented. SD for any value macrophage mixtures were washed three times with RPMI- did not exceed 8% of that value. ND, not determined. Downloaded by guest on October 1, 2021 .

Medical Sciences: Masure Proc. Natl. Acad. Sci. USA 89 (1992) 6523

mid-encoded AC activity, these plasmids were introduced 12 into a mutant strain, BP348, that contains a TnS insertion in x= the chromosomal copy of cyaA. An intact plasmid-borne X copy of cyaA, with 2.4 kb of upstream sequence that includes 8 the intergenic region, and cyaC (pAMN13) expressed en- EI A zyme activity in a bvg-dependent manner in response to 4 MgSO4 (Table 1). In contrast, a plasmid (pAMN10) lacking this upstream sequence but retaining the promoter for cyaA produced only low levels of enzyme activity. This suggested that the deleted region contained cis-acting elements required for the bvg-mediated expression of cyaA. This is in good agreement with the results of lacZ fusion studies character- izing bvg-dependent expression of cyaA (35) and gel retar- dation analysis of the intergenic region (36). Next the cyaA gene lacking the regulatory region was positioned just downstream from the inducible tac promoter of an expression vector (pAMN2). In this case, significant AC activity was detected only in the presence of the exogenous inducer IPTG, regardless of the presence or absence of the modulator MgSO4 (Table 1). Two other features of this Time, hr expression system define other members of the cya operon. Detection of extracellular pAMN2-encoded enzyme con- FiG. 2. In vitro modulation of AC expression in B. pertussis. (A) firms bvg-independent transcription of the chromosomal Growth curve of the parent strain BP536 (-) and two samples of the of and which are for secretion derivative BPRU140 (BP536/pAMN2) with (e) and without (5) 250 copies cyaB, -D, -E, required /iM IPTG. Cultures were grown in liquid medium (26) and at time (15). Second, even though the mutant strain BP348 contains zero were adjusted to a final concentration of 40 mM MgSO4 to an intact chromosomal copy of cyaC and the intergenic modulate the expression ofAC. Results (cfu/ml) were determined by region, the hemolytic phenotype was eliminated when the serial dilution of liquid cultures onto solid medium. (B) Extracellular plasmid-encoded, IPTG-induced enzyme was expressed in AC activity. Samples from liquid cultures described in A (300 pl) the presence of MgSO4 (BPRU176; data not shown). This were removed at timed intervals, centrifuged, and suspended in 0.1% suggests that cyaC, oriented in the opposite direction to the Triton X-100 to solubilize the extracellular associated enzyme. rest of the operon, is regulated in a bvg-dependent manner. Samples were centrifuged again to remove whole cells and the Thus, the plasmid-derived inducible form of the AC enzyme supernatants were assayed for enzyme activity (26, 32) and protein content. Results are presented as units of specific activity (nmol of could be used as a reporter for the expression of cyaA cAMP produced per min per mg of protein). Error bars represent the uncoupled from control by the bvg locus. SEM of duplicate samples. Symbols are the same as in A. In Vitro Modulation of cyaA Expression. The inducible expression vector pAMN2 containing cyaA was introduced dependent regulatory circuit controlling the expression ofAC into a wild-type strain of B. pertussis containing a functional is functional under limiting growth conditions. chromosomal copy of the cya operon. This strain (BPRU140) In Vivo Modulation of cyaA Expression with the Entry of B. expressed AC activity in a bvg-dependent manner, but down- pertussis into Human Macrophages. To determine whether B. modulation could be overcome by the addition of IPTG (Fig. 2). One hour following addition of40mM MgSO4, AC activity 12 of BPRU140 decreased to a basal level that remained con- stant over 4 hr (i.e., one generation time). This