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INFECTION AND IMMUNITY, Oct. 1998, p. 4957–4964 0019-9567/98/$04.0010 Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Vol. 66, No. 10

Expression of the Virulence Plasmid-Carried Apyrase Gene (apy) of Enteroinvasive Escherichia coli and Shigella flexneri Is under the Control of H-NS and the VirF and VirB Regulatory Cascade FRANCESCA BERLUTTI,1 MARIASSUNTA CASALINO,2 CARLO ZAGAGLIA,3 PIERA ASSUNTA FRADIANI,4 PAOLO VISCA,5 AND MAURO NICOLETTI6* Istituto di Microbiologia,1 Dipartimento di Medicina Sperimentale e Patologia,3 and Dipartimento di Biologia Cellulare e dello Sviluppo,4 Sezione di Scienze Microbiologiche, Universita ` di Roma La Sapienza, 00185 Rome, Dipartimento di Biologia, Universita ` Roma Tre, 00146 Rome,2 Istituto Superiore di Sanita `, 00161 Rome,5 and Dipartimento di Scienze Biomediche, Sezione di Microbiologia, Universita ` G. D’Annunzio, 66100 Chieti,6 Italy Received 11 May 1998/Returned for modification 26 June 1998/Accepted 21 July 1998

The transcription of the virulence plasmid (pINV)-carried invasion genes of Shigella flexneri and enteroinvasive Escherichia coli (EIEC) is induced at 37°C and repressed at 30°C. In this work, we report that the O135: K2:H2 EIEC strain HN280 and S. flexneri SFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce apyrase (ATP-diphosphohydrolase), the product of the apy gene. In addition, the S. flexneri strains, but not the EIEC strain, produce a nonspecific phosphatase encoded by the phoN-Sf gene. Both apy and phoN-Sf are pINV-carried loci whose contribution to the pathogenicity of enteroinvasive microorganisms has been hypothesized but not yet established. We found that, like that of virulence genes, the expression of both the apy and the phoN-Sf genes was temperature regulated. Strain HN280/32 (a pINV-integrated avirulent derivative of HN280 which has a severe reduction of virB transcription) expressed the apy gene in a temperature-regulated fashion but to a much lower extent than wild-type HN280, while the introduction of the Dhns deletion in HN280 and in HN280/32 induced the wild-type temperature-independent expression of apyrase. These results indicated that a reduction of virB transcription, which is known to occur in the pINV-integrated strain HN280/32, accounts for reduced apyrase expression and that the histone-like protein H-NS is involved in this regulatory network. Independent spontaneously generated mutants of HN280 and of SFZM53 which had lost the capacity to bind Congo red dye (Crb2) were isolated, and the molecular alterations of pINV were evaluated by PCR analysis. Alterations of pINV characterized by the absence of virF or virB and by the presence of the intact apy locus or intact apy and phoN-Sf loci were detected among Crb2 mutants of HN280 and SFZM53, respectively. While all Crb2 apy1 mutants of HN280 failed to produce apyrase, Crb2 apy1 phoN-Sf1 mutants of SFZM53 lacked apyrase activity but produced a nonspecific phosphatase, like parental SFZM53. Moreover, the introduction of recombinant plasmids carrying cloned virF (pMYSH6504) or virB (pBN1) into Crb2 mutants of HN280 and SFZM53 lacking virF or virB, respectively, fully restored temperature-dependent apyrase expression to levels resembling those of the parental strains. Taken together, our results demonstrate that, as has already been shown for invasion genes, apy is another locus whose expression is controlled by temperature, H-NS, and the VirF and VirB regulatory cascade. In contrast, the temperature-regulated expression of the nonspecific phosphatase does not appear to be under the control of the same regulatory network. These findings led us to speculate that apyrase may play a role in the pathogenicity of enteroinvasive bacteria. The mechanism of pathogenicity of Shigella flexneri and enteroinvasive Escherichia coli (EIEC) is based on the capacity of these strains, once ingested, to reach the colonic mucosa and to invade colonic epithelial cells, leading to intracellular bacterial multiplication, spread to adjacent cells, cell death, and eventually inflammation and ulceration of the colonic mucosa (14, 26). The pathogenicity of enteroinvasive microorganisms is a complex phenomenon which requires the coordinated expression of several genes located both on the virulence plasmid (pINV) and on the chromosome. The expression of pINVcarried virulence genes is modulated by environmental stimuli (temperature, pH, osmolarity, contact with epithelial cells, and so forth) which are sensed by the bacterium (11, 16, 25, 27, 29, 31, 41). Temperature is a key factor, since the transcription of virulence genes is strongly repressed during the growth of en-

teroinvasive bacteria at temperatures below 37°C. The global regulator hns has been shown to direct the temperature-regulated expression of virulence genes by repressing their transcription during growth at 30°C (10, 11, 16, 17). The current model for temperature-dependent regulation suggests that at a nonpermissive temperature, H-NS represses transcription by preventing the binding of the positive regulator VirF to target promoters (10, 17, 31, 42). At the permissive temperature of 37°C, H-NS repression is relieved and VirF binds to the virB and icsA (virG) promoters, thus activating their transcription. The activation of virB expression results in the production of VirB, which serves as a positive effector, inducing the expression of the invasion genes ipa, mxi, and spa (10, 41–43). Moreover, we previously showed that, when pINV is integrated into the host chromosome, H-NS represses the expression of virulence genes by severely reducing virB transcription at 37°C as well (9, 46). Although knowledge of the pathogenicity of enteroinvasive bacteria has been enormously improved during the last 15 years, some molecular aspects of this complex mechanism still

