H-NS Regulation of Virulence Gene Expression in Enteroinvasive ...

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and, with E. coli K-12 strains containing a virF-lacZ gene fusion, by H-NS. This leads us ... Shigella flexneri and enteroinvasive Escherichia coli (EIEC) strains are ...
JOURNAL OF BACTERIOLOGY, Aug. 1995, p. 4703–4712 0021-9193/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 177, No. 16

H-NS Regulation of Virulence Gene Expression in Enteroinvasive Escherichia coli Harboring the Virulence Plasmid Integrated into the Host Chromosome BIANCA COLONNA,1 MARIASSUNTA CASALINO,2 PIERA ASSUNTA FRADIANI,1 CARLO ZAGAGLIA,3† SILVIA NAITZA,1 LIVIA LEONI,1 GIANNI PROSSEDA,1 ANNA COPPO,4 PATRIZIA GHELARDINI,5 AND MAURO NICOLETTI3* Dipartimento di Biologia Cellulare e dello Sviluppo, Sezione di Scienze Microbiologiche,1 and Centro Acidi Nucleici, Consiglio Nazionale delle Ricerche,5 Universita ` di Roma ‘‘La Sapienza,’’ 00185 Rome, Dipartimento di Biologia, Universita ` di Roma III, 00146 Rome,2 Istituto di Medicina Sperimentale, Consiglio Nazionale delle Ricerche, Viale K. Marx, 15, 00156 Rome,4 and Istituto di Medicina Sperimentale, Cattedra di Microbiologia, Universita ` ‘‘G. D’Annunzio,’’ 66100 Chieti,3 Italy Received 24 January 1995/Accepted 11 June 1995

We have previously shown that integration of the virulence plasmid pINV into the chromosome of enteroinvasive Escherichia coli and of Shigella flexneri makes these strains noninvasive (C. Zagaglia, M. Casalino, B. Colonna, C. Conti, A. Calconi, and M. Nicoletti, Infect. Immun. 59:792–799, 1991). In this work, we have studied the transcription of the virulence regulatory genes virB, virF, and hns (virR) in wild-type enteroinvasive E. coli HN280 and in its pINV-integrated derivative HN280/32. While transcription of virF and of hns is not affected by pINV integration, transcription of virB is severely reduced even if integration does not occur within the virB locus. This indicates that VirF cannot activate virB transcription when pINV is integrated, and this lack of expression accounts for the noninvasive phenotype of HN280/32. Virulence gene expression in strains HN280 and HN280/32, as well as in derivatives harboring a mxiC::lacZ operon fusion either on the autonomously replicating pINV or on the integrated pINV, was studied. The effect of the introduction of plasmids carrying virB (pBN1) or virF (pHW745 and pMYSH6504), and of a Dhns deletion, in the different strains was evaluated by measuring b-galactosidase activity, virB transcription, and virB-regulated virulence phenotypes like synthesis of Ipa proteins, contactmediated hemolysis, and capacity to invade HeLa cells. The introduction of pBN1 or of the Dhns deletion in pINV-integrated strains induces temperature-regulated expression or temperature-independent expression, respectively, of b-galactosidase activity and of all virulence phenotypes, while an increase in virF gene dosage does not, in spite of a high-level induction of virB transcription. Moreover, a wild-type hns gene placed in trans fully reversed the induction of b-galactosidase activity due to the Dhns deletion. These results indicate that virB transcription is negatively regulated by H-NS both at 30 and at 37&C in pINV-integrated strains and that there is also a dosedependent effect of VirF on virB transcription. The negative effect of H-NS on virB transcription at the permissive temperature of 37&C could be due to changes in the DNA topology occurring upon pINV integration that favor more stable binding of H-NS to the virB promoter DNA region. At 30&C, the introduction of the high-copy-number plasmid pMYSH6504 (but not of the low-copy-number pHW745) or of the Dhns deletion induces, in strains harboring an autonomously replicating pINV, b-galactosidase activity, virB transcription, and expression of the virulence phenotypes, indicating that, as for HN280/32, the increase in virF gene dosage overcomes the negative regulatory effect of H-NS on virB transcription. Moreover, we have found that virF transcription is finely modulated by temperature and, with E. coli K-12 strains containing a virF-lacZ gene fusion, by H-NS. This leads us to speculate that, in enteroinvasive bacteria, the level of VirF inside the cell controls the temperature-regulated expression of invasion genes.

chromosomal gene hns (virR) has been shown to be a thermoosmotic regulator, since it modulates the expression of pINVencoded virulence genes by repressing transcription when strains are cultured at the nonpermissive temperature (308C) or in low-osmolarity medium (11, 20, 26, 32). Plasmid genes associated with virulence comprise the ipa, mxi, and spa operons that are necessary for invasioin of epithelial cells; the icsA (virG) gene that is essential for intra- and intercellular spread; and the regulatory genes virB and virF (2, 16, 17, 24, 46). virB activates transcription of ipa, mxi, and spa and is in turn regulated by two DNA-binding regulatory proteins, VirF and H-NS (1, 10, 11, 16, 20, 23, 26, 41, 42, 46). Mutations or deletions of hns derepress transcription of invasion genes at 308C only in the presence of VirF (10, 11, 20, 23, 42). VirF positively regulates transcription of virB and icsA in a DNAtopology-dependent manner, while H-NS acts as a negative

Shigella flexneri and enteroinvasive Escherichia coli (EIEC) strains are known to cause disease in humans by very similar mechanisms of pathogenicity. The essential early steps in infection comprise invasion of colonic epithelial cells, bacterial multiplication, and spread to adjacent cells (16, 36). The genes required for the expression of these virulence traits are located on a large virulence plasmid (pINV) and organized in regulons that are controlled by a central regulator (15, 16, 18, 27). The * Corresponding author. Mailing address: Istituto di Medicina Sperimentale, Cattedra di Microbiologia, Facolta` di Medicina e Chirurgia, Universita` ‘‘G. D’Annunzio,’’ Via dei Vestini, 31, 66100 Chieti, Italy. Phone: 0039-871-355279. Fax: 0039-871-355282. † Present address: Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Cattedra di Farmacologia, Universita` di Roma ‘‘Tor Vergata,’’ 00133 Rome, Italy. 4703

