Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6521-6525, July 1992 Medical Sciences

Modulation of adenylate cyclase toxin production as Bordetella pertussis enters human macrophages (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

distinct phenotypes (Vir+ and Vir-) in response to defined medium components. An increased concentration of MgSO4 or nicotinic acid or a decreased temperature produces a Virphenotype (8-11). Genes expressed in the Vir+ state have been termed virulence-activated genes (i.e., those encoding Ptx, Fha, and AC) and those expressed in the Vir- state have been termed virulence-repressed genes (9). Little is known about the in vivo role of phenotypic modulation because of the inability to measure the expression of the regulated genes such as Ptx, Fha, and Fim with sufficient sensitivity and specificity in vivo in response to a changing environment. In contrast, the bvg-regulated AC, with its high specific activity, was chosen as a reporter for the bacterial response to the natural transition of B. pertussis into human macrophages. The cya operon, responsible for the AC (Cya) and hemolytic (Hly) phenotypes, contains five genes, cyaA, -B, -C, -D, and -E (12, 13) with the genetic organization depicted in Fig. 1. Transcription of the structural gene, cyaA, is driven by a single promoter that is dependent on bvg for expression. An additional promoter is present between cyaA and cyaB that is constitutive for the transcription of cyaB, -D, and -E (15). The gene product of cyaA is a calmodulin-stimulated, extracellular AC that also confers the hemolytic phenotype and the ability to increase cAMP in mammalian cells (16-18). The products of the other contiguous genes cyaB, -D, and -E are required for secretion of AC (12). In the opposite orientation to this polycistron and separated by a short, 260-base-pair intergenic region is another gene, cyaC, whose product conveys the hemolytic and invasive properties to the toxin, presumably through covalent modification of AC (19-21).

ABSTRACT During the course of human infection, Bordetella pertussis colonizes sequential niches in the respiratory tract that include intracellular and extracellular environments. In vitro the expression of virulence factors such as the adenylate cyclase toxin is coordinately regulated by the bvg locus, which is an example of a two-component sensory transduction system. With this toxin as a reporter, enzyme activities were compared between a wild-type and an altered strain to determine whether bacterial entry into human macrophages affected gene expression. BPRU140, a strain containing an inducible expression vector, produced enzyme activity independent of bvg. Samples of the parent, the induced, and the uninduced BPRU140 were incubated individually with macrophages for 30 min. Extracellular bacteria were then killed by gentamicin. The number of viable intracellular bacteria and the internalized bacterial enzyme activity were measured over time. By 2.5 hr all samples reached a steady-state concentration of 105 bacteria per 106 macrophages. Following an initial peak of enzyme activity, adenylate cyclase values for the parent and the uninduced BPRU140 decreased to a basal level, while the values for the induced strain remained at least 3-fold greater. Therefore, compared with the persistence of enzyme in the induced strain BPRU140, the decrease in enzyme production by the parent and the uninduced BPRU140 upon entry into macrophages indicates in vivo down-modulation of gene expression. These observations support the hypothesis that sensory transduction contributes to adaptations for bacterial survival in the infected host.

Several genera of pathogenic bacteria, such as the gastrointestinal pathogens Salmonella, Yersinia, Shigella, and Listeria, invade and survive within host cells. Recent reports describing the entry and intracellular survival of Bordetellae from rabbit alveolar macrophages (1) and cultured mammalian cells (2,3) suggest at least two different niches in the host: one extracellular on the ciliated epithelium (4) and another within leukocytes (1,5). The recovery ofbacteria from within rabbit alveolar macrophages in vivo indicates an intracellular state during infection (1). It is assumed that bacteria mount an adaptive response to a changing environment by the coordinate regulation of those genes which convey a selective advantage for survival. The virulence factors of Bordetella pertussis, such as pertussis toxin (Ptx), filamentous hemaglutinin (Fha), fimbriae (Fim), and the adenylate cyclase (AC) toxin, are organized into a regulon. Activation of expression of the genes in this regulon is controlled by the genetic locus bvg, which encodes two proteins, BvgA and BvgS, that are members of the two-component family of bacterial sensory transduction proteins (6, 7). In vitro coordinate regulation by bvg, a process termed phenotypic modulation, produces two

