Immunoglobulin-mediated agglutination of and biofilm formation by Escherichia coli K-12 require the type 1 pilus fiber.

Abstract

The binding of human secretory immunoglobulin A (SIgA), the primary immunoglobulin in the gut, to Escherichia coli is thought to be dependent on type 1 pili. Type 1 pili are filamentous bacterial surface attachment organelles comprised principally of a single protein, the product of the fimA gene. A minor component of the pilus fiber (the product of the fimH gene, termed the adhesin) mediates attachment to a variety of host cell molecules in a mannose inhibitable interaction that has been extensively described. We found that the aggregation of E. coli K-12 by human secretory IgA (SIgA) was dependent on the presence of the pilus fiber, even in the absence of the mannose specific adhesin or in the presence of 25 mM alpha-CH(3)Man. The presence of pilus without adhesin also facilitated SIgA-mediated biofilm formation on polystyrene, although biofilm formation was stronger in the presence of the adhesin. IgM also mediated aggregation and biofilm formation in a manner dependent on pili with or without adhesin. These findings indicate that the pilus fiber, even in the absence of the adhesin, may play a role in biologically important processes. Under conditions in which E. coli was agglutinated by SIgA, the binding of SIgA to E. coli was not increased by the presence of the pili, with or without adhesin. This observation suggests that the pili, with or without adhesin, affect factors such as cell surface rigidity or electrostatic repulsion, which can affect agglutination but which do not necessarily determine the level of bound immunoglobulin.

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INFECTION AND IMMUNITY, Apr. 2004, p. 1929–1938
0019-9567/04/$08.00?0DOI: 10.1128/IAI.72.4.1929–1938.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 4
Immunoglobulin-Mediated Agglutination of and Biofilm Formation by
Escherichia coli K-12 Require the Type 1 Pilus Fiber
Paul E. Orndorff,1Aditya Devapali,2Sarah Palestrant,2Aaron Wyse,2Mary Lou Everett,2
R. Randal Bollinger,2,3and William Parker2*
Department of Microbiology, Pathology, and Parasitology, College of Veterinary Medicine, North Carolina State
University, Raleigh, North Carolina 27606,1and Departments of Surgery2and Immunology,3Duke University
Medical Center, Durham, North Carolina 27710
Received 14 August 2003/Returned for modification 7 November 2003/Accepted 27 December 2003
The binding of human secretory immunoglobulin A (SIgA), the primary immunoglobulin in the gut, to
Escherichia coli is thought to be dependent on type 1 pili. Type 1 pili are filamentous bacterial surface
attachment organelles comprised principally of a single protein, the product of the fimA gene. A minor
component of the pilus fiber (the product of the fimH gene, termed the adhesin) mediates attachment to a
variety of host cell molecules in a mannose inhibitable interaction that has been extensively described. We
found that the aggregation of E. coli K-12 by human secretory IgA (SIgA) was dependent on the presence of the
pilus fiber, even in the absence of the mannose specific adhesin or in the presence of 25 mM ?-CH3Man. The
presence of pilus without adhesin also facilitated SIgA-mediated biofilm formation on polystyrene, although
biofilm formation was stronger in the presence of the adhesin. IgM also mediated aggregation and biofilm
formation in a manner dependent on pili with or without adhesin. These findings indicate that the pilus fiber,
even in the absence of the adhesin, may play a role in biologically important processes. Under conditions in
which E. coli was agglutinated by SIgA, the binding of SIgA to E. coli was not increased by the presence of the
pili, with or without adhesin. This observation suggests that the pili, with or without adhesin, affect factors such
as cell surface rigidity or electrostatic repulsion, which can affect agglutination but which do not necessarily
determine the level of bound immunoglobulin.
The ability of secretory immunoglobulin A (SIgA) to bind
and agglutinate enteric bacteria is thought to be of critical
importance, providing the basis for the immune system’s inter-
actions with enteric bacteria (7, 53, 59). SIgA binding and
agglutination of enteric bacteria prevents the bacteria from
breaching the epithelial barrier, a process termed “immune
exclusion” (59, 60). SIgA may also facilitate biofilm formation
by the normal flora in the large bowel, a process that may aid
in the growth of the normal flora and attenuate growth of
pathogenic microorganisms (5). The SIgA-mediated aggrega-
tion of enteric bacteria (59) and SIgA-mediated biofilm for-
mation by enteric bacteria have been assessed in vitro (5).