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regulator on both genes (10, 42). Moreover, VirF activation of virB transcription was shown to be highly sensitive to changes in DNA topology (42, 43). We have previously shown (48) that the pINV plasmids of the O135:K2:H2 EIEC strain HN280 and of the S. flexneri serotype 5 strain M90T are able to integrate into the host chromosome and that integration severely reduces the expression of pINV-carried virulence genes. pINV-integrated strains grown at 378C are noninvasive, do not produce contact-mediated hemolysis, and lose the ability to bind Congo red. At a frequency of about 1024 to 1023, pINV-integrated strains spontaneously generate excised forms of pINV plasmids by a recA-dependent process. Precisely excised pINV plasmids regain the temperature-regulated expression of the full virulence pattern, while strains with imprecisely excised pINV plasmids are always noninvasive. These results led us to envisage pINV integration into the chromosome as an advantageous strategy for enteroinvasive bacteria to ensure plasmid maintenance and to regulate the expression of pINV-associated genes that could be particularly useful when bacteria are outside mammalian host cells and/or have to face adverse environmental conditions (48). In this work, we have studied the transcription of virF, virB, and hns in wild-type HN280 and in its pINV-integrated derivative strain HN280/32. We have found that while transcription of virF and hns is not affected by integration, transcription of virB is severely reduced, even if integration does not occur within the virB locus. Moreover, virulence gene expression was evaluated under different genetic conditions and by the use of the mxi::lacZ transcriptional operon fusion. The results obtained indicate that (i) in pINV-integrated strains, the repression of virB transcription could be due to changes in DNA topology occurring upon pINV integration which favor a more stable binding of H-NS to the virB promoter, thus preventing virF activation, and that (ii) in strains harboring an autonomously replicating pINV, an increase of the virF gene dosage overcomes the negative regulatory effect of H-NS on virB transcription, inducing expression of virulence phenotypes at the nonpermissive temperature of 308C. In addition, using a virF::lacZ gene fusion, we show that virF transcription is finely modulated by temperature and by H-NS. The importance of such a regulation in the complex mechanism of pathogenicity of enteroinvasive bacteria is discussed. MATERIALS AND METHODS Bacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used are listed in Table 1. Enriched and minimal growth media were Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.), LB medium (35), and M9 minimal salt medium (35). The solid media contained 1.5% agar. Congo red (Sigma Chemical Co., St. Louis, Mo.) was added to Trypticase soy agar to a final concentration of 0.01%. Antibiotics were used at the following concentrations: ampicillin, 25 or 50 mg/ml; chloramphenicol, 30 mg/ml; kanamycin, 30 mg/ml; tetracycline, 5 mg/ml; trimethoprim, 10 mg/ml. b-Galactosidase assays. b-Galactosidase assays were performed on sodium dodecyl sulfate (SDS)-chloroform-permeabilized cells grown in LB broth to mid-log phase as described by Miller (29). Genetic and molecular procedures. Transformation and P1 transduction were carried out by standard methods (9, 29, 39). Isolation of plasmids, restriction digestions, electrophoresis, and purification of DNA fragments were carried out as described by Sambrook et al. (35). DNA fragments from agarose gels were denatured and transferred to nitrocellulose filters. Southern blot hybridizations were performed as previously described (48). DNA probes were 32P labelled by the random priming method (35). Hybridization and washing of the blots were performed under stringent conditions as previously described (48). The probes used were the 1.3-kb HindIII-BamHI fragment of plasmid pBN1 (virB probe) (1), the 1.3-kb HpaII fragment of plasmid pMYSH6504 (virF probe) (33), the 850-bp EcoRI-AccI fragment of plasmid pPLc11 (hns probe) (40), and the 2.6-kb ClaI fragment of plasmid pMC1403 containing the entire lacZ gene and part of the lacY gene (lacZY probe) (5).

J. BACTERIOL. Wild-type EIEC HN280 has been characterized as a lactose-negative strain (30). Since HN280 produces pale-rose colonies when grown for 48 h on MacConkey lactose agar plates, we further analyzed this strain and found that it contains a complete chromosomal lacZYA operon but is impaired in the transport of lactose into the cell (lacY mutant) (8). A Dlac derivative of HN280 was constructed by transduction with P1 grown on strain CSH26 (Table 1). One transductant, named HN570, contained the expected lac deletion (Table 2) and behaved as the parental HN280 both in producing a positive Sereny test and in ability to invade HeLa cell monolayers. The temperature-regulated mxiC::lacZ fusion from the virulence plasmid of strain BS184 (26) was then transferred to pHN280 in strain HN570 by P1 transduction. A kanamycin-resistant transductant, named HN580, was selected for further analysis. pINV localization of the lac operon fusion was confirmed by probing Southern blots of undigested HN580 plasmid DNA with the lacZY probe (data not shown). Northern (RNA) analysis. Bacterial strains were grown in LB broth at 30 or 378C to an optical density at 600 nm of 0.5. Total RNA was extracted as described by Von Gabain et al. (45). Aliquots of total RNA were denatured at 1008C for 5 min in the presence of 2 M formaldehyde and 50% formamide, separated on an agarose gel, and then hybridized as described by Sambrook et al. (35) with the virF, virB, or hns DNA probes described above. Western blots (immunoblots). Western immunoblots were performed as previously described (48). Whole bacterial protein extracts were separated on SDSpolyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose membranes by the procedure of Burnette (4). The blots were probed with mouse monoclonal antibodies (MAbs) H4 and J22, which recognize IpaB and IpaC, respectively (28). MAbs recognizing EIEC antigens were detected by using horseradish peroxidase-labelled goat anti-mouse antibodies as secondary antibodies and visualized by enhanced chemiluminescence. Construction of the virF-lacZ gene fusion. Two primers, designed on the basis of the published virF sequence (34) and identified as F1 (59-CAAATACTTA GCTTGT-39, corresponding to nucleotides 2 to 16) and F2 (59-GCGAACCTT TATATCT-39, corresponding to nucleotides 241 to 226), were used to amplify the 240-bp DNA fragment (bp 2 to 241) of the virF gene of S. flexneri 2a strain YSH6000 contained in the recombinant plasmid pMYSH6504 (33) (Table 1) by PCR. PCR was carried out by using 50 ng of circular template DNA and 1 mM each primer in a 100-ml reaction mixture containing 67 mM Tris-HCl (pH 8.8), 16 mM ammonium sulfate, 1.5 mM MgCl2, 0.01% Tween 20, and 200 mM each deoxynucleoside triphosphate. Twenty-five cycles were used, each cycle comprising 30 s of denaturation at 958C, 3 min of primer annealing at 428C, and 1 min of extension with Taq polymerase at 728C. The PCR-generated fragment of the expected size (240 bp) was recovered from a 1% agarose gel and cloned into the SmaI site of plasmid pMC1403 (5) to generate pFlac1. pMC1403 is a vector suitable for generating gene fusions, since the first eight codons of the aminoterminal end of the lacZ gene were removed and a unique SmaI site has been incorporated adjacent to the eighth codon of the lacZ gene (5). The 240-bp PCR-generated fragment contains the transcriptional and transductional regulatory signals of the virF gene and the codons for the first five amino acids of the VirF protein. Thus, the expression of the hybrid VirF-LacZ protein in pFlac1 is under the control of the virF regulatory region. Nucleotide sequencing performed by linear PCR with the F1 and F2 oligonucleotides as primers by using the Circumvent Thermal Cycle Deoxy Sequencing Kit (New England Biolabs Inc., Beverly, Mass.), as recommended by the manufacturer, confirmed that no error had occurred in cloning the 240-bp amplified fragment in plasmid pFlac1. Virulence assays. Sereny tests (38) were performed with adult albino guinea pigs, as previously described (48). Experiments to measure bacterial invasion of HeLa cells were performed as previously described at a multiplicity of infection of about 100 bacteria per HeLa cell (48). Contact-mediated hemolysis was detected as previously described (48).

