JOURNAL

OF

BACTERIOLOGY, Feb. 1992,

p.

1360-1369

Vol. 174, No. 4

0021-9193/92/041360-10$02.00/0 Copyright C) 1992, American Society for Microbiology

Amino Acid Substitutions in Naturally Occurring Variants of Ail Result in Altered Invasion Activity KAREN B. BEER' AND VIRGINIA L. MILLER' 2*

Department of Microbiology and Molecular Genetics,' Molecular Biology Institute,2 University of California at Los Angeles, 405 Hilgard Avenue, Los Angeles, California 90024-1489 Received 3 September 1991/Accepted 5 December 1991

Yersinia enterocolitica is the causative agent of a variety of gastrointestinal syndromes ranging from acute enteritis to mesenteric lymphadenitis. In addition, systemic infections resulting in high mortality rates can occur in elderly and immunocompromised patients. More than 50 serotypes of Y. enterocolitica have been identified, but only a few of them commonly cause disease in otherwise healthy hosts. Those serotypes that cause disease have been divided into two groups, American and non-American, based on their geographical distributions, biotypes, and pathogenicity. We have been studying two genes, inv and ail, from Y. enterocolitica that confer in tissue culture assays an invasive phenotype that strongly correlates with virulence. Some differences between the American and non-American serotypes at the ail locus were noted previously and have been investigated further in this report. The ail locus was cloned from seven Y. enterocolitica strains (seven different serotypes). Although the different clones produced similar amounts of Ail, the product of the ail gene from non-American serotypes (AilNA) was less able to promote invasion by Escherichia coli than was the product of the ail gene from American serotypes (AilA). This difference is probably due to one or more of the eight amino acid changes found in the derived amino acid sequence for the mature form of AilNA compared with that of AilA. Seven of these changes are predicted to be in cell surface domains of the protein (a model for the proposed folding of Ail within the outer membrane is presented). These results are discussed in relation to the growing family of outer membrane proteins, which includes Lom from bacteriophage A, PagC from Salmonella typhimurium, and OmpX from Enterobacter cloacae.

Yersinia enterocolitica is the causative agent of a variety of gastrointestinal syndromes whose severity is dependent on the age and immunocompetence of the host (6, 13). In young children, infection with Y enterocolitica presents as acute enteritis, while adolescents and young adults often present with a pseudoappendicular syndrome. More-severe infections with high mortality rates can occur, but these are usually seen only with elderly and immunocompromised patients. Y. enterocolitica is also associated with a variety of postinfection complications that include erythema nodosum and reactive arthritis. Transmission of Y. enterocolitica is generally believed to be by consumption of contaminated food or water (6, 13), but several cases of septic shock due to transfusion of blood contaminated with Y. enterocolitica have recently been reported (8, 24, 52). Over 50 serotypes of Y. enterocolitica have been identified, but most of them do not commonly cause disease in otherwise healthy hosts (6, 12). Those serotypes that do cause disease (pathogenic serotypes) differ in their geographical distribution and their pathogenicity. The American serotypes (08, 04,32, 013a,13b, 018, 020, and 021) are isolated from patients with human diseases in the United States, while the non-American serotypes (03, 09, and 05,27) are the most common causes of human disease outside the United States. However, in recent years, the geographical barriers have been breaking down (4, 7, 21, 26). A dramatic illustration of this was an outbreak in Atlanta of yersiniosis due to a non-American serotype 03 strain during the winter of 1988 to 1989; acquisition of the infecting organism was associated with contact with raw chitterlings

These two groups of serotypes differ not only in their geographical distributions but also in their biochemical characteristics, behavior in animal models, and clinical manifestations (6, 12). American serotype strains are able to cause lethal infections in adult mice, but non-American serotype strains can do so only if the mice are overloaded with iron or desferrioxamine B mesylate (42). In addition, the American strains can cause conjunctivitis in experimental animals, while non-American strains do not. Strains of the American serotypes also reportedly cause a more-acute infection than do non-American serotype strains. Interestingly, the postinfection sequelae observed with Y. enterocolitica have been associated only with infections by non-American serotype strains (03 and 09) (6). Both groups of pathogenic Y. enterocolitica carry a plasmid of -70 kb that is highly conserved among the pathogenic yersiniae and has been shown to be required for full expression of the virulence phenotype (12, 18, 59). The pathogenic serotypes of Y. enterocolitica (i.e., those that are associated with disease) are also able to invade tissue culture cells, whereas nonpathogenic Y. enterocolitica is inefficient at invading tissue culture cells (27, 35, 45, 55). This property is encoded primarily by chromosomal genes, as evidenced by the ability of Y. enterocolitica cured of the virulence plasmid to invade cells in vitro and to cross the intestinal epithelia of animals (40, 43, 56). Two genes, inv and ail, which can each promote invasion of tissue culture cells when expressed by noninvasive Escherichia coli, have been cloned from Y. enterocolitica serotype 08 (34). The inv gene is homologous (77% identical amino acids) to the inv gene previously cloned from Yersinia pseudotuberculosis (22, 23, 58), but the product of the Y. enterocolitica inv gene (invasin) is only 92 kDa as opposed to 103 kDa for the invasin from Y pseudotuberculosis (38, 58). Although se-

