Virulence Plasmids of Enteroinvasive Escherichia coliand Shigella ...

INFECTION AND IMMUNITY, Mar. 1991, 0019-9567/91/030792-08$02.00/0

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Vol. 59, No. 3

792-799

Virulence Plasmids of Enteroinvasive Escherichia coli and Shigella flexneri Integrate into a Specific Site on the Host Chromosome: Integration Greatly Reduces Expression of Plasmid-Carried Virulence Genes CARLO ZAGAGLIA,1 MARIASSUNTA CASALINO,2 BIANCA COLONNA,2 CINZIA CONTI,3 ATTILIO CALCONI,2 AND MAURO NICOLETTIl*

Istituto di Medicina Sperimentale, Cattedra di Microbiologia, Universita "G. D'Annunzio," 63100 Chieti, and Dipartimento di Biologia Cellulare e dello Sviluppo, Sezione di Scienze Microbiologiche,2 and Istituto di Microbiologia, Universitd di Roma "La Sapienza," 00185 Rome, Italy Received 3 August 1990/Accepted 12 November 1990

The ability of enteroinvasive Escherichia coli and Shigellaflexneri to cause disease depends on the presence of a large virulence plasmid (pINV). In this report we show that pHN280, the pINV of the 0135:K-:Henteroivasive strain E. coli HN280, and pWRIOO, the pINV of S. flexneri serotype 5 strain M9OT, are able to integrate into a specific site on the host chromosome. pINV-integrated HN280 and M9OT strains required methionine (Met-) to grow in minimal medium, were noninvasive, did not produce contact-mediated hemolysin, and had lost the ability to bind Congo red (Crb-) at 37°C. Immunoblots of whole bacterial extracts from pHN280-integrated HN280 derivatives revealed that integration severely reduced the expression of ipa and virG (icsA) plasmid genes. Met- HN280 and M9OT derivative strains spontaneously generated Met' revertants that either contained excised forms of pINV or had lost pINV. Restriction analysis of excised pINVs showed that they either were virtually identical to parental pINVs (precise excision) or had suffered some deletion (imprecise excision). Precisely excised pINVs expressed the full pattern of virulence, whereas imprecisely excised pINVs were always Crb- and noninvasive. The revertion to Met' was shown to be recA dependent, indicating that homologous plasmid and chromosomal DNA sequences are involved in the integration-excision process. The maintainance of pINV through integration and downregulation of its virulence genes may represent an advantageous mechanism for enteroinvasive bacteria, particularly when they are outside host cells and/or have to face adverse environmental conditions. In this paper we present evidence that pINVs of 0135: K-:H- EIEC strain HN280 (26) and of S. flexneri M9OT (33) can integrate into and precisely excise from a specific site (metB) of the host chromosome. Independent pINVintegrated derivatives of HN280 and M9OT all required methionine (Met-) to grow in minimal medium and did not express plasmid-encoded virulence genes. Met- derivatives of both strains spontaneously generated Met+ revertants that either harbored excised derivatives of pINV or had lost pINV. Revertion to methionine prototrophy of pINV-integrated HN280 derivative strains was found to be recA dependent, thus indicating that recombination between the homologous plasmid and chromosomal DNA sequences mediates integration-excision events. The data are discussed in view of understanding of the complex regulatory mechanisms of virulence gene expression in enteroinvasive strains.

