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Journal of Bacteriology, May 2003, p . 3214-3217, Vol . 185,
No . 10
Alpha-Toxin Is Required for Biofilm Formation by Staphylococcus aureus
Nicky C . Caiazza and G . A . O'Toole*
Department of Microbiology and Immunology, Dartmouth Medical School, Hanover,
New Hampshire
Received 9 May 2002/ Accepted 24 February 2003
Staphylococcus aureus is a common pathogen associated with nosocomial
infections . It can persist in clinical settings and gain increased
resistance to antimicrobial agents through biofilm formation .
We have found that alpha-toxin, a secreted, multimeric, hemolytic
toxin encoded by the hla gene, plays an integral role in biofilm
formation . The hla mutant was unable to fully colonize plastic
surfaces under both static and flow conditions . Based on microscopy
studies, we propose that alpha-hemolysin is required for cell-to-cell
interactions during biofilm formation .
Biofilms are surface-associated, sessile bacterial communities . A
mature biofilm is formed when planktonic cells initially colonize a
surface, aggregate and/or grow into multicellular colonies, and embed
themselves in an exopolysaccharide matrix . Staphylococcus aureus
is capable of biofilm formation, which increases its persistence and
boosts its levels of antimicrobial resistance (5),
and biofilms of this organism have been observed on surfaces ranging
from intravascular catheters to pacemaker leads (17,
18) . Genetic analyses of staphylococci have shown that
the progression of biofilm development consists of two steps: initial
cell-to-surface interactions followed by cell-to-cell interactions (9) .
Recent reports have shown autolysin (10), teichoic
acids (8), and surface proteins such as Bap to be
integral to the initial stages of colonization (4) .
The ica locus, which is required for the synthesis of the
polysaccharide intracellular adhesin (PIA), plays a role in
subsequent cell-to-cell interactions (3, 14) .
The accessory gene regulator (agr) is a two-component regulatory
system in S . aureus that has been implicated in biofilm
formation—an agr mutant is a hyper-biofilm-forming strain (22) .
To study biofilms of S . aureus, we took the approach of
examining known downstream targets regulated by the agr system
and determining their impact on biofilm formation . We show that one
of these targets, alpha-hemolysin, a 34-kDa protein that causes host
cell lysis by heptamerizing upon insertion into eukaryotic cell
membranes, plays a role in biofilm formation (21,
23) . Mutants defective in alpha-hemolysin
production failed to form biofilms under both static and flow
conditions, and strains lacking alpha-hemolysin have an apparent
defect in cell-to-cell interactions .
Figure 1A shows the results of a biofilm assay wherein
bacteria were grown at 37°C in tryptic soy broth (TSB) and 0.2%
glucose for 8 h, as described by Heilmann et al . (9,
11) . The level of bacterial adhesion, as
quantified by crystal violet staining, is
 3-fold
lower for the hla mutant than for the wild type (strains are
described in Table 1) . The alpha-hemolysin-deficient strain
was also defective for biofilm formation when compared to the
wild type at 16 h (data not shown) . Plasmid pDU1212 contains a
wild-type copy of the hla gene, and when this plasmid is introduced
into the hla::erm strain, biofilm formation is induced to a
level above that of even the wild-type strain (Fig . 1B),
whereas the vector control pNC1 has no effect on biofilm formation .
It has been shown previously that the supernatant of S . aureus
DU1090/pDU1212 (hla+) contains 2.5- and 110-fold more
hemolytic activity (in hemolytic units per milliliter) than wild-type
and hla::erm strains, respectively (1) .
Thus, the level of alpha-hemolysin may correlate with the level of
biofilm formation .
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FIG . 1 . The hla mutant is defective in biofilm formation . (A)
Biofilm formation phenotypes . Biofilm formation by the wild type (WT;
S . aureus 8325-4) and a hla::erm mutant (S . aureus
DU1090) was quantitated . Crystal violet was used to stain cells adhering
to polystyrene after 8 h of growth at 37°C . (B) Complementation of the
hla::erm mutant . Biofilms formed on polystyrene (8 h at
37°C) were analyzed for an hla::erm mutant harboring a
vector control plasmid (pNC1) or a plasmid providing a wild-type copy of
the hla gene (pDU1212 [hla+]).
