INFECTION AND IMMUNITY, Aug. 2009, p. 3234–3243
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 8
Sab, a Novel Autotransporter of Locus of Enterocyte
Effacement-Negative Shiga-Toxigenic Escherichia coli
O113:H21, Contributes to Adherence and
Sylvia Herold, James C. Paton, and Adrienne W. Paton*
Research Centre for Infectious Diseases, School of Molecular and Biomedical Science,
University of Adelaide, Adelaide, South Australia 5005, Australia
Received 8 January 2009/Returned for modification 20 February 2009/Accepted 21 May 2009
Shiga-toxigenic Escherichia coli (STEC) strains cause serious gastrointestinal disease, which can lead to
potentially life-threatening systemic complications such as hemolytic-uremic syndrome. Although the produc-
tion of Shiga toxin has been considered to be the main virulence trait of STEC for many years, the capacity to
colonize the host intestinal epithelium is a crucial step in pathogenesis. In this study, we have characterized
a novel megaplasmid-encoded outer membrane protein in locus of enterocyte effacement (LEE)-negative
O113:H21 STEC strain 98NK2, termed Sab (for STEC autotransporter [AT] contributing to biofilm forma-
tion). The 4,296-bp sab gene encodes a 1,431-amino-acid protein with the features of members of the AT protein
family. When expressed in E. coli JM109, Sab contributed to the diffuse adherence to human epithelial (HEp-2)
cells and promoted biofilm formation on polystyrene surfaces. A 98NK2 sab deletion mutant was also defective
in biofilm formation relative to its otherwise isogenic wild-type parent, and this was complemented by
transformation with a sab-carrying plasmid. Interestingly, an unrelated O113:H21 STEC isolate that had a
naturally occurring deletion in sab was similarly defective in biofilm formation. PCR analysis indicated that sab
is present in LEE-negative STEC strains belonging to serotypes/groups O113:H21, O23, and O82:H8. These
findings raise the possibility that Sab may contribute to colonization in a subset of LEE-negative STEC strains.
Shiga-toxigenic Escherichia coli (STEC) strains are promi-
nent food-borne pathogens that cause watery or bloody diar-
rhea and hemorrhagic colitis, which can progress to the life-
threatening hemolytic-uremic syndrome (HUS) (15, 21, 29). In
order to establish and maintain an infection, STEC strains are
equipped with a diverse array of virulence factors. Among
these factors, Shiga toxin has been considered to be a sine qua
non of virulence, as reviewed previously (21, 29). However,
attachment of STEC to the human intestinal mucosa is a crit-
ical first step in pathogenesis. Many STEC strains, including
those of the highly prevalent O157:H7 serotype, carry the locus
of enterocyte effacement (LEE) pathogenicity island, which
encodes the capacity to produce attaching and effacing (A/E)
lesions on the intestinal epithelium, similarly to those pro-
duced by enteropathogenic E. coli strains (11, 35). These STEC
strains are often referred to as enterohemorrhagic E. coli
(EHEC), although this classification is ill defined. A/E lesions
are characterized by ultrastructural changes including the re-
modeling of the host cell cytoskeleton and intimate attachment
of the bacteria to the cell surface (11, 35). The process of the
generation of A/E lesions involves the expression of the eae
gene, which encodes intimin, an outer membrane surface ad-
hesin, and the delivery of the intimin receptor Tir and several
other effector proteins into host cells via the LEE-encoded
type III secretion apparatus (reviewed in references 5 and 11).
However, many STEC isolates from cases of severe disease,
including HUS, lack the LEE locus yet are clearly capable of
efficient colonization of the human gut (28, 29). Several can-
didate adhesins have been identified in these strains, including
the megaplasmid-encoded autoagglutinating adhesin Saa (26),
the long polar fimbriae encoded by the lpf operon (10) (two
distinct homologues of which are also present in STEC
O157:H7 strains [39, 40]), and the immunoglobulin-binding
protein EibG, which contributes to a chain-like adherence
phenotype on HEp-2 cells (18). Tarr et al. (38) also previously
identified Iha, a homologue of Vibrio cholerae IrgA, which
promotes the adherence of STEC O157:H7 to HeLa cells and
is widely distributed in LEE-positive and LEE-negative strains.
STEC O157:H7 strains also produce a type IV pilus, HCP (47),
and an E. coli common pilus, ECP (30), both of which contrib-
ute to in vitro adherence to intestinal epithelial cells. Addi-
tional putative adhesins from LEE-positive STEC strains in-
clude Efa1, which mediates the attachment of O111:NM STEC
strains to Chinese hamster ovary cells (23). In addition, Torres
et al. (41) previously identified a calcium-binding and heat-
extractable AT protein of EHEC, termed Cah, which mediates
aggregation and participates in biofilm formation. Recently,
Wells et al. (45) also characterized the EHEC-encoded AT
protein EhaA, which contributes to adherence to primary bo-
vine epithelial cells (but not HeLa cells) and promotes biofilm
formation as well.
The AT proteins referred to above belong to a rapidly grow-
ing family of gram-negative surface proteins that are exported
* Corresponding author. Mailing address: School of Molecular and
Biomedical Science, University of Adelaide, Adelaide, South Australia
5005, Australia. Phone: 61-8-83037552. Fax: 61-8-83033262. E-mail:
?Published ahead of print on 1 June 2009.
across the periplasmic space and either attached to the exter-
nal face of the outer membrane or released by proteolysis into
the environment (13). These large proteins share a character-
istic structure comprising three distinct domains, namely, an
N-terminal signal peptide, a divergent functional passenger
domain (?-domain), and a conserved C-terminal domain
which forms a ?-barrel pore in the outer membrane (13, 46).