time course x of down-modulation was identical to that of the parental a 8 strain BP536 and is consistent with bvg-dependent down- modulation of AC expression measured at the level of tran- 4 A, scription (37). In contrast, AC activity remained 4-fold higher in BPRU140 when induced with IPTG, despite the presence of 40 mM MgSO4. Thus, this strain expressed AC activity due to the chromosomal copy of the gene, in a bvg-dependent manner leading to a 4-fold reduction in enzyme activity with .2 the addition of MgSO4, but expression of AC activity due to the vector was of but rather under pAMN2 independent bihg V: control of the tac promoter. ¢a) In Vitro Modulation of cyaA Expression Under Slow Growth Conditions. Little is known about the in situ state of bacterial growth upon colonization of the host or the effect of growth rate on the ability to phenotypically modulate gene expres- sion. Bacteria may have limited access to nutrients within intracellular compartments. Therefore, a decrease in AC 2 expression by the bacteria with the transition to inside the Time, hr macrophage could be attributable to either down-modulation via the bvg locus or a decrease in expression in response to FIG. 3. In vitro modulation of AC expression under growth- limiting growth conditions. To assure that reduced growth limiting conditions. (A) Growth curve of BPRU140 grown in com- rate per se would not affect bvg-dependent AC expression, plete (26) (in, ) or growth limiting (E, a) medium in the absence (-, a) or presence (a, e) of 40 mM MgSO4. To phenotypically modulate bacteria were to essential nutrient and subjected deprivation the bacteria, cultures initially grown in the absence of MgSO4 were evaluated for the ability to phenotypically modulate the switched to medium containing the divalent cation or, alternatively, expression of the AC. The presence or absence of the cultures grown in the presence of MgSO4 were switched to medium modulator MgSO4 altered the expression of AC in normal or devoid of this component. (B) AC activities of cultures described in slow-growth conditions (Fig. 3). Therefore, the bvg- A (determined as described in Fig. 2). Downloaded by guest on October 1, 2021 6524 Medical Sciences: Masure Proc. Nat!. Acad. Sci. USA 89 (1992) pertussis modulates expression ofAC upon entry into human macrophages, bacterial AC enzyme activity was monitored in parallel with bacterial invasion and survival in macrophages. Three samples were studied. (i) The parent strain, BP536, which contains only a bvg-regulated chromosomal copy of cyaA, was compared with two controls. (ii) BPRU140, which contains an IPTG-inducible plasmid-borne copy of cyaA in a wild-type background, was assayed as a positive control to assess induced AC production independent ofbvg. (iii) Killed BP536 was assayed as a negative control to establish basal AC activity associated with internalized bacteria not actively producing the enzyme. Bacterial AC activity within the macrophage was assessed by destroying extracellular en- zyme with trypsin and liberation of the internalized bacterial enzyme by lysis of the macrophages that contained ingested bacteria. Intracellular bacteria invasion and survival were defined as colony-forming units obtained following gentami- cin treatment of a bacteria/macrophage mixture. The parental strain (BP536) and BPRU140 bound macro- 8- phages in a ratio of10:1 (107 bacteria per 106 macrophages per ml; Fig. 4A). After the addition of gentamicin (100 ,ug/ml), bacterial killing progressed until a steady-state concentration of 105 bacteria per ml was reached by 2.5 hr. Bacteria refractory to the bactericidal activity of gentamicin after 2.5 hr were taken to represent the intracellular pool of bacteria. Similar results were obtained with polymixin B (100 &g/ml) (data not shown). Ninety-five percent of the macrophages remained viable during the course of the experiment as 