RESULTS Transcription of virB, virF, and hns in pINV-integrated strains. We have recently reported that pHN280, the pINV plasmid of EIEC HN280, is able to integrate into the host chromosome and that pINV-integrated strains are noninvasive (48). In order to understand the mechanism by which pINVintegrated strains modulate expression of virulence genes, we performed Northern blot analysis to evaluate the transcription of the regulatory genes virB, virF, and hns (virR) (20, 23, 41, 42, 46). Equivalent amounts of total RNA extracted from strains harboring an autonomously replicating pINV and from pINVintegrated strains were denatured, separated on 1.2% agarose gels, and hybridized with the virB-, virF-, and hns-specific probes (see Materials and Methods). We have previously shown that, by a recA-dependent process, pINV-integrated strains spontaneously generated excised forms of pINV that expressed the full pattern of virulence (48).

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TABLE 1. Bacterial strains and plasmids Strain or plasmid

Strains HN280 HN280/32 HN280/10 HN321 HN570 BS184 HN580 HN580/10 HN580/32 HN580/321 HN580/322 HN680 HN680/32 HN780 HN780/32 CSH26 MC4100 HN4104 GC226 TP504 Plasmids pHN280 pHN580 pBN1 pHW745 pMYSH6504 pMC1403 pFlac1 pPLc11 pDIA510 a

Relevant characteristicsa

Source or reference(s)

Wild-type EIEC strain of serotype O135:K2:H2; contains virulence plasmid pHN280; invasive pHN280-integrated derivative of strain HN280; noninvasive recA1 derivative of strain HN280; invasive; Tcr recA1 derivative of strain HN280/32; noninvasive; Tcr Dlac derivative of strain HN280; invasive Derivative of S. flexneri 2a strain 2457T that contains a temperature-regulated mxiC::lacZ operon fusion (vir-83::MudI1734) in the mxiC gene of the virulence plasmid; noninvasive; lactose positive; Kmr Derivative of HN570 in which mxiC::lacZ operon fusion of strain BS184 has been transferred to virulence plasmid pHN280 (named pHN580); noninvasive; lactose positive; Kmr recA1 derivative of strain HN580; noninvasive; lactose positive; Kmr Tcr Derivative of HN580 containing pHN280 mxiC::lacZ integrated into chromosome; noninvasive; lactose negative; Kmr recA1 derivative of strain HN580/32; noninvasive; lactose negative; Kmr Tcr pHN280 mxiC::lacZ-excised derivative of HN580/32; noninvasive; lactose positive; Kmr D(hns tdk adhE oppABCD)118 zch-506::Tn10 derivative of strain HN280; invasive; Tcr D(hns tdk adhE oppABCD)118 zch-506::Tn10 derivative of strain HN280/32; invasive; Tcr D(hns tdk adhE oppABCD)118 zch-506::Tn10 derivative of strain HN580; noninvasive; lactose positive; Kmr Tcr D(hns tdk adhE oppABCD)118 zch-506::Tn10 derivative of strain HN580/32; noninvasive; lactose positive; Kmr Tcr E. coli K-12 F2 ara D(lac pro)thi E. coli K-12 F2 araD139 D(argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 pstF25 rbsR D(hns tdk adhE oppABCD)118 zch-506::Tn10 derivative of MC4100; Tcr srl::Tn10 derivative of recA1 E. coli K-12 strain KL1699; Tcr E. coli K-12 F2 leuB6 serB1203 thi-1 zch-506::Tn10 zdd-230::Tn9 D(hns tdk adhE oppABCD)118 Cmr Tcr

7, 48 48 48 48 This study 26

Virulence plasmid of strain HN280 pHN280 derivative containing mxiC::lacZ operon fusion of virulence plasmid of strain BS184; Kmr pMYSH6003 replicon vector (pBR322-derived vector) carrying virB gene of S. flexneri 2a strain YSH6000 virulence plasmid pMYSH6000; Tpr pHSG595 replicon vector (pSC101-derived vector) carrying virF gene of S. sonnei HW383 virulence plasmid pSS120; Cmr pMYSH6001 replicon vector (pBR322-derived vector) carrying virF gene of S. flexneri 2a strain YSH6000 virulence plasmid pMYSH6000; Apr Kmr pBR322 replicon vector carrying E. coli K-12 lac operon; Apr pMC1403 replicon vector containing virF-lacZ gene fusion; Apr pTZ18R replicon vector carrying E. coli K-12 hns gene; Apr pBR322 replicon vector containing 0.9-kb EcoRI-SnaBI DNA fragment carrying entire E. coli hns gene; Apr

48 This study

This study This study This study This This This This This

study study study study study

This study 29 48 This study 48 3

1 46 33 5 This study 40 3

Tcr, tetracycline resistant; Kmr, kanamycin resistant; Apr, ampicillin resistant; Cmr, chloramphenicol resistant; Tpr, trimethoprim resistant.