This unique protein structure provides all the information re-
quired for transport to the cell surface, with the N-terminal
sequence directing the protein to the periplasm via the sec
pathway and the C-terminal domain mediating the transloca-
tion of the passenger domain to the external surface (14). The
various ?-domains confer a broad range of functions and/or
phenotypes including aggregation, biofilm formation, adher-
ence, invasion, serum resistance, and protease or esterase ac-
tivity (7, 12).
Research in our laboratory has focused on the identification
of novel virulence factors of LEE-negative STEC strains asso-
ciated with human disease. In this study, we describe the iden-
tification and characterization of a member of the AT family
produced by hypervirulent LEE-negative O113:H21 STEC
strain 98NK2, which confers adherence to human epithelial
cells and mediates biofilm formation.
MATERIALS AND METHODS
Bacterial strains, plasmids, and routine molecular biological techniques. The
E. coli strains and plasmids used in this study are listed in Table 1. Cells were
routinely grown in Luria-Bertani (LB) medium with or without 1.5% Bacto agar.
All bacteria were grown at 37°C except the temperature-sensitive strain
98NK2(pkD46). When required, antibiotics were used at 50 ?g/ml for kanamycin
and 100 ?g/ml for ampicillin. SOC medium (20 g/liter L-tryptone, 5 g/liter yeast
extract, 20 mM glucose, 8.6 mM NaCl, 2.5 mM KCl, 20 mM MgSO4) was used
as the recovery medium for transformants.
Routine DNA methods such as restriction, ligation, and transformation of
DNA were carried out as described previously by Maniatis et al. (19). Nucleotide
sequences were derived from E. coli K-12 or E. coli O113:H21 strain 98NK2 and
strain E41 sequences deposited in the National Center for Biotechnology Infor-
mation (NCBI) database. Purification of PCR products and isolation of plasmid
DNA were carried out using a MinElute PCR purification kit (Qiagen) and a
Qiaprep Spin minikit (Qiagen), respectively. All oligonucleotide primers were
obtained from Sigma Genosys. Restriction enzymes (New England Biolabs) were
used as recommended by the manufacturer.
Mutagenesis of 98NK2 and plasmid constructs. The deletion of the LH0147
(sab) gene and insertion of a kanamycin resistance cassette were performed using
the Lambda Red recombinase system as described previously (9). In order to do
so, a kanamycin resistance gene was amplified from plasmid pKD4 using the
Expand Long Template PCR system (Roche) and primer pairs d-LH0147-F
TGTAGGCTGGAGCTGCTTC-3?) and d-LH0147-R (5?-GGGTGTTCTGAC
TAG-3?). The resultant purified PCR product was electroporated into E. coli
strain 98NK2(pkD46). Kanamycin-resistant, ampicillin-sensitive recombinants
were checked by PCR using the respective flanking region primers and kanamy-
cin-specific primers K1, K2, and Kt, which were described previously (9). Suc-
cessful mutagenesis was confirmed by PCR and sequence analysis. The absence
or reduced size of PCR products obtained using primer pairs LH0147-f (5?-GG
TGGATACAGCAGGTAATG-3?) and LH0147-r (5?-TATCTCACCACCTGC
LH0147-fla-r (5?-CACCGACGGAGAAATTACCC-3?), respectively, confirmed
that all megaplasmid copies lacked the entire sab gene. Confirmed mutants were
The complete sab gene was also amplified from 98NK2 genomic DNA using
primer pair LH0147-BamHI (5?-CCCGGATCCGGAAACTCCAAGAGTATT
GC-3?) and LH0147-EcoRI (5?-CCCGAATTCCCTTGCTTTTCCCTGTTACC-
3?). The resultant PCR product was purified, digested with BamHI and EcoRI,
and ligated into BamHI and EcoRI restriction sites of pBluescript SK(?) to
generate pBsab. The plasmid was then transformed into E. coli JM109 and
confirmed by sequence analysis. For complementation of the 98NK2sab::kan
mutant, plasmid pBsab was introduced by transformation to generate
Purification of Sab and preparation of anti-Sab serum. Purification of Sab was
carried out using a Qiaexpressionist kit (Qiagen). In order to construct an
N-terminal His6-Sab fusion protein lacking the signal sequence and the translo-
cator domain, the region from nucleotides 94 to 2676 was amplified using the
Expand Long Template PCR system (Roche) and primer pair R1-LH0147-F
(5?-GGGGGATCCGAGGTTGATGAAGCTCCGGTG-3?) and R1-LH0147-R
(5?-GGGGCATGCCAGGTCACCGATATTTGCCGC-3?) with restriction sites
BamHI and SphI, respectively. The purified PCR product was digested and
ligated into pQE30 (Qiagen). Correct in-frame insertion was confirmed by re-
striction and sequence analysis. The recombinant plasmid (pQESab) was trans-
formed into the expression host E. coli M15(pREP4), and large-scale purification
of Sab was performed as follows. A log-phase LB broth culture of E. coli
M15(pREP4)(pQESab) containing 100 ?g/ml of ampicillin and 25 ?g/ml kana-
mycin was induced by the addition of 2 mM isopropyl-?-D-thiogalactopyranoside
TABLE 1. Bacteria and plasmids used in this study
E. coli strain or plasmid Descriptiona
STEC O113:H21 (HUS isolate)
STEC O113:H21 (food isolate)
E. coli K-12 derivate
JM109 carrying pB; Apr
JM109 carrying pBsab; Apr
98NK2 sab deletion mutant; Knr
98NK2sab::kan carrying pB; KnrApr
98NK2sab::kan with pBsab; KnrApr
E. coli K-12 derivate
M15(pREP4) carrying pQESab; Apr
pBluescript II SK(?)
sab cloned into pBluescript II SK(?)
pQE30 with nucleotides 94 to 2676 of sab
kan template for mutagenesis
Red recombinase expression vector
aKnr, kanamycin resistance; Apr, ampicillin resistance.
