Molecular Miorobiology (1992) 6(22), 3427-3437

A plasmid-encoded type IV fimbrial gene of enteropathogenic Escheriehia coli associated with localized adherence M. S. Donnenberg,^'^ J. A. Girbn,^ J. P. Nataro^ and J. B. Kaper^ ^Medicai Services, Department of Veterans' Affairs Medicai Center and Division of Infectious Diseases, and ^Center for Vaccine Deveiopment, Division of Geographic Medicine, Department of fvfedicine. University of f^aryiand School of fVledicine, Baltimore, Maryland 21201, USA.

Summary Enteropathogenic Escheriehia co//(EPEC) form adherent microcolonies on the surface of tissue culture celts in a pattern termed localized adherence. Localized adherence requires the presence of a large EPEC adherence factor (EAF) plasmid. Recently a bundleforming pilus has been described in EPEC possessing the EAF plasmid. An analysis of 22 non-invasive EPEC inphoA mutants revealed that seven have insertions in the EAF plasmid and are incapable of iocaiized adherence. We report here the mapping of the inphoA insertions in these mutants. The nucleotide sequence of the gene interrupted in these JnphoA mutants {bfpA) was determined and found to correspond to the A/-terminal amino acid sequence of the major structural protein of the bundle-forming pilus. The bfpA gene bears sequence similarities to members of the type IV fimbrial gene family and encodes a potential site for processing by a prepilin peptidase. A plasmid containing bfpA as the only open reading frame directs the synthesis of a protein recognized by antiserum raised against the bundle-forming pilus. Tv\phoA mutants at this locus are unable to synthesize BfpA, but synthesis is restored by Introduction of a plasmid containing the cloned gene. The minimum fragment of DNA required to restore iocaiized adherence is considerably greater than that required to restore BfpA synthesis. BfpA expression, as assessed by alkaline phophatase activity in bfpAvJnphoA mutants, is affected by temperature and growth medium. These studies describe an EPEC plasmid-encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence. Received 10 June. 1992, revised and accepted 14 August, 1992. "For correspondence. Tel. (410) 328 7560; Fax (410) 328 8700.

Introduction Enteropathogenic Escheriehia eoli (EPEC) are a leading cause of diarrhoea among infants in many developing countries {Cobeljic efa/., 1989; Craviotoefa/., 1991; Kain etai, 1991; Gomes etai, 1989; Echeverria etai, 1991; Levine et ai, 1988). Distinctive properties of this organism include the ability to adhere to tissue culture cells in a localized pattern (Scaletsky et ai, 1984), the ability to attach intimately to epithelial cells, the ability to efface host microvilli (Moon et ai, 1983) and induce profound alterations in cytoskeletal components (Knutton et ai, 1989; Finlay et ai, 1992). and the capacity for cellular invasion (Miliotisera/., 1989; Andradeefa/., 1989; Donnenberg ef ai, 1989; Francis etai, 1991). Cravioto er al. {1979) were the first to demonstrate that, unlike most E eoli, EPEC are abie to adhere to tissue culture cells. Rather than covering the entire epithelial cells, EPEC form adherent microcolonies in a pattern known as localized adherence (Scaletsky etai, 1984). The ability to perform localized adherence is dependent on the presence of a 50-70MDa 'EPEC adherence factor' (EAF) plasmid, common to classic EPEC strains (Nataro er a/., 1985,1987). Loss of this plasmid is associated with loss of localized adherence ability and, according to some (Baldini et ai, 1983) but not all (McConnell et ai, 1989) investigators, transfer of this plasmid to non-adherent laboratory E coli strains confers localized adherence ability. Furthermore, a plasmid-cured EPEC derivative causes significantly less diarrhoea in volunteers than the wild-type strain from which it was derived (Levine et ai, 1985). Various insertion and deletion derivatives of pMAR2, the EAF plasmid of EPEC O127:H6 strain E2348/69, were constructed in efforts to identify the EPEC adherence factor responsible for localized adherence (Baldini ef ai, 1986). Plasmid pMAR15, containing a c. 38 kb product of a Sau3A partial digest of pMAR2, represents the minimum contiguous DNA sequence confirmed to bestow localized adherence ability upon E eoli HB101. Spanning this fragment are two separate regions, separated by plasmids pJPN14 and pJPN101, which, while inactive individually, are capable of acting in trans to confer localized adherence (Nataro etai, 1987). In further studies both pJPN14 and pJPNIOl were subjected to mini-kan transposon mutagenesis (J. P. Nataro, unpublished). Of 55 mini-kan