Shigella flexneri and enteroinvasive Escherichia coli (EIEC) are organisms known to cause dysenteric enteritis in humans by a similar, if not identical, complex mechanism of pathogenicity (23). The virulence of these strains depends on the presence of both chromosomal genes and a large virulence plasmid (pINV) (14, 23, 33). pINVs represent a family of highly related plasmids. They are functionally interchangeable between Shigella and EIEC strains (31), they share significant DNA homologies among each other (13, 30), and they all specify FIIA incompatibility (23, 39). Several virulence traits are encoded by pINV genes, including (i) the ability to invade and multiply within epithelial cells (33), (ii) the ability to spread infection intracellularly and to adjacent cells (3), (iii) the expression of several antigens implicated in the entry phenotype (2, 12, 41), (iv) the synthesis of a contact-mediated hemolysin (34), (v) the binding of the dye Congo red (Crb+ phenotype) (22), and (vi) the inhibition of bacterial growth (Igr+ phenotype) (36). Interestingly, it has been shown that the expression of the virulence phenotype of Shigella spp. is regulated by the growth temperature. A chromosomal regulatory gene (virR) (24) specifies a repressor molecule that blocks the expression of several unlinked virulence genes at 30°C (22, 23). Modulation of the expression of virulence genes by temperature might be advantageous for Shigella and EIEC microoganisms, since it would allow a considerable economization of biosynthesizing energies outside the susceptible host. *

MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used are listed in Tables 1 and 2, respectively. EIEC strain HN280 was isolated during a study on diarrheal diseases in children in Somalia (26). It has been serotyped 0135:K-:H- and cross-reacts with polyvalent anti-S. flexneri antiserum. It does not ferment lactose, it is lysine decarboxylase negative, and it is able to grow as prototroph in M9 minimal salt medium (26). Media and chemicals. Enriched and minimal growth media were Trypticase soy broth (BBL Microbiology Systems,

Corresponding author. 792

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TABLE 1. Bacterial strains

Species and

Sereny test

phenotype'

auxotrophy

HN280

EIEC 0135

+

+

-

Wild-type strain (26)

HN280/32 HN280/33 HN280/34 HN280/35 HN280/36

EIEC 0135

-

-

+

Independent pINV-integrated derivatives of HN280 (this study)

HN280/10

EIEC 0135

+

+

-

recAl derivative of strain HN280 (this study)

HN321 HN331 HN341

ElEC 0135

-

+

+

recAl derivatives of strains HN280/32, HN280/33, and HN280/34, respectively (this study)

HN322 HN333

EIEC 0135

+

+

-

pINV-excised derivatives of HN280/32 and HN280/33; they carry plasmids pHN322 and pHN333, respectively (this study)

MC4100

E. coli K-12

-

-

-

Plasmidless laboratory strain (39)

MC4100(pHN290) MC4100(pHN32) MC4100(pHN33)

E. coli K-12

-

+

-

Transconjugants carrying Tnl-labeled plasmids pHN290, pHN32, and pHN33, respectively (this study)

M9OT

S. flexneri 5

+

+

-

Wild-type strain (33)

HN1045 HN1046

S. flexneri 5

-

-

+

Independent pRSM4 transformants of M9OT (this study)

HN1045/131

S. flexneri 5

+

+

-

pINV-excised derivative of HN1045; it carries plasmid pHN131 and has lost pRSM4 (this study)

GC2226

E. coli K-12

-

-

-

srl::TnJO derivative of the recAl

Strain

serotype

Crb Igr

Methionine

strain KL1699

(L. Paolozzi) a Crb and Igr phenotypes were observed when bacteria were grown at 37°C.

Cockeysville, Md.), LB medium (19), brain heart infusion broth, and M9 minimal salt medium (19). Congo red (Sigma Chemical Co., St. Louis, Mo.) was added at 0.01% to Trypticase soy agar to determine Congo red binding (Crb phenotype). Carbon sources (0.2%), amino acids or amino acid precursors (50 ,ug/ml), and vitamins (2 ,ug/ml) were added to M9 medium when required. Rifampin (6 ,ug/ml), ethidium bromide (25 ,ug/ml), and acridine orange (70 ,ug/ml) were used in plasmid-curing experiments (25). Genetic procedures. Conjugation, transformation, and P1 vir transduction were carried out by standard methods (8, 18, 25, 38). Mobilization of nonconjugative pINVs in E. coli K-12 strains was accomplished by the use of the temperature-sensitive conjugative plasmid pTH10 (42). The recAl mutation was transferred into various strains by P1 vir cotransduction with the tetracycline resistance marker of TnJO inserted in the sri locus of strain GC2226 (Table 1). Tetracycline-resistant transductants were tested for the presence of the recAl mutation by sensitivity to UV light. Compatibility experiments were conducted by transforming competent cells with plasmid pRSM4 (Table 2). pRSM4 transformants, derivatives of HN280, and M9OT were tested for maintainance of both the incoming and the resident pINV plasmid. Molecular procedures. Chromosomal DNA was prepared by the procedure of Silhavy et al. (38). Rapid plasmid DNA extractions were performed by the method of Kado and Liu