1 2 3 4 determined by visual examination for trypan blue exclusion. Time, hr Induction of cyaA with IPTG in the mutant strain BPRU140 did not affect the survival of these bacteria. To kill the Fio. 4. Survival and AC activity of B. pertussis within human bacteria prior to incubation with the macrophages, a sample macrophages. (A) Survival of bacteria following uptake into macro- of BP536 was pretreated for 2 hr with gentamicin at 100 phages. Bacteria were mixed with human macrophages and, 30 mi ,ug/ml. This led to a four-log reduction of surviving bacteria later, exposed to gentamicin (100 Iug/ml, arrow) to kill extracellular bacteria. Samples were removed at timed intervals, macrophages compared with bacteria that were not pretreated. The bvg- were lysed, and the solution was plated to quantitate viable intra- strain BP537 does not express any known virulence deter- cellular bacteria. Values are representative of experiments con- minant. This strain bound less avidly to macrophages than ducted in triplicate. The parent BP536 (n) was compared with killed the bvg+ strains (2 x 105 cfu/ml vs. 1 x 107 cfu/ml). Those BP536 (treated with gentamicin for 2 hr prior to mixture with that were bound did not survive treatment with gentamicin macrophages) (0), isogenic BPRU140 (BP536 carrying the plasmid- and macrophage challenge (103 cfu/ml). This is consistent borne, IPTG-inducible cyaA) treated (e) or untreated (o) with 250 with other studies describing the inability ofavirulent strains ,uM IPTG (beginning 10 min prior to admixture with macrophages), to attach to human macrophages (38) or invade other cells (2, and BP537 (bvg-) (x). (B) AC activity associated with internalized Bacterial in strain was not observed bacteria. Symbols are as described in A. AC activity associated with 3). multiplication any internalized bacteria was determined by trypsin treatment of the over the 4-hr time course of the experiment. This is not bacteria/macrophage mixture to destroy enzyme activity outside the unexpected given the 4- to 6-hr doubling time observed for macrophage, followed by quenching of the trypsin with soybean the growth of B. pertussis in liquid medium (Fig. 3). There- trypsin inhibitor, lysis of the macrophage, and quantitation of fore, virulent B. pertussis is able to survive challenge by enzyme activity. Values are means ofduplicate samples presented as human macrophages in this invasion assay. units of specific activity (nmol of cAMP produced per min per mg of AC Activity Associated with the Entry of B. pertussis Into protein). SD for any value did not exceed 7%. Results are represen- Human Macrophages. There were clearly two components to tative of experiments conducted in triplicate. the internalized AC activity identified from the bacteria/ macrophage mixtures. Within 30 min of the attachment of form ofthe enzyme, produced AC activity 5-fold greater than wild-type BP536 or BPRU140, comparable peaks ofbacterial the basal values of the killed parental strain. Thus, after 1.5 AC activity were measured in the macrophage lysates (Fig. hr AC values were taken to be indicative ofenzyme produced 4B). Since this peak could be reproduced by killed BP536 by bacteria within the macrophage. (pretreated with gentamicin) the initial peak ofAC activity for Between 2 and 4 hr AC activity for all samples reached a all toxin-containing strains was attributed to internalization steady-state value. The wild-type parent and the uninduced ofpreexisting toxin regardless ofthe viability ofthe bacteria. mutant responded to internalization with low AC values By 1.5 hr, this peak ofinitial AC activity decreased to a basal comparable to those of the nonviable bacteria (Fig. 4B). value for the nonviable BP536, which is not actively produc- When the AC values were plotted as a function ofactivity per ing new enzyme. It seems unlikely that the basal AC values colony-forming unit over