Phase-variable outer membrane proteins in Escherichia coli

IMMUNOLOGY AND MEDICAL MICROBIOLOGY

FEMS Immunology and Medical Microbiology 16 (1996) 63-76 ELSEVIER

MiniReview

Phase-variable outer membrane proteins in Escherichia coli

Peter Owen * , Mary Meehan, Helen de Loughry-Doherty, Ian Henderson

Department oj Microbiology, Moyne Institute of Preuentiue Medicine, Trinity College Dublin, Dublin 2, Ireland

Received 22 April 1996; accepted 19 June 1996

Abstract

Escherichia coli contains at least two phase-variable proteins in its outer membrane. One, termed antigen 43 (Ag43), is

the product of the metastable flu gene located at min 43.6 on the E. coli chromosome and is responsible for colony form variation and for autoaggregation in liquid media. Ag43 is composed of two proteinaceous subunits, a43 and p43 in 1:I stoichiometry. (y43 (apparent M, 60,000) is surface expressed, extends beyond the O-side chains of smooth lipopolysaccharide and is bound to the cell surface through an interaction with p43 (apparent M, 53,000), itself an integral,

heat-modifiable, outer membrane protein. cy43 shows limited N-terminal sequence homology with certain enterobacterial

adhesins, and notable sequence homology with AIDA-1, an adhesin of diffuse-adhering E. coli. In addition, (y43 contains an RGD motif and a consensus sequence for an (autoproteolytic?) aspartyl protease active site. Expression of Ag43 is subject to reversible phase variation - in liquid minimal medium, the rates of variation from Ag43+ to Ag43- states and from Ag43- to Ag43+ states being = 2.2 X 1O-3 and = 1 X 10V3, respectively. Phase switching of Ag43 is regulated by DNA

methylation (deoxyadenosine methylase (dam) mutants being ‘locked OFF’) and by OxyR (oayR mutants being ‘locked ON’). It is proposed that OxyR acts as a repressor of Ag43 transcription by binding to unmethylated GATC sites in the regulatory region of the gene. In some strains, Ag43 may also undergo antigenic variation. A 94 kDa immunocrossreactive outer membrane protein, showing similar rates of phase variation, has additionally been detected for some enteropathogenic and uropathogenic strains of E. co/i. This 94 kDa protein can be proteolytically cleaved in situ with trypsin to yield two membrane-bound products with M,s and properties similar to those of ff43 and p43. Results suggest that Ag43 may

represent one of a family of antigenically-related high-& adhesins which are synthesized as polyprotein precursors. Some members may be processed and presented on the cell surface as bipartite protein complexes (as Ag43). Others can remain uncleaved.

Keywords: Phase variation; Outer membrane proteins: Escherichia coli; Antigen 43; flu

1. Introduction fimbriae, wall-associated proteins, capsules, lipo-

Phase and antigenic variation are well-established properties of a nu.mber of bacterial surface antigens/virulence determinants including flagella,

polysaccharide (LPS), and envelope-associated/outer membrane proteins (see Tables 1 and 2). The selec- tive advantage imparted to bacteria displaying these types of variation range from immune avoidance, enhanced survival in changing environments, to fa-

* Corresponding author. ‘Tel: +353 (I) 608 1188; fax: +353

(1) 679 9294.

cilitated spread of infection [1,2]. In Gram-negative bacteria, much attention has focused on the phase-

0928-8244/96/$15.00 Copyright 0 1996 Published by Elsevier Science B.V. All rights reserved. PII SO928-8244(96>00069-7

64 P. Owen et al./ FEMS Immunology and Medical Microbiology 16 (1996) 63-76

Table 1

Some bacterial surface antigens undergoing phase/antigenic variation

Antigen Organism Frequency (ON + OFF) Mechanism

Flagella

Type 1 fimbriae

P-fimbriae

987P fimbriae

Type IV pili

Serospecific agglutinins

M protein

Aggregation substance

Variable lipoproteins

LPS epitopes

Capsule

Salmonella spp.

Escherichia coli Escherichia coli Escherichia coli Neisseria gonorrhoeae Neisseria meningitidis Bordetella pertussis Group A streptococci

Enterococcus faecalis Mycoplasma hyorhinis Haemophilus influenzae Neisseria spp.

10-3-10-5

1.05 x 10-s

2.6 x lo--’

2.7 x 1o-3

10-3

10-3-10-6 10-3-10-4

10-z

site specific inversion

Dam and Lrp control of inversion

Dam and Lrp control of transcription

unknown

strand slippage during translation and

post translational glycosylation

BvgAS control of transcription

VirR regulated?

TraEl control of transcription

strand slippage during transcription

strand slippage during translation

strand slippage?