* Corresponding author. Mailing address: Dipartimento di Scienze Biomediche, Sezione di Microbiologia, Universita` G. D’Annunzio, Via dei Vestini, 31, 66100 Chieti, Italy. Phone: 39-871-3555279. Fax: 39871-3555282. E-mail: [email protected]. 4957

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INFECT. IMMUN. TABLE 1. Bacterial strains and plasmids

Strain or plasmid

Strains HN280 HN280/32 HN280-3 and -8 HN280-16 and -35 HN680 HN680/32 SFZM53 SFZM53-1 SFZM53-11 SFZM53-15 M90T BS176 454 Plasmids pBR322 pMYSH6504 pBN1 a

Source or reference(s)

Relevant characteristicsa

Wild-type EIEC strain of serotype O135:K2:H2; contains virulence plasmid pHN280; apy1 pHN280-integrated derivative of strain HN280; noninvasive; apy1 Independent spontaneous Crb2 derivatives of strain HN280; pINV carries apy, virB, and icsA and lacks virF Independent spontaneous Crb2 derivatives of strain HN280; pINV carries apy, virF, and icsA and lacks virB D(hns tdk adhE oppABCD)118 zch-506::Tn10 derivative of strain HN280; invasive; apy1 Tcr D(hns tdk adhE oppABCD)118 zch-506::Tn10 derivative of strain HN280/32; invasive; apy1 Tcr Wild-type invasive S. flexneri strain of serotype 4; apy1 phoN-Sf1 Independent spontaneous Crb2 derivative of strain SFZM53; pINV carries apy, virB, and icsA and lacks virF and phoN-Sf Independent spontaneous Crb2 derivative of strain SFZM53; pINV carries apy, phoN-Sf, and virB and lacks virF and icsA Independent spontaneous Crb2 derivative of strain SFZM53; pINV carries apy, phoN-Sf, virF, and icsA and lacks virB Wild-type invasive S. flexneri strain of serotype 5; apy1 phoN-Sf1 pINV-cured derivative of strain M90T; noninvasive Wild-type invasive S. flexneri strain of serotype 2a; apy1 phoN-Sf1

7, 9; 46 This This 9 9 6 This

Cloning plasmid vector; Apr Tcr pBR322-derived vector carrying the virF gene; Apr Kmr pBR322-derived vector carrying the virB gene; Tpr

33 32 1

this study study study

study

This study This study 34 35 2

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

need to be elucidated. For instance, it has been shown that the presence of intracellular bacteria dramatically affects host cell metabolism; S. flexneri-infected HeLa cells show biochemical characteristics typical of cells undergoing metabolic stress, including a decrease in total deoxynucleoside triphosphate (dNTP) levels (12, 23, 49). The pINV-carried apy gene, which encodes apyrase (ATP-diphosphohydrolase), an enzyme belonging to class A of bacterial acid phosphatases (4, 20, 39), was recently identified in virulent Shigella spp. and in related EIEC but not in noninvasive E. coli. A possible role of apyrase in the dramatic decrease in dNTP levels in host cells during intracellular multiplication has been suggested (4, 23). Based on its periplasmic localization and on its enzymatic activity, it has been proposed that apyrase could be considered a general cytotoxin, possibly involved, directly or indirectly, in damaging cellular metabolism and eventually in host cell death (4, 23). The hypothesis that apyrase could be considered a virulenceassociated protein and therefore might play a role in pathogenesis is further supported by the observations that the production of apyrase was found to be temperature dependent and that apyrase was absent in an S. flexneri 2a spontaneously derived mutant unable to bind Congo red dye (Crb2) (4). Moreover, the phoN-Sf gene, encoding a nonspecific phosphatase (another class A phosphatase), was recently identified on pINV of virulent Shigella spp. and EIEC (39, 44). Apyrase and the nonspecific phosphatase differ in their specific activities toward different substrates and thereby can be easily distinguished in bacterial sonic extracts by the zymogram assay (4, 20, 39, 44). Even though specific roles have not yet been assigned, the presence of apy and phoN-Sf on pINV of Shigella spp. and EIEC raises the possibility that these two genes play an important role in the physiology of enteroinvasive bacteria. In this work, we demonstrate that the O135:K2:H2 EIEC strain HN280 produces apyrase, while S. flexneri SFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce both apyrase and a nonspecific phosphatase. Like that of other virulence genes, the expression of the apyrase gene was found to be controlled at the transcriptional level by temperature, H-NS, and the VirF and VirB regulatory cascade. We also