Aggregation of bacteria by IgA can be blocked in vitro by
antisera specific for the heavy or for the light chains of IgA,
indicating that the agglutination is specific for the IgA mole-
cule (59). SIgA-mediated biofilm formation by Escherichia coli
in vitro is also specific, since biofilm formation can be mediated
by SIgA and by mucin but not by the absence of protein or by
IgG, albumin, hemoglobin, secretory chain, or the Fab and Fc
domains of SIgA (5).
There are a number of bacterial components that are im-
portant in autoaggregation and biofilm formation by E. coli.
These components include colanic acid (13), type 1 pili (52),
curli (49), and antigen 43 (12, 31). However, the potential role
of these molecules in bacterial aggregation and biofilm forma-
tion in the gut remains to be determined; curli and colanic acid
are not expressed well under conditions found in the mamma-
lian gut (34, 41, 42, 50). In addition, the importance of antigen
43 and type 1 pili for biofilm formation is dependent on culture
conditions (12). Further, the biofilm-forming effects of antigen
43 may only be important in the absence of type 1 pili (25).
Perhaps most importantly, SIgA can mediate aggregation and
biofilm formation by E. coli under conditions in which E. coli
does not typically form aggregates or biofilms. The observation
that SIgA and mucus, common components of the intestinal
milieu, could facilitate biofilm formation suggests that mi-
crobes in the gut probably do not need to produce all necessary
components for biofilm formation in order to form a biofilm.
Such an idea is not unprecedented, as formation of bacterial
biofilms by bacteria associated with plant roots is facilitated at
least in part by molecules secreted by the plant (6, 15, 20).
Thus, if indeed IgA and mucus are involved in biofilm forma-
tion or aggregation of bacteria in the gut, it is of substantial
interest to determine which bacterial components may be re-
quired to facilitate these interactions. At least one study has
indicated that the interaction between SIgA and E. coli may
depend on type 1 pili (61).
Type 1 pili are filamentous proteinaceous appendages pro-
duced by several members of the Enterobacteriaceae. In Esch-
erichia coli, type 1 pili have been conclusively associated with
the ability to infect extraintestinal sites (30, 43). However, the
involvement of the pili in intestinal colonization is less certain
(4, 29, 44). Type 1 pili have been studied extensively with
regard to their genetics, biosynthesis, and ability to bind re-
ceptor molecules on a variety of eucaryotic cells in a mannose-
inhibitable manner (43). Although the pili are made principally
* Corresponding author. Mailing address: Duke University Medical
Center, Box 2605, Department of Surgery, Durham, NC 27710. Phone:
(919) 681-3886. Fax: (919) 681-7263. E-mail: bparker@duke.edu.
1929

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of a single protein monomer, the product of the fimA gene,
several minor protein components are also incorporated (23,
47). These are most often found at the ends of pili and are
organized into fibrillar structures (28). One of the minor com-
ponents, the product of the fimH gene (FimH, termed the
adhesin), binds directly to receptor molecules (32).
A variety of receptors on eucaryotic cells (17, 18, 26) and
molecules of interstitial spaces (48, 55) are bound by the ad-
hesin. This binding, and even intermolecular adhesin binding
(24), is characterized by its sensitivity to mannose inhibition. In
the absence of a functional fimH gene product, piliated E. coli
appear to lack all of the colonization and host cell binding
properties associated with type 1 piliation (24, 29, 30). A role
of the fimbrial fiber itself in bacterium-host interactions (apart
from being required for adhesin presentation) has been spec-
ulated upon (43) but never supported experimentally (29).
Type 1 pili are recognized by SIgA and mucins (37), mole-
cules present in high abundance along the mucosal barrier.
The binding of SIgA to enteric bacteria has been shown to
decrease the ability of those bacteria to breach the intestinal
barrier (59, 60). Thus, interactions between type 1 pili and
SIgA may help reduce chronic inflammation in the intestine
and aid in the initial colonization of the host. Indeed, IgA-
deficient individuals appear to be colonized with E. coli that
exhibit reduced type 1 pilus expression (16).