Haenwphilus influenzae recombinational deletion and Ret-dependent amplification

Data compiled from Refs. [ 1,46-581.

variable fimbrial adhesins [3-51. The presence and variable outer membrane protein, together with its

properties of several phase-variable outer membrane initial characterization, have been documented [9-l 11

proteins have also been documented but, for the and reviewed [ 12,131. The present article will focus

main, these are restricted to bacteria such as Neisse- on more recent developments concerning the pres-

ria gonorrhoeae, N. meningitidis, Bordetella pertus- ence, properties and likely significance of this and sis, Haemophilus injluenzae and Borrelia spp. (see other related phase-variable enterobacterial outer

Table 2). membrane proteins.

A number of years ago, workers in this laboratory began a systematic immunochemical analysis of the

envelopes of Escherichia coli ML308-225 013:068:H- [6-81. During the course of these studies, a novel bipartite protein, termed antigen 43 (Ag43), was identified. The circumstances surround-

ing the discovery of this somewhat unique phase-

2. Biochemical properties of Ag43

Ag43 appears to be an Escherichia-specific antigen. It is a major cellular component, can be present in copy numbers up to = 50,0OO/cell, and is located

Table 2

Phase-variable outer membrane proteins of Gram-negative bacteria

Organism Protein Function Mechanism of phase variation

Escherichia coli

Neisseria gonorrhoeae, Neisseria meningitidis Neisseria meningitidis Haemophilus influenzae Bordetella pertussis

Borrelia hermsii Borrelia burgdorferi

antigen 43 adhesion? Dam and OxyR control of transcription?

94 kDa protein adhesion? Dam and OxyR control of transcription?

Opa adhesion/invasion strand slippage during translation and recombination

OPC adhesion/invasion strand slippage during transcription

OapA adhesion? unknown

FHA adhesion BvgAS control of transcription

BrkA adhesion/serum resistance BvgAS control of transcription

QmpQ nutrient transport? BvgAS control of transcription

Vmp lipoproteins immune evasion non-reciprocal recombination

Osp lipoproteins immune evasion non-reciprocal recombination

Abbreviations: FHA, filamentous haemagglutinin.

Data compiled from Refs. [l 1,19,52,59-701.

P. Owen et al. / FEMS Immunology and Medical Microbiology 16 (1996) 63-76 65

exclusively in the outer membrane [lo]. Ag43 is composed of two chemically and immunologically distinct protein subunits (termed cy43 and p43> in 1:l stoichiometry, i.e. 043:p43 (Fig. 1). The (y43 subunit has an apparent M, of 60,000 following

sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). p43 shows pronounced heat-modifiability and migrates with apparent M,s of

37,000 if heated to temperatures I 70°C prior to SDS-PAGE and 53,000 if heated at higher temperatures (see Fig. 3, lanes 4-6). This is reminiscent of

the behaviour of several other outer membrane proteins (notably OmpA) and is probably indicative of a

12 3

FepA*

8 mpF,

o$z

460 453

Fig. 1. Subunit composition of Ag43. Lanes 1 and 3 contain,

respectively, Triton X-lOO-EDTA extracts of “C-labelled outer

membranes of E. coli ML308-225 (15 pg protein) and immune

complexes precipitated from the same by anti-Ag43 immuno-

globulins. Lane 2 contains a mixture of material shown in lanes 1

and 3. Samples were analyzed by SDS-PAGE and the identities

and apparent molecular masses (in kDa) of the 043 and p4’ subunits and other salient membrane proteins are indicated at the

side of the autoradiogram.

monomer with extremely stable p-structure. The (Y 43

and p43 polypeptides are not linked by disulphide bonds, and do not possess detectable carbohydrate, identifiable co-factors, acyl groups or enzyme activity, at least as judged by standard histochemical

assays [ 101. The hypothesis that (Y 43 and p43 form an anti-

genie complex in situ [lo] is supported by (a) immunoprecipitation reactions conducted under non-de- naturing conditions on Triton X- 1 OO-solubilized envelopes in which subunit-specific antisera always result in coprecipitation of similar amounts of o43 and p43 (Figs. 1 and 2B) [lo]; (b) reconstitution experiments in which an (Y 43: p43 complex can be regenerated from mixtures composed of purified (y43 and /343-containing membranes stripped of the ff43 subunit [ 111; (c) the detection of an unstable (Yap: p”” complex (M, = 115,000) capable of dissociating to give 043 and p43 monomers in Western immuno-

blotting experiments conducted on outer membranes solubilized in SDS at 2O”C-30°C [14,15]; (d) cross- linking reactions.

These latter cross-linking experiments, involving the use of both non-cleavable and cleavable homobi- functional reagents (spans 1. l- 1.2 nm), were’ performed in association with Western immunoblotting and test-tube immunoprecipitation experiments (Fig. 2A and B, respectively) in which subunit specific

anti-a43 and anti-p43 immunoglobulins [ 11,161 were used to selectively view interactions involving Ag43. Cross-linking results in the appearance of numerous additional immunoreactive bands in the M, range