3428

M. S. Donnenberg. J. A. Giron, J. P. Nataro and J. B. Kaper

pMAR2

^s

pJPNIOl

pMSD201 S B

pJPNIOl

101.35

1

f f 101.27 101.36

101.5

I

I

10

15

MINI-KAN INSERTS

insertions in pJPN14, five abolished the abiiity to confer localized adherence upon HB101{pJPN101). Similarly, three of 40 mini-kan insertions in pJPNIOl inactivated the ability to confer localized adherence upon HB101{pJPN14). Thus these experiments suggest that multiple loci required for localized adherence exist on plasmid pMAR2. The molecular nature of localized adherence is not understood. Among previous candidate adhesins are a 32kDa outer membrane protein (OMP) {Scaletsky et ai, 1988) later reported to be OmpF (Chart and Rowe, 1989), and a 94 kDa OMP (Levine et ai, 1985; McConnell et ai, 1989), later shown to be intimin, the product of the eaeA (formerly eae) gene (Jerse and Kaper, 1991). Currently, the most promising candidate for the EPEC adherence factor is the recently described bundle-forming pilus (BFP) purified from EPEO 0111:NM strain B171 after passage on solid medium containing sheep blood (Giron et ai, 1991). These fimbriae tend to aggregate, forming bundles that can be visualized by transmission electron microscopy. Furthermore, expression of the fimbrial gene product is induced in tissue culture medium and under these conditions the bacteria tend to clump (Vuopio-Varkila and Schoolnick, 1991). Bundle-forming pili (BFP) have thus far been identified only in EAF plasmid-containing and not plasmid-cured strains. The W-terminal amino acid sequence of the major fimbrial protein resembles that of the Vibrio eholerae toxin-coregulated pilus (TCP) and members of the type IV fimbria! family (Shaw and Taylor, 1990). However, like TCP, BFP lacks the A/-methylphenylalanine found at the amino terminus of other type IV fimbriae. The isolation of non-invasive EPEC TnphoA mutants

Fig. 1. Map of pJPNIOl. The position of pJPNI 01 on the circular map of EPEC plasmid pMAR2 and tts relation to the EAF gene probe (Nataro et ai, 1985) are shown. Restriction endonuclease sites for Sail (S) and SamHI (B) are shown on the linear map of pJPNIOl. Sequences derived from the pJBK68 vector, depicted as dotted lines, are only shown in part. Sites of selected mini-kan insertions are shown as black circles. The names of corresponding pJPNIOl derivative plasmids are listed below the sites of mini-kan insertion. The SamHI fragment containing bfpA and cloned in pBR322 to create pMSD201 is also indicated.

KILOBASES

has resulted in the identification of several classes deficient in various stages of the infectious process (Donnenberg efa/., 1990). Among these classes are tv*/o groups of mutants that have lost the ability to perform localized adherence. One group consists of WJQ mutants with insertions within 300 bp of each other on the EPEC chromosome, indicating that genes necessary for localized adherence are not confined to the EAF plasmid. Seven other mutants have InphoA insertions located within 500 bp of each other on the EAF plasmid. These mutants do not adhere to HEp-2 cells when incubated for the traditional 3h period. When the incubation is extended to 6h, bacteria begin to adhere, but not in a localized pattern. In the current study, we demonstrate that the plasmid gene interrupted by TnphoA in these latter mutants encodes the major structural protein of the EPEC bundle-forming pilus, currently the leading candidate for the elusive EPEC adherence factor.