Therefore, we compared virB transcription in pINV-integrated strain HN280/32 and in its recA derivative HN321 with that in the parental invasive strain HN280 and in its recA derivative HN280/10. Figure 1 shows a Northern blot with strains grown either at 378C (Fig. 1A) or at 308C (Fig. 1B) and hybridized with the virB probe. At 378C, virB transcription undergoes an ;20-fold reduction in the pINV-integrated strains. As expected, transcription of virB in HN280 and HN280/10 is temperature regulated (Fig. 1B). Moreover, there are no significant differences in the level of virB transcription between RecA1 and RecA2 strains, indicating that excision events in strain HN280/32 produced negligible effects on virB transcription. On the other hand, transcription of virF is not affected by the integration of the pINV plasmid, since the levels of mRNA reacting with the virF probe appear to be the same in Northern blots of HN280 and HN280/32 (Fig. 2A). However, the amount of virF mRNA found in strains grown at 308C is about onethird the amount observed at 378C (Fig. 2A). We did not find

any significant differences in the levels of virF transcription in RecA1 and RecA2 strains (data not shown). Neither pINV integration nor growth temperature affects transcription of the hns gene (Fig. 2B). These results indicate that the severely reduced expression of invasion-associated genes occurring in pINV-integrated strains is due to the reduced transcription of virB. pINV does not integrate into the virB locus. To ascertain whether the reduced level of virB transcription in pHN280integrated strains might be ascribed to pINV integration, we hybridized HindIII- or EcoRI-digested total DNA from strains HN280 and HN280/32 with the virB probe. This probe recognizes a 2.0-kb fragment in both HindIII digests (Fig. 3, right panel, lanes 1 and 2) and an 8.1-kb fragment in both EcoRI digests (lanes 3 and 4), indicating that pINV integration does not alter virB. An 8.1-kb EcoRI fragment harboring virB exists also in the virulence plasmid pMYSH6000 (1). Since this fragment contains almost 3 kb of DNA upstream of the virB gene

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TABLE 2. Expression of b-galactosidase by mxiC::lacZ operon fusion mutants Strain

Relevant genotype

CSH26 HN570 BS184 HN580 HN580/10 HN580(pBN1) HN580(pHW745) HN580(pMYSH6504) HN780 HN780(pDIA510) HN580/32 HN580/321 HN580/322 HN580/32(pBN1) HN580/32(pHW745) HN580/32(pMYSH6504) HN780/32 HN780/32(pDIA510)

Dlac (E. coli K-12) Dlac (EIEC) pmxiC::lacZ (S. flexneri) pmxiC::lacZ (EIEC) recA1 pvirB pvirF pvirF Dhns pmxiC::lacZ phns mxiC::lacZ (chromosome) recA1 mxiC::lacZ (chromosome) pmxiC::lacZ (plasmid) pvirB pvirF pvirF Dhns mxiC::lacZ (chromosome) phns

b-Galactosidase activitya 308C

378C

2 2 39 27 23 56 42 323 270 5 3 5 25 20 10 18 167 5

2 2 532 336 325 609 355 404 320 90 3 5 350 80 45 60 167 7

a Units of b-galactosidase are as defined by Miller (29). Results are averages for four independent experiments. Standard errors were less than 10%. pBN1-, pHW745-, and pMYSH6504-transformed recA derivative strains HN580/10 and HN580/321 expressed levels of b-galactosidase similar to those obtained with transformed RecA1 strains HN580 and HN580/32.

(1), we can argue that the virB-containing 8.1-kb EcoRI fragment is well conserved in pHN280 and pMYSH6000 and rule out that pINV integration might have occurred also within the 3-kb DNA region upstream of the virB gene of pHN280. Expression of a mxiC::lacZ fusion in autonomously replicating pINV plasmids and in integrated pINV plasmids. To better evaluate the expression of virB-regulated pINV-encoded virulence genes in pINV-integrated strains, we transferred the temperature-regulated mxiC::lacZ transcriptional operon fusion from the virulence plasmid of S. flexneri 2a strain BS184

FIG. 1. Northern hybridization analysis of RNA from bacterial strains grown at 378C (A) and at 308C (B). The blots were probed with the 1.3-kb HindIIIBamHI fragment from plasmid pBN1 (virB probe). Each lane was loaded with 10 mg of total RNA. The relative amounts of RNA loaded in each lane were estimated by visualization of the rRNA by ethidium bromide staining.

FIG. 2. Northern hybridization analysis of RNA from bacterial strains grown at 37 and at 308C. The blots were probed with the 1.3-kb HpaII fragment of plasmid pMYSH6504 (virF probe) (A) or with the 850-bp EcoRI-AccI fragment of plasmid pPLc11 (hns probe) (B). The relative amounts of RNA loaded in each lane were estimated by visualization of the rRNA by ethidium bromide staining.

(26) to pHN280 by P1 transduction, thus generating strain HN580 (see Materials and Methods). HN580 shows temperature-regulated expression of b-galactosidase comparable to that of BS184 (Table 2) and, as expected, is noninvasive. Integration of the pHN280 mxiC::lacZ plasmid (pHN580) into the chromosome was induced by growing HN580 in LB broth containing 4 mg of rifampin per ml (48). From rifampin-treated bacteria, we obtained eight independent lactose-negative derivatives that apparently had lost pHN580. As expected for pINV-integrated strains (48), two of them, at frequencies of 1024 to 1023, spontaneously generated lactose-positive seg-

FIG. 3. Southern blot analysis of EIEC HN280 and HN280/32 DNAs digested with HindIII or EcoRI. Total DNAs (leftmost panel) from HN280 and HN280/32 were digested with HindIII (lanes 1 and 2, respectively), or with EcoRI (lanes 3 and 4, respectively), separated by electrophoresis on an 0.8% agarose gel, blotted onto nitrocellulose, and then probed with the 1.3-kb HindIII-BamHI fragment of plasmid pBN1 (virB probe) (rightmost panel).