80,000-400,000, the most prominent of which has an apparent M, 210,000 (Fig. 2A, lanes 2-4; Fig. 2B, lane 2). This band is distinct from the various multimers observed following cross-linking of purified undenatured LY 43 (Fig. 2A, lane 5). Interest- ingly, reductive cleavage of the disulphide cross- bridge within these high-M, complexes results not only in the elimination of the high-M, bands and the regeneration of the subunit monomers but also in the

appearance of an 80 kDa polypeptide (see Fig. 2B, lane 4) identified from its N-terminal amino acid

sequence (Q’EPTDTPVSXDXIVVTXAi9) as the iron-enterochelin receptor, FepA [ 171. More detailed examination reveals that complexes in the M, 115,000 region contain LY’~ and /343 cross-linked as heterodimers and that the dominant cross-linked

66 P. Owen et al. / FEMS Immunology and Medical Microbiology 16 (1996) 63-76

species (M, 210,000) represents an Ag43:FepA

complex [15]. The significance of this latter observation is unclear, but may have more to do with

restricted lateral mobility [ 181 and structural similarities between two major integral outer membrane

A 12345678910

115b _

80,

B1234

(210

455

4115

proteins (FepA and /343) than with any functional interaction.

An indication of the region of ff43 interacting with /?43 comes from studies in which a variety of

proteases can be shown to C-terminally cleave purified (y43 to give a 48 kDa product. In contrast, envelope-associated CY 43 is relatively resistant to

exogenously-supplied proteases, irrespective of whether the host cell bears either smooth (Sl-LPS or rough (RI-LPS. Thus, (y43 appears to be anchored to

P 43 in such a way as to effectively protect its

C-terminus from proteolysis [ 151.

Both subunits have been purified to homogeneity. CYST, but not p43, can be selectively and almost

quantitatively released from E. coli outer membranes by brief heating to 60°C (see Fig. 8). Purifica-

Fig. 2. Cross-linking and nearest neighbour analysis of Ag43.

Panel A: Outer membranes (lanes l-4 and 6-9) and purified (Y 43

(lanes 5 and 10) were incubated with DSP (dithiobissuccinimidyl

propionate) at the following protein:DSP ratios, viz. 1:0 (lanes 1

and 6), 1O:l (lanes 2 and 7); 5:l (lanes 3 and 8); 2: 1 (lanes 4 and

9); and 2O:l (lanes 5 and 10). Cross-linked outer membranes (25

pg protein) and cross-linked (Y 43 (2 p,g protein) were solubilized

in taemmli sample buffer [44] in the absence (lanes l-5) and

presence (lanes 6-10) of 2-mercaptoethanol. Samples were re-

solved on SDS-PAGE gels containing 7.5% (w/v) acrylamide and

were probed by Western immunoblotting [16] using anti-(u43

immunoglobulins. Note that (a), as anticipated, uncrosslinked

membranes contain only one immunoreactive band corresponding

to the a43 subunit (lanes 1 and 6) and (b) additional high-M,

bands induced by crosslinking (lanes 2-5) disappear and the a43

subunit is regenerated when cross-bridges are cleaved with 2-mer-

captoethanol (lanes 7-10). Panel B: Immune complexes precipi-

tated by anti-cu43 sera from Triton X-lOO-EDTA extracted un-

cross-linked (lanes 1 and 3) and cross-linked (lanes 2 and 4)

14C-labelled outer membranes were analyzed by SDS-PAGE using

a 7.5% (w/v) acrylamide separating gel and fluorography. Sam-

ples were solubilized in Laemmli sample buffer [44] in the

absence (lanes 1 and 2) and presence (lanes 3 and 4) of 2-mer-

captoethanol. Note that (a) subunit specific anti-o43 immuno-

globulins can precipitate both 04’ and p4’ from undenatured

membrane preparations (lanes 1 and 31, (b) the profile of immuno- precipitated polypeptides observed for cross-linked membranes

(lane 2) is very similar to that obtained in Western immuno-

blotting experiments (Panel A, lane 31, (c) the 80 kDa FepA protein is precipitated from cross-linked but not from uncross-lin-

ked membranes (lanes 3 and 4) or by preimmune serum (not shown). In Panels A and B the identities of (Yap, p43 and FepA

and the molecular masses in kilodaltons of some of the main

immunoreactive cross-linked complexes are indicated at the side

of the immunoblot/fluorogram.

P. Owen et al. / FEMS Immunology and Medical Microbiology 16 f 1996) 63-76 67

- 411, m

Fig. 3. Purification of p4J as monitored by SDS-PAGE. Lane 1,

SDS extract of heat-stripped Triton X-lOO-Mg’+ insoluble outer

membranes (5 p,g protein); lane 2, pooled fractions following

Sephacryl S-300 gel filtration (5 pg protein): lane 3, soluble

material obtained following butanol precipitation (4 pg protein);

lanes 4 and 5, butanol-ins~oluble material (4 pg protein): lane 6,

p43 following preparative SDS-PAGE (2 pg protein). Prior to

electrophoresis, samples were heated for 5 min at either 100°C

(lanes l-4 and 6) or 50°C (lane 5). The identities of the 53 kDa

(p) and 37 kDa (p’) forms of p43, and some salient outer

membrane proteins are indicated at the side of the gel.

tion to apparent homogeneity is then readily achieved by gel filtration and ion-exchange chromatography. Purified cr43 (pZ= 4.6; polarity of 49.2%) elutes as a monomer of appare:nt kf, 50,000 [ 111. Purification of /?43 (polarity 47.9%) is more difficult, but can be achieved [14] by detergent solubilization of heat-

stripped outer membranes bearing peptidoglycan, removal of peptidoglycan and associated porins and lipoproteins, gel filtr.ation in presence of SDS, cold butanol precipitation, and preparative SDS-PAGE (Fig. 3).