show that pINV integration into the host chromosome severely reduces apy expression at 37°C, this inhibitory effect being reversed by the introduction of a Dhns deletion which induces temperature-independent apy expression. Finally, we provide evidence that the expression of the nonspecific phosphatase (phoN-Sf) in S. flexneri is also temperature regulated but is not controlled by the same regulatory network as that governing the expression of virulence genes. MATERIALS AND METHODS Bacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used are listed in Table 1. Growth media were Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.), Terrific broth medium (33), and Luria broth medium (33). The solid media contained 1.5% agar. Congo red (Sigma Chemical Co., St. Louis, Mo.) was added at 0.01% to Trypticase soy agar to determine Congo red binding (Crb phenotype). Antibiotics were used at the following concentrations: ampicillin, 100 mg/ml; kanamycin, 30 mg/ml; tetracycline, 5 mg/ml; and trimethoprim, 10 mg/ml. Genetic and molecular procedures. Plasmids were introduced into EIEC and S. flexneri strains by electroporation. Isolation of plasmids, restriction digestion, electrophoresis, and purification of DNA fragments were carried out as described by Sambrook et al. (33). Rapid plasmid DNA extractions were performed by the method of Kado and Liu (18). Southern blot hybridizations were performed as previously described (9). The probe was a 1,124-bp PCR-generated fragment encompassing the region from positions 2232 to 1892 relative to the ATG translation start codon of the apy gene. The primers used to amplify the probe were designed on the basis of the available apy sequence (GenBank accession no. U04539) and were designated FB30 (59-CATCATAATCAAGAG ACAAAACG-39, corresponding to nucleotides 9 to 31) and FB31 (59-TTTTCT GCTTCTGCCGCA-39, corresponding to nucleotides 1132 to 1115). Amplification was carried out by use of a model 480 DNA thermal cycler (Perkin-Elmer, Foster City, Calif.) with total DNA from strain HN280 as the template. The PCR mixture contained 13 PCR buffer (Boehringer Mannheim Biochemicals), 0.2 mM each dNTP, 25 to 50 pmol of each primer, 20 ng of template DNA, 2 U of Taq polymerase (Boehringer), and 5% glycerol in a total volume of 50 ml and was overlaid with mineral oil. Samples were subjected to 25 cycles, each cycle comprising 30 s of denaturation at 95°C, 3 min of primer annealing at 53°C, and 1 min of extension with Taq polymerase at 72°C. The PCR-generated fragment of the expected size was visualized and, when required, recovered from a 1% agarose gel and 32P labelled by the random priming method (33). Hybridization and washing of the blots were performed under stringent conditions as previously described (9). Apyrase and nonspecific phosphatase assays. Apyrase and nonspecific phosphatase activities in whole-cell extracts were detected by the zymogram technique (4, 39). Bacteria grown overnight in Terrific broth at 30 or 37°C were