Natural antibodies, i.e., antibodies occurring without any
known history of sensitization to the relevant corresponding
antigen, often bind to bacterial antigens and are, at least to
some degree, elicited by exposure to the normal microbial flora
(8, 56). Based on our current understanding of natural anti-
bodies, it might be thought that the binding of these antibod-
ies, including natural SIgA, to bacteria is dependent on the
specificity of the antigen binding site of the antibody. On the
other hand, some studies have suggested that the binding of
SIgA to E. coli may depend on the mannose-specific interac-
tions between the adhesin of the type 1 pili and mannose
residues expressed on SIgA. For example, Moshier et al. found
that expression of type 1 pili enhanced adhesion to surfaces
coated with SIgA or with mucins (37). This adhesion was in-
hibited by mannose, a finding consistent with the idea that
mucin and SIgA may provide receptors for binding of type
1pili. Wold and colleagues found that the sIgA2-mediated ag-
glutination of E. coli expressing type 1 pili can be inhibited by
mannose (agglutination titer reduced from 128 to 2 in the
presence of 43 mM ?-CH3Man) (61). This finding is expected,
since IgA2 carries oligosaccharide receptors for type 1 pili (61).
However, Wold et al. also found that, like IgA2-mediated
agglutination, IgA1-mediated agglutination of E. coli express-
ing type 1 pili is inhibited by mannose (agglutination titer
reduced from 64 to 4 in the presence of 43 mM ?-CH3Man)
(61). This finding is unexpected since, in contrast to IgA2, IgA1
apparently does not contain mannose residues that might be
recognized by the adhesin of the type 1 pili (1, 2). Thus, it
remains unclear to what extent the binding of the mannose
specific adhesin to mannose residues on SIgA is important in
the binding of SIgA to E. coli.
In the present study, the interactions between human SIgA
and various strains of E. coli expressing pili with adhesin, pili
without adhesin, or no pilus and no adhesin were evaluated.
Measures of this interaction included (i) direct binding be-
tween antibody and E. coli, (ii) antibody-mediated aggregation
of E. coli, and (iii) antibody-mediated biofilm formation by E.
coli. These studies provide strong support for the idea that, in
addition to previously described adhesin-dependent interac-
tions, the fiber of the type 1 pili of E. coli has a strong adhesin-
independent effect on the aggregation of E. coli by SIgA. This
adhesin-independent effect is potentially due to alterations in
the surface topology of the E. coli as a result of pilus expres-
sion. These findings provide new insight into the mechanisms
underlying the interactions between type 1 pili and SIgA and
thus into the potential role of type 1 pili in colonization of the
gut.
MATERIALS AND METHODS
Materials. Minimal essential Eagle medium (MEM) and alkaline phospha-
tase-conjugated, affinity-isolated goat antibodies specific for human ?-chain were
obtained from Sigma Chemical Co. (St. Louis, Mo.). Sodium pyruvate, HEPES,
and nonessential amino acids (MEM-Amino Acids) were obtained from Gibco-
BRL (Grand Island, N.Y.). Solvable was purchased from Perkin-Elmer Life
Sciences (Boston, Mass.). Uniformly labeled [14C]glucose was obtained from
Amersham (Piscataway, N.J.). Wire screen (150 mesh, 94 mm) cell screen filters
were obtained from Bellco Glass, Inc. (Vineland, N.J.).
IgM was purified from normal human plasma by using the euglobulin precip-
itation technique as follows. Outdated human plasma was obtained from the
American Red Cross (Charlotte, N.C.) and stored at ?85°C until use. After it
thawed, the plasma was clarified by centrifugation at 6,500 ? g for 30 min at 4°C.
The plasma was then diluted with 20 parts of cold distilled water and incubated
overnight at 4°C. The mixture was again centrifuged at 6,500 ? g for 30 min at
4°C, and the precipitated euglobulin fraction was collected. The pellet was
dissolved in phosphate-buffered saline (PBS), and the IgM was separated from
the other proteins on a 2.5-cm (internal diameter)-by-90-cm (length) Sepharose
CL-4B column. Purified IgM was stored in 10% glycerol and flash frozen until
use.