VOL. 77, 2009LEE-NEGATIVE STEC AUTOTRANSPORTER ADHESIN3235
(IPTG) (Inalco Pharmaceutical, CA) and incubated for 3 h with shaking at 37°C.
The cells were harvested by centrifugation (6,000 ? g for 15 min at 4°C) and
resuspended in 10 ml sonication buffer (50 mM Na2HPO4, 1 M NaCl, 40 mM
imidazole, 1 mM phenylmethylsulfonyl fluoride, 10 ?g/ml pepstatin, 10 ?g/ml
leupeptin). Cells were lysed using an SLM Aminco French pressure cell at 12,000
lb/in2. The cell lysate was then centrifuged at 100,000 ? g for 1 h at 4°C, and the
supernatant was loaded onto a Ni-nitrilotriacetic acid (HisLink; Promega) col-
umn preequilibrated with 10 ml sonication buffer at a rate of 15 ml/h. After
washing the column with 15 ml of wash buffer (50 mM Na2HPO4, 300 mM NaCl,
10% glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 1 ?g/ml pepstatin, 1 ?g/ml
leupeptin), the protein was eluted using 30 ml of a 0 to 500 mM imidazole
gradient. Ten 3-ml fractions were collected and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Peak fractions were
pooled, and purified protein was stored in phosphate-buffered saline (PBS)
containing 50% glycerol at ?20°C.
To generate antiserum, five male BALB/c mice were immunized by intraper-
itoneal injection of three 10-?g doses of purified Sab in alum adjuvant at 1-week
intervals, as described previously (26). Mice were exsanguinated by cardiac
puncture 1 week after the third immunization, and serum was stored at 4°C.
E. coli membrane isolation. E. coli strains were grown in LB broth, inner and
outer membranes were isolated, and proteins in the latter were fractionated on
the basis of solubility in 1% Zwittergen 3-14 (Calbiochem), as described previ-
ously by Veiga et al. (43).
Western blot analysis. Bacterial lysates and protein fractions were separated
by SDS-PAGE using a 4 to 20% Bis-Tris gradient gel (Invitrogen) according to
the manufacturer’s instructions. Loadings were standardized on the basis of
initial CFU for whole bacterial lysates (108CFU per lane) or total protein.
Immunodetection was performed following transfer onto polyvinylidene difluo-
ride membranes (Hybond; Amersham). Membranes were blocked for 30 min
with 1? Tris-buffered saline–0.05% Tween 20 containing 5% skim milk and
probed with mouse anti-Sab serum, followed by incubation with goat anti-mouse
immunoglobulin G conjugated to alkaline phosphatase (Bio-Rad). Bands were
detected using a chromogenic nitroblue tetrazolium–5-bromo-4-chloro-3-indolyl
HEp-2 and Henle 407 cell adherence assay. HEp-2 or Henle 407 cells in
Dulbecco’s modified Eagle’s medium (DMEM; Gibco) (with 10% fetal calf
serum [FCS]) were seeded onto 24-well tissue culture plates and used when
approximately 90% confluent. E. coli cells were grown overnight in LB medium
at 37°C and diluted to a density of approximately 104CFU/ml in DMEM
(without FCS or antibiotics). Washed monolayers were infected with 1 ml bac-
terial suspension and incubated for 3 h at 37°C (in 5% CO2). Following this step,
culture medium was removed, and monolayers were washed four times with PBS
to remove nonadherent bacteria. To detach and lyse cells, monolayers were
treated with 100 ?l of 0.025% trypsin–0.02% EDTA and 400 ?l of 0.025% Triton
X-100. Aliquots and serial dilutions thereof were plated onto LB agar to deter-
mine the total number of adherent bacteria (CFU/well). In parallel, strains were
incubated likewise but without epithelial cells, and growth of bacteria was mon-
itored by measurements of CFU/well. Results were expressed as a percentage of
adherent bacteria relative to total bacteria for each strain. Assays were carried
out in quadruplicate on two different days.
For Giemsa staining, 50% confluent HEp-2 cells grown on coverslips in 24-
well tissue trays were washed and infected with approximately 106CFU of the
appropriate strain suspended in 1 ml DMEM. After incubation at 37°C for 3 h,
monolayers were washed four times with PBS, fixed with 70% methanol for 10
min, and stained with Giemsa stain. Finally, washed coverslips were mounted
onto glass slides and examined by light microscopy.
ELISA. Surface expressions of Sab in 98NK2 and various recombinant strains
were compared using a whole unfixed bacterial cell enzyme-linked immunosor-
bent assay (ELISA), as described elsewhere previously (1), with the following
modifications. Strains were grown overnight in LB medium with the appropriate
antibiotics at 37°C and subcultured in the same medium for 2 h. The equivalent
of 1 ? 108bacteria was then pelleted by centrifugation (4,000 ? g for 10 min at
4°C), washed once with PBS, and blocked in 200 ?l PBS–10% FCS for 30 min.