3. Sequence analysis of Ag43

There is no significant homology between the N-terminal sequence of ,B 43 (P ’ TNVTLASGATW- NlPDNATVQSV23..:l and other published sequences. However, the N-terminus of (y43 (A’DIVVHPGET-

VNGGTLANHDNQ**..) contains a stretch of six residues (in bold) which is also present in the N- termini of the major subunits of several enterobacterial fimbriae [11,19]. Moreover, V8 cleavage of denatured cy43 generates, amongst other species, 3 low-M, peptides (hf,s 3,000-9,500) with distinct

N-terminal sequences [20], each showing about 50% homology with internal sequences within AIDA-1, an outer-membrane-protein adhesin of diffuse adhering E. coli [21]. These features have recently been confirmed and extended following cloning by poly-

merase chain reaction of the DNA encoding the cy43 subunit [22]. Nucleotide sequence analysis of the cloned product predicts an M, 49,634 protein displaying: (a) 30% overall identity (72.5% similarity) with AIDA- [21] and 22% overall identity (70% similarity) with the phase-variable filamentous haemagglutinin from Bordetellu pertussis [23]; (b) a repetitive motif (TV.N.GG.Q.V...G.A..) not dissimi- lar to that observed in AIDA- [21]; (cl an RGD motif implicated in the binding of human integrins [24]; (d) a stretch of amino acids (...LLADSGAAVS- CT...) compatible with the consensus observed for an aspartyl protease active site [25].

4. Ag43 is surface expressed

Ag43 is clearly a major surface antigen for E. coli. Its presence on the cell surface of wild-type strains bearing S-LPS can be readily demonstrated by progressive immunoadsorption experiments [8], immunofluorescence microscopy and by immuno-

gold labelling (Figs. 4 and 5). Results of Western blot analysis performed on serum adsorbed with

Al 2 Bl 23

Fig. 4. Surface expression of the Ag43 subunits as demonstrated

by immunoadsorption and Western immunoblotting. Anti-Ag43

serum was incubated with buffer (lanes Al and Bl) or with an

equal volume of either E. coli ML308-225 cells (lane A2) or

doubling concentrations of E. coli C600 cells (lanes B2 and B3).

Following removal of agglutinated cells, the various adsorbed sera

were used at equivalent dilutions in Western immunoblotting [ 161

experiments resolving outer membranes of E. coli ML308-225. The positions of the o43 and p43 subunits are indicated. Note

that cells bearing R-LPS adsorb antibodies to both subunits (lanes

Bl-B3) whereas cells bearing S-LPS selectively adsorb anti-o

immunoglobulins (lanes Al -A2).


intact cells (Fig. 4) and of direct immunofluorescence studies using subunit specific sera [ 151 have

antibody in strains bearing S-LPS, whereas both

additionally established that only (Y 43 is accessible to subunits are accessible in strains expressing R-LPS. It will be recalled that most major outer membrane

A2

P. Owen et al. / FEMS Immunology and Medical Microbiology 16 (1996163-76 69

proteins (e.g. porins and OmpA) with known

polypeptide domains on the trans (outer) surface of the outer membrane are poorly accessible to antibody, bacteriophage and colicins in strains bearing

lengthy O-antigen side chains [26,27]. This suggests a more fibrous structure for CX~~, facilitating its penetration of the 0-polysaccharide repeats and expression on the cell surface. The tram domains of

P 43, an integral membrane protein, probably reside closer to the membrane surface, and only become

unmasked in strains bearing truncated R-LPS

molecules.

5. Ag43 undergoes phase variation

A notable property of Ag43 is its ability to undergo reversible phase variation i.e. ON + OFF -+ ON [ 11,121. This can be demonstrated readily following comparative .analysis of Ag43+ and Ag43- variants by colony immunoblotting, immunofluorescence microscopy, immunogold labelling and by Western immunoblotting (Figs. 5 and 9). Im- munofl uorescence experiments further confirm that 80-90% of cells derived from Ag43+ colonies express the antigen on their surface whereas only a minority (l-5%) of cells derived from Ag43- colonies do so. Screening of progeny derived from

both positive and negative variants of E. coli ML308-225 grown in succinate minimal media indi- cates‘ that rates of phase variation from Ag43+ to Ag43- states and from Ag43- to Ag43+ states approximate to 2.2 .X 10m3 and 1 X 1K3, respectively. The corresponding rate of switch (Ag43+ + Ag43-) for cells grown in L-broth is about four-fold higher. These values are similar to those cited for other phase-variable surface structures (see Table 1).