VOL. 66, 1998 washed twice with sterile saline, concentrated in the same medium to an A600 of >40, and then disrupted by sonication by three 30-s bursts with an ultra-Sonifier (Soniprep 150; MSE, Loughborough, England). Cell debris was removed by centrifugation at 10,000 3 g for 10 min, and 15-ml aliquots of sonicated extracts were boiled for 5 min in Laemmli’s buffer (21) without 2-mercaptoethanol and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, apyrase activity was detected as described by Bhargava et al. (4). Briefly, polyacrylamide gels were incubated for 3 h at 37°C in several changes of renaturation buffer (50 mM Tris-HCl [pH 7.0], 1% [vol/vol] Triton X-100) to obtain renaturation of apyrase. After renaturation, the gels were equilibrated for 1 h in several changes of 100 mM Tris-HCl (pH 7.5) and incubated for 30 min at approximately 10°C in 100 mM Tris-HCl–10 mM EDTA–1 mM ATP; apyrase activity was visualized by immersing the gels in a 4:1 (vol/vol) freshly prepared solution of acidified ammonium molybdate (5 mM ammonium molybdate, 0.12 M sulfuric acid) and 10% (wt/vol) ascorbic acid. Apyrase activity was indicated by the appearance on the gels after 10 min of incubation at room temperature of a blue precipitate located at a migration distance corresponding to the Mr of the polypeptide component of the renaturated enzyme. Nonspecific phosphatase activity was determined as previously described (39). Briefly, after electrophoresis, the enzyme was renatured by incubation of the gels for 3 h at 37°C in several changes of renaturation buffer (50 mM sodium acetate buffer [pH 6.0], 1 mM MgCl2, 1% [vol/vol] Triton X-100). The gels were then equilibrated for 1 h in several changes of 100 mM sodium acetate buffer (pH 6.0). Nonspecific phosphatase activity was revealed by the addition of 0.25 mM 5-bromo-4-chloro-3-indolylphosphate to the equilibration buffer. Enzymatic activity was indicated by the presence after overnight incubation at 37°C of blue bands located at the expected migration distance of the enzyme. Apyrase and nonspecific phosphatase activities were quantified by scanning of the developed gels with an Ultrascan XL laser densitometer (LKB/Pharmacia) and are expressed as arbitrary absorbance units (AU). PCR analysis. The primers used to amplify the virF, virB, virG, and phoN-Sf genes from total DNA preparations of EIEC and S. flexneri strains were designed on the basis of available sequences and were designated KK9 (59-GAGGAGG TTTCTATC-39, corresponding to nucleotides 527 to 541) and KG19 (59-CTTT GCTGCATGATG-39, corresponding to nucleotides 844 to 830) for the virF sequence (GenBank accession no. M29172), KF26 (59-GCGAAAGTCACTCG TC-39, corresponding to nucleotides 746 to 761) and KG20 (59-CCATCATGC CGCATCC-39, corresponding to nucleotides 1426 to 1411) for the virB sequence (GenBank accession no. X14340), GU23 (59-GAAAAGTTGCGGTCTC-39, corresponding to nucleotides 327 to 342) and GT18 (59-AGGTAATTCTCCGGC C-39, corresponding to nucleotides 642 to 627) for the icsA (virG) sequence (22), and PSHN (59-CCTTTGTTTTAGCATCTTCTG-39, corresponding to nucleotides 6 to 26) and TSHN (59-TTTCCGAGAGTGGTAAAGG-39, corresponding to nucleotides 1003 to 985) for the phoN-Sf sequence (GenBank accession no. D82966). The primers used to amplify the 1,124-bp fragment of apy and the cycling conditions for PCR amplification are described above. Annealing was performed at 42°C for virF, at 43°C for virB, at 48°C for icsA, and at 53°C for phoN-Sf amplification. RNA blot analysis. Bacterial strains were grown in Luria broth at 30 or 37°C to an A600 of >0.6. Total RNA was extracted by a modification of the hot phenol method as previously described (9) and quantified spectrophotometrically at 260 nm (A260). The quality of each RNA preparation was checked by visualization of rRNA bands in ethidium bromide-stained agarose gels electrophoresed under nondenaturing conditions. For Northern blot analysis, total RNA (20-mg samples) was denatured at 100°C for 5 min in the presence of 2 M formaldehyde and 50% formamide, separated on a formaldehyde-morpholinepropanesulfonic acid (MOPS)-agarose gel, transferred to Hybond-N membranes (Amersham Corp.), and then hybridized with a 672-bp PCR-generated DNA fragment (apy probe) as described by Sambrook et al. (33). Two primers were designed to amplify the 672-bp internal portion of the apy DNA coding region from a total DNA preparation of HN280 and were designated AGZ17 (59-CTGAAGGCAGAAGGTT TTCT-39, corresponding to nucleotides 310 to 329) and AGZ18 (59-TTATGGG GTCAGTTCATTGGT-39, corresponding to nucleotides 981 [the last nucleotide of the TAA stop codon] to 961) for the apy sequence (GenBank accession no. U04539). Primer annealing was performed at 50°C. The PCR-generated fragment was recovered from an agarose gel and 32P labelled by the random priming method (33). For quantitative transcript estimations, dot blot analysis was performed. RNA samples (10, 5, and 2.5 mg) were suspended in denaturing solution (73 SSC [1X SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 14% formaldehyde) and heated for 5 min at 65°C. RNA dilutions were applied in duplicate to Hybond-N membranes under vacuum and hybridized with the 32P-labelled 672-bp apy probe as previously described (33). After hybridization and washing, dried membranes were analyzed in an Instant Imager electronic autoradiographer (Camberra Packard) in order to quantify the amount of bound probe. Results were adjusted by subtracting the background level for the filters and were normalized by probing duplicate filters with the 32P-labelled rrnB probe (7.5-kb BamHI fragment) from plasmid pKK3535 (5).

EXPRESSION OF THE apy GENE

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RESULTS Physical organization of the apy locus in EIEC and S. flexneri. The apy gene was recently located in a 2.1-kb HindIII fragment of pINV of both S. flexneri and EIEC (4) and in the 43.1-kb SalI A fragment of pINV of S. flexneri YSH6000 (serotype 2a) (38). Based on the published apy sequence (4), two primers were designed (see Materials and Methods) to detect by PCR the presence of the apy gene in a panel of EIEC and S. flexneri strains. The expected 1,124-bp fragment was amplified from total DNAs of the O135:K2:H2 EIEC strain HN280 as well as of S. flexneri SFZM53, M90T, and 454, belonging to serotypes 4, 5, and 2a, respectively (Table 1). The PCR-generated fragment of HN280 was then used as a probe in Southern hybridization experiments with pINV DNA as well as with HindIII-digested total DNA preparations. The 32 P-labelled probe recognized pINV DNA of strains HN280, SFZM53, M90T, and 454 and chromosomal DNA of strain HN280/32, a pINV-integrated derivative of strain HN280. Moreover, the probe hybridized with a 2.1-kb DNA fragment from HindIII genomic digests of all of these strains, independently of whether pINV was autonomously replicating or chromosomally integrated (data not shown). These results indicate that apy is located on pINV of all of the strains examined and that the apyrase DNA region is well conserved among EIEC and S. flexneri strains of different serotypes and geographical origins. Phosphatase activities of EIEC and S. flexneri. The apyrase and nonspecific phosphatase activities of EIEC and S. flexneri strains were evaluated by the zymogram technique. Equivalent amounts of protein extracts were subjected to polyacrylamide gel electrophoresis. Enzymatic activities were detected by specific staining and quantified by densitometric scanning of developed gels as outlined in Materials and Methods. EIEC strain HN280 and S. flexneri SFZM53, M90T, and 454 all exhibited specific ATP-hydrolyzing (apyrase) activity, albeit at different levels. Interestingly, the enzymatic activity was dramatically reduced when the strains were cultured at 30°C (Fig. 1 and Table 2). To determine whether the expression of apyrase was controlled by temperature at the transcriptional level, Northern hybridization and dot blot analysis of total RNAs from strains grown at 30 and 37°C were performed with the 672-bp PCR-generated fragment internal to the apy DNA coding region as a probe (apy probe; see Materials and Methods). In agreement with the apyrase activity data, the RNA blot results demonstrated that the transcription of apy in HN280, SFZM53, M90T, and 454 was strictly regulated by temperature, being at least 12-fold higher at 37°C than at 30°C (Fig. 1B). Moreover, S. flexneri SFZM53, M90T, and 454 but not EIEC strain HN280 or BS176, a pINV-cured derivative of S. flexneri M90T, also produced a nonspecific phosphatase encoded by the phoN-Sf gene (44). Like that of apyrase, the expression of the nonspecific phosphatase was also found to be regulated by temperature (Table 3). The finding that the expression of apyrase and the expression of nonspecific phosphatase were both regulated by growth temperature, being repressed at 30°C and activated at 37°C, prompted us to examine whether the expression of these two genes might be under the control of the effector genes virF and virB and of the nucleoid-associated protein H-NS, which are known to regulate the expression of virulence genes ipa, mxispa, and icsA (virG) in a temperature-dependent fashion (10, 16, 17, 42). Effect of hns on apy expression in strains harboring autonomously replicating or chromosomally integrated pINV. We previously showed that pINV of EIEC strain HN280 is able to