time (Fig. 5), the wild-type parent, obtained with the nonviable bacteria represent activity pro- BP536, produced only basal AC activity that was consistently duced by the small number of surviving cells (y102 bacteria), only one-third that obtained with the induced strain and these basal values are probably due to the residual BPRU140. These observations demonstrate down-modula- activity from the initial internalization of bacteria that deliv- tion of AC expression in the parent strain with entry into ers AC to the macrophage. This interpretation is consistent macrophages and suggest a setting in which phenotypic with numerous studies using purified toxin, which is capable modulation, so well studied in vitro, also occurs in vivo. of increasing cAMP in a wide variety of eukaryotic cells Failure to down-modulate would have produced AC values (16-18). During the same time period, the positive control, comparable to those obtained with the induced strain. The BPRU140 induced with IPTG to express the plasmid derived ability of the parent bacteria to alter AC expression with the Downloaded by guest on October 1, 2021 Medical Sciences: Masure Proc. Natl. Acad. Sci. USA 89 (1992) 6525

1.00 2. Ewanowich, C. A., Melton, A. R., Weiss, A. A., Sherburne, R. K. & Peppler, M. S. (1989) Infect. Immun. 57, 2698-2704. i 0 0.75 3. Lee, C. K., Roberts, A. L., Finn, T. M., Knapp, S. & Mekal- anos, J. J. (1990) Infect. Immun. 58, 2516-2522. C Xx u 4. Tuomanen, E. & Weiss, A. (1985) J. Infect. Dis. 152, 118-125. 0.50 5. Steed, L. L., Setareh, M. & Friedman, R. L. (1991) J. Leuk. u E Biol. 50, 321-330. ra 0.25 6. Scarlato, V., Prugnola, A., Arico, B. & Rappuoli, R. (1990) U Proc. Nati. Acad. Sci. USA 87, 6753-6757. 7. Stibitz, S. & Yang, M. S. (1991) J. Bacteriol. 173, 4288-4296. 2 3 4 8. Idigbe, E. O., Parton, R. & Wardlaw, A. C. (1981) J. Med. Time, hr Microbiol. 14, 409-418. 9. Knapp, S. & Mekalanos, J. J. (1988) J. Bacteriol. 170, 5059- FIG. 5. In vivo modulation of cyaA expression with the entry of 5066. B. pertussis into human macrophages. AC activity associated with 10. Lacey, B. W. (1960) J. Hyg. 58, 57-93. viable bacteria internalized within human macrophages was mea- 11. Melton, A. R. & Weiss, A. A. (1989) J. Bacteriol. 171, 6206- sured as described in Fig. 4. Units of AC specific activity (nmol of 6212. cAMP per min per mg of protein) from the experiment depicted in 12. Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A. & Danchin, Fig. 4 are plotted as a function of cfu over time. The parent BP536 A. (1988) EMBO J. 7, 3997-4004. (-) was compared with isogenic BPRU140 (BP536 carrying the 13. Glaser, P., Ladant, D., Sezer, O., Pichot, F., Ullmann, A. & plasmid-borne, IPTG-inducible cyaA) treated (e) or untreated (o) Danchin, A. (1988) Mol. Microbiol. 2, 19-30. with 250 IPTG. 14. Weiss, A. A., Hewlett, E. L., Myers, G. A. & Falkow, S. .uM (1983) Infect. Immun. 42, 33-41. transition to inside the macrophage is presumably mediated 15. Laoide, B. M. & Ullmann, A. (1990) EMBO J. 9, 999-1005. by the products of the 16. Confer, D. L. & Eaton, J. W. (1982) Science 217, 948-950. bvg locus despite potential growth- 17. Friedman, E., Farfel, Z. & Hanski, E. (1987) Biochem. J. 243, limiting conditions. This argument is strengthened by the 145-151. results in Fig. 3, which demonstrate modulation of AC 18. Shattuck, R. L. & Storm, D. R. (1985) Biochemistry 24, 6323- expression in vitro by the bacteria even under limiting growth 6328. conditions. It is certainly possible that the stimulus for this 19. Berry, E. L., Weiss, A. A., Ehrmann, I. E., Grey, M., Hew- down-modulation is contained within the milieu of the en- lett, E. L. & Goodwin, M. (1991) J. Bacteriol. 