Neither growth on glucose, in high osmolarity (0.3 M NaCl) or in the presence of high or low Fe2+, affects either switching frequency or levels of ex-

pression as judged by both Western immunoblotting and immunofluorescence experiments. However, in common with a number of bacterial virulence determinants [28,29], expression of Ag43 is affected by temperature - growth at 24°C causing a 4- to 5-fold

decrease in levels of expression but little apparent change in switch frequencies [15].

Diverse and complex molecular mechanisms are involved in phase variation of bacterial surface components (Tables 1 and 2) [l-3]. Recent studies have indicated that Ag43 may add to this evolving list of mechanisms [19]. Thus, as assessed by a combina- tion of colony and Western immunoblotting and by immunofluorescence microscopy, a variety of well-

defined regulatory mutants (cp.p, cya, fis, gyrA, himA, hns, hul, hu2, h-p, recA, rpoS, and topA) all show levels and patterns of expression of Ag43 analogous to wild-type strains. A role for the leucine response protein (Lrp) in Ag43 expression is further eliminated in experiments in which addition of leucine and alanine to the growth medium show little effect on Ag43, but cause a 32- to 16-fold reduction in the (Lrp-regulated) expression of IS99 fimbriae. Significantly, however, as judged by the above screening techniques, strains carrying mutations in deoxyadenosine methylase (Dam) [30] lack all trace of Ag43, i.e. dam mutants are ‘locked OFF’ for

expression of Ag43. In contradistinction, oxyR mutants constitutively express Ag43 and show no evidence of phase switch, i.e. they are ‘locked ON’ for Ag43 expression. Introduction of the dam mutation into oxyR derivatives fails to affect the ‘locked ON’ expression of Ag43. Based on this and other evidence a tentative model for the regulation of Ag43 has been proposed [19]. In this model, competition

between OxyR (a LysR-type transcriptional activator [29]) and Dam for unmethylated GATC sites in the regulatory region of the gene leads to phase variation. Methylation of GATC sites by Dam prevents OxyR (but not RNA polymerase) from binding, and

Fig. 5. Ag43 of E. coli IvIL308-225 undergoes phase variation. Panel A: colony immunoblots performed using anti-cu4’ sera on Ag43+ (Al) and Ag43- (A2) variants following growth in succinate minimal medium [45] and subculture onto L-agar. Positive ( + ve), negative

(- ve), and sectored (S) colonies are indicated. Panel B: corresponding immunofluorescent photomicrographs performed using anti-a43 sera

and fluorescein-labelled secondary antibody of the Ag43+ (Bl) and Ag43- (B2) variants shown in Panel A. Panel C: transmission electron

micrograph of cells following frozen-thin sectioning and labelling with anti-a43 conjugated to 20 nm gold particles. The bar represents 0.2

km. Note the presence wIthin all panels of a minority population expressing the opposite phenotype.


123456

.+_-_ _ _

.i 2 3. 4 16. 6

Fig. 6. Ag43 displays antigenic heterogeneity. (y43 subunits puri-

fied [ 1 l] from the envelopes of five uropathogenic E. coli isolates

and of E. coli ML308-225 (lanes 1-6, respectively) were screened

by Western immunoblotting [ 161 (panel A) and by rocket immuno-

electrophoresis (RIE, panel B) against antibodies raised to purified

cr43 from E. coli ML308-225. Note that weak reactions are

observed for some preparations (Nos. 1, 3 and 5) following RIE

and that preparations showing two a43 homologues by Western

immunoblotting (Nos. 2 and 4) generate a single major RIE band

which can be shown by precipitate excision [9] to correspond only

to the lower of the two M, forms.

transcription proceeds to give phase ON cells. Bind- ing of the OxyR repressor to unmethylated GATC sites physically excludes RNA polymerase leading to the transcriptionally inactive (phase OFF) state [19]. Although competition for unmethylated GATC sites as a basis of regulation of antigen expression is not

new (e.g. competition between Lrp and Dam affects

123456

pup gene expression [4]), to the authors’ knowledge this is the first example of one involving OxyR, a protein better known for its ability to act as a transcriptional activator of genes important in oxidative stress [29,31].

A recent survey of enteropathogenic (EPEC) and uropathogenic strains has found evidence for molecular heterogeneity in Ag43 expression. Thus, some

strains produce proteins which bear all the hallmarks of cy43 subunits (i.e. they have M,s in the 60,000 range, can be selectively detached and purified from outer membranes following brief heating at 60°C have closely similar N-terminal amino acid se-

quences and react in Western immunoblots with anti-a 43 sera) but possess slightly different M,s and/or antigenicities (see Figs. 6 and 7). Notable in this respect is the presence in some strains of homologues which react with anti-a43 sera in denatured form (i.e. in Western immunoblots) but not in undenatured form (i.e. following immunodiffusion, rocket immunoelectrophoesis or test tube precipitation reac-

tions; Fig. 6). Whether such differences are reflective of rapid antigenic variation of the type manifested by the gonococcal Opa proteins (Table 2) and extend additionally to the anchoring ( p43) subunit remains

to be established.