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INFECT. IMMUN.

FIG. 1. Zymogram analysis of apyrase activity (A) and RNA hybridization analysis (B) of O135:K2:H2 EIEC strain HN280 and S. flexneri SFZM53 (serotype 4), M90T (serotype 5), and 454 (serotype 2a) grown at 30 and 37°C. The conditions used to renature and to visualize apyrase activity are described in Materials and Methods. MW, prestained molecular weight markers (Bio-Rad, Hercules, Calif.); sizes (in thousands) are shown to the right of panel A. Northern blots were probed with the apy probe, a 32P-labelled 672-bp PCR-generated DNA fragment (internal to the apy coding region) described in Materials and Methods. Each lane was loaded with 20 mg of total RNA. The specific b-emission value of each RNA sample (counts per minute per microgram of RNA), determined by electronic analysis of RNA dot blots, is given within a circle below each lane. The electrophoretic mobilities of the 23S, 16S, and 5S rRNA fractions are indicated to the right of panel B.

integrate into the host chromosome and that the integration inhibits the expression of pINV-carried virulence genes by severely reducing virB transcription (9, 46). The inhibition of virB transcription occurring upon pINV integration is probably due to changes in the DNA topology which favor more stable binding of H-NS to the virB promoter, since the introduction of a Dhns deletion in pINV-integrated strains restores virB transcription at both 30 and 37°C, leading to temperature-independent expression of virulence genes (9). To ascertain whether apy expression is under the control of the same regulatory network as that governing the expression of virulence genes, we assayed sonic extracts and performed RNA blot analysis of strains HN680 and HN280/32, a Dhns deletion derivative and a pINV-integrated derivative of wild-type EIEC strain HN280 (9), respectively, and of strain HN680/32, a Dhns derivative of strain HN280/32 (9), cultured at 30 and 37°C. The results obtained (Fig. 2 and Table 2) indicated that apy expression is negatively regulated by H-NS at the transcriptional level, since the Dhns mutant HN680 showed deregulated, temperature-independent apy expression. Integration of pINV into the chromosome of HN280/32 caused a strong reduction of apy expression (approximately threefold lower than that of parental HN280) at 37°C, while introduction of the Dhns mutation into HN280/32 (strain HN680/32) increased the expression of apyrase at both 30 and 37°C. Taken together, these results indicate that the reduced virB transcription occurring upon pINV integration (9) leads to reduced apyrase expression and that H-NS represses apy transcription in the pINV-integrated strain grown at both 30 and 37°C. Expression of apyrase but not of nonspecific phosphatase is under the control of the VirF and VirB regulatory cascade. To investigate the control mechanisms for apy expression, we isolated and analyzed by PCR 66 and 35 spontaneous, independently derived Crb2 mutants of EIEC strain HN280 and of S. flexneri SFZM53, respectively. The Crb2 phenotype occurs at a low frequency in S. flexneri and EIEC and arises as a consequence of molecular alterations (deletions, insertions, and curing) of pINV (8, 36, 38). Structural alterations of pINV encompassing apy, virB, virF, and/or icsA were detected in

TABLE 2. Expression of apyrase activity by EIEC and S. flexneri strains Strain

Relevant genotype

HN280 HN680 HN280-3 HN280-3(pMYSH6504) HN280-8 HN280-8(pMYSH6504) HN280-16 HN280-16(pBN1) HN280-35 HN280-35(pBN1) HN280/32 HN680/32 SFZM53 SFZM53-1 SFZM53-1(pMYSH6504) SFZM53-15 SFZM53-15(pBN1) M90T BS176 454