After removing the blocking reagent, bacteria were incubated with mouse anti-
Sab diluted 1:200 in 10% FCS in PBS for 1 h at room temperature, washed three
times with PBS, and resuspended in 200 ?l 10% FCS in PBS containing goat
anti-mouse immunoglobulin G-alkaline phosphatase conjugate (1:5,000, enzyme
immunoassay grade; Bio-Rad). After incubation at 37°C for 1 h, bacteria were
washed three times with PBS, resuspended in 300 ?l substrate buffer (1 M
diethanolamine, 3 mM NaN3, and 1 mM MgCl2containing p-nitrophenyl phos-
phate) (one tablet dissolved in 5 ml substrate buffer; Sigma), and transferred
onto a 96-well plate (Sarstedt). Following 1 h of incubation at 37°C, the A405was
measured. Each strain was analyzed in triplicate.
Protein binding assay. Purified Sab was concentrated using a Nanosep instru-
ment (Pall Corporation) as recommended by the manufacturer and labeled with
Oregon Green 488 using a FluoReporterOregon Green 488 protein labeling kit
(Molecular Probes) according to the manufacturer’s instructions. For protein-
binding assays, HEp-2 cells were grown on coverslips in 24-well plates until 50%
confluent. Medium was then removed, HEp-2 cells were washed twice with PBS,
and fresh DMEM was added. Cells were exposed to 0 or 20 ?g/ml purified
labeled Sab for 2.5 h at 37°C. Unbound protein was removed by washing with
PBS three times. Cells were fixed with formalin (10% in PBS) for 10 min and
washed with PBS and water. Samples were mounted onto glass slides using
Prolong Gold antifade and cured for 2 h. All samples were viewed using a 60?
1.4-numerical-aperture oil objective in an Olympus IMT-2 inverted microscope
coupled to a Bio-Rad MRC-600 dual-laser confocal microscope, as described
Immunofluorescence microscopy. The equivalent of 1 ? 108CFU of log-phase
bacteria grown in LB medium or DMEM was pelleted by centrifugation (4,000 ?
g for 10 min at 4°C), washed once with PBS, and fixed in 500 ?l 3.7% parafor-
maldehyde in 0.85% saline for 15 min at room temperature. Sterile coverslips
were placed into 24-well plates and coated with 200 ?l 10% poly-L-lysine for 1
min. Fifty microliters of fixed bacteria was applied onto the coverslip by centrif-
ugation (2,000 ? g for 5 min), and unbound bacteria were removed by washing
once with PBS. Bacteria were incubated with polyclonal mouse anti-Sab (1:100 in
PBS–10% FCS) for 15 min, washed three times with PBS, and incubated with
Alexa 594-conjugated donkey anti-mouse secondary antibody (Molecular
Probes) for another 15 min. Finally, coverslips were washed three times with
PBS, mounted with Prolong Gold antifade reagent, and visualized by fluores-
Biofilm assay on polystyrene surfaces. Biofilm formation assays on polystyrene
surfaces using 96-well Maxisorb plates (Nunc) were carried out as described
previously (42, 44), with the following modifications. Briefly, 200 ?l of 1:40-
diluted cultures of each strain grown overnight was inoculated into 96-well plates
(24 wells for each strain), incubated for 18 h at 37°C, washed twice with 300 ?l
PBS to remove unbound cells, and stained with 200 ?l 0.1% crystal violet for 5
min. Unbound stain was removed by washing twice with PBS, and quantification
of biofilm formation was carried out by the addition of 200 ?l of 96% ethanol and
measurement of the A570.
Identification of an ORF of E. coli O113:H21 98NK2 encod-
ing a predicted AT. In a previous study comparing adherence
phenotypes of different LEE-negative STEC isolates, we ob-
served that an O113:H21 food isolate, strain MW10, exhibited
significantly lower adherence to Henle 407 cells than other
O113:H21 isolates from HUS cases, such as strain 98NK2 (27).
Human-virulent O113:H21 STEC strains are known to carry
putative adherence-associated genes such as saa and iha on
their megaplasmid (pO113) (26, 38). We therefore used the
known DNA sequence of pO113 from another O113:H21
STEC strain (EH41) (22) to design PCR primers to detect the
presence of these putative adhesin genes as well as several
other pO113 genes of unknown function in adherent versus
nonadherent strains (98NK2 and MW10, respectively). Inter-
estingly, the only difference noted was the absence in MW10 of
a PCR product generated from an open reading frame (ORF)
designated LH0147 in the published sequence (data not
shown). Further PCR and sequence analyses confirmed that
the regions of pO113 DNAs from 98NK2 and EH41 containing
this ORF and flanking regions are essentially identical, but the
LH0147 ORF in MW10 contains a deletion (result not shown).
This raised the possibility that a lack of a functional LH0147
product might account for the adherence defect in MW10.