(15). Plasmid DNA preparations used in restriction enzyme digestions were performed by a modification of this technique (13). Restriction digestions and electrophoresis were performed as described previously (8). DNA fragments from agarose gels were denatured and transferred to nitrocellulose filters as described by Maniatis et al. (19). Probes were labeled with [a-32P]dATP by nick translation and used in filter hybridization as previously described (7). Western blots. Western immunoblots were performed essentially as described by Baudry et al. (2). Whole bacterial protein extracts separated on sodium dodecyl sulfate-polyacrylamide gels were electrophoretically transferred to nitrocellulose membranes by the procedure of Burnette (4). After transfer, diluted (1:250) serum from a monkey experimentally infected with S. flexneri M9OT was used to assay expression of major virulence-associated peptides. Antibodies recognizing shigella or EIEC antigens were detected with radioiodinated protein A. Virulence assays. Sereny tests (37) were performed with adult albino guinea pigs. Strains that elicited keratoconjunctivitis within 96 h were considered invasive. Invasiveness was also assayed in HeLa cells essentially as described by Sansonetti et al. (34). In brief, nonconfluent monolayers of HeLa cells grown in 12-well Costar culture dishes were infected with 1 ml of exponentially growing bacteria that had been grown at 37 or 42°C at a multiplicity of infection of about 100 bacteria per HeLa cell. After centrif-

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TABLE 2. Plasmids used in this study Plasmid

Source or derivation

pHN280

pINV of EIEC strain HN280 (26)

pHN281

Cryptic plasmid of HN280 (26)

pHN322

pHN333

Independent precisely excised derivatives of pHN280 (this study)

pHN290 pHN32 pHN33

pTH10-mobilized, Tnl-labeled derivatives of pHN280, pHN322, and pHN333, respectively (this study)

pWR100

pINV of S. flexneri M9OT (33)

pHN131

Precisely excised derivative of pWR100 (this study)

pTH10

Thermosensitive replication mutant of plasmid RP4, it carries Tnl (42)

pCHR312

pMY6003 carrying a 4.8-kb HindIlI fragment comprising the ipa gene region of the virulence plasmid of S. flexneri 2a strain YSH6000 (35)

pRSM4

pUC19 carrying a 550-bp fragment of pWR110 that contains a functional RepFIIA basic replicon (38)

ugation, dishes were incubated for 15 min at 37 or 42°C. Wells were then washed three times with Earle balanced salt solution, covered with 2 ml of fresh Earle minimal essential medium (GIBCO Laboratories, Grand Island, N.Y.) with 50 jig of gentamicin (Sigma) per ml, and incubated for 90 min more. The monolayers were washed five times with Earle balanced salt solution, fixed in methanol, stained with Giemsa stain, and examined by light microscopy for invasion of cells. Synthesis of contact-mediated hemolysin by bacterial strains grown in Trypticase soy broth at 37 or 42°C was monitored as described by Sansonetti et al. (34). Contact hemolysis activity was measured spectrophotometrically by reading the optical density at 545 nm. The Igr phenotype was determined by measuring the sizes of the colonies and by comparing them with those of the virulent wild type (36). Assay for bacterial multiplication within HeLa cells. Intracellular multiplication was evaluated essentially as described by Sansonetti et al. (34). HeLa cells were infected and centrifuged as described above. Washed wells were then covered with 2 ml of fresh minimal essential medium containing gentamicin (50 ,uglml) and incubated at 37 or 42°C. The number of CFU was determined for each lysed monolayer suspension every hour for 5 h from the end of the 15-min infection period. Intracellular multiplication has been quantitated as the ratio between the number of CFU recovered from lysed monolayer suspensions and the number of CFU initially added to the monolayers for each time point. RESULTS