Wild-type EIEC Dhns DvirF DvirF pvirF DvirF DvirF pvirF DvirB DvirB pvirB DvirB DvirB pvirB apy (chromosome) Dhns Wild-type S. flexneri serotype 4 D(virF phoN-Sf) D(virF phoN-Sf) pvirF DvirB DvirB pvirB Wild-type S. flexneri serotype 5 pINV cured Wild-type S. flexneri serotype 2a

a

Apyrase activitya at: 30°C

37°C

0.146 0.665 0.034 0.123 0.072 0.187 0.022 0.113 0.016 0.086 0.090 0.718 0.144 0.026 0.095 0.034 0.103 0.134 ND 0.053

0.925 0.946 0.042 0.754 0.145 0.691 0.028 0.654 0.036 0.806 0.238 0.796 0.736 0.112 1.168 0.052 0.851 0.665 ND 0.692

Ratio of apyrase activity (37°C/30°C)

6.3 1.4 1.2 6.1 2.0 3.7 1.3 5.8 2.2 9.4 2.6 1.1 5.1 4.3 12.3 1.5 8.3 5.0 13.0

Expressed as AU obtained by scanning of developed zymograms as described in Materials and Methods. Results are averages for at least four independent experiments. Standard errors were less than 15%. ND, not detectable.

EXPRESSION OF THE apy GENE

VOL. 66, 1998

dicating that the expression of phoN-Sf is not under the control of the VirF and VirB regulatory cascade.

TABLE 3. Expression of nonspecific phosphatase by S. flexneri strains

Strain

M90T BS176 SFZM53 SFZM53-11 SFZM53-15 454

Relevant genotype

Wild-type S. flexneri serotype 5 pINV cured Wild-type S. flexneri serotype 4 D(virF icsA) DvirB Wild-type S. flexneri serotype 2a

Nonspecific phosphatase activitya at:

4961

Ratio of apyrase activity (37°C/30°C)

30°C

37°C

0.226

1.198

5.3

ND 0.153

ND 0.978

6.4

0.104 0.093 0.136

1.056 1.128 0.942

10.1 12.1 6.9

a Expressed as AU obtained by scanning of developed zymograms as described in Materials and Methods. Results are averages for at least four independent experiments. Standard errors were less than 15%. ND, not detectable.

Crb2 mutants by PCR with pairs of specific oligonucleotide primers (see Materials and Methods for details). Since nonspecific phosphatase activity was detected only in S. flexneri strains, Crb2 mutants of SFZM53 were also assayed for the presence of phoN-Sf. The absence of specific amplified fragments in PCRs was suggestive of the loss of related genes on the rearranged pINV. A specific apy-amplifiable fragment was obtained from 38 (57.6%) of the 66 Crb2 mutants of HN280 and from 30 (85.7%) of the 35 Crb2 mutants of SFZM53. When sonic extracts were assayed, none of these HN280 or SFZM53 Crb2 mutants produced apyrase activity at detectable levels (data not shown). To confirm the presence of an intact apy locus in the Crb2 mutants, Southern blot analysis was performed on HindIII genomic digests of 15 randomly selected Crb2 mutants for each strain with the 1,124-bp PCR-generated fragment as a hybridization probe for the apy locus. A hybridization pattern indistinguishable from that of parental wildtype strains was evident in all of the Crb2 mutants analyzed (data not shown). Moreover, the molecular alterations found more frequently in Crb2 derivatives which yielded the apy amplicon were the loss of virF and/or virB or the insertion sequence-like insertional inactivation of virF (28, 38). The analysis of Crb2 mutants of HN280 and of SFZM53 that harbor an entire apy locus but that fail to express apyrase argues against the possibility that virF is directly involved in activating apy expression. In fact, independent Crb2 mutants of HN280 and of SFZM53 harboring pINV alterations characterized by the absence of the virB locus and by the presence of intact apy and virF loci did not express apyrase activity. To gain further insight into the regulatory mechanism of apy expression, plasmids pMYSH6504 (virF) and pBN1 (virB) (Table 1) were separately introduced into Crb2 mutants, and transformants were assayed for apyrase activity by the zymogram assay as well as for apy expression by RNA blot analysis as shown in Table 2 and Fig. 3. The introduction of pMYSH6504 (virF) into Crb2 mutants HN280-3, HN280-8, and SFZM53-1 (they all lack virF) and of pBN1 (virB) into Crb2 mutants HN280-16, HN280-35, and SFZM53-15 (they all lack virB) (Table 1) restored temperature-regulated apy transcription and expression of apyrase activity at levels comparable to those of the isogenic wild-type strains. From these results it can be concluded that the expression of apyrase is under the control of the VirF and VirB regulatory cascade. Interestingly, Crb2 mutants SFZM53-11 (lacking virF) and SFZM53-15 (lacking virB) (Table 1) both produced a wild-type temperature-regulated nonspecific phosphatase (Table 3), in-

DISCUSSION The ability of S. flexneri and EIEC to successfully infect humans is due in part to the presence of a large plasmid (pINV) which encodes virulence determinants enabling these facultative intracellular pathogens to invade and to multiply within colonic epithelial cells and to disseminate intra- and intercellularly (14, 15, 34). The pINV-carried virulence genes are organized in regulons and have been shown to be located in noncontiguous SalI DNA fragments, encompassing only one-fifth of the total length of pINV DNA (14, 15, 24, 36, 37, 44). Virulence gene expression is a complex process which imposes a remarkable metabolic burden on the bacterial cell; multiple environmental stimuli are perceived by enteroinvasive bacteria and transduced into transcriptional responses, leading to a fine-tuning of the expression of virulence genes (11, 13, 16, 25, 27, 29, 31, 41). Temperature is a very important signal, and virF and virB have been found to form a regulatory cascade responsible for the induction of invasion genes. At 37°C, the positive regulator VirB activates transcription of the ipa locus (essential for entry into susceptible cells) and of the mxi and