6. Ag43 is the product of the jlu gene

Located at min 43.6 on the E. coli chromosome is a metastable gene called flu. This gene is often cited

7 8 9_ 10 11 12

Fig. 7. Presence of cross-reacting membrane proteins in enteropathogenic strains of E. cofi. Cell envelopes (25 kg protein) prepared in the

presence of a cocktail of protease inhibitors from E. coli ML308-225 (lanes 1, 5, 9 and 12) and EPEC strains of serotypes 044 (NCTC

9044; lane 2). 018ac (NCTC 10863; lane 31, 0128 (NCTC 9708; lane 41, 026 (NCTC 9026; lane 6), 0126 (NCTC 8622; lane 7). 0142

(NCTC 10089; lane 81, 0111 (NCTC 8007; lane lo), 0114 (NCTC 9114; lane 11) were analyzed by SDS-PAGE and Western immunoblotting [16] using antibodies raised to purified (Y 43 from E. coli ML308-225 (anti-a.43 immunogobulins). The positions of LY 43, the 94 kDa protein, and some of the 18 standard M, proteins used are indicated at the side of the immunoblot, only the relevant part of

which is shown.


as a prime example of phase variation in prokary- otes. The flu locus was originally identified by Diderichsen in 1980 and shown to be responsible for colony form variation in many E. coEi strains. Diderichsen established that Flu- variants can give rise to large, rough, irregular colonies and to autoaggregation in liquid media, whereas Flu+ variants produce smaller, smoother ‘colonies and do not au- toaggregate [32]. Up till now, little additional

progress [33] has been made in characterizing the flu gene or identifying the product responsible for colony form variation. There was original speculation [32] that (type 1) fimbriae may be involved but this has

proved unfounded. In these respects, a major break- through has been the recent identification of Ag43 as

the likely product of the flu gene [19]. Thus, by screening the Kohara lambda-mini set

gene bank [34] with an ff43-gene probe, the DNA encoding Ag43 has bbeen located to the region (min 43) of the E. coli chromosome encompassing jlu. That j7u encodes A.g43 has been established by correlating the behaviour of thousands of authentic

E. coliflu variants in colony immunoblotting, West- em immunoblotting, and immunofluorescence experiments conducted using anti-Ag43 and subunit- specific antisera. Unfortunately, the following rather

confusing relationship exists between the Flu phenotype [32] and Ag43 expression, viz. Flu- = Ag43+ and Flu+ = Ag43 - [ 191. In order to minimize further confusion, and mindful of the admittedly serendipi- tous incorporation of the appropriate map position into the original name [6,10], we will persevere with the term Ag43, at least until a function for this unique outer membrane protein has been determined

with some certainty.

7. Function of Ag43

As yet we cannot unambiguously ascribe a func-

tion to Ag43. The most obvious candidate in view of its surface expression, amino acid sequence homology, integrin-binding motif, involvement in autoaggregation and phase variability would be a novel type of adhesin. The h4, of the complex and of its subunits are, of course, far outside the normal range for the major subunits of E. coli fimbriae. However, the combined IV&S and properties of (Y 43 and p 43

are not unlike those of (precursor forms of) certain afimbrial colonization factors associated with bacterial outer membranes, e.g. the AIDA-1, intimin and Tsh proteins of E. coli [21,3.5,36], the IcsA and SepA proteins of Shigellajlexneri [37,38], the bordetella pertactins [39] and the Hap [40] and Hsr [41] proteins from H. influenzae and Helicobacter muste- Zae, respectively. Certainly, Ag43 does not possess any morphologically recognisable or regular repeat- ing structure that is discernible either by negative staining [ 10,l I] or by deep etching of Ag43+ and Ag43- variants [15]. This latter observation would tend to rule out an earlier suggestion that Ag43 may represent an enterobacterial S-layer [lo].

Preliminary experiments have provided some evidence for a role in adhesion. Thus, Ag43+ variants

were shown to adhere to cultured Hep-2 cells significantly (6- to g-fold) better than Ag43- variants. Furthermore, adhesion occurred in a mannose resistant fashion and could be inhibited in a dose response manner by purified (Yap. Related haemagglutination experiments conducted with a range of ery-

throcytes (from 12 species) and yeast cells were less illuminating. Both Ag43+ and Ag43- variants displayed patterns of weak mannose-sensitive haemagglutination compatible with the low amounts of type 1 fimbriae demonstrable for these strains. The bal-

ance of evidence suggested that if Ag43 causes haemagglutination, it has a pattern similar to that of type 1 fimbriae [15]. It should be stressed that there is a problem in performing meaningful adhesion studies with strains in which the putative adhesin undergoes phase variation, since results are compro- mised by the presence within any variant population

of a significant minority expressing the opposite phenotype. This problem should be circumvented with the generation of isogenic strains locked ON and locked OFF for Ag43 expression [ 191.