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regants with the same plasmid content and the same level of b-galactosidase as parental HN580 had. The b-galactosidase levels of one pHN580-integrated strain (HN580/32) and of one excised segregant (HN580/322) are reported in Table 2. These results indicate that pINV integration dramatically reduces the expression of the mxiC::lacZ operon fusion. Effect of virB, virF, and hns on the expression of the mxiC:: lacZ operon fusion in the pINV-integrated strain HN580/32. To study the expression of the mxiC::lacZ operon fusion, we transformed the parental strain HN580, the pHN580-integrated strain HN580/32, and their recA-derivatives HN580/10 and HN580/321 (Table 1) with plasmids carrying virB (pBN1) or virF (pHW745 and pMYSH6504). We also introduced a Dhns deletion into HN580 and into HN580/32 using a P1 grown on the E. coli K-12 strain TP504 (Table 1). Since the b-galactosidase activities of RecA1 and RecA2 strains transformed with plasmid vectors carrying virB or virF were very similar, only the RecA1 strains are shown in Table 2. The introduction of the high-copy-number plasmid pBN1 (virB) in HN580 and in HN580/32 increases the expression of b-galactosidase at 378C (609 and 80 Miller units [U], respectively) but not at 308C (Table 2). It has been reported recently that at 308C, very low levels of virB mRNA are also produced in wild-type S. flexneri 2a strain YSH6000 transformed with pBN1, while wild-type expression of virB (and thus of invasion genes) at 308C is achieved by introducing a plasmid vector carrying virB under the control of the tac promoter (41). This indicates that even if the gene is cloned in a high-copy-number plasmid, the expression of virB is still temperature regulated. The lower level of b-galactosidase activity observed at 378C in HN580/32(pBN1) indicates that probably other factors, besides pBN1-mediated virB expression, might contribute to promote wild-type expression of the mxiC::lacZ operon fusion in pINV-integrated strains. We increased the level of virF transcription by transforming HN580 and HN580/32 with pHW745, a low-copy-number plasmid, or with pMYSH6504, a high-copy-number plasmid. Determination of the copy number (data not shown) revealed that the copy number of pMYSH6504 was at least three to five times greater than that of pHW745. As shown in Table 2, at 308C, a temperature at which transcription of virB is known to be repressed by H-NS (42), we obtained derepressed expression of b-galactosidase in HN580 transformed with pMYSH 6504 (323 U) but not with pHW745 (42 U). On the other hand, introduction of pHW745 or pMYSH6504 into HN580/32 only moderately increases the enzymatic activity in strains grown at 378C. Interestingly, the introduction of the Dhns mutation in HN580/32 (strain HN780/32) significantly increases the expression of b-galactosidase both at 30 and at 378C, and, as expected (20, 26), strain HN780 (Dhns derivative of HN580) shows temperature-independent expression of the reporter gene (Table 2). Moreover, we introduced the recombinant plasmid pDIA 510, a pBR322-derived plasmid carrying a 0.9-kb EcoRI-SnaBI DNA fragment that encodes the entire wild-type hns gene (3), in strains HN780 and HN780/32. As shown in Table 2, pDIA 510 fully complemented the hns deletion since HN780(pDIA 510) shows temperature-regulated expression of b-galactosidase activity, while HN780/32(pDIA510) produces only low levels of enzymatic activity, either at 30 or at 378C, closely resembling parental hns1 strain HN580/32. This indicates that the absence of H-NS allows transcription, either at 30 or at 378C, of the mxiC::lacZ fusion in pINV-integrated strains. virF activation of virB transcription in pINV-integrated strains. In S. flexneri strains harboring an autonomously replicating pINV, VirF positively activates transcription of virB at

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FIG. 4. Northern hybridization analysis of RNA from bacterial strains grown at 378C (A) and at 308C (B). The blots were probed with the 1.3-kb HindIIIBamHI fragment of plasmid pBN1 (virB probe). The relative amounts of RNA loaded in each lane were estimated by visualization of the rRNA by ethidium bromide staining.

378C (1, 41, 42). To better evaluate the role of virF in promoting virB transcription, we performed Northern blot analysis of total RNA from strains HN280 and HN280/32 using the virB probe. The introduction of pMYSH6504 (virF) in HN280 or in HN280/32 dramatically increases the level of virB mRNA, both at 30 and at 378C (Fig. 4). Now, since at 308C pMYSH6504 induces high-level b-galactosidase activity in HN580, but not in HN580/32 (Table 2), these results suggest that pINV integration influences the pMYSH6504-induced virB-dependent expression of the mxiC::lacZ fusion in HN580/32. Effect of extra copies of virB and virF, and of an hns deletion, on contact-mediated hemolysis and on capacity to invade HeLa cells. In order to confirm and to better define the results obtained with the mxiC::lacZ operon fusion (Table 2) and in the Northern blot experiments (Fig. 1 and 4), we tested virBtransformed (pBN1), virF-transformed (pHW745 and pMYSH 6504), and hns-deleted mutants of strains HN280 and HN280/ 32 for the expression at 30 and at 378C of virB-regulated phenotypes, the production of contact-mediated hemolysis, and the capacity to invade HeLa cell monolayers. Contact-mediated hemolysis is known to be mediated by the products of three ipa genes, IpaB, IpaC, and IpaD (28), and HeLa cell invasiveness requires the coordinated expression of the entire set of plasmid virulence genes (10, 16, 42). The results are reported in Table 3. As expected, HN280 shows a temperatureregulated expression of both virulence phenotypes, while HN280/32 fails to produce hemolytic activity or to invade HeLa cells at either temperature.

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TABLE 3. Gene dosage effects of virB, virF, and hns on expression of contact-mediated hemolysis and capacity to invade HeLa cells of wild-type HN280 and of pINV-integrated derivative strain HN280/32 Contact hemolysisa Strain

Relevant genotype

HN280 HN280(pBN1) HN280(pHW745) HN280(pMYSH6504) HN680 HN280/32 HN280/32(pBN1) HN280/32(pHW745) HN280/32(pMYSH6504) HN680/32

pINV (plasmid) pvirB pvirF pvirF Dhns pINV (plasmid) pINV (chromosome) pvirB pvirF pvirF Dhns pINV (chromosome)

a b

HeLa cell invasion (%)b

308C

378C

308C

378C

,1 5 14 110 80 ,1 ,1 ,1 15 65

100 130 83 70 85 ,1 70 28 48 75

,1 2 1 93 68 ,1 ,1 ,1 10 55

85 90 40 42 70 ,1 90 20 30 60

Values represent the percentage of mean absorbance obtained for wild-type strain HN280 at 378C. Percentage of infected HeLa cells.