FIG. 2. Zymogram analysis of apyrase activity (A) and RNA hybridization analysis (B) of O135:K2:H2 EIEC strain HN280 derivatives HN680 (Dhns), HN280/32 (pINV integrated), and HN680/32 (Dhns; pINV integrated) grown at 30 and 37°C. The conditions used to renature and to visualize apyrase activity are described in Materials and Methods. MW, prestained molecular weight markers (Bio-Rad); sizes (in thousands) are shown to the right of panel A. Northern blots were probed with the apy probe, a 32P-labelled 672-bp PCR-generated DNA fragment (internal to the apy coding region) described in Materials and Methods. Each lane was loaded with 20 mg of total RNA. The specific b-emission value of each RNA sample (counts per minute per microgram of RNA), determined by electronic analysis of RNA dot blots, is given within a circle below each lane. The electrophoretic mobilities of the 23S, 16S, and 5S rRNA fractions are indicated to the right of panel B.

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FIG. 3. Zymogram analysis of apyrase activity (A) and RNA hybridization analysis (B) of HN280-16 and SFZM53-15 (Crb2 mutants of O135:K2:H2 EIEC strain HN280 and of S. flexneri SFZM53 [serotype 4], respectively) and of pBN1 (virB) transformants grown at 30 and 37°C. The conditions used to renature and to visualize apyrase activity are described in Materials and Methods. MW, prestained molecular weight markers (Bio-Rad); sizes (in thousands) are shown to the right of panel A. Northern blots were probed with the apy probe, a 32P-labelled 672-bp PCR-generated DNA fragment (internal to the apy coding region) described in Materials and Methods. Each lane was loaded with 20 mg of total RNA. The specific b-emission value of each RNA sample (counts per minute per microgram of RNA), determined by electronic analysis of RNA dot blots, is given within a circle below each lane. The electrophoretic mobilities of the 23S, 16S, and 5S rRNA fractions are indicated to the right of panel B.

spa loci (which encode an entry-associated secretion type III apparatus) (10, 41, 42). Transcription of virB is, in turn, regulated by the action of two counteracting DNA-binding regulatory proteins, VirF and H-NS. VirF is a member of the AraC family which positively regulates virB and icsA transcription at 37°C, while H-NS is a histone-like negative regulator which prevents VirF-mediated activation of virB and icsA transcription at a low temperature (30°C) (10, 25, 45). Moreover, when pINV is integrated into the host chromosome, H-NS acts as a transcriptional repressor even at 37°C (9). As a consequence of this complex regulatory network, S. flexneri and EIEC strains show temperature-regulated expression of invasion genes, and virB transcription has been proposed as the key step for the thermoregulation of pINV-carried virulence genes (41, 42). In spite of the progress made in the understanding of the regulatory aspects of virulence gene expression, complete elucidation of the mechanisms of pathogenicity of S. flexneri and EIEC has not been achieved yet. In this respect, it has been reported that the presence of intracellular S. flexneri dramatically affects host cell metabolism (23). Even if the fate of infected cells can vary depending on the host cell type (12,

INFECT. IMMUN.

48, 49), cellular damage, lysis, and induction of programmed cell death are the final outcomes of a not yet fully elucidated process following intracellular multiplication. A decrease in total dNTP levels has been associated with the presence of intracellular bacteria, which might act either by directly altering the cellular metabolism or by scavenging dNTP molecules through the synthesis of specific dNTP-hydrolyzing enzymes which are released within the infected host cell (4, 23). Recently, the discovery on pINV of S. flexneri and EIEC of two genes encoding low-molecular-weight phosphatases, ATPdiphosphohydrolase (apyrase) and a nonspecific phosphatase, was reported (4, 44). The genes, apy and phoN-Sf, have been located on the distant 43.1-kb SalI A fragment and on the 9.6-kb SalI I fragment, respectively, of the virulence plasmid of S. flexneri YSH6000 (serotype 2a) (4, 36, 38, 44). Both genes encode periplasmic enzymes with biochemical characteristics of class A of acid phosphatases (19, 30, 39, 40). The present work was undertaken to investigate the regulation of phosphatases in enteroinvasive bacteria. We showed that the O135:K2:H2 EIEC strain HN280 produces apyrase, while S. flexneri strains of different serotypes and origins produce a nonspecific phosphatase in addition to apyrase, and that the expression of both enzymatic activities is regulated by temperature at the transcriptional level (Fig. 1 and Tables 2 and 3). The finding that both enzymes are temperature regulated led us to consider the possibility that the expression of apy and phoN-Sf could be under the control of the same regulatory network (hns, virF, and virB) as that governing the temperature-regulated expression of virulence genes in enteroinvasive bacteria. To test this hypothesis, we first studied apyrase expression in strains HN680, HN280/32, and HN680/32. HN280/ 32 is a noninvasive pINV-integrated derivative of wild-type EIEC strain HN280 which is unable to express pINV-carried virulence genes because of a severe reduction of virB transcription (9). HN680 and HN680/32 are Dhns derivatives of HN280 and of HN280/32, respectively. We previously showed (9) that the introduction of the Dhns deletion in HN280 activates virB transcription at the nonpermissive temperature of 30°C and, by consequence, induces the temperature-independent expression of virulence genes. The same Dhns mutation restores the expression of invasion genes, and thus invasiveness, when introduced into pINV-integrated strain HN280/32 cultured at either 30 or 37°C (9). As for pINV-carried virulence genes, we found that pINVintegrated strain HN280/32 showed a severe reduction of apy expression compared to parental EIEC strain HN280 (Fig. 2 and Table 2). Moreover, HN680 and HN680/32 showed temperature-independent apy transcription and expression of apyrase (Fig. 2 and Table 2). These results demonstrated that hns regulates apy expression by negatively controlling the transcription of the virB gene at 30°C and that the reduced expression of virB at 37°C in pINV-integrated strain HN280/32 results in poor apyrase expression. To confirm the effect of H-NS on apyrase expression, we also compared apyrase activities in HN280 and HN280/32, both carrying a silenced hns allele obtained by the insertion of transposon Tn5-CM within the hns locus (9). As expected, both HN280 and HN280/32 Tn5-CM::hns transductants produced temperature-independent apyrase activity, as did Dhns strains HN680 and HN680/32 (3). To further characterize the regulation of apy expression, we isolated independently derived Crb2 mutants of wild-type EIEC strain HN280 and of S. flexneri SFZM53. No Crb2 mutants of HN280 or SFZM53 showed apyrase activity, and molecular alterations encompassing virF and/or virB were detected in most HN280 and SFZM53 Crb2 derivatives which