(26). *

Corresponding author. 1360

AMINO ACID SUBSTITUTIONS IN Ail VARIANTS

VOL. 174, 1992

1361

TABLE 1. Y enterocolitica strain list Ail

Strain

Serotype

TCla

MAb reaction

Ail-cc

Ail-Bc

ail cloned

Reference

American serotype 8081 634-83 Y137 655-83 657-83 9286-78 658-83 2440-87 Y295

08 04,32 013a,13b 018 020 020 021 08 08

+ + + + + + +

+ + +

+ + + + + +

pVM102 pKB5

+ +

+ + + + + +

+ + + + + + +

40 35 51 35 35 2 35 35 35

Non-American serotype 642-83 MC51 6771/84 2452-87 MC32 637-83 2517-87 2455-87

09 03 03 03 03 05,27 09 01,2,3

+ + + + + + + +

Nonpathogenic serotype 9291-78

06

-

+ +

+ + +

-

+ + + + + + + +

-

-

-

-

pKB10 pKB6

pKB4

pKB3 pKB2 pKB7

35 35 35 35 35 35 35 35 2

Phenotype as previously reported (35). b Results of reactivity tests of either whole-cell or membrane fractions with the monoclonal antibody (MAb) 2B2 to Ail are summarized from Fig. 1 and 5A. c Ail-C and Ail-B are probes derived from the ail structural gene and downstream sequences, respectively, and are described in Materials and Methods. Data summarize published data regarding the abilities of these probes to hybridize to genomic DNA in Southern analysis of the indicated strains (35). d The plasmid carrying ail from the indicated strain in the cloning vector pBR322. a

quences homologous to inv can be detected in all Yersinia spp. (35), these sequences appear to be functional only in pathogenic Y enterocolitica and Y pseudotuberculosis (39). It is not known if the inv gene of Yersiniapestis is functional. The second invasion gene cloned from Y enterocolitica 08, ail, encodes a membrane protein of 17 kDa (33). In contrast to invasin, which promotes a high level of invasion of most tissue culture cell types tested, Ail shows tissue specificity (34). Ail promotes a high level of invasion of CHO cells but only a low level of invasion of HEp-2 cells (34). Sequences homologous to ail are found in all pathogenic species of Yersinia including the pathogenic serotypes of Y enterocolitica, Y pestis, and Y. pseudotuberculosis (35). However, ail and surrounding sequences are not found in nonpathogenic serotypes of Y enterocolitica or nonpathogenic species of Yersinia (35). The genetic basis for the observed differences between American and non-American strains of Y enterocolitica is not known, but some clues are beginning to emerge. For example, Carniel et al. (10, 11) observed that Y pestis, Y pseudotuberculosis, and American serotypes of Y enterocolitica produce two high-molecular-weight outer membrane proteins whose synthesis is repressed by iron. These proteins are not synthesized by non-American strains. The gene for one of these iron-repressible proteins has been cloned and was shown by Southern analysis to be present only in the highly pathogenic species mentioned above (11). Another difference between American and non-American strains is that an IS3-like sequence is found in American serotype strains (35). One copy of this IS3-like sequence is located within 50 bp of the 3' end of ail in the American serotypes. This IS3-like sequence is not found in nonAmerican strains (35). To further define potential differences at the ail locus of American and non-American serotype strains, ail was

cloned from seven Y. enterocolitica strains (seven different serotypes) and analyzed as to nucleotide sequence, derived amino acid sequence, and ability to confer an invasive phenotype on E. coli HB101. The results indicate that the product of the ail gene from non-American serotypes (ailNA) is less able to promote invasion by E. coli than is the product of the ail gene from American serotypes (ailA), raising the possibility that these differences could account in part for the differences in pathogenicity of these two groups. A model for the folding of Ail within the outer membrane is proposed and discussed in relation to the other members of the Ail gene family. MATERIALS AND METHODS Bacterial strains. Bacterial strains were maintained at -70°C in Luria broth (LB) (30) medium containing 25% (vol/vol) glycerol or on LB agar plates. Y enterocolitica strains are listed in Table 1; tissue culture invasion (TCI), Ail-C probe, and Ail-B probe data are summarized from Miller et al. (35). E. coli HB101 [F- hsdS20 (rB- mB-) recA13 ara-14 proA2 lacYl galK2 rpsL20 xyl-5 mtl-l] (1), and E. coli LE392 [F- thi-I thr-1 leuB6 lacYl (rk- Mk+) tonA21 supE44] were maintained on LB agar plates or on LB agar plates containing the appropriate antibiotics for plasmid maintenance. Antibiotics were used at 100 and 15 ,ug/ml for ampicillin and tetracycline, respectively. Nucleic acid purification and probe preparation. Chromosomal DNA was isolated as previously described (29). Plasmid DNA was isolated by the alkaline lysis method (28) or by Qiagen columns (Qiagen, Chatsworth, Calif.). DNA probes were prepared as follows. Plasmid DNA was digested with the appropriate restriction endonucleases, and the fragments were separated by electrophoresis through a 0.7% agarose gel. The DNA fragments were purified from the agarose gel