MATERIALS AND METHODS Bacterial Strains and Media. Escherichia coli strains used were DH5a, which is F-080dlacZA(lacZYA-argF)U169 recAl endAl hsdRJ7 (rK-mK+) supE44 A- thy-i gyrA relAl (Bethesda Research Laboratories), and SM10, which is thi, thr, leu, su111, RP4-2-Tc::Mu (22). B. pertussis strains used were BP348, a cyaA::TnS derivative of BP338 (12, 14); BP536, a spontaneous naladixic acid-resistant derivative of BP338 (23); BP537, a spontaneous bvg- derivative of BP536 (23); and BPRU44, a cya::TnStacl derivative of BP338 (20). BPRU176, -178, and -182 are derivatives of BP348 that contain plasmids pAMN2, -10, and -13, respectively (Fig. 1). The conjugal transfer of plasmids from E. coli to B. pertussis was performed as described (23, 24). E. coli were grown in Luria-Bertani (LB) liquid or solid medium (25). B. pertussis were grown in Stainer Scholte (SS) medium (26) or on Bordet-Gengeou (BG) blood agar plates. To phenotypically modulate B. pertussis in vitro, bacteria were grown in medium supplemented with 40 mM MgSO4. To cultivate B. pertussis under growth-limiting conditions, bacterial suspensions were incubated in SS medium lacking a

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Abbreviations: AC, adenylate cyclase; IPTG, isopropyl .6-D-

thiogalactopyranoside; cfu, colony-forming unit(s).

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cyaoperon

Th5 oi BP348 A B

B

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pAMN2O

Proc. Natl. Acad. Sci. USA 89 (1992)

K

cyaA K

cyaA FIG. 1. Structural organization of the cya locus and plasmid constructions. The cya operon consists of a polycistron that contains cyaA, -B, -D, and -E. cyaC is in the opposite orientation and is separated by a 260-base-pair intergenic region. The TnS chromosomal insertion in the mutant strain BP348 (14) resides in cyaA at the position indicated (12). Restriction sites are indicated by vertical lines (B, BamHI; K, Kpn I) and triangles represent promoters. Ptac, tac promoter.

supplement of essential components required for growth (26). Antibiotics when required were used at the following concentrations: ampicillin, 100 ,ug/ml; chloramphenicol, 34 ,ug/ml for E. coli or 10 pAg/ml for B. pertussis; and streptomycin, 300 ,g/ml. When indicated, isopropyl f3-Dthiogalactopyranoside (IPTG) was added at 250 ,ug/ml. Plasmid Construction and DNA Manipulation. To identify the bvg-dependent regulatory region for cyaA and create tac promoter fusions, a 6.1-kilobase (kb) BamHI-Kpn I fragment that contains the gene and its promoter but lacks any upstream sequence was subcloned from pRM003 (27) and inserted into the corresponding sites of the broad-host-range cloning vectors pMMB207 and pMMB208 (28) to generate pAMN10 and pAMN2, respectively (Fig. 1). These plasmids constitute a pair in which cyaA is in opposite orientations to the tac promoter of the expression vector, creating inducible (pAMN2) and noninducible (pAMN10) vectors. Plasmid pAMN13, which contains cyaA, its promoter, and 2.4 kb of upstream sequence spanning the intergenic region and cyaC, was created by subcloning a 2.4-kb BamHI fragment (20) into pAMN10. Plasmids derived from pUC derivatives were isolated by the alkaline hydrolysis method (29), whereas plasmids derived from pMMB207 and pMMB208 were isolated by a boiling method (30). Recombinant DNA manipulations were performed by standard protocols. DNAmodifying enzymes were obtained from United States Biochemical. AC Assay. Extracellular cell-associated AC was isolated from whole bacteria as a detergent extract (31). Production of cAMP was measured as described (32). Units of specific enzyme activity are presented as nmol ofcAMP produced per min per mg of total protein. Samples were assayed in the presence of 2.5 ,uM calmodulin, and protein concentrations were determined by the Coomassie blue G250 method (33). Bacteria--Macrophage Invasion Assay. Human monocytes were isolated from buffy coats and harvested from Teflon beakers after 5-10 days and suspended at 2 x 106 per ml in RPMI-1640 medium (GIBCO) (34). Frozen stocks of isogenic strains of B. pertussis were grown for 1-2 days on selective BG medium. Bacterial cells were harvested from the plates and resuspended in RPMI-1640 and adjusted to an OD620 of 0.5 [109 colony-forming units (cfu)/ml]. At zero time, 1 ml of the bacterial cell suspension was mixed with 1 ml of macrophages (2 x 106 per ml) and placed on a tumbler at 3TC for 30 min. Macrophages and bound bacteria were separated from unbound bacteria by centrifugation for 10 min at 800 rpm in a Beckman Accuspin centrifuge. The bacteria/ macrophage mixtures were washed three times with RPMI-