The 4,296-bp ORF LH0147 from E. coli 98NK2 is located
upstream of the pO113 ehx locus, which encodes the EHEC
hemolysin (Fig. 1A). LH0147 encodes a 1,431-amino-acid (aa)
(146-kDa) protein with features characteristic of members of
3236HEROLD ET AL.INFECT. IMMUN.
the AT protein family. This, as well as the functional properties
of ORF LH0147 presented below, led us to designate the gene
product of LH0147 as Sab (for STEC AT mediating biofilm
formation). In silico analysis using SignalP 3.0 (3) and Pfam (2)
indicated the presence of a signal sequence with a predicted
cleavage site after aa 24 and a large N-terminal passenger
domain containing four Hep-Hag repeats and five Him motifs,
as depicted in Fig. 1B. These motifs are often found in bacte-
rial invasins and hemagglutinins, respectively (42). After cleav-
age of the putative signal sequence, the theoretical molecular
mass of mature Sab is 142 kDa. The C-terminal region (aa
1353 to 1431) comprises a putative translocator domain, which
exhibits the features of the pfam03895 superfamily represented
by the trimeric AT YadA of Yersinia enterocolitica. Moreover,
the region from aa 847 to 1431 has multidomain similarity with
the C termini of Haemophilus influenzae Hia-like proteins (Fig.
1B). The trimeric ATs are a novel AT family characterized by
a very short translocator domain that forms stable trimers in
the outer membrane. All members of the trimeric AT family
examined to date appear to have adhesin-like activity, mediat-
ing adherence to eukaryotic cells and extracellular matrix pro-
teins, such as collagen and fibronectin, or binding to circulating
host factors, such as immunoglobulins or complement-inhibi-
tory proteins (7, 17). BlastP analysis indicated that Sab is most
similar to an AT adhesin of Actinobacillus pleuropneumoniae
(GenBank accession number YP_001968314), which exhibits
33% identity and 48% similarity (15% gaps) to aa 601 to 1431
of Sab. Furthermore, aa 601 to 1010 of Sab exhibit 30% iden-
tity and 48% similarity (with 12% gaps) to the collagen adhesin
EmaA of Aggregatibacter actinomycetemcomitans (accession
number AAQ22366), another member of the trimeric AT fam-
ily. However, at the nucleotide sequence level, the entire sab
gene showed negligible homology to any other entries in the
Presence of sab in other STEC strains. The prevalence of
sab in a selection of STEC strains from our collection was then
determined by PCR analysis by using a primer pair that am-
plifies a 163-bp product from within the region of the sab ORF
encoding the passenger domain. Both LEE-positive and LEE-
negative strains were tested, and the distribution of sab was
compared with those of eae (a marker for LEE) and the known
STEC megaplasmid-borne genes ehxA, saa, and subA (Table
2). The sab gene was not present in any of the LEE-positive
strains tested, nor was sab present in four LEE-negative STEC
strains which also lacked any of the other known plasmid-
borne genes, consistent with absence of a megaplasmid alto-
gether. The remaining 14 LEE-negative strains were all posi-
tive for ehxA, indicating the likely carriage of a megaplasmid.
Of these strains, six were positive for sab by PCR: four of these
strains were of serotype O113:H21, one was of serotype O82:
H8, and one was of serotype O23. These strains were all pos-
itive for the three other plasmid-carried putative virulence
genes tested (ehxA, saa, and subA) (Table 2). Several of the
sab-negative strains carried two or three of the other genes,
implying that sab is associated with a specific subset of LEE-
negative STEC strains.
Cloning of the sab gene and construction of a 98NK2 sab
deletion mutant. PCR was used to amplify a 4,368-bp fragment
containing the complete sab ORF, and this was cloned as a
BamHI/EcoRI fragment into pBluescript SK(?) to obtain
pBsab. The construct was then transformed into E. coli JM109
A 98NK2 sab-negative derivate was constructed by the de-
letion of the sab gene and insertion of a kanamycin cartridge
using the Lambda Red recombinase system, as described in
Materials and Methods. Successful mutagenesis was confirmed
by PCR and sequence analysis. The confirmed mutant was
designated 98NK2sab::kan. To complement the 98NK2 sab
deletion mutant, pBsab and the empty vector control were
transformed into 98NK2sab::kan to generate 98NK2sab::
kan(pBsab) and 98NK2sab::kan(pB), respectively.
Expression and location of Sab. BlastP analysis of the ma-
ture Sab protein predicted that Sab belongs to the AT family
and, hence, should be exposed on the bacterial surface. To
FIG. 1. Genetic organization of the sab gene and domain structure of the Sab protein. (A) The sab locus is flanked on one side by LH0146
(encoding a putative OmpA family lipoprotein) and the ehx locus (encoding STEC hemolysin) and on the other side by LH0148 (unknown
function). The region shown corresponds to nucleotides 114000 to 124000 of the pO113 sequence from STEC EH41 (GenBank accession number
NC_007365). (B) The domain structure of the Sab protein (aa 1 to 1431) was predicted using SignalP (3), Pfam (2), and NCBI Protein BLAST
VOL. 77, 2009 LEE-NEGATIVE STEC AUTOTRANSPORTER ADHESIN3237
confirm this, and to examine Sab expression, Sab from 98NK2
was purified as an N-terminal His6-Sab fusion protein lacking
the signal sequence and the translocator domain, as described
in Materials and Methods. The purified protein was used
to raise a polyclonal mouse antiserum, enabling Western
immunoblot analysis of cell lysates of wild-type 98NK2,
98NK2sab::kan, 98NK2sab::kan(pB), 98NK2sab::kan(pBsab),
JM109(pB), and JM109(pBsab). Immunoreactive bands were
not seen in unconcentrated lysates of 98NK2, suggesting low
baseline levels of expression of Sab, at least in vitro (result not
shown). However, strong immunoreactive bands with sizes of
approximately 160 kDa and ?260 kDa, as well as several other
minor species, were seen in lysates of 98NK2sab::kan(pBsab)
but not 98NK2sab::kan(pB) (Fig. 2A). The same two immu-
noreactive species were seen in lysates of JM109(pBsab) ex-
cept that the larger species predominated and there was an
additional smear of immunoreactive material with even greater
molecular size. However, there were no immunoreactive bands
in the JM109(pB) lysate (Fig. 2A). To overcome the inability to
detect Sab in wild-type 98NK2, we analyzed a French press
lysate of concentrated cells from a 500-ml culture grown in
DMEM. A clear immunoreactive band of approximately 160
kDa, as well as higher-molecular-mass material, was detected
in the lysate of 98NK2 but not in that from the otherwise
isogenic mutant 98NK2sab::kan (Fig. 2B). To further probe the
cellular location of anti-Sab-reactive material, inner and outer
membranes from 98NK2 and 98NK2sab::kan were prepared,
and proteins were solubilized, as described in Materials and
Methods. These extracts, along with the insoluble pellet re-
maining after detergent extraction of the outer membrane
fraction, were subjected to SDS-PAGE and Western blotting.