For purification of SIgA, unneeded human milk from anonymous donors was
obtained from the Duke University Medical Center Pediatric Intensive Care
Unit. Milk from 10 to 20 donors was pooled and stored at ?80°C until use. SIgA
was purified from the pooled milk by size exclusion chromatography on a 2.5-cm
(internal diameter)-by-90-cm (length) Sepharose CL-4B column. Purified SIgA
was stored in 10% glycerol and flash frozen until use. Purification on the Sepha-
rose column resulted in a greater loss of IgA2 than of IgA1, since the IgA2 eluted
later, resulting in contamination of part of the IgA2 fraction with other milk
proteins. The IgA preparation was determined to contain 85% IgA1 and 15%
IgA2 based on two independent assessments. First, 84% of the IgA preparation
was cleaved with Igase (specific for IgA1). Second, quantitative analysis of the
elution profile from the Sepharose column, which contains the IgA1 peak fol-
lowed by the IgA2 peak (partially resolved), was conducted. The absorbance
profile at 280 nm was fitted to Gaussian distributions, and the areas under the
curve were calculated by using the program GRAMS/32 (version 5.10; Galactic
Industries Corp., Salem, N.H.). Based on this analysis, our preparations con-
tained 80 to 85% IgA1, and 50 to 60% of the IgA2 was discarded during the
preparation.
Before use, purified SIgA or IgM was thawed and dialyzed against two changes
of PBS, followed by a final dialysis against MEM (for measuring biofilm forma-
tion) or against PBS (for measuring aggregation). For measurement of biofilm
formation, the purified immunoglobulin was then sterilized by filtration through
a 0.2-?m-pore-size filter.
Bacterial strains and growth conditions. All bacterial strains utilized in the
present study were derivatives of E. coli K-12 (Table 1). Strains were propagated
in L broth and L agar under conditions previously described (51). For plasmid
containing strains, growth medium was supplemented with chloramphenicol (20
?g/ml). For agglutination studies and biofilm formation, bacteria were grown in
MEM supplemented with 100 mM HEPES, 0.1 mM MEM-Amino Acids, and 1.0
mM sodium pyruvate prior to use.
Genetic techniques. Bacterial transformation with circular plasmid DNA was
as described by Lederberg and Cohen (33). Plasmid-encoded alleles were intro-
duced into the E. coli chromosome by recombination after transformation of a
recBC sbcB strain of E. coli with linearized plasmid DNA as described by Orn-
dorff et al. (46). Transfer of chromosomal alleles was accomplished via P1
transduction as described by Miller (36) by using P1 vir (46).
1930 ORNDORFF ET AL.INFECT. IMMUN.

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Piliation assays. Piliated E. coli were detected in bacterial agglutination assays
that used type 1-specific antiserum (51). Strains producing pili with a functional
FimH adhesin were detected in hemagglutination assays with guinea pig eryth-
rocytes as previously described (51). Under the conditions used in our assays, all
piliated strains had approximately the same level of piliation per cell as indicated
by bacterial agglutination titer and electron microscopic examination of nega-
tively stained preparations (51).
SIgA-mediated agglutination measurements. Bacteria were grown in minimal
medium for 16 h at 37°C in an air-tight 15-ml container. The number of bacteria
in each tube was quantified by using a Beckman Coulter Multisizer II Coulter
counter with a 50-?m aperture. A correction factor was used to determine
absolute numbers of bacteria, since it was found that the counter with a 50-?m
aperture detects 1.0% of E. coli (absolute numbers of bacteria based on CFU
found by serial dilution). The agglutination reaction was carried out at room
temperature with 4.9 ? 108bacteria per ml and 0.5 mg of SIgA/ml in PBS.
The agglutination of bacteria was monitored by using a Coulter Multisizer II
counter with a 50-?m aperture. To assess the number of agglutinated bacteria,
100 ?l of the mixture of bacteria in 0.5 mg of SIgA/ml was diluted into 10.0 ml
of sterile filtered saline. For this dilution, large-orifice graduated pipette tips
(USA Scientific, Ocala, Fla.) were used to avoid breaking up aggregates. The
numbers of aggregates between 3 to 6 ?m and the number ?6 ?m in diameter
were recorded as a function of time. These two size ranges were selected for
several reasons. First, it was difficult to monitor the presence of aggregates
smaller that 3 ?m because the unaggregated bacteria interfered with the mea-
surement of these smaller aggregates. Second, it was decided to monitor the
formation of large (?6-?m) aggregates separately because (i) aggregates larger
than 6 ?m formed later during the agglutination reaction compared to smaller
(?6 ?m) aggregates; (ii) even though there were small numbers of large aggre-
gates compared to the number of smaller aggregates, each large aggregate
represented a substantial amount of bacteria [given the number of bacteria
within a spherical aggregate varies approximately as (4/3)?r3, the volume of a
sphere]; and (iii) large aggregates were not formed when the agglutination
reaction was not as vigorous. Thus, monitoring the formation of large aggregates
(?6 ?m) separately provided additional information, whereas the measurement
of smaller aggregates (3 to 6 ?m) provided an approximate measure of the
absolute number of total aggregates.
