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Modulation of Adenylate Cyclase Toxin Production As Bordetella Pertussis Enters Human Macrophages (Bacterial Invasion/Gene Expression) H

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Modulation of Adenylate Cyclase Toxin Production As Bordetella Pertussis Enters Human Macrophages (Bacterial Invasion/Gene Expression) H 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 ABSTRACT During the course of human infection, 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, enzyme 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 bacteria 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 calmodulin-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 secretion 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. Escherichia coli 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 Bordetella pertussis, 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 proteins, 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 macrophage 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.
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