1640 and resuspended in a fresh 2 ml of RPMI-1640 plus gentamicin (100 ,g/ml) to kill extracellular bacteria. Internalized bacteria were determined at timed intervals by plating serial dilutions of a macrophage lysate on BG medium. The lysate was prepared by pelleting and resuspension of a 100-,lI sample of the bacterial/macrophage mixture with phosphatebuffered saline containing 0.1% Triton X-100. Determination of the Internalized Bacterial AC Activity. Trypsin treatment has been shown to completely destroy any bacteria-associated AC activity (31). Thus to distinguish AC activity derived from internalized bacteria from that of extracellular bacteria, samples of the bacteria/macrophage suspension were treated with trypsin to destroy any enzyme activity outside the macrophage. Samples from the bacteria/ macrophage mixture were removed at timed intervals, adjusted to a final trypsin concentration of 20 ,ug/ml, and incubated for 10 min at 37°C. After a 5-min incubation with a 20 molar excess of soybean trypsin inhibitor, the macrophages were lysed as described above, and the samples were frozen and subsequently assayed for AC activity. AC activity measured in the macrophage lysates was derived from the bacteria and not the endogenous eukaryotic enzyme since enzyme activity in a lysate of macrophages challenged with a cya- strain, BPRU44, was three log units less than with the Cya+ strains (data not shown).

RESULTS AND DISCUSSION Identification of a Regulatory Region for the Expression of

AC and Construction of an Inducible Gene (cyaA). In vitro expression of the AC in a derivative of a wild-type strain, BP536, is mediated by the bvg locus and can be downmodulated by the addition of MgSO4 (40 mM) to the culture medium of growing B. pertussis (Table 1). BP536 expresses 30-fold less enzyme activity in the presence of MgSO4. In contrast, the bvg- strain BP537 expresses only a low level enzyme activity regardless of the presence or absence of MgSO4. B. pertussis may down-modulate the expression of the AC as the bacteria make the transition from outside to inside the macrophage in a manner similar to that observed in vitro with MgSO4. To test this hypothesis it was necessary to create a strain in which expression of the structural gene cyaA was inducible and independent of bvg regulation. The expression of enzyme activity in this strain would serve as a basis for comparison (positive control) to the predicted decreased expression in the parent strain. Plasmid-borne copies of the cyaA gene were constructed first to identify the bvg-dependent regulatory region and second to place the cyaA gene under the control of the inducible tac promoter. (Plasmid constructions are depicted in Fig. 1.) To test plasTable 1. The bvg-dependent and IPTG-induced expression of AC from B. pertussis Specific activity, nmol of cAMP per min per mg of protein No MgSO4 Strain Description addition MgSO4 IPTG + IPTG BP536 6.15 0.22 6.44 0.12 bvg+ BP537 0.15 ND ND 0.19 bvgBP348 0 ND 0 ND cyaA::Tn5 BPRU182 BP348/pAMN13 4.2 ND 0.66 ND BPRU178 BP348/pAMN10 0.61 0.51 0.61 0.17 0.17 6.08 5.19 BPRU176 BP348/pAMN2 0.43 Bacteria were grown for 48 hr on selective BG agar in the presence or absence of 40 mM MgSO4 and 250 AuM IPTG as indicated. Extracellular AC was solubilized from whole bacteria in 0.1% Triton X-100 and assayed for the production of cAMP (25, 32). Mean values of two independent determinations are presented. SD for any value did not exceed 8% of that value. ND, not determined.