1362

BEER AND MILLER

slices by using Geneclean (BiolOl, La Jolla, Calif.). The purified fragments were then labeled with [32P]dCTP by the random primer method as previously described (16). The Ail-C probe, a 1.2-kb ClaI-AvaI fragment, contains the ail structural gene, and the Ail-B probe, a 900-bp AvaI-Clal fragment, contains an IS3-like sequence (35). The Ail-C probe gives the same results in Southern and Northern (RNA) analyses as a probe containing only sequences internal to the ail structural gene (57). The Ail-A probe is a 500-bp AvaI-AvaI fragment located downstream of Ail-B. DNA restriction enzymes, T4 DNA ligase, and Klenow were purchased from New England BioLabs (Beverley, Mass.) and used according to the instructions of the manufacturer. Calf intestinal alkaline phosphatase was purchased from Boehringer Mannheim (Indianapolis, Ind.). Southern hybridization analysis. Chromosomal DNA was digested to completion with the appropriate restriction endonuclease, and the fragments were separated by electrophoresis through a 0.7% agarose gel. The separated DNA fragments were transferred to nitrocellulose (Schleicher & Schuell, Inc., Keene, N.H.) by the method of Southern (49). Hybridizations were performed at medium stringency, and the nitrocellulose filters were washed as described previously (35). Colony blots using 541 filters (Whatman) were performed as described elsewhere (19) and hybridized under medium stringency as described above. Cloning of ail. The ail locus was cloned from seven of the Y enterocolitica strains listed in Table 1 either by cosmid cloning (strains 637-83 and 657-83) or by cloning isolated fragments with asymmetric ends directly into pBR322 (strains 634-83, 2440-87, 642-83, MC32, and 245587). Cosmid cloning into pLAFR2 digested with BamHI was performed essentially as described previously (28) using Y. enterocolitica chromosomal DNA fragments of 17 to 35 kb generated by partial Sau3A digestion. The ligation reaction was then packaged in bacteriophage X (Stratagene, La Jolla, Calif.) and used to infect E. coli LE392. Transfectants were selected on LB-tetracycline plates, and cosmids carrying ail were identified by colony blot hybridization with the Ail-C probe. Smaller fragments containing ail were subsequently subcloned into pBR322. For the remainder of the strains, an ail-containing EcoRI-HindlIl fragment was identified by Southern analysis, and DNA fragments of the appropriate size were purified from agarose slices by using Geneclean (BiolOl) and ligated to pBR322 digested with EcoRI and HindIII. The ligation mixture was transformed into E. coli HB101, and ail-containing recombinant plasmids were identified by colony blot with the Ail-C probe. DNA sequencing. The ail gene and surrounding DNA were sequenced from double-stranded plasmid DNA by using the dideoxy method of Sanger et al. (44) as modified for use with Sequenase (U.S. Biochemicals). Four different oligonucleotide primers corresponding to bp 563 to 546 (VM1 5' CAC TCGCAGCGTACACAT 3'), 864 to 848 (VM2 5' GCGGC CCCAGTAATGG 3'), 1066 to 1048 (VM3 5' GGGGTTC ACTTCACTCAGG 3'), and 783 to 798 (VM4 5' GGGGCC ATCTTTCCGC 3') of the nucleotide sequence of the serotype 08 ail reported by Miller et al. (33) were used. Tissue culture cells and the TCI assay. Human laryngeal epithelial (HEp-2) and chinese hamster ovary (CHO) cells were maintained and prepared for the TCI assay as previously described (17). Quantitative TCI assays, using gentamicin for selection of intracellular bacteria, were performed as described previously (35). Attachment of bacteria was assessed by determining the total number of cell-associated bacteria as previously described (35).

J. BACTERIOL.

Western blot (immunoblot) analysis. Membrane proteins were prepared from Y. enterocolitica and E. coli as previously described (38, 46). Whole-cell extracts were prepared from 1.5 ml of cultures grown at 37°C with aeration in LB. The cells were collected by centrifugation and washed twice with 10 mM Tris (pH 8.0). The cells were resuspended in 100 RI of 10 mM Tris (pH 8.0), boiled for 5 min, and then cooled to ambient temperature before 2 U of DNase (Promega Biotec) was added. The samples were digested with DNase at 37°C for 2 h and then stored at -20°C. The extracts, either whole cell or membrane, were mixed with sodium dodecyl sulfate-sample buffer containing dithiothreitol with or without 10 M urea and boiled for 5 min, and then the proteins were separated by electrophoresis through a polyacrylamide gel (25, 47). The proteins were then transferred to nitrocellulose and incubated with the indicated primary antibody as described elsewhere (9). After incubation with the primary antibody, immunoblots were incubated with a secondary antibody (either goat anti-rabbit or goat anti-mouse) conjugated to alkaline phosphatase. Antibody binding was visualized by incubating the filter with 5-bromo-4-chloro-3-indolylphosphate and p-Nitro Blue Tetrazolium chloride as previously described (5). Monoclonal antibody 2B2 (immunoglobulin G2a) to Ail was kindly provided by J. Bliska. Ail purified from E. coli pVM102 as described elsewhere (33) and eluted from a polyacrylamide gel was used to immunize a New Zealand White rabbit by standard techniques (20).

RESULTS Identification of Ail in Y. enterocolitica. Previous studies clearly indicated that Ail from Y. enterocolitica 8081 (serotype 08) was produced by E. coli carrying ail recombinant plasmids and could be found in the membrane fraction (33). To determine if Ail was synthesized by and localized to the same compartment in 18 different Y. enterocolitica strains (11 different serotypes), membrane fractions were isolated and analyzed by immunoblot with an Ail-specific monoclonal antibody (2B2). All American serotypes tested produced a membrane protein that reacted with this monoclonal antibody (see Fig. 1 and SA; data not shown; summarized in Table 1). This immunoreactive protein had the same mobility as Ail purified from E. coli carrying the ail recombinant plasmid pVM103 (Fig. 1). Strain 9291-78, previously shown not to carry ail sequences (35), did not have any protein that reacted with 2B2. In contrast to the American serotype isolates, the non-American serotype isolates did not react with monoclonal antibody 2B2 even though they are known to carry ail-homologous sequences (Fig. 1). Whole-cell extracts of the non-American serotype isolates were also tested but did not have any 2B2 reactive proteins (see Fig. SA). A number of possibilities could account for this unexpected result: the ail gene in these strains may not be transcribed, the ail mRNA may not be translated, or the non-American serotype Ail may lack the epitope recognized by 2B2. To test the first of these possibilities, total RNA was isolated from the strains listed in Table 1 and analyzed by Northern dot blot with the Ail-C probe. The two negative controls, Y. enterocolitica 9291-78 and E. coli pBR322, did not synthesize mRNA that hybridized to this probe. All other strains produced ail mRNA with homology to the Ail-C probe (data not shown). Several of these strains were also examined by Northern blot with the Ail-C probe and were found to produce the same-size ail mRNA as American serotype strain 8081 (data not shown). Thus, it would appear that the failure to detect Ail in Western analysis from

AMINO ACID SUBSTITUTIONS IN Ail VARIANTS

VOL. 174, 1992 1

+

2

+

3 4

at

@0 Y.