Biofilm growth measurements. Biofilms were grown in 12-by-75-mm Becton
Dickinson 5-ml polystyrene round-bottom Falcon tubes. To initiate bacterial
growth in the tubes, the tubes were incubated for 16 h under aerobic conditions
with minimal medium (1.0 ml) that was inoculated with bacteria. Failure of
particular strains to grow biofilms was not due simply to failure to adhere during
this initial incubation, since planktonic (nonadherent) growth was observed in all
tubes with all strains for the duration of all experiments. After the initial 16-h
incubation, the medium was slowly drained from the tube, and fresh medium
containing 25 nCi of14C/ml was then slowly added with or without 0.5 mg of
SIgA/ml. The steel needle from a 16-gauge, 5.25-in. catheter (BD Angiocath;
Becton-Dickinson, Sandy, Utah) coupled to a syringe was used to remove and
add liquid to all tubes. To add or remove liquid, the angiocath was placed at the
bottom of the tube, and the tube was kept in an upright position. In tubes
containing no SIgA, 0.5 mg of bovine serum albumin/ml was added to maintain
the same protein concentration in all tubes. During the next 2.5 days, the medium
was changed four times at 4- to 18-h intervals. (Shorter time intervals were used
as the biofilms became thicker and used nutrients at a faster rate.) Tubes
containing no SIgA were changed at the same time intervals as tubes continuing
SIgA. For each change, tubes were gently washed three times with PBS by slowly
removing and adding solution by using a 16-gauge, 5.25-in. angiocath. This
washing method was effective for removing almost all of the nonadherent bac-
teria.
The incorporation of14C by bacteria was used as a measure of the amount of
bacteria and was taken to be proportional to the number of bacteria. For this
measurement, tubes were washed three times as described above, and some
bacteria were removed from the tubes by vortexing at maximum speed for 30 s
in 1 ml of PBS. The suspended bacteria were removed, and the remaining
adherent bacteria were removed by vortexing them at maximum speed for 30 s
with 100 mg of washed, sterilized sand in 1 ml of PBS. The suspended bacterial
cells were washed three times with PBS, resuspended in 50 ?l of deionized water,
dissolved in 500 ?l of Solvable, and diluted into 10 ml of scintillation fluid
(Packard Hionic-Fouor). The amount of14C was then quantified by using a
Wallac 1409 liquid scintillation counter. Using a serial dilution of14C-labeled
bacteria, we found the results of this method to be linear (r2? 0.99) over greater
than a thousandfold range (from 85,000 disintegrations per minute [dpm] to 40
dpm). The presence of CFU was not used as a measure of activity since we could
not separate completely the bacteria within biofilms with confidence without
using conditions that might kill some of the bacteria.
Binding of SIgA to immobilized E. coli. The binding of SIgA to immobilized E.
coli was measured by enzyme-linked immunosorbent assay (ELISA) as follows.
E. coli samples were washed with PBS, and 109E. coli organisms in 60 ?l of PBS
with 0.2% NaN3were added to each well of a Costar 96-well assay plate (flat
bottom, polystyrene high binding). After an overnight incubation at 4°C, wells
were washed four times with 200 ?l of PBS/well and blocked with 0.5% human
serum albumin in PBS for 1 h at room temperature. After this incubation, the
blocking agent was discarded, and the wells were washed three times with 200 ?l
of PBS/well. SIgA (0.5 mg/ml; 50 ?l/well) diluted in PBS was added, followed by
incubation for 1.5 h at 4°C. The wells were then washed three times with PBS,
TABLE 1. E. coli strains and plasmids used in this study
Strain or plasmidRelevant propertiesa
Source and/or reference
Strains
MG1655
ORN225
F???