Results flapping the locations of TnphoA insertions on the EAF plasmid In an earlier study, seven non-invasive TnphoA mutants of E2348/69 were found to have plasmid insertions that eliminated the ability to perform localized adherence (Donnenberg efa/., 1990). The SamHI fragment from each mutant, containing upsteam flanking DNA and a portion of TnphoA including the phoA and neo genes, was cloned into pACYCI 84. The size of the cloned fragments ranged from 5.45 to 5.85 kb, representing 450-850 bp of EPEC DNA upstream of the site of transposon insertion. When

EPEC type IV fimbrial gene 3429 grown on medium containing a chromogenic substrate for aikaline phosphatase, E. eoli DH5a clones containing each SamHI fusion junction were strongly positive regardless of the orientation of the insert. This observation indicates that the mutated gene lacks a SamHI site 5' prime to the site of the TnphoA insertions and that the cloned DNA upstream of the fusion junctions might include the entire 5' sequence of the interrupted gene, together with its promoter. The SamHI fragment from one mutant, containing 4970 bp from the left side of TnphoA and approximately 800 bp of cloned EPEC DNA, was used as a DNA probe to analyse fragments of the EAF plasmid by Southern hybridization. The probe hybridized to a 5.5kb BamH\ fragment found in EAF plasmid pMAR2 and plasmid pJPNIOl, but not pJPN14. This SamHI fragment was cloned in pBR322 to create pMSD201 and analysed by restriction endonuclease mapping. The cloned fragment corresponds to one of the sites at which mini-kan insertions eliminate the ability of pJPNIOl to bestow localized adherence upon HB101(pJPN14) {Fig. 1).

Table 1. Complementation of mutant 31-6-1(1) by pJPNIOl and derivatives to restore localized adherence. At least 200 epithelial cells were counted per well. Data from the first of duplioate wells are shown. Similar results were obtained with strain 10-1-1(1).

Strain E2348/69 31-6-1(1) 31-6-1(1) pJPNIOl 31-6-1(1) pJPNIOl .5 31-6-1(1) pJPNIOl .27 31-6-1(1) pJPNIOl .35 31-6-1(1) pJPNIOl .36

Per cent cells with 5:5 bacteria 58.8 0 22.5 0.5 1-5 6.5 1.5

No. bacteria per epithelial cell (mean ± SD)

No. bacteria per cluster (mean ± SD)

28.8 0.1 5.7 0.1 0.2 1.6 0.2

39.3 1.5 20.5 1.5 2.5 17.3 4.38

± ± ± ± ± ± ±

30.0 0.4 12.1 0.5 0.98 7.3 1.0

± 17.7 ±0.5 ± 13.8 ± 1.2 ± 2.4 ± 17.3 ± 5.3

adherence (data not shown). Thus, it appears that the ability to complement these TnphoA mutants and restore localized adherence is associated with between 5 and 15 kb of DNA downstream of the site of the transposon insertions.

Complementation of loealized adherenee by fragments of the EAF plasmid

The nucleotide sequence of the bfpA gene

Plasmid pMSD201, containing the SamHI fragment of pJPNIOl that hybridized with the fusion junction probe, was introduced into TnphoA mutants 10-1-1(1) and 31-6-1(1) by electroporation. The resulting transformants were negative for localized adherence ability. The fragment cloned in pMSD201 includes approximately 5 kb downstream of the sites of TnphoA insertion in these mutants. In view of the strong alkaline phosphatase activity of the cloned SamHI fusion junctions it seems unlikely that the failure of pMSD201 to complement the mutants is due to lack of expression of the cloned gene. Therefore the much larger plasmid pJPNIOl was transferred into each mutant by triparental conjugation. This plasmid, which contains approximately 15 kb downstream of the sites of transposon insertions, restored localized adherence activity to both mutants (Figs 1 and 2, Table 1). In contrast, plasmid pJPNIOl.5, a JPN101 derivative containing a mini-kan insertion that maps to the same SamHI fragment as the TnphoA insertions, was incapable of restoring localized adherence to either mutant (Figs 1 and 2, Table 1). Plasmids pJPN101.27 and pJPNIOl .36, which contain mini-kan insertions much further downstream, were able partially to complement both mutants. Furthermore, pJPNIOl.35, which contains a mini-kan insertion in vector sequences, was also able to complement both mutants. None of these plasmids was able to restore localized adherence to strain JPN15, an E2348/69 derivative cured of pMAR2, confirming that additional plasmid sequences are required for localized