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mid-encoded AC activity, these plasmids were introduced into a mutant strain, BP348, that contains a TnS insertion in the chromosomal copy of cyaA. An intact plasmid-borne copy of cyaA, with 2.4 kb of upstream sequence that includes the intergenic region, and cyaC (pAMN13) expressed enzyme activity in a bvg-dependent manner in response to 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 characterizing bvg-dependent expression of cyaA (35) and gel retardation 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 expression system define other members of the cya operon. Detection of extracellular pAMN2-encoded enzyme confirms bvg-independent transcription of the chromosomal copies of cyaB, -D, and -E, which are required for secretion (15). Second, even though the mutant strain BP348 contains an intact chromosomal copy of cyaC and the intergenic region, the hemolytic phenotype was eliminated when the plasmid-encoded, IPTG-induced enzyme was expressed in the presence of MgSO4 (BPRU176; data not shown). This suggests that cyaC, oriented in the opposite direction to the rest of the operon, is regulated in a bvg-dependent manner. Thus, the plasmid-derived inducible form of the AC enzyme could be used as a reporter for the expression of cyaA uncoupled from control by the bvg locus. In Vitro Modulation of cyaA Expression. The inducible expression vector pAMN2 containing cyaA was introduced into a wild-type strain of B. pertussis containing a functional chromosomal copy of the cya operon. This strain (BPRU140) expressed AC activity in a bvg-dependent manner, but downmodulation could be overcome by the addition of IPTG (Fig. 2). One hour following addition of 40 mM MgSO4, AC activity of BPRU140 decreased to a basal level that remained constant over 4 hr (i.e., one generation time). This time course of down-modulation was identical to that of the parental strain BP536 and is consistent with bvg-dependent downmodulation of AC expression measured at the level of transcription (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 the addition of MgSO4, but expression of AC activity due to the vector pAMN2 was independent of bihg but rather under control of the tac promoter. 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 expression. Bacteria may have limited access to nutrients within intracellular compartments. Therefore, a decrease in AC expression by the bacteria with the transition to inside the macrophage could be attributable to either down-modulation via the bvg locus or a decrease in expression in response to limiting growth conditions. To assure that reduced growth rate per se would not affect bvg-dependent AC expression, bacteria were subjected to essential nutrient deprivation and evaluated for the ability to phenotypically modulate the expression of the AC. The presence or absence of the modulator MgSO4 altered the expression of AC in normal or slow-growth conditions (Fig. 3). Therefore, the bvg-

Proc. Natl. Acad. Sci. USA 89 (1992)

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Time, hr FiG. 2. In vitro modulation of AC expression in B. pertussis. (A) Growth curve of the parent strain BP536 (-) and two samples of the derivative BPRU140 (BP536/pAMN2) with (e) and without (5) 250 /iM IPTG. Cultures were grown in liquid medium (26) and at time zero were adjusted to a final concentration of 40 mM MgSO4 to modulate the expression of AC. Results (cfu/ml) were determined by serial dilution of liquid cultures onto solid medium. (B) Extracellular AC activity. Samples from liquid cultures described in A (300 pl) were removed at timed intervals, centrifuged, and suspended in 0.1% Triton X-100 to solubilize the extracellular associated enzyme. Samples were centrifuged again to remove whole cells and the supernatants were assayed for enzyme activity (26, 32) and protein content. Results are presented as units of specific activity (nmol of cAMP produced per min per mg of protein). Error bars represent the SEM of duplicate samples. Symbols are the same as in A.

dependent regulatory circuit controlling the expression of AC is functional under limiting growth conditions. In Vivo Modulation of cyaA Expression with the Entry of B. pertussis into Human Macrophages. To determine whether B. 12 x

a 8

A,

4

.2 V:

¢a)

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FIG. 3. In vitro modulation of AC expression under growthlimiting conditions. (A) Growth curve of BPRU140 grown in complete (26) (in, ) or growth limiting (E, a) medium in the absence (-, a) or presence (a, e) of 40 mM MgSO4. To phenotypically modulate the bacteria, cultures initially grown in the absence of MgSO4 were switched to medium containing the divalent cation or, alternatively, cultures grown in the presence of MgSO4 were switched to medium devoid of this component. (B) AC activities of cultures described in A (determined as described in Fig. 2).