Whatever its precise function, Ag43 is certainly expressed by a wide variety of E. coli strains from laboratory workhorses [lo] to pathogens and also in vivo. Thus, a survey of thirteen classical EPEC strains (serotypes 018ac, 026,044,055,086,0111, 0114, 0119, 0125-0128 and 0142) by colony immunoblotting and immunofluorescence microscopy using anti-a43 antibodies revealed the presence in ten of cross-reacting phase-variable surface component(s). Western blot analysis of cell

72 P. Owen el al. / FEMS Immunology and Medical Microbiology 16 (1996) 63-76

envelopes derived from positive variants showed that at least eight of these strains possessed heat-releasa- ble, cross-reactive proteins in the M, range 54,000- 60,000. Some strains additionally possessed an M, 94,000 protein (see Fig. 7 and below). Furthermore, in a recent survey of 96 uropathogenic strains of E. coli, a significant percentage (60%) was found to react positively when infected urines were examined

by immunofluorescence microscopy. These isolates in turn displayed the presence of phase-variable, cross-reacting polypeptides in the appropriate M, range following subculture and Western immunoblotting with anti-cu43 sera.

8. Other phase-variable outer membrane proteins

An interesting and possibly significant observation is the demonstration in some enterobacterial pathogens of additional phase-variable outer membrane proteins of higher M,. Thus, the aforemen- tioned survey of EPEC revealed the presence in four strains of a 94 kDa protein which cross-reacted

strongly with anti-a 43 immunoglobulins. The 94

Al 234 61 2

Fig. 8. Properties of the 94 kDa cross-reacting protein. Panel A:

outer membranes of E. coli 044 were incubated at 4°C or at 60°C

for 5 min prior to centrifugation (48,000X g for 1 h). Pellets (25

p,g protein) and supernatant fractions (at equivalent volume load-

ings) were then analyzed by SDS-PAGE and Western immuno-

blotting. Lanes 1 and 2, pellet and supernatant fractions, respec-

tively, obtained from control (unheated) membranes. Lanes 3 and

4, pellet and supernatant fractions respectively obtained from

membranes heated at 60°C. Panel B: outer membranes (2.5 pg

protein) of E. coli 044 were heated to 100°C (lane 1) or to 55°C

(lane 2) in Laemmli sample buffer [44] prior to analysis by

SDS-PAGE and Western immunoblotting [16]. The position of

043, the 94 kDa protein and heat-modified products are indicated

at the side of the immunoblots, which were both developed using

anti-a43 immunoglobulins. Note that cr43 but not the 94 kDa

protein is released from outer membranes following heating to

60°C (panel A) and that the 94 kDa protein shows altered mobility

following heating to 55°C cf. 100°C (panel B).

c!* - ,L^

.; ,,<p

1’ ,.‘.Sa !I.

6123 4 5676 910

Fig. 9. The 94 kDa cross reacting protein undergoes phase varia-

tion. Lysates were formed directly from individual colonies of E. coli ML308-225 (lanes Al, A2, Bl-B3), E. cofi 0142 (lanes

A3-AlO) and E. coli 044 (lanes B4-BlO) which had given either

strong positive reactions (lanes Al, A3-A7, Bl, B4-B7) or weak

negative reactions (lanes A2, A8-AlO, B2, B3, BS-BlO) follow-

ing colony immunoblotting with anti-o43 immunoglobulins. Sam-

ples (15 pg protein) were then analyzed by SDS-PAGE and

Western immunoblotting [16] using anti-o43 antibodies. The posi-

tion of cy43 and of the 94 kDa proteins are indicated at the side of

the immunoblots. Note the absence of the 94 kDa protein from

negative colony variants of E. coli 0142 (panel A), and of the

o43 subunit from negative colony variants of E. coli ML308-225

and E. coli 044 (panel B).

kDa protein is located in the outer membrane as judged from analysis of membrane fractions har- vested following isopycnic gradient centrifugation but, unlike the cy43 subunit, cannot be heat-released

from cell envelopes. It reacts poorly with anti-/343 sera but, like /I 43, is heat-modifiable, migrating with apparent M,s of 90,000 and 74,000 if heated at 50°C for 15 min prior to electrophoresis (Fig. 8).

The phase variability of the 94 kDa protein is best demonstrated in a strain such as E. coli 0142 which

lacks cross-reacting polypeptides in the M, range anticipated for the o43 subunit (see Fig. 7). Here, excellent correlation exists between the results of colony immunoblotting, immunofluorescence and Western immunoblotting experiments conducted with cross-reacting anti-cl! 43 antibodies. Thus, a majority of cells in a positive colony variant react with anti- (y43 serum in immunofluorescence experiments and display the 94 kDa protein following Western immunoblotting. Colonies reacting weakly following immunoblotting possess a minority population (I-

P. Owen et al./ FEMS Inwnunology and Medical Microbiology 16 (1996) 63-76 73

2%) which react in immunofluorescence and possess

only trace levels of the 94 kDa protein on Western blots (see Fig. 9A). Comparative analysis by immunofluorescence of ,E. coli ML308-225 (Ag43+ 94 kDa_1, E. coli 0142 (Ag43- 94 kDa+) and corresponding derivatives carrying the dam mutation suggests that both the frequency and mechanism of phase switch of the two antigens are similar.