Using primers complementary to sequences at each end of TnphoA, the DNA sequences beyond the cloned upstream and downstream fusion junctions were determined for four of the mutants. Primers complementary to the distal sequences obtained were synthesized and used to complete the sequence analysis on both strands. A 579 bp open reading frame (ORF) was identified, interrupted in each mutant by an in-frame TnphoA insertion (Fig. 3). Two methionine residues are found near the 5' end of the sequence, the second at position six relative to the first. The first methionine is preceded at nucleotide - 1 1 by the sequence TAAGGA, which is exactly complementary to the 3' terminal sequence of 16S rRNA and could represent a ribosome-binding site (Ringquist etai, 1992). Preceding this site by 12 residues is the sequence TATTAAT, which is similar to the consensus for a - 1 0 promoter sequence; however no - 3 5 sequence match is found further upstream. The region - 4 to + 7 relative to the second methionine is compatible with a translation intiation site, but no apparent ribosome-binding site precedes it. However, this region is preceded by both a putative - 1 0 promoter site (TATAAG, overlapping the possible ribosome-binding site associated with the first methionine) and a putative - 3 5 site (GTGACC, ending 18 residues before the start of the putative - 1 0 sequence). Thus, the nucleotide sequence yields conflicting data regarding potential sites for initiation of transcription and translation. A 28 bp region of dyad symmetry predicting an 11 bp stem with a six-base loop is found

3430

M. S. Donnenberg, J. A. Giron, J. P. Nataro and J. B. Kaper

EPEC type IV fimbrial gene TAG TTT TAA GAT TAT TCC GTG ACC TAT TAA TAC GGG GGT TTT ATA

45

AGG AAA ACA GTT TTT ATG Met I GAA AAA GGT CTG TCT TTG Glu Lys Gly Leu Ser Leu

GTT TCT AAA ATC ATG AAT AAG AAA TAC Val Ser Lys lie Met Asn Lys Lys Tyr

90

ATT GAA TCT GCA ATG GTG CTT GCG CTT lie Glu Ser Ala Met Val Leu Ala Leu

13 5

GCT GCC ACC GTT ACC GCA GGT GTG ATG TTT TAC TAC CAG TCT GCG Ala Ala Thr Val Thr Ala Gly Val Met Phe Tyr Tyr Gin Ser Ala

180

TCT GAT TCC AAT AAG TCG CAG AAT GCT ATT TCA GAA GTA ATG Ser Asp Ser Asn Lys Ser Gin Asn Ala lie Ser Glu Val Met 29-2-1(1) GCA ACG TCT GCA ATT AAT GGT CTG TAT ATT GGG CAG ACC AGT Ala Thr Ser Ala lie Asn Gly Leu Tyr lie Gly Gin Thr Ser

AGC Ser

225

TAT Tyr

2 70

AGT GGA TTG GAC TCA ACG ATT TTA CTT AAC ACA TCT GCA ATT CCG Ser Gly Leu Asp Ser Thr lie Leu Leu Asn Thr Ser Ala lie Pro

315

GAT AAT TAC AAA GAT ACA ACA AAC AAA AAA ATA ACC AAC CCA TTT Asp Asn Tyr Lys Asp Thr Thr Asn Lys Lys lie Thr Asn Pro Phe

360

GGG GGG GAA TTA AAT GTA GGT CCA GCA AAC AAT AAC ACC GCA TTT Gly Gly Glu Leu Asn Val Gly Pro Ala Asn Asn Asn Thr Ala Phe

405

GGT TAC TAT CTG Gly Tyr Tyr Leu 6-8-1(1) AGT CTT GCA ACC Ser Leu Ala Thr

ACG CTT ACC AGG TTG GAT AAA GCG GCA TGT GTT Thr Leu Thr Arq Leu Asp Lys Ala Ala Cys Val

450

TTG AAC TTA GGT ACT TCA GCG AAA GGC TAC GGT Leu Asn Leu Gly Thr Ser Ala Lys Gly Tyr Gly