pHN280 is the pINV of EIEC strain HN280. EIEC strain HN280 displayed a fully virulent phenotype. It produced a positive Sereny test. When grown at 37 or 42°C (but not at 30°C), it was able to penetrate and to multiply within HeLa cells, produced Crb+ Igr+ colonies, and elaborated a contact-mediated hemolysin. Agarose gel electrophoresis re-

vealed that HN280 harbored two large plasmids: pHN280 (260 kb) and pHN281 (160 kb). pHN280 was shown to be pINV, because spontaneous Crb- Igr- derivatives correlated with deletions only on this plasmid and because, when the two plasmids were separately mobilized in E. coli MC4100 by plasmid pTH10 (42), only MC4100 harboring pHN280::Tnl displayed a Crb+ Igr+ phenotype and was able to penetrate and to multiply within HeLa cells. Integration of pHN280 into the host chromosome. Since spontaneous pINV-cured derivatives of HN280 did not show up after the plasmid content of more than 500 independent Crb- Igr- colonies was examined, we performed curing experiments with rifampin, ethidium bromide, or acridine orange. From rifampin-treated HN280 we obtained five independent Crb- Igr- derivatives (HN280/32, HN280/33, HN280/34, HN280/35, and HN280/36; Table 1) that apparently harbored only pHN281. Unexpectedly, at a frequency of about 10-3 to 10-4, they all generated Crb+ Igr+ segregants that presented the same plasmid content as parental HN280. To test whether pHN280 might have integrated into the host chromosome (where it was unable to express the Crb+ Igr+ phenotype), we examined the growth requirements of the five Crb- Igr- derivatives on M9 minimal medium plates. Although on this medium the parental strain HN280 grew as a prototroph, the five derivatives did not. M9 medium supplemented with different amino acids and vitamins allowed us to determine that all five strains required methionine (Met-) for growth. To identify the specific step that was blocked in the methionine biosynthetic pathway, the five strains were streaked on M9 plates supplemented with the four different methionine precursors. Since growth was restored only in the presence of cystathionine and homocysteine but not in the presence of homoserine or o-succinyl-homoserine, we conclude that the putative integration site of the virulence plasmid was in the metB gene and affected the synthesis of cystathionine--y-synthase (6). Excision of pHN280 from the metB gene restores the Crb+ Igr+ phenotype and is correlated with the reappearance of the Met+ phenotype. If integration of the virulence plasmid into the host metB gene was responsible for the methionine auxotrophy of these strains, then pINV excision should lead to the coordinate reappearance of both the prototrophic phenotype and the virulence plasmid. To test this prediction, individual overnight broth cultures of five isolated colonies each of HN280/32, HN280/33, and HN280/34 were extensively washed in M9 and plated on M9 plates. Methionineindependent (Met+) revertants were obtained at a frequency of about 2 x 1i0 to 8 x 10-4. Twelve revertants from each broth culture were purified on the same M9 plates and tested for Crb Igr phenotype and plasmid DNA content. One-third of the Met+ revertants displayed a Crb+ Igr+ phenotype, and they were shown to contain a plasmid apparently identical in size to pHN280 (Table 3). The remaining Met+ revertants presented a Crb- Igr- phenotype, and they contained either a plasmid smaller than pHN280 (but distinct from pHN281) or no plasmid. These results indicated that excision occurred at relatively high frequency, producing different excised forms of the virulence plasmid. To determine whether excision was dependent on the host Rec system, we constructed HN280/321, HN280/331, and HN280/341 by transduction with P1 vir grown on strain GC2226 (Table 1). Unexpectedly, the introduction of the recAl mutation conferred a Crb+ phenotype, and for this reason we could calculate excision frequency only by scoring Met+ segregants. Since we could not obtain prototrophic revertants (frequency of reversion to Met+,