The situation is sl:ightly more complex and less easy to interpret in strains such as E. coli 044 which

possesses both Ag43 and 94 kDa proteins (see Fig. 7). Here, as judged by immunofluorescence and immunoblotting using anti+ 43 serum, bacteria display at least three distinct levels of expression, the 94 kDa protein appearing to switch independently of the immunodominant, cross-reactive Ag43 (see Fig.

9B). A notable feature of the 94 kDa protein is its

sensitivity to exogenous proteases (Fig. lOA). It can be readily cleaved in situ with trypsin to generate two membrane-associated products, viz. (a) a 55 kDa protein which, like CYST, can be selectively released

from outer membranes by heat shock at 60°C (Fig. 10B); (b) a heat-modifiable product (M, 50,000) similar to p 43. In s.trains such as E. coli 044

(Ag43+ 94 kDa+ 1, the LY and p homologues pro- duced by controlled proteolysis are clearly distinct (at least in M, and possibly antigenicity) from the endogenous c4!43 and p43 subunits. It should be stressed that, at present, there is no evidence for a precursor-product relationship between the 94 kDa

protein and Ag43. Thus, the 94 kDa protein is a stable feature of outer membrane profiles, all at- tempts to stimulate its endogenous cleavage to native

Ag43 subunits being unsuccessful. The above considerations apart, there is evidence

that Ag43 is synthesized initially as a high-M, polyprotein precursor. The DNA encoding /343 is contiguous with the 3’-end of (y43 DNA [22]. Fur- thermore, cy43 contains an aspartyl protease active site [22] possibly responsible for rapid and efficient processing. This may <explain the consistent inability to detect putative polyprotein precursors of Ag43 in cells of E. coli ML308-225 or K12 derivatives lyzed in the presence of a cocktail of protease inhibitors. It seems not unlikely that Ag43 is synthesized, ex- ported, and processed. in a manner reminiscent of gonococcal IgAl protease [42]. Certainly, Ag43

A 1 23456 Bl 234

Fig. 10. An (Y 43 homologue can be generated from the 94 kDa

cross-reacting protein by proteolysis. Panel A: cell envelopes of

E. coli 0142 were treated for 10 min at 20°C with trypsin at the

following envelope:trypsin protein ratios, viz. l:O, 1000: 1; 500: I;

2oO:l 100~1 and 5O:l (lanes 1-6, respectively). Proteolysis was

stopped by boiling in Laemmli sample buffer [44] and samples (25

pg envelope protein) immediately analyzed by SDS-PAGE. Panel

B: outer membranes of E. co/i 044 were treated with trypsin at a

membrane:trypsin protein ratio of 1OO:l and proteolysis stopped

by the addition of a cocktail of protease inhibitors. Trypsin-treated

membranes were then incubated for 5 min at either 4°C (lanes 1

and 2) or 60°C (lanes 3 and 4) prior to centrifugation (48,000 X g

for 1 h). Pellets (lanes 1 and 3) and supematant fractions (lanes 2

and 4) were immediately analyzed at equivalent volume loadings

by SDS-PAGE. The positions of the 94 kDa protein, its 54 kDa

degradation product, and tx4j are indicated at the side of the

corresponding Western immunoblots developed using anti-tr43

immunoglobulins. Note that the 54 kDa degradation product (a)

reacts weakly with anti-o43 immunoglobulins (panel A) and (b)

has an apparent M, similar but not identical to cy43 and, like CX~~,

becomes detached from envelopes at 60°C (panel B).

seems to have features (such as a C-terminal domain rich in /?-structure, few disulphide bonds, C-terminal processing and a protease active site) which are characteristic of members of the IgAl protease-like autotransporters. It should be noted that this class of

high-M, proteins includes colonization factors such as AIDA-1, Tsh, pertactin, Hap and Hsr [38,43].

9. Conclusions

Antigen 43 appears to be one of a family of phase-variable and antigenically related adhesins (?> which bear structural similarities to the IgAl protease class of autotransporters. Some members of the Ag43 family have cell surface expression as a ma- ture 94 kDa uncleaved polyprotein (as in E. coli

0142). Others appear to be post-translationally (auto?)processed and expressed on the cell surface as


an (Y~~: /343 bipartite protein complex, e.g. Ag43 of E. coli ML308-225. A bipartite structure might, in certain circumstances, facilitate release of the adhe- sive antigenic subunit ((Y 43), thus perhaps priming

eukaryotic receptors for subsequent interaction with P43-bearing cells or provide another mechanism of immune evasion.

Acknowledgements

This work was supported in part by a grant (HRR 32-91) from the Health Research Board of Ireland. The authors would like to express their appreciation to J. Beesley and U. Sleytr for performing the immunogold labelling and freeze-etch analysis, respectively.

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Phase-variable outer membrane proteins in Escherichia coli