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carried an intact apy locus, indicating that apy expression is under the control of the VirF and VirB regulatory cascade. This conclusion was confirmed by the restoration of temperature-dependent apyrase expression upon introduction of the cloned virF or virB gene, respectively, into the virF- or virBdefective Crb2 mutants of HN280 and of SFZM53. These findings clearly demonstrate that apy is another virBregulated gene whose expression is under the control of the VirF and VirB regulatory cascade. Further experiments are needed to ascertain whether virB activates apyrase expression directly or indirectly through positive control of an unknown apy-specific effector. On the other hand, temperature-regulated expression of the nonspecific phosphatase was evident in Crb2 mutants of S. flexneri SFZM53 (Table 3), indicating that, unlike that of apyrase, the temperature-regulated expression of the nonspecific phosphatase escapes the control of the regulatory cascade. Moreover, since a Dhns mutant of S. flexneri M90T (serotype 5) still produces temperature-regulated nonspecific phophatase activity at levels similar to those in the parental strain (3), we conclude that H-NS is not involved in the regulation of phoNSf expression. Experiments are under way to elucidate the regulatory pathway leading to the temperature-regulated expression of the nonspecific phosphatase in S. flexneri. In conclusion, the data reported in this study indicate that the expression of apy but not that of phoN-Sf is under the regulatory control of temperature, of hns, and of the VirF and VirB regulatory cascade. Whether periplasmic apyrase acts as a generalized cytotoxin (e.g., by directly damaging host cell metabolism), is involved in the utilization of exogenous nucleotides which must be dephosphorylated to nucleosides to cross the impermeable cytoplasmic membrane (47), or plays a role in some unknown bacterial metabolic function not directly involved in pathogenesis is still an open question; additional experiments are required to precisely assess the role of apyrase in the mechanism of pathogenicity of S. flexneri and EIEC. However, the findings that (i) apy transcription is coregulated with that of invasion genes; (ii) the highest level of expression of apyrase activity occurs at entry into the late exponential growth phase in both EIEC and S. flexneri strains (3); and (iii) the DNA region encompassing apy is conserved in all four Shigella species and in EIEC (4) and can be found without structural variation among virulent enteroinvasive isolates from different sources strongly suggest that the expression of apyrase may be important, especially when bacteria are inside susceptible host cells, probably during the phase of intracellular multiplication.

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ACKNOWLEDGMENTS We thank C. Sasakawa for plasmids pBN1 and pMYSH6504, P. J. Sansonetti for S. flexneri M90T and BS176, and M. L. Bernardini for S. flexneri 454. We also thank A. Calconi and A. Petrucca for expert technical assistance. This work was supported by MURST grant 60% and in part by Consiglio Nazionale delle Ricerche (contracts 95.01704.CT04 and 98.00412.CT04). REFERENCES 1. Adler, B., C. Sasakawa, T. Tobe, S. Makino, K. Kamatsu, and M. Yoshikawa. 1989. A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri. Mol. Microbiol. 3:627–635. 2. Barzu, S., A. Fontaine, P. J. Sansonetti, and A. Philipon. 1996. Induction of a local anti-IpaC antibody response in mice by a Shigella flexneri 2a vaccine candidate: implications for use of IpaC as a protein carrier. Infect. Immun. 64:1190–1196. 3. Berlutti, F., M. Casalino, and M. Nicoletti. Unpublished data. 4. Bhargava, T., S. Datta, V. Ramakrishnan, R. K. Roy, K. Sankaran, and

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