MG1655 except fimB?-tetR fimE::IS1 (stably expresses type 1 pili);
Hag?Bag?
ORN225 except fimH?-neoR (stably expresses nonhemagglutinating
type 1 pili); Bag?Hag?
MG1655 except ?(fimBEAICDFGH) ?neoR; Hag?Bag?
3
P1 transduction from strain
ORN202b
P1 transduction from strain
ORN133 (35)
P1 transduction from strain
ORN172 (62)
22
ORN226
ORN227
ORN201thr-1 leuB thi-1 ?(argF-lac)U169 malAI xyl-7 ara-13 mtl-2 gal-6 rpsL
fhuA2 supE44 pilG recA13 ?(fimBEAICDFGH); Hag?Bag?
Plasmids
pACYC184
pSH2
P15A; CmrTcr
pACYC184 fimBEAICDFGH; Cmr(Hag?Bag?when resident in
strain ORN201)
KpnI-SalI fimH deletion mutant of pSH2; entire fimH gene
deleted (Hag?Bag?when resident in strain ORN201)
9
21
pORN30721
aHag?, Capable of hemagglutinating guinea pig erythrocytes as described in the text; Bag?, bacterial agglutination in polyclonal rabbit antiserum raised against
purified type 1 pili as described in the text; Cmr, chloramphenicol resistant; Tcr, tetracycline resistant.
bStrain ORN202 was constructed by first inserting a XhoI DNA fragment containing the tetR gene from Tn10 into the ClaI site in fimB on pSH2 in vitro, creating
pORN314. (The ClaI site in fimB had been converted to an XhoI site via linker insertion mutagenesis [45] after partial digestion of pSH2 with ClaI.) Linearization of
the plasmid with SalI, followed by transformation ORN117 (?fimA?-lacZ fimE::IS1 recBC sbcB) (46), produced a pool of recombinants, some of which had the desired
fimE::IS1 fimB?-tetR alleles and were in the locked ON phase (stably transcribing the fimA?-lacZ gene). Such recombinants were identified by phenotypic screening,
and the genotype was confirmed by DNA hybridization tests by methods previously described (57). P1 transductions were subsequently used to position the fimE::IS1
fimB?-tetR alleles adjacent to fimA in ORN115 (46), creating the ORN202 donor strain used to construct the stably piliated version of MG1655 (ORN225) used here.
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alkaline phosphatase-conjugated goat antibodies specific for human ?-chain
were added, and the wells were incubated for 1 h. The wells were then washed
three times with PBS, and 100 ?l of a developing solution consisting of 1.0 mg of
p-nitrophenyl phosphate/ml in 100 mM diethanolamine–0.5 mM MgCl2–0.2%
NaN3(pH 9.5) was added. The A405was determined by using an EL 340
BioKinetics reader (Bio-Tek Instruments, Winooski, Vt.). The absorbance in
wells containing no E. coli was taken to be background and was subtracted from
the total.
Binding of SIgA to suspended (nonimmobilized) E. coli. To determine the
amount of SIgA bound by suspended (nonimmobilized) E. coli, the E. coli
samples were incubated with SIgA and washed, and the bound SIgA was quan-
tified. For this purpose, ca. 2.7 ? 109E. coli was added to a final volume of 1.5
ml of PBS containing 1.0 mg of SIgA. The bacteria were incubated at room
temperature for 2 h and then washed three times with PBS. Bacteria were
solubilized by boiling for 12 min in 180 ?l of sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) sample buffer containing 44% (vol/vol)
?-mercaptoethanol. Then, 20 ?l of this preparation was loaded per lane onto a
4 to 20% acrylamide gradient gel, separated electrophoretically, and transferred
to a polyvinylidene difluoride membrane. Membranes were blocked overnight
with 1% bovine serum albumin in PBS and then incubated for 1 h at room
temperature with alkaline-phosphatase conjugated, affinity-purified goat anti-
body specific for human ?-chain in blocking buffer. Blots were washed and
developed as described above. Bands corresponding to SIgA were digitized and
quantified by fitting peak intensities to Gaussian distributions by using the pro-
gram GRAMS/32 (version 5.10; Galactic Industries Corp.). To determine abso-
lute amounts of SIgA in a given lane, the areas under the peaks obtained from
unknown samples were compared to a standard curve made with purified SIgA
of known concentration. All standard curves obtained by using this method had
a correlation coefficient (r2) greater than 0.99.