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pertussis modulates expression of AC 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 enzyme 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 gentamicin treatment of a bacteria/macrophage mixture. The parental strain (BP536) and BPRU140 bound macrophages in a ratio of 10:1 (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 determined by visual examination for trypan blue exclusion. Induction of cyaA with IPTG in the mutant strain BPRU140 did not affect the survival of these bacteria. To kill the bacteria prior to incubation with the macrophages, a sample of BP536 was pretreated for 2 hr with gentamicin at 100 ,ug/ml. This led to a four-log reduction of surviving bacteria compared with bacteria that were not pretreated. The bvgstrain BP537 does not express any known virulence determinant. This strain bound less avidly to macrophages than the bvg+ strains (2 x 105 cfu/ml vs. 1 x 107 cfu/ml). Those that were bound did not survive treatment with gentamicin and macrophage challenge (103 cfu/ml). This is consistent with other studies describing the inability of avirulent strains to attach to human macrophages (38) or invade other cells (2, 3). Bacterial multiplication in any strain was not observed over the 4-hr time course of the experiment. This is not unexpected given the 4- to 6-hr doubling time observed for the growth of B. pertussis in liquid medium (Fig. 3). Therefore, virulent B. pertussis is able to survive challenge by human macrophages in this invasion assay. AC Activity Associated with the Entry of B. pertussis Into Human Macrophages. There were clearly two components to the internalized AC activity identified from the bacteria/ macrophage mixtures. Within 30 min of the attachment of wild-type BP536 or BPRU140, comparable peaks of bacterial AC activity were measured in the macrophage lysates (Fig. 4B). Since this peak could be reproduced by killed BP536 (pretreated with gentamicin) the initial peak ofAC activity for all toxin-containing strains was attributed to internalization of preexisting toxin regardless of the viability of the bacteria. By 1.5 hr, this peak of initial AC activity decreased to a basal value for the nonviable BP536, which is not actively producing new enzyme. It seems unlikely that the basal AC values obtained with the nonviable bacteria represent activity produced by the small number of surviving cells (y102 bacteria), and these basal values are probably due to the residual activity from the initial internalization of bacteria that delivers AC to the macrophage. This interpretation is consistent with numerous studies using purified toxin, which is capable of increasing cAMP in a wide variety of eukaryotic cells (16-18). During the same time period, the positive control, BPRU140 induced with IPTG to express the plasmid derived

8-

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Time, hr Fio. 4. Survival and AC activity of B. pertussis within human macrophages. (A) Survival of bacteria following uptake into macrophages. Bacteria were mixed with human macrophages and, 30 mi later, exposed to gentamicin (100 Iug/ml, arrow) to kill extracellular bacteria. Samples were removed at timed intervals, macrophages were lysed, and the solution was plated to quantitate viable intracellular bacteria. Values are representative of experiments conducted in triplicate. The parent BP536 (n) was compared with killed BP536 (treated with gentamicin for 2 hr prior to mixture with macrophages) (0), isogenic BPRU140 (BP536 carrying the plasmidborne, IPTG-inducible cyaA) treated (e) or untreated (o) with 250 ,uM IPTG (beginning 10 min prior to admixture with macrophages), and BP537 (bvg-) (x). (B) AC activity associated with internalized bacteria. Symbols are as described in A. AC activity associated with internalized bacteria was determined by trypsin treatment of the bacteria/macrophage mixture to destroy enzyme activity outside the macrophage, followed by quenching of the trypsin with soybean trypsin inhibitor, lysis of the macrophage, and quantitation of enzyme activity. Values are means of duplicate samples presented as units of specific activity (nmol of cAMP produced per min per mg of protein). SD for any value did not exceed 7%. Results are representative of experiments conducted in triplicate.