N 0)

5

6 7 8 9 10 11 12 13 14 15 16

1 t

*I +

*

+

+ ++

non- American serotypes

-0

c ~0~~~~0

@0

+

-

-

+

C'X

L

X

-

N ,

°

m C

FIG. 1. Western analysis of membrane fractions from ail' Y enterocolitica using monoclonal antibody 2B2. Membrane fractions were prepared as described in Materials and Methods from overnight cultures of the indicated bacteria grown in LB with aeration at 37°C. Ail (lanes 11 and 12) was purified from E. coli(pVM103) as previously described (33). The presence (+) and absence (-) of urea in the sample buffer are indicated. Ail tends to run as a single band only after being boiled with urea. The lanes contain samples from the following strains: 1, 9291-78; 2, 642-83; 3, MC51; 4, 6771-84; 5, 2452-87; 6, MC32; 7, 637-83; 8, 2517-87; 9, 2455-87; 10, 8081c; 11 and 12, purified Ail; 13 and 14, E. coli(pVM103); 15 and 16, E. coli(pBR322). S, protein standards. Plasmid pVM103 carries the ail gene from Y enterocolitica 8081c (34).

1363

non-American serotypes is not due to an inability to transcribe the gene. To address the other possibilities, the ail gene was cloned and analyzed from several of these Y. enterocolitica strains. Cloning and characterization of ail from different serotypes of Y. enterocolitica. The ail locus was cloned from seven additional strains of Y. enterocolitica as described in Materials and Methods. All recombinant clones, including the original 08 serotype clone pVM102 (34), have inserts of the ail locus in pBR322; the recombinant plasmids and corresponding strains are listed in Table 1. Partial restriction maps of the clones are shown in Fig. 2, along with the locations of ail and Ail-B hybridizing fragments. A 1.2-kb AvaI-ClaI fragment containing ail was conserved in all the clones, and nucleotide sequencing data indicated that other additional restriction endonuclease sites within this fragment were also conserved (see below). However, there was considerable restriction endonuclease site heterogeneity both upstream and downstream of ail. Confirming previous observations indicating that an IS3like element is unique to American serotypes (35), the Ail-B probe, which contains an IS3-like sequence, hybridized only to the American serotype clones; in each case, this IS3-like sequence was located adjacent to the 3' end of ail. Southern hybridization analysis of genomic DNA using Ail-A, a 500-bp probe immediately downstream of Ail-B on pVM102 (the 08 ail clone), had previously indicated that these sequences were present in both American and non-American serotype strains (32). Therefore, the ail clones were examined for hybridization to Ail-A. Despite the presence of several kilobases of downstream sequences in these clones, no sequences homologous to Ail-A were found in the nonAmerican serotype ail clones (data not shown). These results suggested that there may be changes between the two

SEROTYPE, PLASMID A-08.

C

ALPKA

_A

.C

AL

x

A

C

I

A

pVI102

(8081) A-020. pB10

ILLp

(657-83) A-o4.32. p135

H

(63-83) A-08. pXB

NA-O1.2.3. p1B7

c Li

(2440-87)

F

C

AL

c

H

A

(2455-87) NA-0.27. pXB2

KA )I

iP

A

.c

H

P

A

.c

AV

c

I

(637-83) NA-03. p1B3

c

i

(1C32)

NA-09. p134

X

(642-83) 1 kb

FIG. 2. Restriction maps of ail clones. Shown are partial restriction maps of ail loci from different Y enterocolitica strains cloned into pBR322. The plasmid designation is shown on the left along with the serotype and pathogenic group (American [A] or non-American [NA]). The strain from which the clone originated is indicated in parentheses. Plasmid pVM102 was previously described (34); it does not have the AvaI site present in the other clones. However, plasmid pVM103 from the same strain contains a larger insert with additional sequences 5' of ail which does have an Aval site in the same relative position as that indicated in the other clones. C, ClaI; A, AvaI; H, HindIll; E, EcoRI; P, PstI. Stippled boxes indicate the locations of ail; slashed boxes indicate the fragments that hybridized to the Ail-B probe.