A smear of high-molecular-mass immunoreactive material was
detected in both the soluble and insoluble outer membrane
protein fractions of 98NK2, and in the case of the boiled
insoluble fraction, a faint band with a mass of approximately
160 kDa was also seen. No immunoreactive bands were de-
tected in the inner membrane protein fraction of 98NK2 or in
any of the fractions from 98NK2sab::kan (Fig. 2C). In order to
verify appropriate fractionation, replicate filters were also
probed with an antibody specific for Iha, which is known to be
located exclusively in the outer membrane (38). Immunoreac-
tive bands of the expected sizes were seen in the outer mem-
brane fractions of both 98NK2 and 98NK2sab::kan but not in
any of the other fractions (data not shown). Collectively, these
data show that the expression of the sab gene in either the
98NK2 or JM109 background results in the production of a
160-kDa protein species, which is slightly larger than that pre-
dicted by sequence analysis (142 kDa). Given the high speci-
FIG. 2. Western blot analysis using anti-Sab. Strains were grown in
LB medium (A and C) or DMEM (B) at 37°C, and lysates or protein
extracts were prepared and separated by SDS-PAGE, blotted, and
probed with anti-Sab, as described in Materials and Methods.
(A) Whole-cell lysates. Lanes: 1, 98NK2sab::kan(pB); 2, 98NK2sab::
kan(pBsab); 3, JM109(pB); 4, JM109(pBsab). (B) French press lysates.
Lanes: 1, 98NK2; 2, 1:10 dilution of 98NK2; 3, 98NK2sab::kan. (C) In-
ner and outer membrane fractions. Lanes: 1, 98NK2; 2, 98NK2sab::
kan. The arrow indicates the mobility of the 160-kDa Sab species.
TABLE 2. Presence of sab and other STEC virulence genes in
various E. coli strains
Presence of gene:
aSTEC strains were isolated either from foods or from the feces of patients
with uncomplicated diarrhea (D), bloody diarrhea (BD), microangiopathic he-
molytic anemia and thrombocytopenia (M/T), or HUS.
bDetermined previously by PCR (24, 26).
cDetermined by PCR using primers LH0147-f (5?-GGTGGATACAGCAGG
TAATG-3?) and LH0147-r (5?-TATCTCACCACCTGCTATCG-3?) (163-bp
dND, not determined.
eOnt, O nontypeable; OR, O rough.
3238HEROLD ET AL.INFECT. IMMUN.
ficity of the antiserum, the higher-molecular-mass immunore-
active material is likely to represent Sab multimers, as
previously observed for other AT proteins (1, 32, 42). Bands
with reduced masses detected in 98NK2sab::kan(pBsab) and
JM109(pBsab) are presumably a consequence of proteolytic
degradation during the isolation process.
Surface exposure of Sab. Surface exposure of Sab was then
examined by indirect immunofluorescence microscopy with
anti-Sab, in the first instance using cultures of wild-type strain
98NK2 and its isogenic mutant. The total expression level in
98NK2 was too low in either LB medium or DMEM to enable
detection by immunofluorescence (data not shown). However,
strong immunofluorescence labeling was observed in cultures
of 98NK2sab::kan(pBsab) but not in cultures of 98NK2sab::
kan(pB) (Fig. 3). The accessibility of Sab on whole unfixed
cells to exogenous antibody was also examined by ELISA (Fig.
4). This showed significantly greater labeling in 98NK2 cells
than in 98NK2sab::kan cells (P ? 0.01), while labeling of
98NK2sab::kan(pBsab) cells was significantly greater again
(P ? 0.005). In contrast, there were no significant differences in
labeling among the three strains when whole-cell ELISAs were
performed using the same dilution of either nonimmune
mouse serum or anti-Iha as the primary antibody (result not
shown). Collectively, these findings confirm that Sab is exposed
on the surface of intact 98NK2 cells and is accessible to exog-
enous antibody. However, the baseline level of expression in
wild-type cells is low compared to that in 98NK2 derivatives
carrying sab on a multicopy plasmid.