form of the enzyme, produced AC activity 5-fold greater than the basal values of the killed parental strain. Thus, after 1.5 hr AC values were taken to be indicative of enzyme produced by bacteria within the macrophage. Between 2 and 4 hr AC activity for all samples reached a steady-state value. The wild-type parent and the uninduced mutant responded to internalization with low AC values comparable to those of the nonviable bacteria (Fig. 4B). When the AC values were plotted as a function of activity per colony-forming unit over time (Fig. 5), the wild-type parent, BP536, produced only basal AC activity that was consistently only one-third that obtained with the induced strain BPRU140. These observations demonstrate down-modulation of AC expression in the parent strain with entry into macrophages and suggest a setting in which phenotypic modulation, so well studied in vitro, also occurs in vivo. Failure to down-modulate would have produced AC values comparable to those obtained with the induced strain. The ability of the parent bacteria to alter AC expression with the

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Proc. Natl. Acad. Sci. USA 89 (1992)

1.00

i 0 0.75 C X

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FIG. 5. In vivo modulation of cyaA expression with the entry of B. pertussis into human macrophages. AC activity associated with viable bacteria internalized within human macrophages was measured as described in Fig. 4. Units of AC specific activity (nmol of cAMP per min per mg of protein) from the experiment depicted in Fig. 4 are plotted as a function of cfu over time. The parent BP536 (-) was compared with isogenic BPRU140 (BP536 carrying the plasmid-borne, IPTG-inducible cyaA) treated (e) or untreated (o) with 250 .uM IPTG.

transition to inside the macrophage is presumably mediated by the products of the bvg locus despite potential growthlimiting conditions. This argument is strengthened by the results in Fig. 3, which demonstrate modulation of AC expression in vitro by the bacteria even under limiting growth conditions. It is certainly possible that the stimulus for this down-modulation is contained within the milieu of the endocytic vesicles of the macrophage encountered by the bacteria with internalization. Survival of B. pertussis upon challenge with human macrophages has been attributed to expression of adhesins and virulence factors in the Vir+ state (39). If the regulatory circuit between the bvg and the cya loci serves as a paradigm for gene regulation of other members of the vir regulon, then the results reported here suggest that the natural progression of events in the disease may require that B. pertussis phenotypically changes to a Vir- state upon entry into the macrophage. This hypothesis is supported by studies with animal models for the disease. A mutant with a transposon insertion in vrg6 (a vir-repressed gene) was less able to colonize the lung and trachea in a mouse model for respiratory infection (40). Similar studies also demonstrated that the AC (the product of a vir-activated gene) is absolutely required for colonization (41). Therefore, B. pertussis has developed a survival strategy based on the coordinate regulation of genes including the cya operon that results in two distinct phenotypes. In the Vir+ state the bacteria adhere and colonize the host in several niches. Thereafter access to another environment, the alveolar macrophage, leads to the Virstate, cessation of toxin production, and establishment of intracellular bacteria that remain sequestered from host defenses. The ability of the bacteria to sense its environment and switch between two phenotypes confers a selective advantage for B. pertussis in promoting successful and persistent survival within the host. I thank Drs. E. Tuomanen and C. A. Butler for many thoughtful discussions, support, and critical reading of the manuscript. I thank Anne Naughton for her expert technical assistance and Drs. Jeff Weiser and Emil Gotschlich for their encouragement. This work was supported by National Institutes of Health Grants R29 A132024-01 and RO1 A123459 and by a Career Scientist Award from the I. T. Hirshl Trust.

1. Saukkonen, K., Cabellos, C., Burroughs, M., Prasad, S. & Tuomanen, E. (1991) J. Exp. Med. 173, 1143-1149.

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Modulation of adenylate cyclase toxin production as Bordetella pertussis enters human macrophages.

During the course of human infection, Bordetella pertussis colonizes sequential niches in the respiratory tract that include intracellular and extrace...
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