49 5

GTT AAT ATC TCT GGC GAA AAT AAC ATT ACA TCA TTT GGT AAT AGC Val Asn lie Ser Gly Glu Asn Asn H e Thr Ser Phe Gly Asn Ser 31-6-1(1) GCT GAT CAG GCT GCT AAA TCG ACT GCT ATT ACT CCT GCT GAA GCG Ala Asp Gin Ala Ala Lys Ser Thr Ala lie Thr Pro Ala Glu Ala

540

Fig. 3. The nucleotide sequenoe of bfpA. A potential ribosome-binding site is underlined. A 3' region of dyad symmetry is indicated by the arrows. The first amino acid of mature BfpA is indicated by the vertical arrow. The sites of TnphoA rnsenion for three mutants are displayed above the corresponding sequences. These sequence data will appear in the EMBUGenBank/ DDBJ Nucleotide Sequence Data Libraries under the accession number Z12295.

585

GCA ACT GCA TGT AAA AAT ACT GAT TCA ACC AAT AAA GTT ACA TAT Ala Thr Ala Cys Lys Asn Thr Asp Ser Thr Asn Lys Val Thr Tyr

6 30

TTT ATC AAG TAA TCT ACA TAA TAC GCC CTG TTT TGG GCG TAT TAT Phe Het Lys » •.

67 5

ATG GGA GGT ATA TGT GAG GAC AGT AAT CCT TTT TTT TTG TCC TTT

720

TGG TAT CCT GTT TTA CTC AGG CAC AGG

747

beginning six nucleotides after the termination codon, representing a possible site for translational regulation. The predicted amino acid sequence near the 5' end of this ORF corresponds almost perfectly to the W-terminal amino acid sequence of the BFP of strain B171; 26 of 28 residues are identical (Giron er ai, 1991). Using the first amino acid of the B171 proteins as the W-terminus, the predicted mature product of the sequenced E2348/69 locus is 18730 daltons, in excellent agreement with the estimate of 18.5 tc 19.5kDa for B171 BFP obtained by electrophoresis (Giron et ai, 1991; Vuopio-Varkila and Schoolnik, 1991); therefore we designate this locus bfpA. A search of the GenBank Data Library revealed amino acid sequence similarities to the TCP of V. eholerae and to pili of Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, and Baeteroides nodosus, all of whioh are members of the type IV fimbrial family (Shaw and Taylor, 1990). Except for tcpA, which shows similarities throughout, the sequence similarities are concen-

3431

trated at the 5' ends of the genes. At the nucleotide level, there is 41 % identity between tcpA and bfpA of E2348/69. The first amino acid of the putative mature protein is leucine, which differs from the W-methylphenylalanine found in most members of the type IV fimbrial family. The mature protein is preceded by an atypical highly basic hydrophilic leader sequence characteristic of the group. Unlike that of TCP, and like other members of the family, this sequence is short (8 or 13 residues, depending on which methionine is the site of translation initiation). In addition, the amino acids immediately preceding the cleavage site are lysine and glycine, which are typical of this fimbria! family. Following the predicted processing site, nucleotides coding for Leu-Ser-Leu-lle-Glu are found. These residues are highly similar to the consensus sequence of Phe-Thr-Leu-lle-Glu found in proteins processed by prepilin peptidase of P. aeruginosa and other members of the type IV fimbrial family (Nunn and Lory, 1992).

3432

/W. S. Donnenberg, J. A. Giron, J. P. Nataro and J. B. Kaper

10

•H

tent with a molecular mass 2.0kDa greater than that of mature BfpA. Thus the inability of 31-6-1(1)(pMSD201) to perform localized adherence may be due, in part, to a failure to process BfpA correctly. The size difference between the bands is consistent with an unprocessed prepiiin corresponding to initiation of translation at the first methionine residue in the ORF (predicted Mr ^ 20269).

a

o

(Bo

-I

n —'

7776 cloned bfpA gene encodes a protein antigenically related to BFP

30

T 36

40

Temperature (C) Fig. 4. The effect of temperature on expression of BfpA-alkaline phosphatase fusion proteins. The results of triplicate values from one experiment are shown. Error bars represent standard deviations.