In agreement with the results of b-galactosidase assays (Table 2), the introduction of the hns deletion into HN280 (strain HN680) induces expression of the virulence phenotypes at 308C, as expected, while the hns deletion in HN280/32 (strain HN680/32) restores expression of the virulence phenotypes at both temperatures. Increasing the number of copies of the virB or the virF gene, by transforming HN280 with pBN1 as well as with pHW745 or pMYSH6504, gives results that also parallel the expression of the mxiC::lacZ fusion: HN280(pBN1) shows a considerable increase in contact-mediated hemolysis at 378C, and HN280(pMYSH6504), but not HN280(pHW745), is fully invasive at the nonpermissive temperature of 308C. Surprisingly, the presence of pHW745 or pMYSH6504 leads to some inhibition in the capacity of HN280 to invade HeLa cells and, to a lesser extent, in the expression of contact-mediated hemolysis. Unexpectedly, considering the low-level expression of b-galactosidase induced in HN280/32, the introduction of pBN1 or pHW745 in HN280/32 induces temperature-regulated expression of the virulence phenotypes to an extent that for HN280/ 32(pBN1) closely resembles that obtained with HN280. A further increase of the VirF level (pMYSH6504) leads to the expression of virulence phenotypes at both temperatures, a result consistent with the above-reported temperature-independent induction of virB transcription under these conditions (Fig. 4). Expression of virB and expression of invasion genes are regulated by different levels of VirF. To better evaluate the findings on the expression of contact-mediated hemolysis and of the capacity to invade HeLa cells (Table 3), Western blots of whole bacterial extracts from strains HN280, HN280/32, HN280(pBN1), HN280/32(pBN1), HN280(pMYSH6504), and HN280/32(pMYSH6504) grown at 30 or 378C were probed with MAbs H4 and J22, which specifically recognize IpaB and IpaC, respectively (28) (Fig. 5). As expected, in HN280 the Ipa proteins are expressed only at 378C, whereas in HN280/32 only a faint IpaC band is visible at 378C. The introduction of pBN1 in HN280 slightly increases the amount of the two proteins at 378C, while in HN280/32 it induces temperature-regulated production of IpaB and IpaC to almost the wild-type level. The introduction of pMYSH6504 in HN280 makes the expression of the Ipa proteins temperature independent. The amount of the two proteins increases at 378C in comparison with the amount in HN280 without the plasmid. IpaC is also detectable in HN280/32(pMYSH6504) grown either at 30 or at 378C, albeit at a lower level than that in HN280(pMYSH6504), again indicating that the pMYSH6504-induced increase in virB

mRNA produces a different effect on the expression of the virB-regulated ipa operon in strains harboring an autonomously replicating pINV or an integrated pINV. hns modulates transcription of virF. Our finding that in HN280 the expression of virF is thermoregulated (see above) prompted us to evaluate the effect of hns on the expression of virF. To this end, we constructed plasmid pFlac1, which harbors a virF-lacZ gene fusion, as described in Materials and Methods. The 240-bp virF sequence contains three start sites, which we regarded as the most probable ones on the basis of

FIG. 5. Western immunoblotting to determine expression of ipaB and ipaC genes. Equivalent amounts of whole-cell protein extracts were resolved by SDSpolyacrylamide gel electrophoresis, electroblotted onto nitrocellulose, and probed with MAbs H4 and J22, which specifically recognized IpaB and IpaC proteins, respectively, as described in Materials and Methods. (A) Strains grown at 378C; (B) strains grown at 308C.

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TABLE 4. hns-dependent expression of b-galactosidase of VirF-LacZ hybrid protein b-Galactosidase activityb

Straina

MC4100 MC4100(pMC1403) HN4104(pMC1403) MC4100(pFlac1) HN4104(pFlac1)

308C

378C

4 2 2 64 569

5 2 3 210 463

Units of b-galactosidase are as defined by Miller (29). Details regarding the construction of the strains, as well as the recombinant plasmid pFlac1, are given in Table 1 and in Materials and Methods. a b

the known molecular size of VirF (34). pFlac1 and the vector pMC1403 were introduced separately by transformation into the E. coli K-12 strain MC4100 and into its Dhns derivative HN4104. The b-galactosidase activities determined for strains grown at 30 and at 378C are reported in Table 4. pMC1403 does not induce production of b-galactosidase. At 308C, the expression of the reporter gene in HN4104(pFlac1) increases about ninefold in comparison with that in MC4100(pFlac1), whereas at 378C the increase is about twofold. Moreover, the b-galactosidase activity of MC4100(pFlac1) grown at 308C is about one-third that at 378C. DISCUSSION The current model for the temperature-regulated expression of the pINV-encoded virulence genes ipa, mxi, spa, and icsA (virG) in S. flexneri and EIEC strains suggests that at 378C the VirF protein acts as a positive transcriptional activator of the virB and icsA genes (10, 42). Transcription of virB and icsA is negatively regulated by H-NS and cannot take place at a low temperature (308C) (10, 42). VirF binds in a DNA-topologydependent manner to a specific DNA region of the virB promoter (nucleotides 2117 to 217), and, at a low temperature, H-NS binds to nucleotides 220 to 120 of the virB promoter, encompassing the transcriptional start site (42). At 378C virB is transcribed; its product induces expression of ipaBCDA, mxi, and spa genes; and the bacterium is fully invasive (10, 41, 42). The nucleotide sequence of the virF gene has shown that VirF shares significant homology with the positive transcriptional regulators belonging to the AraC family (42). The hns gene has been proposed as a global regulatory gene, since mutations in a number of different genes (drdX, bglY, osmZ, pilG, and virR) are allelic to hns (14, 19, 21) and control the expression of a variety of unlinked E. coli, S. flexneri, and EIEC operons including pap, cfa, proVWZ, virB, and icsA, in response to temperature or to medium osmolarity (10, 13, 14, 22, 26, 32, 44). H-NS is a 16-kDa histone-like protein that is believed to be a major constituent of the E. coli and S. flexneri nucleoids and is present in about 20,000 copies per cell (20, 21). H-NS binds to DNA with a quite loose sequence specificity, but it preferentially interacts with curved regions and its effect as a regulatory protein may be either negative or positive, controlling the expression of several totally unrelated promoters (33). The effect of H-NS on transcription can be direct, with H-NS acting as a transcriptional repressor, or indirect, with the protein influencing the level of chromosomal DNA superhelicity (11, 14, 19, 21, 31, 42, 44). As a result of the above-described complex regulatory network, S. flexneri and EIEC strains show a temperature-regulated expression of invasion genes, and the virB transcriptional stage has been proposed as the main target for the thermoreg-