1364

BEER AND MILLER

groups in this downstream region aside from a simple insertion of an IS3-like element. Sequence analysis of ail clones. The nucleotide sequences of ail and surrounding regions in each of the clones were determined (Fig. 3). The nucleotide sequences of ailA, including 5' and 3' flanking sequences, from American serotype strains were identical to the published 08 sequence (33) except that the 04,32 sequence had a single nucleotide change within the coding region. In contrast, the nonAmerican serotype sequences were identical to each other but exhibited considerable sequence divergence from the American serotype sequence (5% change). There were 21 nucleotide changes in the ail coding region, 9 of them resulting in altered amino acid residues. The nucleotide sequences of these two groups diverged significantly 30 bp downstream of ail. As expected, the sequence downstream of the site of divergence in ailA had several features in common with members of the IS3 family of insertion sequences (48). These include a TGA at the terminus and a cluster of C residues followed by an A+T-rich region. An alignment of the American (AilA)- and non-American (AilNA)-derived amino acid sequences of Ail is shown in Fig. 4. The two proteins were each 178 amino acids in length with typical procaryotic N-terminal signal sequences and were 95% identical (97% similar). There were nine amino acid differences from the 08 Ail sequence from strain 8081; one of these differences was in the signal sequence. Six of the remaining eight amino acid changes were clustered in the C-terminal third of the protein. Protein products of ail clones. These clones were then analyzed by in vitro transcription-translation reactions to determine if they are capable of directing the synthesis of Ail. All clones directed the synthesis of a novel polypeptide that comigrated with Ail encoded by pVM102 (data not shown). Previous analysis had indicated that this is the product of ail (33). To confirm that Ail was expressed from E. coli carrying recombinant clones and had the same antibody reactivity as Ail from Y. enterocolitica, Western analysis using either the monoclonal antibody 2B2 (Fig. 5A) or a polyclonal antibody (Fig. 5B) was done on whole-cell extracts. Again, it was observed that ARlNA, whether expressed in E. coli or Y enterocolitica, did not react with 2B2. All clones (ailA and ailNA) and all ail-containing Y enterocolitica reacted with a polyclonal antibody raised against Ail, whereas E. coli carrying pBR322 and the ail mutant Y enterocolitica 9291-78 did not react with this antibody. These results indicated that Ail is synthesized in comparable amounts by both ailA and ailNA strains, and thus the lack of reactivity with 2B2 is probably due to an altered epitope in ABlNA. Invasion phenotype of ail clones in E. coli. E. coli HB101 carrying pVM102 was considerably more invasive for tissue culture cells than the background level observed with HB101 carrying the cloning vector pBR322 (Table 2) (34). This phenotype was more prominent in CHO cells than in HEp-2 cells, although HB1O1(pVM102) attached equally well to both cell types (34). Therefore, the invasion of HEp-2 and CHO cells by recombinant E. coli expressing the different ail clones was examined (Table 2). All clones conferred an invasive phenotype on E. coli with the same tissue culture cell specificity previously observed. However, E. coli carrying the ailA clones were five- to sixfold more invasive than E. coli carrying the ailNA clones. This difference does not appear to be due to a defect in attachment, since both groups of clones attached well to the tissue culture cells. The average levels of attachment to HEp-2 and CHO cells,

J. BACTERIOL.

respectively, were 21.7 and 37.1% for non-American serotype clones and 21.6 and 35% for American serotype clones. The isoleucine-to-threonine change at residue 165 in ailA from serotype 04,32 had no effect on invasion by E. coli carrying this clone.

DISCUSSION The ail gene and the region surrounding it are of interest for a number of reasons. First, the presence of ail is highly correlated with virulence in humans and with invasion of mammalian cells in tissue culture assays (35). Second, a large region surrounding ail is unique to pathogenic serotypes of Y. enterocolitica (35). Third, there are differences between the two major pathogenic groups (American and non-American) of Y. enterocolitica at this locus. One of these differences, the presence of an IS3-like element near ailA but not ailNA, was previously reported (35); other differences at this locus are presented in this report. Fourth, Ail is a member of a growing family of proteins which includes the Salmonella typhimurium virulence protein PagC (41). AilA and Al1NA differ antigenically, as evidenced by the difference in reactivity of monoclonal antibody 2B2 to Ail from American and non-American serotypes. Pursuing these initial observations, we cloned ail from seven different serotypes of Y. enterocolitica (four non-American and three American), determined the ail nucleotide and derived amino acid sequences, and assayed the abilities of these clones to confer an invasive phenotype on E. coli HB101. All of these clones directed the synthesis of a protein that reacts with polyclonal antibody to purified Ail. Restriction endonuclease mapping and DNA sequence analysis indicated that the region immediately upstream of ail is conserved among both groups of serotypes but that the region downstream of ail differs considerably between the two groups. Although E. coli carrying ailA or ailNA clones adhered well to HEp-2 and CHO cells, the ailA clones promoted five- to sixfold more invasion of these tissue culture cells by E. coli than did the ailNA clones. For the following reasons, we believe that this reduction in invasiveness of E. coli ailNA is due to specific alterations in the protein sequence rather than to alterations in levels of expression or localization. The cloned ail genes are all present in the same cloning vector (pBR322) and, with the possible exception of pKB5 (an American serotype 04,32 clone that promotes a high level of invasion), direct the synthesis of comparable amounts of protein in in vitro transcription-translation reactions. In addition, Western analysis indicated that comparable amounts of Ail were produced by all clones and all strains. Both AilA and AiINA have typical signal sequences, and both are found in the membrane fraction of either Y. enterocolitica or recombinant E. coli expressing Ail. Together, these results suggest that the eight amino acid changes found in the mature form of AilNA compared with AilA are responsible for the decreased invasion phenotype of E. coli carrying ailNA. It is possible that these amino acid changes drastically alter protein folding or topology in the membrane, but if this were the case, then the AiINA would probably not promote invasion by E. coli at all rather than simply result in reduced invasiveness. Also, the hydrophobicity profiles of AilA and AilNA are very similar, and most (seven) of the amino acid changes are predicted to fall in cell surface domains (see below). The observation that pathogenic Y. enterocolitica can be separated into two distinct classes with respect to the ail genes suggests that the changes at this locus precede

1365

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08 08 020 04,32 01,2,3 05,27 03 09

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FIG. 4. Derived amino acid sequences of Ail for the indicated serotypes. The top line is from the previously published sequence of Ail from Y. enterocolitica 8081 (33). Dashes indicate identical amino acids. Underlined amino acids at the N terminus indicate the location of the signal

sequence.