BFP expression The availability of bfpAwTnphoA mutants is convenient for the study of BFP expression because the activity of the fusion proteins produced can be measured easily. The alkaline phosphatase activity of mutants was studied therefore under a variety cf conditions known tc affect the expression of various fimbriae. Growth temperature had a dramatic effect on fusion protein activity (Fig. 4). Unlike tepA (Parsot and Mekalanos, 1990), bfpA is expressed maximally at normal body temperature. Very tittle activity was detected at 25°C or 40°C. Interestingly, growth in tissue culture medium was associated with a marked increase in expression of the fusion protein (Table 2). As shown previously (Vuopio-Varkila and Schoolnik, 1991) and confirmed in E2348/69 by immunobiotting lysates of E2348/69 grown in L broth and EMEM, BfpA is produced at increased levels in tissue culture media (Fig. 5A). Next we examined lysates of TnphoA mutants grown in Eagle's Minimal Essential Medium (EMEM) for the presence of BFP. As shown in Fig. 5A, BfpA was absent from mutant 31-6-1(1), but was produced by 31-6-1(1) when the cloned bfpA gene was reintroduced to 31-6-1(1) on plasmid pMSD201. Therefore plasmid pMSD201, which is unable to restore localized adherence to either mutant, can complement 31-6-1(1) for BfpA production. These results are consistent with the mapping studies which show that localized adherence requires far more than the bfpA locus. Interestingly, the BFP antiserum recognizes two bands in 31-6-1(1)(pMSD201), a smaller minor band with an apparent molecular mass of 19.1 kDa (the same as BfpA) and a larger and predominant band that is consis-

A 766 bp fragment containing the bfpA gene of E2348/69 was amplified by polymerase chain reaction (PCR) and cloned in both orientations into plasmid pCRIOOO (Mead ef ai, 1991). For plasmid pMSD205 the gene is in the correct orientation with respect to the T7 promoter on the vector, whereas the insert in pMSD206 is in the opposite orientation. Each plasmid was introduced by transformation into BL21(DE3) containing pLysS. This strain contains a prophage-encoded inducible T7 RNA polymerase gene under the control of a lac promoter. It also contains a plasmid encoding T7 lysozyme that serves to limit expression of genes under the control of T7 promoters in the presence of low levels of T7 RNA polymerase (Das, 1990). Whole-cell lysates of bacteria grown in the presence and absence of inducer were subjected to SDS-PAGE and immunobiotting with anti-BFP antiserum. Immunoreactive proteins of 21.1 and 19.1 kDa were detected in extracts of strains containing the bfpA gene cloned in either orientation either in the presence or absence of inducer (Fig. 5B). Expression of this product was greatly enhanced in the presence of Inducer. In the case of pMSD206, the increased level of BfpA in the presence of inducer may be due to the longer incubation period which allowed increased cell growth. Since cell growth ceases soon after induction of the T7 polymerase only in cells containing target genes, this explanation does net hold for the strain carrying pMSD205 (Studier and Moffatt, 1986). The smaller immunoreactive protein runs at the same rate as the BfpA protein, while the larger product

Table 2. Effect of growth medium on expression of BfpA-alkaline phosphatase fusion proteins. Data are presented as the mean + standard deviation of triplicate values. Alkaline Phosphatase Units (per Afloo culture unit) Medium

E234a'69

10-1-1(1)

31-6-1(1)

LB EMEM

0.89 ± 0.09 1.76 ± 3.66

4.11 ±0.35 106.62 ± 41.22"

3.98 ±0.17 78.85 ± 6.45"

a. P = 0.01 vs. LB. b. P < 0.001 vs. LB.

EPEC type IV fimbrial gene 3433

z

o

3" UJ

a.

^

CO

B

-

-4- -

-f

-

+

IPTG

S S IS S ^1 C^ J

^

n

CM

A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence.

Enteropathogenic Escherichia coli (EPEC) form adherent microcolonies on the surface of tissue culture cells in a pattern termed localized adherence. L...
3MB Sizes 0 Downloads 0 Views