Sab contributes to epithelial cell adherence. Many ATs,
especially members of the trimeric AT family, mediate adher-
ence to epithelial cells. Accordingly, we examined the adher-
ence of JM109(pBsab) or JM109(pB) to human epithelial cell
lines (HEp-2 and Henle 407 cells) using a quantitative assay
(see Materials and Methods). A significant increase in the level
of adherence to HEp-2 cells was observed for JM109(pBsab)
relative to that of JM109(pB) (20.2% versus 3.0% of the total
number of bacteria, respectively; P ? 0.05) (Fig. 5A). A similar
trend was observed for Henle 407 cells [9.9% versus 4.8% of
the total numbers of bacteria were adherent for JM109(pBsab)
and JM109(pB), respectively], although this difference did not
reach statistical significance (Fig. 5A). Interestingly, no differ-
ence in adherence was observed when similar experiments
were conducted with Hct-8 (human colonic) cells (data not
Examination of Giemsa-stained monolayers after 3 h of
incubation revealed a diffuse pattern of adherence for
JM109(pBsab) but not for JM109(pB), as shown in Fig. 5B.
Interaction of purified Sab with HEp-2 cells. To determine
whether Sab binds directly to human cell lines, HEp-2 cell
monolayers were incubated with Oregon Green-labeled Sab in
DMEM at 37°C. After 2.5 h of incubation, unbound proteins
were washed, and samples were examined with a dual-laser
confocal microscope. Substantial binding of Sab was observed
Sab mediates biofilm formation. To assess whether Sab pro-
motes biofilm formation, wild-type MW10, 98NK2, 98NK2sab::
kan, 98NK2sab::kan(pBsab), 98NK2sab::kan(pB), JM109(pB),
and JM109(pBsab) were tested for their capacities to form
biofilms on polystyrene surfaces. As shown in Fig. 7, significant
biofilm formation was observed for 98NK2, whereas biofilm
formation was negligible for the otherwise isogenic mutant
98NK2sab::kan (P ? 0.005) as well as sab-negative STEC
strain MW10 (P ? 0.005). 98NK2sab::kan(pBsab) also exhib-
ited significantly increased levels of biofilm formation rela-
tive to that of 98NK2sab::kan(pB) (P ? 0.005), as did
JM109(pBsab) relative to that of JM109(pB) (P ? 0.005).
FIG. 3. Immunofluorescence microscopy. Log-phase cultures of
98NK2sab::kan(pBsab) (A) and 98NK2sab::kan(pB) (B) were formalin
fixed and labeled with anti-Sab followed by Alexa 594-conjugated
donkey anti-mouse secondary antibody (see Materials and Methods).
The fluorescent image for each strain is accompanied by the phase-
contrast image for the corresponding field.
FIG. 4. Surface expression of Sab on whole unfixed cells judged by
use of ELISA. Cells from log-phase cultures of 98NK2, 98NK2sab::
kan, and 98NK2sab::kan(pBsab) were probed with anti-Sab and sec-
ondary goat anti-mouse immunoglobulin G-alkaline phosphatase an-
tibody, as described in Materials and Methods. Surface expression was
quantitated by measuring the A405. Data are means ? standard devi-
ations of data from three independent experiments (**, P ? 0.01;***,
P ? 0.005 by Student’s unpaired, two-tailed t test [relative to
VOL. 77, 2009LEE-NEGATIVE STEC AUTOTRANSPORTER ADHESIN 3239
Although Shiga toxin is the definitive virulence factor of
STEC, the capacity to compete with commensal flora and
colonize the human intestinal epithelium is critical for
pathogenesis. The mechanism by which LEE-positive STEC
strains adhere intimately to the enterocyte surface has been
studied extensively, whereas the mechanism(s) by which
LEE-negative STEC strains adhere to the intestinal mucosa
is less well understood. Nevertheless, the importance of
adhesins is reflected in the broad range of such proteins
discovered in LEE-positive as well as in LEE-negative
STEC strains, including Saa, (26), Lpf (10, 39, 40), EibG
(18), Iha (38), Efa1 (23), Cah (41), ECP (30), HCP (47), and
EhaA (45). Nearly all LEE-positive as well as many LEE-
negative STEC strains carry megaplasmids, which encode
putative accessory virulence factors, including some of the
above-mentioned proteins. Significantly, the megaplasmids
of highly virulent LEE-negative STEC strains such as O113:
H21 strains EH41 and 98NK2 are much larger than those of
the classical LEE-positive STEC O157:H7 strains and en-
code a broad range of additional virulence factors, many of
which appear to be unique to LEE-negative strains (22, 25).
In this study, we have characterized one such putative viru-
lence factor, the AT Sab, which is present in some but not all
LEE-negative STEC serotypes/groups including multiple iso-
lates of O113:H21 and single isolates of O23 and O82:H8. In
both 98NK2 and EH41, the sab gene is located on megaplas-
mid pO113, approximately 1.3 kb downstream of the hemoly-
sin locus ehx. All sab-positive STEC strains tested were also
positive for ehxA as well as the autoagglutinating adhesin gene
FIG. 5. Adherence assays. (A) Quantitative adherence of JM109-
(pBsab) and JM109(pB) to HEp-2 and Henle 407 cells (see Materials and
Methods). Data correspond to means ? standard deviations of data from
quadruplicate assays from two independent experiments. (*, P ? 0.05 by
Student’s unpaired, two-tailed t test). (B) Giemsa staining of adherent
bacteria on HEp-2 cell monolayers visualized by light microscopy.
FIG. 6. Protein-binding assay. HEp-2 monolayers were exposed to 0 ?g/ml (A) or 20 ?g/ml (B) Oregon Green-labeled Sab protein for 2.5 h
at 37°C. Unbound protein was removed by washing, and fixed cells were viewed by confocal microscopy.