Binding of SIgA to bacterial antigens as determined by immunoblotting.
Approximately 3 ? 1010bacteria were solubilized in 200 ?l of SDS sample buffer
containing 25% (vol/vol) ?-mercaptoethanol. Then, 20 ?l of a one-to-five dilu-
tion of this preparation in SDS sample buffer was loaded per lane onto a 4 to
20% acrylamide gradient gel, separated electrophoretically, and transferred to a
polyvinylidene difluoride membrane. Membranes were blocked overnight with
0.5% human serum albumin and 0.1% Tween 20 in PBS, followed by incubation
for 2 h at room temperature with 100 ?g of human SIgA/ml. Blots were washed
three times with PBS and then incubated for 1 h with alkaline phosphatase-
conjugated, affinity-purified goat antibody specific for human ?-chain in blocking
buffer. Finally, blots were washed three times with PBS and then developed with
0.33 mg of nitroblue tetrazolium and 0.165 mg of BCIP (5-bromo-4-chloro-3-
indolylphosphate)/ml in 100 mM Tris-HCl–100 mM NaCl2–5.0 mM MgCl2at pH
9.5.
RESULTS
SIgA-mediated agglutination of various E. coli strains in the
presence or absence of 25 mM ?-CH3Man. The extent to which
SIgA-mediated agglutination of E. coli was dependent on the
?Man was evaluated. To this end, the agglutination of various
E. coli strains by SIgA in the presence or absence of mannose-
specific adhesin was measured by using a Coulter counter as
described in Materials and Methods. Controls with no SIgA
failed to agglutinate, indicating that agglutination was indeed
specific for SIgA. As shown in Fig. 1, E. coli expressing pili with
adhesin was agglutinated by SIgA. Similarly, E. coli expressing
pili without the adhesin was also agglutinated by SIgA. In
contrast, the E. coli expressing no pili and no adhesin was not
agglutinated by SIgA, indicating that the presence of the pili in
the absence of the adhesin can facilitate SIgA-mediated agglu-
tination. Consistent with this idea, the presence of 25 mM
?-CH3Man did not prevent the SIgA-mediated agglutination
of either E. coli expressing pili plus adhesin or E. coli express-
ing pilus only (Fig. 1).
In addition to experiments with 25 mM ?-CH3Man to inhibit
binding of the adhesin to E. coli, some experiments were con-
ducted in which 100 mM ?-CH3Man was utilized. Consistent
with previous results (61), the presence of 100 mM ?-CH3Man
inhibited 96% of the SIgA-mediated agglutination (deter-
mined at 17.5 h with the number of aggregates greater than 3
?m as a measure of aggregation) of E. coli expressing pili with
adhesin. However, 100 mM ?-CH3Man also inhibited 90% of
the SIgA-mediated agglutination of E. coli expressing pili with-
out the mannose-specific adhesin. This latter finding indicated
that at least some of the inhibition of SIgA-mediated aggrega-
tion observed in the presence of 100 mM ?-CH3Man was not
specific for the mannose-specific adhesin.
Secretory IgA-mediated biofilm formation by various
strains of E. coli. Under conditions in which free floating or
free-swimming (planktonic) bacteria are repeatedly washed
away, E. coli and other microorganisms form adherent plaques,
FIG. 1. Agglutination of various strains of E. coli in the presence of
SIgA. The numbers and sizes of particles after the addition of 0.5 mg
of SIgA/ml to various strains of E. coli (ORN225, ORN226, and
ORN227; see Table 1) were measured as a function of time with a
Coulter counter. For presentation purposes, smaller aggregates (3 to 6
?m) (A) were plotted separately than larger aggregates (?6 ?m) (B),
which formed slower than the smaller aggregates. Experiments were
performed in duplicate, and the standard errors are shown.