173, 720-726. docytic vesicles of the macrophage encountered by the 20. Saukkonen, K., Sande, S. & Masure, H. R. (1991) in Proceed- bacteria with internalization. ings of the Sixth International Symposium on Pertussis, ed. Manclark, C. R. (Dept. Health and Human Services, Bethesda, Survival of B. pertussis upon challenge with human mac- MD), DHHS Publ. No. (FDA) 90-1164, pp. 267-274. rophages has been attributed to expression of adhesins and 21. Issartel, J. P., Koronakis, V. & Hughes, C. (1991) Nature virulence factors in the Vir+ state (39). If the regulatory (London) 351, 759-761. circuit between the bvg and the cya loci serves as a paradigm 22. Simon, R., Priefer, U. & Piihler, A. (1983) BiolTechnol. 1, for gene regulation of other members of the vir regulon, then 784-791. the results reported here suggest that the natural progression 23. Relman, D. A., Domenighini, M., Tuomanen, E., Rappuoli, R. of events in the disease may require that B. & Falkow, S. (1989) Proc. Nati. Acad. Sci. USA 86,2637-2641. pertussis phe- 24. Stibitz, S., Black, W. & Falkow, S. (1986) Gene 50, 133-140. notypically changes to a Vir- state upon entry into the 25. Miller, J. F. (1972) Experiments in Molecular Genetics (Cold macrophage. This hypothesis is supported by studies with Spring Harbor Lab., Cold Spring Harbor, NY). animal models for the disease. A mutant with a transposon 26. Masure, H. R., Donovan, M. G. & Storm, D. R. (1991) Meth- insertion in vrg6 (a vir-repressed gene) was less able to ods Enzymol. 195, 137-152. colonize the and trachea in a mouse model for respira- 27. Masure, H. R., Au, D. C., Gross, M. K., Donovan, M. G. & tory infection (40). Similar studies also demonstrated that the Storm, D. R. (1990) Biochemistry 29, 140-145. AC (the product of a vir-activated gene) is absolutely required 28. Morales, V. M., Backman, A. & Bagdasarian, M. (1991) Gene for colonization (41). Therefore, B. pertussis has 97, 39-47. developed 29. Birnboim, H. C. & Doly, J. (1979) Nucleic Acids Res. 7, a survival strategy based on the coordinate regulation of 1513-1518. genes including the cya operon that results in two distinct 30. Holmes, D. S. & Quigley, M. (1981) Anal. Biochem. 114, phenotypes. In the Vir+ state the bacteria adhere and colo- 193-199. nize the host in several niches. Thereafter access to another 31. Masure, H. R. & Storm, D. R. (1989) Biochemistry 28, 438- environment, the alveolar macrophage, leads to the Vir- 442. state, cessation of toxin production, and establishment of 32. Salomon, Y., Londos, C. & Rodbell, A. M. (1974) Anal. intracellular bacteria that remain sequestered from host de- Biochem. 58, 541-548. fenses. The ability of the bacteria to sense its environment 33. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. and switch a 34. Wright, S. & Silverstein, S. C. (1982) J. Exp. Med. 156, between two phenotypes confers selective 1149-1164. advantage for B. pertussis in promoting successful and per- 35. Goyard, S. & Ullmann, A. (1991) FEMS Microbiol. Lett. 77, sistent survival within the host. 251-256. 36. Huh, Y. J. & Weiss, A. A. (1991) Infect. Immun. 59, 2389- I thank Drs. E. Tuomanen and C. A. Butler for many thoughtful 2395. discussions, support, and critical reading of the manuscript. I thank 37. Scarlato, V. & Rappuoli, R. (1991) J. Bacteriol. 173, 7401-7404. Anne Naughton for her expert technical assistance and Drs. Jeff 38. Relman, D., Tuomanen, E., Falkow, S., Golenbock, D. T., Weiser and Emil Gotschlich for their encouragement. This work was Saukkonen, K. & Wright, S. D. (1990) Cell 61, 1375-1382. supported by National Institutes of Health Grants R29 A132024-01 39. Saukkonen, K., Cabellos, C., Burroughs, M., Prasad, S. & and RO1 A123459 and by a Career Scientist Award from the I. T. Tuomanen, E. (1991) J. Exp. Med. 173, 1143-1149. Hirshl Trust. 40. Beattie, D. T., Shahin, R. & Mekalanos, J. J. (1992) Infect. Immun. 60, 571-577. 1. Saukkonen, K., Cabellos, C., Burroughs, M., Prasad, S. & 41. Goodwin, M. S. & Weiss, A. A. (1990) Infect. Immun. 58, Tuomanen, E. (1991) J. Exp. Med. 173, 1143-1149. 3445-3447. Downloaded by guest on October 1, 2021