4709

ulation of pINV-encoded invasion genes (41). Though the molecular mechanisms still have to be elucidated, hns certainly plays a crucial role, since transposon-induced mutations and/or deletions of this gene allow transcription of virB and icsA at 308C (10, 41, 42). The present investigation shows that virB transcription is severely reduced in the pINV-integrated EIEC strain HN280/32 in comparison with transcription in wild-type HN280 (Fig. 1) and that the lack of expression is not due to the recombinational event leading to pINV integration (Fig. 3). To study the expression of virB, we assayed b-galactosidase activity in strains harboring a mxiC::lacZ operon fusion on an autonomously replicating pINV (strain HN580) or on an integrated pINV (strain HN580/32). When we increased the virB or virF gene dosage or introduced a Dhns deletion into HN580 and HN580/32, we obtained substantially different results. At 378C the introduction of the high-copy-number plasmid pBN1 (virB) in HN580/32 significantly increases the b-galactosidase activity, albeit at a level lower than that in HN580 (Table 2). On the other hand, HN280/32 transformed with pBN1 expresses, in a temperatureregulated manner, almost wild-type levels of IpaB and IpaC proteins (Fig. 5); lyses erythrocytes to an extent of about 70% of the HN280 level; and invades HeLa cells with the same efficiency as HN280 does (Table 3). Since at 378C HN280/32 produces the same level of virF mRNA as the wild-type strain does (Fig. 2), our results indicate that at 378C VirF cannot activate virB transcription in pINV-integrated strains while VirF-induced transcription of the virB gene carried by pBN1 induces temperature-regulated expression of the invasive phenotype in HN280/32. The apparent discrepancy in b-galactosidase activity between HN580/32(pBN1) and HN580, together with the almost wild-type expression of the invasive phenotype in HN280/32(pBN1), might indicate (i) that the expression of the mxiC gene might be differently regulated in pINV-integrated strains or might simply reflect different mxiC::lacZ gene dosages between the autonomously replicating pINV and the integrated pINV and (ii) that the reduced expression of the mxiC gene is still sufficient to allow full expression of the invasive phenotype. The introduction of virF into HN580/32, either on the lowcopy-number plasmid pHW745 or on the high-copy-number plasmid pMYSH6504, induces only a moderate increase in b-galactosidase activity at 378C (Table 2), in spite of the pMYSH6504-induced high-level expression of virB mRNA in HN280/32, both at 30 and at 378C (Fig. 4). Moreover, HN280/32 transformed with pHW745 or pMYSH6504 exhibits low levels of Ipa proteins (Fig. 5) and of contact-mediated hemolysis and invades HeLa cells, although with low efficiency (Table 3). These results indicate that in HN280/32, virB transcription depends on the level of VirF and suggest that the different topological status of the integrated pINV together with an increased VirF level might induce conditions that favor VirF binding within the virulence regulon to sites recognized only when the pINV plasmid is integrated, interfering with the correct expression of the virB-regulated virulence phenotypes. The introduction of the Dhns deletion in HN580/32 (strain HN780/32) induces high-level expression of the reporter gene both at 30 and at 378C. Moreover, strain HN680/32, the Dhns derivative of strain HN280/32, produces contact-mediated hemolysis and invades HeLa cells, albeit at a lower extent relative to that of HN280 grown at 378C (Table 3). These results indicate that in pINV-integrated strains, H-NS plays a key role in regulating the expression of invasion genes by negatively controlling the expression of the virB gene, both at 30 and at 378C.

4710

COLONNA ET AL.

To confirm these results, we have isolated two independent Tn5-CM (37) transposon-induced mutants of the pINV-integrated strain HN580/32 that express b-galactosidase activity at 30 and at 378C. Molecular mapping of the two mutants and of their respective HN280/32 chloramphenicol-resistant (Cmr) transductants indicated that the Tn5-CM transposon is inserted within the hns locus (6). Moreover, the two Cmr HN280/32 transductants both produce, at 30 and at 378C, contact-mediated hemolysis and invaded HeLa cells at the same extent as the Dhns strain HN780/32 did (data not shown), reinforcing our conclusions on the crucial role of hns in repressing virulence gene expression in pINV-integrated strains. We speculate that changes in DNA topology, occurring upon pINV integration, may produce conditions that at 378C reduce the efficiency of VirF as a virB activator through a more effective binding of H-NS to the virB promoter. The effect of an increased virB and virF gene dosage or of the introduction of a Dhns deletion was also studied with strains harboring an autonomously replicating pINV. While at 378C pBN1 (virB) induces in HN580 a significant increase in b-galactosidase activity, the introduction of pMYSH6504 (virF) or the presence of a Dhns deletion induces temperatureindependent expression of the reporter gene. When we assayed virB transcription in HN280 we found that, as for HN280/32, pMYSH6504 dramatically increases the level of virB mRNA, both at 30 and at 378C (Fig. 4). Moreover, at 308C HN280 (pMYSH6504) produces Ipa proteins, exhibits contact-mediated hemolysis, and invades HeLa cells to an extent similar to that of HN280 grown at 378C (Fig. 5 and Table 3) and is able to spread intracellularly and to infect adjacent cells. Cell-tocell spreading is known to occur only at 378C and requires the expression of the icsA gene, which, like virB, is regulated positively by virF and negatively by hns (10, 42). This indicates that in HN280 an increased virF gene dosage induces temperatureindependent expression of icsA, as well as of virB. On the other hand, at 378C HN280(pMYSH6504) produces even higher levels of virB mRNA and of IpaB and IpaC proteins than it does at 308C, but unexpectedly the expression of contact-mediated hemolysis and of HeLa cell invasiveness is considerably repressed (Table 3). We speculate that this inhibitory effect could be due to an excess of VirB and VirF that at 378C might interfere with the coordinated expression of the invasive phenotype. Nevertheless, to ascertain if the virF gene dosage effect, which induces deregulated expression of virB and of the virB-dependent virulence phenotypes, also occurs in other enteroinvasive bacteria, we transformed the S. flexneri serotype 5 strain M90T (36) with either pHW745 or pMYSH6504. As with HN280, we found that at 308C only pMYSH6504-transformed cells show levels of contact-mediated hemolysis and HeLa cell invasiveness comparable to those of the parental strain M90T grown at 378C (49). As already reported for other microorganisms (16) and in agreement with the data obtained by measuring the activity of the reporter gene, the introduction of a Dhns deletion in HN280 (strain HN680) induces temperature-independent expression of contact-mediated hemolysis and of the capacity to invade HeLa cells (Table 3), albeit to a somewhat lower level than in wild-type HN280 at 378C. This most probably reflects the fact that the Dhns deletion, according to our observations, induced more slowly growing colonies and slightly increased pINV instability (10). Recently, it has been reported that at 308C H-NS causes repression of invasion genes by blocking transcription initiation from the virB promoter (42). The data obtained in this study indicate that the negative regulatory effect of H-NS on virB transcription can be efficiently counteracted by increasing