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or biofilms, on various surfaces (11). Biofilm formation may be
facilitated by factors produced by the bacteria themselves or
may be facilitated by the addition of factors such as SIgA that
mediate interbacterial interactions. Thus, SIgA-mediated bio-
film formation by E. coli can be used as an independent marker
for the interaction between E. coli and SIgA. E. coli expressing
pili and adhesin formed biofilms in the presence of SIgA but
not in the absence of SIgA (Fig. 2). Similarly, E. coli expressing
pili with no adhesin formed biofilms in the presence of SIgA
but not in the absence of SIgA (Fig. 2A). Thus, biofilm for-
mation by these bacteria was mediated by SIgA. In contrast, E.
coli expressing no pili did not form SIgA-mediated biofilms
(Fig. 2A), indicating that the pili, even without the presence of
adhesin, are able to mediate biofilm formation by SIgA. Quan-
titative evaluation of the biofilms formed revealed that, in the
absence of SIgA, the amount of biofilm formation decreased
by 99% in E. coli expressing pili plus adhesin and by 97% in E.
coli expressing pili only. However, the SIgA-mediated biofilm
formed by E. coli expressing pili with adhesin contained ?10-
fold more radiolabeled material than the SIgA-mediated bio-
film formed by E. coli expressing pili without adhesin (Fig. 2B).
This observation suggests that the presence of the adhesin
plays an important role but is not absolutely required in the
interaction between SIgA and E. coli.
The study required to remove biofilms from the polystyrene
tubes provided further evidence that the interaction between
SIgA and the adhesin was important in biofilm formation. A
total of 91% of the SIgA-mediated biofilms formed by E. coli
expressing pili without adhesin could be removed by vortexing
without the use of sand as an abrasive. In contrast, only 35% of
the SIgA-mediated biofilm formed by E. coli expressing pili
with adhesin could be removed by vortexing without sand, the
rest requiring sand to facilitate removal. This observation pro-
vides further evidence that the presence of the adhesin plays an
important role but is not absolutely required in the interaction
between SIgA and E. coli.
The experiments described above (Fig. 2A, B) were con-
ducted with chromosomal mutants of E. coli in which the
expression of the adhesin (FimH) was eliminated by deletion
of the terminal 22 codons of the fimH gene. Deletion of the
terminal 22 codons eliminated functional expression of the
adhesin as measured by the mannose-dependent agglutination
of guinea pig erythrocytes and by the failure of anti-adhesin
antibodies to bind (21). However, to ensure that the results
shown in Fig. 2 were not due to sIgA binding by a hypothetical
truncated adhesin (FimH), we conducted additional experi-
ments in which E. coli containing a recombinant plasmid car-
rying the genes for just the pilus fiber (lacking the fimH gene
entirely) was compared to an E. coli carrying the genes for
complete piliation (pilus plus adhesin). E. coli containing just
the cloning vector was used and expressed no pili (see Table 1).
The results (Fig. 2B) revealed that there was no discernible
difference between the plasmid containing mutant lacking the
entire fimH gene and the chromosomal mutant with a deletion
of part of the fimH gene. The chromosomal mutant set was
used exclusively for the remainder or the experiments.
Role of pili and adhesin in the interaction between IgM and
E. coli. To determine whether pili without adhesin might also
facilitate interactions between E. coli and a polyvalent immu-
noglobulin other than SIgA, the role of pili in the agglutination
FIG. 2. SIgA-mediated biofilm formation of E. coli as a function of
pilus and adhesin expression (A) Experiments with chromosomal mu-
tants. Tubes A contain no bacteria, tubes B contain E. coli (chromo-
somal mutants) without pilus or adhesin (ORN225; Table 1), tubes C
contain E. coli with pilus but no adhesin (ORN226; Table 1), and tubes
D contain E. coli with pilus and adhesin (ORN227; Table 1).
(B) Quantification of the SIgA-mediated biofilm growth by various E.
coli expressing pili with adhesin, pili without adhesin, or neither pili
nor adhesin. Chromosomal mutants were the same used in the exper-
iment described in Fig. 2A. Plasmid constructs expressing pili with
adhesin (ORN201 containing pSH2; Table 1), pili without adhesin
(ORN201 containing pORN307; Table 1), or neither pili nor adhesin
(ORN201 containing pACY184; Table 1) were also used. Biofilm
growth did not occur in the absence of immunoglobulin (see Fig. 3),
indicating that the biofilm growth was mediated by SIgA. The growth
is shown on a log scale. Growth was quantified by measuring the
amount of14C-glucose incorporated into the biofilm as described in
Materials and Methods. Experiments were performed in duplicate,
and the standard errors are shown.
VOL. 72, 2004 MEDIATION OF SIgA EFFECTS BY TYPE 1 PILUS FIBER1933