3240 HEROLD ET AL.INFECT. IMMUN.
saa and the subtilase cytotoxin gene subA; the latter genes are
located 20 and 28 kb, respectively, downstream of sab on
pO113. However, the absence of sab in several other LEE-
negative STEC strains that carry one or more of the other
megaplasmid-borne genes tested (ehxA, saa, or subA) under-
scores the heterogeneity of STEC strains with respect to their
complement of megaplasmid-encoded accessory virulence
Western blot analysis indicated that the level of expres-
sion of the Sab protein in wild-type 98NK2 is low under the
in vitro conditions employed in this study (growth in LB
medium or DMEM). Indeed, it was detectable only when
concentrated cell lysates were examined. It is possible that
Sab is expressed at high levels in vivo, although we found no
difference in sab gene expression levels by real-time reverse
transcription-PCR when cells were grown in LB medium or
DMEM in the presence or absence of HEp-2 cells (data not
shown). Nevertheless, the introduction of sab on a high-
copy-number plasmid in either the JM109 or 98NK2sab::kan
background resulted in much higher levels of expression.
SDS-PAGE and Western blotting results suggested that the
protein is capable of forming multimers. Moreover, Sab was
present exclusively in the outer membrane protein fraction
and was exposed on the surface of intact E. coli cells, as
judged by the accessibility to exogenous antibody using im-
munofluorescence or ELISA.
The surface localization of Sab is consistent with our obser-
vation that it confers the capacity to adhere diffusely to HEp-2
cells when expressed in JM109 cells. Moreover, the purified
fluorescently labeled passenger domain of Sab bound to the
surface of HEp-2 cells, suggesting that Sab may act as a direct
adhesin rather than as an indirect facilitator of adherence. Sab
also appears to be largely responsible for the capacity of wild-
type 98NK2 cells to form a biofilm on polystyrene surfaces. In
contrast, biofilm formation was negligible in 98NK2sab::kan as
well as in the poorly adherent STEC food isolate MW10, which
lacks an intact sab gene. Furthermore, the biofilm formation
defect in 98NK2sab::kan was complemented by transformation
with pBsab but not with empty vector. Likewise, biofilm for-
mation by JM109(pBsab) was significantly greater than that by
The C terminus of Sab shares homology with the proto-
typic trimeric ATs Hia and YadA, and given its mobility on
SDS-PAGE gels, which is indicative of multimerization, its
location in the outer membrane, its exposure on the E. coli
surface, and its unequivocal role in adherence to epithelial
cells and biofilm formation, we hypothesize that Sab is also
a member of the trimeric AT family. Several other ATs were
previously reported to promote biofilm formation, for ex-
ample, Cah (41), EhaA (45), UpaG (42), Ag43 (8), and
AIDA (34). The capacity to form biofilms may enhance the
survival of pathogens in different environmental niches,
such as in food products or in the gastrointestinal tracts of
humans or animal reservoirs of infection. It is particularly
noteworthy that even the low baseline level of sab expres-
sion in wild-type 98NK2 was sufficient to confer a substantial
level of biofilm formation, and this was completely abolished
in the otherwise isogenic mutant 98NK2sab::kan. Other
studies have shown that the trimeric AT proteins YadA,
NhhA, BadA, and UpaG promote binding to extracellular
matrix proteins, such as fibronectin, laminin, or collagen
(31, 33, 37, 42). However, we found no evidence that either
purified Sab protein or E. coli JM109 carrying sab was ca-
pable of binding to any of these extracellular matrix proteins
(result not shown). Some ATs have also been shown to
mediate autoagglutination, but this was not observed for Sab
(result not shown).
In summary, we have shown that the STEC megaplasmid-
encoded putative trimeric AT family protein Sab promotes ad-
The distribution of sab, although based on an analysis of a rela-
tively small number of clinical and environmental STEC isolates,
indicates that Sab, like the autoaggregative adhesin Saa (26), is
associated with LEE-negative STEC strains. Thus, both adhesins
the capacity to form A/E lesions on enterocytes. It is also note-
worthy that apart from encoding Sab and Saa, as well as addi-
tional putative accessory virulence factors including subtilase cy-
totoxin (SubAB) (25) and two additional SPATE family secreted
serine protease ATs, EspP (4) and EpeA (16), the megaplasmids
of highly virulent STEC O113:H21 strains 98NK2 and EH41 are
self-transmissible (36). Thus, the assembly of such a diverse pay-
load of virulence factors onto a single mobile DNA element is
likely to have contributed to the evolution of hypervirulent LEE-
negative STEC strains.
This work was supported by program grant 284214 and project grant
565359 from the National Health and Medical Research Council of
Australia (NHMRC) (to J.C.P. and A.W.P.). S.H. is a recipient of a
German Research Foundation (Deutsche Forschungsgemeinschaft)
research fellowship; J.C.P. is an NHMRC Australia Fellow.
We are also grateful to Kerrie May and Marcin Grabowicz for
helpful discussions and to Ursula Talbot for technical assistance.
FIG. 7. Biofilm formation on polystyrene plates. The indicated
strains were grown in 96-well polystyrene plates for 18 h. Plates were
then washed and stained with crystal violet (see Materials and Meth-
ods). Biofilm formation was quantitated by measuring the A570after
solubilization of the stained bacteria. Data are means ? standard
deviations (n ? 24) (***, P ? 0.005 by Student’s unpaired, two-tailed
t test [relative to the respective negative control]).
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