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Invasion assays were performed as described in reference 35. All plasmids listed were carried in E. coli HB101, and all ail clones were cloned into plasmid pBR322. b Invasion is expressed as follows: percent invasion = 100 x (number of gentamicin-protected bacteria/number of bacteria added). These are the avarages of duplicate samples and are representative of several experiments. Numbers in parentheses represent the fold increase over background [invasion seen with HB101(pBR322)]. a

changes in serotype. Similar observations have been noted for other loci (10, 11, 14). Thus, it appears that the separation into American and non-American groups reflects the clonal variation of pathogenic Y. enterocolitica. Recently, the sequences of a number of proteins (Lom, PagC, OmpX) similar to Ail have been reported. Lom is an outer membrane protein of no known function encoded by bacteriophage X and expressed in X lysogens of E. coli (3). PagC is an outer membrane protein from S. typhimurium (41); mutations in pagC result in a decrease in macrophage survival and a decrease in virulence (31). OmpX is an outer membrane protein from Enterobacter cloacae (50); the funcA

TABLE 2. Invasion phenotype of E. coli carrying ail clones from different serotypesa

Yersinia

FIG. 5. Western analysis of whole-cell extracts from Y. enterocolitica and recombinant E. coli. (A) Monoclonal antibody 2B2 was used as the primary antibody; (B) polyclonal antibody to Ail was used as the primary antibody. Proteins from whole-cell extracts were separated on a 10% Laemmli gel (25) in panel A or a 12% Tricene gel (47) in panel B. Ail was purified from E. coli(pVM103) as described elsewhere (33). NA and A refer to non-American and American, respectively. Lane 1 contains purified Ail. Lanes 2 through 10 contain whole-cell extracts from E. coli carrying the following plasmids: lane 2, pBR322; lane 3, pKB2; lane 4, pKB3; lane 5, pKB4; lane 6, pKB7; lane 7, pKB5; lane 8, pKB6; lane 9, pKB10; lane 10, pVM102. Lanes 11 through 19 contain whole-cell extracts from Y enterocolitica strains 8081c (lane 11) 657-83 (lane 12), 2440-82 (lane 13), 634-83 (lane 14), 2455-87 (lane 15), 642-83 (lane 16), MC32 (lane 17), 637-83 (lane 18), and 9291-78 (lane 19).

tion of this protein is unknown, but overproduction of OmpX results in decreased levels of the porins OmpF and OmpC. Lomi, PagC, and OmpX have 31.5, 36.8, and 43.5% amino acid identity with Ail, respectively. There are several interesting things to note about this family. Two of the family members, Ail and PagC, are believed to be virulence factors. The functions of the other two family members are unknown, but conceivably they could also be virulence factors. Lom is encoded by a phage (3), and the ail gene in some strains is found near an IS3-like element (35), suggesting an association of these genes with mobile genetic elements; this is in keeping with their fairly broad distribution within members of the family Enterobacteriaceae. Despite the high degree of similarity between these proteins throughout their length, there is as yet no known common function. lom and pagC when cloned into E. coli do not confer an attachment or invasion phenotype as does ail (37). This raises the question of the significance of the observed similarities. Based on the guidelines put forth by Tommassen (53), a model for the folding of OmpX within the outer membrane has been proposed (50). If the same guidelines are applied to Ail or the other family members, a very similar model is obtained (Fig. 6). In this model, the membrane-spanning domains of Ail are predicted to be amphipathic ,-sheets, with one side having a high average hydrophobicity (0.713) and the other side having a lower average hydrophobicity (-0.025) on the normalized scale of Eisenberg et al. (15). According to this model, seven of the eight amino acid changes in the mature protein of AilNA are predicted to be on the cell surface (Fig. 6). If one looks at the alignment of the family members in the predicted membrane, periplasmic, and cell surface domains, a fairly striking pattern emerges (Fig. 6). The average amino acid identity to Ail in the membrane-spanning domains ranges from 46.1% for Lom to 60.7% for OmpX. In contrast, the level of identity to Ail of the predicted cell surface domains is very low, ranging from 6.1% for Lom to 11.8%

VOL. 174, 1992

AMINO ACID SUBSTITUTIONS IN Ail VARIANTS

1367

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50.7% 46.1% 41.7% 28.3% 11.8% 9.8% 6.1% FIG. 6. Model for the folding of Ail within the outer membrane. The primary amino acid sequence of Ail from Y enterocolitica 8081 (American serotype) is shown as it is hypothesized to be folded in the outer membrane. The circled amino acids are those that are altered in the sequences of Ail from non-American serotypes compared with those of Ail from American serotypes. OM, outer membrane.

for OmpX. Because these are the regions of the proteins that interact with the host (or environment), it is thus perhaps not surprising that no common function has been found for this family of proteins. One would predict that family members with similar functions would have more-extensive similarity in the cell surface domains. The identity between the known Ail family members may represent a conserved structural motif important for localization in the outer membrane, since the conserved domains occur primarily in the membranespanning domains. Supporting this proposition is the fact that the terminal membrane-spanning domains of these proteins each contain a motif (alternating hydrophobic amino acids with Tyr-X-Phe at the C terminus) that is highly conserved among outer membrane proteins (54) including, among others, the porins of E. coli, PII of Neisseria gonorrhoeae, and VirG of Shigella flexnen. It has been proposed that this is an important signal for outer membrane localization (54). The second predicted membrane domain of this family is of particular interest. Seven of the 10 amino acids of this domain are identical in all family members (G-NKYRYE-), and 3 of these conserved residues are charged (underlined). The extreme conservation of this segment raises the possibility that this domain is important for outer membrane targeting or for interaction with a second protein that may be involved in localization or function of this family. This sequence was not found in other outer membrane proteins; thus, it is probably not a general signal for outer membrane localization. Obviously, further testing of this model is necessary both in terms of overall topology within the membrane and function of the highly conserved domain. However, it would suggest that a "parental" gene spread throughout the Enterobacteriaceae (and beyond?) by association with mobile genetic elements. Once in a new strain background, the gene diverged to fulfill different functions while retaining similar structures. This model, in conjunction with the sequence