J. BACTERIOL.

the virF gene dosage. These results are in agreement with those of Tobe et al. (41), who have reported that at 308C, in a virF deletion derivative of the hns1 S. flexneri 2a strain YSH6000 transformed with plasmid vectors carrying virF, the level of virB mRNA increases in accord with the virF gene dosage and have excluded the possibility that the temperature-regulated virB expression is due to growth temperature-dependent alteration of the DNA binding properties of the two regulatory proteins H-NS and VirF. This raises the possibility that VirF activation of virB transcription might be the key regulatory step controlling temperature-regulated virulence gene expression in enteroinvasive bacteria. In fact, here we report that in HN280 the expression of virF is temperature regulated (we estimated the level of virF mRNA at 308C to be approximately one-third of that observed at 378C), and a very similar result (one-quarter reduction) has also been reported for S. flexneri 2a strain YSH6000 (41). This has led us to speculate that the reduction in virF expression occurring at 308C might be the limiting factor governing the thermoregulated expression of virB and to examine the effect of hns on the expression of virF. Using a translational gene fusion between the virF promoter and the lacZ reporter gene, we confirmed that virF expression is modulated by the growth temperature and discovered that it is also negatively regulated by hns (Table 4). In this respect, it is tempting to speculate that hns thermoregulates virulence gene expression by finely modulating the transcription of virF and that the temperature-independent expression of the virB-regulated virulence phenotype in hns mutants might be due to induced virF transcription rather than simply to the absence of H-NS. How do different levels of VirF regulate the expression of virulence genes in enteroinvasive bacteria? Dagberg and Uhlin (10) raised the hypothesis that VirF could be viewed as a transcriptional regulator which has the property of alleviating and/or counteracting the ‘‘silencing’’ effect of H-NS. Our results support this hypothesis, and we suggest that at 308C the reduced level of VirF is not sufficient to overcome the negative regulatory effect of H-NS and thus to activate virulence gene expression in enteroinvasive microorganisms. Several explanations of how virF thermoregulates virB transcription are possible. It has been reported that an increase in the level of the H-NS protein in the cell enhances repression of the ipa and icsA operons, particularly at a low growth temperature (10). Moreover, H-NS is known to influence DNA superhelicity (11, 14, 19, 21) and VirF binds to the virB promoter in a DNA-topology-dependent manner (42). Thus, VirF and H-NS might compete for binding to the virB promoter. At 308C, H-NS binding could be favored because of the lower level of VirF and could induce topological changes affecting the ability of VirF to recognize its specific upstream target sequence. Again, the AraC family of DNA-binding proteins are known to form dimers and to control gene expression by binding to multiple sites within the promoter regions (12, 25, 47). The upstream region of the virB promoter that is recognized by VirF is large enough to host more than one VirFbinding site (23, 42). If more than one site needs to be bound by VirF, the reduced amount of VirF at 308C might fail to saturate all sites and to promote transcription of virB. Alternatively, VirF might need to form multimeric complexes in order to promote transcription of virB, and the formation of these complexes might be strictly dependent on the concentration of VirF within the cell. In conclusion, the data reported here indicate that the expression of pINV-encoded virulence genes is differently regulated in wild-type and in pINV-integrated EIEC strains and that hns plays an important role in such a regulation. Virulence

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VOL. 177, 1995

modulation of human pathogens in response to environmental stimuli and/or to different stages of the infectious process may be relevant for bacterial survival and multiplication within the host. In this respect, although the specific mechanism leading to pINV integration has not been elucidated, we would like to consider pINV integration as another strategy by which enteroinvasive bacteria modulate the expression of pINV-encoded virulence genes.

19.

20.

21.

ACKNOWLEDGMENTS We are indebted to P. J. Sansonetti for kindly providing mouse MAbs H4 and J22. We thank C. Gualerzi for plasmid pPLc11; C. Sasakawa for plasmids pBN1, pMYSH6504, and pCHR83; H. Watanabe for plasmid pHW745; M. J. Casadaban for plasmid pMC1403; A. T. Maurelli for strain BS184; and P. Lejeune for strain TP504 and plasmid pDIA510. We also thank A. Calconi and R. Topazi for expert technical assistance. We are grateful to G. Micheli for critical reading of the manuscript. This work was supported by the Consiglio Nazionale delle Ricerche Target Program on Biotechnology and Bioinstrumentation, by MURST Progetto Controllo della Patogenicita` Microbica, and, in part, by the Foundation ‘‘Istituto Pasteur-Fondazione Cenci Bolognetti.’’

22.

23. 24.

25. 26.

1.

2.

3. 4.

5.

6. 7.

8. 9. 10. 11. 12.

13.

14. 15. 16. 17. 18.

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