analysis of the naturally occurring variants of ail, will help direct our future efforts in identifying and analyzing the functional domains of Ail and other members of its family. ACKNOWLEDGMENTS We thank J. Bliska for providing Y enterocolitica Ail monoclonal antibodies and C. Collins, J. Miller, J. Pepe, M. Wachtel, and S. Valone for critically reading the manuscript. This work was supported by Public Health Service grant A127342 to V. L. Miller from the National Institutes of Health. V. L. Miller is a Pew Scholar in the Biomedical Sciences. REFERENCES 1. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 2. Baker, P. M., and J. J. Farmer III. 1982. New bacteriophage typing system for Yersinia enterocolitica, Yersinia kristensenii, Yersinia frederiksenii, and Yersinia intermedia: correlation with serotyping, biotyping, and antibiotic susceptibility. J. Clin. Microbiol. 15:491-502. 3. Barondess, J. J., and J. Beckwith. 1990. A bacterial virulence determinant is encoded by lysogenic coliphage X. Nature (London) 346:871-874. 4. Bissett, M. L., C. Powers, S. L. Abbott, and J. M. Janda. 1990. Epidemiological investigations of Yersinia enterocolitica and related species: sources, frequency, and serotype distribution. J. Clin. Microbiol. 28:910-912. 5. Blake, M. S., K. H. Johnson, G. J. Russell-Jones, and E. C. Gotschlich. 1984. A rapid, sensitive method for detection of alkaline phosphatase conjugated anti-antibody on Western blots. Anal. Biochem. 136:175-179. 6. Bottone, E. J. 1977. Yersinia enterocolitica: a panoramic view of a charismatic microorganism. Crit. Rev. Microbiol. 5:211-241. 7. Bottone, E. J. 1983. Current trends of Yersinia enterocolitica isolates in the New York City area. J. Clin. Microbiol. 17:63-67. 8. Bufill, J. A., and P. S. Ritch. 1989. Yersinia enterocolitica serotype 03 sepsis after blood transfusion. N. Engl. J. Med. 320:810.

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9. Burnette, W. N. 1981. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112:195-203. 10. Carniel, E., D. Mazigh, and H. I. Mollaret. 1987. Expression of iron-regulated proteins in Yersinia species and their relation to virulence. Infect. Immun. 55:277-280. 11. Carniel, E., 0. Mercereau-Puijalon, and S. Bonnefoy. 1989. The gene coding for the 190,000-Dalton iron-regulated protein of Yersinia species is present only in the highly pathogenic strains. Infect. Immun. 57:1211-1217. 12. Cornelis, G., Y. Laroche, G. Ballingad, M.-P. Sory, and G. Wauters. 1987. Yersinia enterocolitica, a primary model for bacterial invasiveness. Rev. Infect. Dis. 9:64-87. 13. Cover, T. L., and R. C. Aber. 1989. Yersinia enterocolitica. N. Engl. J. Med. 321:16-24. 14. Delor, I., A. Kaeckenbeeck, G. Wauters, and G. R. Cornelis. 1990. Nucleotide sequence of yst, the Yersinia enterocolitica gene encoding the heat-stable enterotoxin, and prevalence of the gene among pathogenic and nonpathogenic yersiniae. Infect. Immun. 58:2983-2988. 15. Eisenberg, D., E. Schwarz, M. Komaromy, and R. Wall. 1984. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J. Mol. Biol. 179:125-142. 16. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. 17. Finlay, B. B., and S. Falkow. 1987. A comparison of microbial invasion strategies of Salmonella, Shigella, and Yersinia species. UCLA Symp. Mol. Cell. Biol. 64:227-243. 18. Gemski, P., J. R. Lazere, and T. Casey. 1980. Plasmid associated with pathogenicity and calcium dependency of Yersinia enterocolitica. Infect. Immun. 27:682-685. 19. Gergen, J. P., R. H. Stern, and P. C. Wenseink. 1979. Filter replicas and permanent collections of recombinant DNA plasmids. Nucleic Acids Res. 7:2115-2136. 20. Harlowe, E., and D. Lane. 1988. Antibodies, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 21. Ichinohe, H., M. Yoshioka, H. Jukushima, S. Kaneko, and T. Maruyama. 1991. First isolation of Yersinia enterocolitica serotype 08 in Japan. J. Clin. Microbiol. 29:846-847. 22. Isberg, R. R., and S. Falkow. 1985. A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli K-12. Nature (London) 317:262-264. 23. Isberg, R. R., D. L. Voorhis, and S. Falkow. 1987. Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell 50:769-778. 24. Janot, C., M. E. Briquel, F. Streiff, and J. C. Burdin. 1989. Infectious complications due to transfusion acquired Yersinia enterocolitica. Transfusion 29:372-373. 25. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277:680-685. 26. Lee, L. A., A. R. Gerber, D. R. Lonsway, J. D. Smith, G. P. Carter, N. D. Puhr, C. M. Parrish, R. K. Sikes, R. J. Finton, and R. V. Tauxe. 1990. Yersinia enterocolitica 0:3 infections in infants and children, associated with the household preparation of chitterlings. N. Engl. J. Med. 322:984-987. 27. Lee, W. H., P. H. McGrath, P. H. Carter, and E. L. Eichie. 1977. The ability of some Yersinia enterocolitica strains to invade HeLa cells. Can. J. Microbiol. 23:1714-1722. 28. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 29. Mekalanos, J. J. 1983. Duplication and amplification of toxin genes in Vibrio cholerae. Cell 35:253-263. 30. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 31. Miller, S. I., A. M. Kukral, and J. J. Mekalanos. 1989. A two component regulatory system (phoP and phoQ) controls Salmo-

32. 33. 34. 35.

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nella typhimurium virulence. Proc. Natl. Acad. Sci. USA 86: 5054-5058. Miller, V. L. Unpublished results. Miller, V. L., J. B. Bliska, and S. Falkow. 1990. Nucleotide sequence of the Yersinia enterocolitica ail gene and characterization of the Ail protein product. J. Bacteriol. 172:1062-1069. Miller, V. L., and S. Falkow. 1988. Evidence for two genetic loci from Yersinia enterocolitica that can promote invasion of epithelial cells. Infect. Immun. 56:1242-1248. Miller, V. L., J. J. Farmer III, W. E. Hill, and S. Falkow. 1989. The ail locus is found uniquely in Yersinia enterocolitica serotypes commonly associated with disease. Infect. Immun. 57: 121-131. Miller, V. L., and J. J. Mekalanos. 1984. Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR. Proc. Natl. Acad. Sci. USA 81:3471-3475. Miller, V. L., S. I. Miller, J. J. Barondess, and J. Beckwith. Unpublished results. Pepe, J. C., and V. L. Miller. 1990. The Yersinia enterocolitica inv gene product is an outer membrane protein that shares epitopes with Yersinia pseudotuberculosis invasin. J. Bacteriol. 172:3780-3789. Pierson, D. E., and S. Falkow. 1990. Nonpathogenic isolates of Yersinia enterocolitica do not contain functional inv-homologous sequences. Infect. Immun. 58:1059-1064. Portnoy, D. A., S. L. Moseley, and S. Falkow. 1981. Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis. Infect. Immun. 31:775782. Pulkkinen, W. S., and S. I. Miller. 1991. A Salmonella typhimurium virulence protein is similar to a Yersinia enterocolitica invasion protein and a bacteriophage lambda outer membrane protein. J. Bacteriol. 173:86-93. Robins-Browne, R. M., and J. K. Prpic. 1985. Effects of iron and desferrioxamine on infections with Yersinia enterocolitica. Infect. Immun. 47:774-779. Robins-Browne, R. M., S. Tzipori, G. Gonis, J. Hayes, M. Withers, and J. K. Prpic. 1985. The pathogenesis of Yersinia enterocolitica infections in gnotobiotic piglets. J. Med. Microbiol. 19:297-308. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. Schiemann, D. A., and J. A. Devenish. 1952. Relationship of HeLa cell infectivity to biochemical, serological, and virulence characteristics of Yersinia enterocolitica. Infect. Immun. 35: 497-506. Schnaitman, C. A. 1971. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J. Bacteriol. 108:545-552. Schogger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368-379. Schwarz, E., M. Kroger, and B. Rak. 1988. IS150: distribution, nucleotide sequence and phylogenic relationships of a new E. coli insertion element. Nucleic Acids Res. 16:6789-6802. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Stoorvogel, J., M. J. A. W. M. van Bussel, J. Tommassen, and J. A. M. van de Klundert. 1991. Molecular characterization of an Enterobacter cloacae outer membrane protein (OmpX). J. Bacteriol. 173:156-160. Tacket, C. O., J. P. Narain, R. Sattin, J. P. Lofgren, C. Konigsberg, R. C. Redtorff, A. Rausa, B. R. Davis, and M. L. Cohen. 1984. A multistate outbreak of infections caused by Yersinia enterocolitica transmitted by pasteurized milk. JAMA 251:483-486. Tipple, M. A., L. A. Bland, J. J. Murphy, M. J. Arduino, A. L. Panililio, J. J. Farmer III, M. A. Tourault, C. R. Macpherson, J. E. Menitove, A. J. Grindon, P. S. Johnson, R. G. Strauss, J. A. Bufill, P. S. Ritch, J. R. Archer, 0. C. Tablan, and W. R.

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Jarvis. 1990. Sepsis associated with transfusion of red cells contaminated with Yersinia enterocolitica. Transfusion 30:207213. 53. Tommassen, J. 1988. Biogenesis and membrane topology of outer membrane proteins in Escherichia coli, p. 352-373. In J. A. F. Op den Kamp (ed.), Membrane biogenesis. NATO ASI series, vol. H16. Springer-Verlag KG, Berlin. 54. Truyve, M., M. Moons, and J. Tommassen. 1991. Carboxyterminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein. J. Mol. Biol. 218:141-148. 55. Une, T. 1977. Studies on the pathogenicity of Yersinia enterocolitica. II. Interaction with cultured cells in vitro. Microbiol. Immunol. 21:365-377.

56. Vesikari, T., T. Nurmi, M. Maki, M. Skurnik, C. Sundqvist, K. Granfors, and P. Gronroos. 1981. Plasmids in Yersinia enterocolitica 03 and 09: correlation with epithelial adherence in vitro. Infect. Immun. 33:870-876. 57. Wachtel, M. R., and V. L. Miller. Unpublished results. 58. Young, V. B., V. L. Miller, S. Falkow, and G. K. Schoolnik. 1990. Sequence, localization and function of the invasin protein of Yersinia enterocolitica. Mol. Microbiol. 4:1119-1128. 59. Zink, D. L., J. C. Feeley, J. G. Wells, C. Vanderzant, J. C. Vickery, W. D. Roof, and G. A. O'Donovan. 1980. Plasmidmediated tissue invasiveness in Yersinia enterocolitica. Nature (London) 283:224-226.

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Amino acid substitutions in naturally occurring variants of ail result in altered invasion activity.

Yersinia enterocolitica is the causative agent of a variety of gastrointestinal syndromes ranging from acute enteritis to mesenteric lymphadenitis. In...
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