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| TABLE 1 . Strains and plasmids
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PIA, encoded by the ica genes, has been shown to be required
for biofilm formation by S . aureus (3,
14, 15); therefore, we investigated PIA
production in the wild-type and hla::erm strains . PIA
was extracted from cells grown in TSB supplemented with 0.2% glucose
(the medium used for biofilm assays), serial twofold dilutions were
spotted onto nitrocellulose, and Western blotting was performed as
previously described by Cramton et al . (3) by using
antibody to PIA/PNAG [ß(1-6)-N-acetylglucosamine] (16) .
A wild-type PIA-producing strain (113) and an isogenic ica
mutant (113 ica::Tc) served as controls . No difference in the
levels of PIA production between the wild-type and alpha-toxin mutant
strains was observed (data not shown) . Furthermore, in 10 clinical
S . aureus strains analyzed (22), no correlation
between PIA production and alpha-hemolysis was observed . We
also investigated the ability of a multicopy dose of hla (plasmid
pDU1212) to rescue the biofilm formation defect of an ica mutant .
Neither pDU1212 (hla+) nor the vector control (pNC1) had
any effect on the biofilm formation phenotype of the ica
mutant (data not shown) .
To better understand the nature of the biofilm-deficient phenotype
of the hla::erm mutant, phase-contrast microscopy was employed
to observe and compare levels of surface attachment at 8 h in
24-well polystyrene plates (Costar, Corning, N.Y.) (Fig . 2) .
This assay was similar to the 96-well plate assay (described in
references 9 and 11) with the exception
that nonadherent cells were removed by aspiration . For the wild-type
strain, microcolonies (dark regions) were found scattered evenly
throughout the field of view but were not present in the fields of
view for the hla::erm mutant and the vector control .
The strain carrying the plasmid pDU1212 (hla+) in
the hla::erm background exhibited more robust biofilm
formation than even the wild type—the entire surface was covered in a
dense mass of microcolonies . Therefore, the crystal violet staining
data presented in Fig . 1B correlates with the
microscopy data presented in Fig . 2 .
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FIG . 2 . Direct visualization of attachment phenotypes . Bacteria were
inoculated onto 24-well plates, incubated for 8 h at 37°C, and then
analyzed by phase-contrast microscopy . Dark areas are the adherent
bacteria, and the light grayish regions represent the surface of the
24-well plate . The magnification is x1,050 .
WT, wild type.
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In a physiological setting, such as the surface of a catheter,
biofilms may exist and persist under conditions of flow . To mimic
these conditions in vitro, S . aureus biofilms were grown under
conditions of constant flow (40 ml/h) by using 0.1x
TSB as the growth medium in the flow cell system described by
Christensen et al . (2) . Overnight cultures of S .
aureus were diluted 1:1,000 in 0.1x
TSB, 300 µl of diluted cells was injected into the flow cell chamber,
and the cells were allowed to acclimate for 15 min before being
subjected to flow . Figure 3A shows that by 4 h the
wild type had attached to the surface of the flow chamber and begun
to form large macrocolonies . After 8 h of constant flow, the
wild-type macrocolonies became larger and more numerous . In addition,
the surface area between macrocolonies was completely covered by a
monolayer of cells . By 24 h, wild-type macrocolonies had increased in
size and density to the point of completely filling the flow chamber .
The architecture of the wild-type biofilm at 24 h consisted of
densely packed circular macrocolonies outlined by narrow, light
regions that were the channels between the macrocolonies . In contrast
to the wild type, the hla::erm mutant attached to the
surface as a sparse monolayer, failed to exhibit macrocolony
formation even at 24 h, and lacked any discernible architecture .
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FIG . 3 . Phenotypes of cells under flow conditions . Biofilms of the
wild-type (WT; S . aureus 8325-4) and the hla::erm
mutant (S . aureus DU1090) strains were grown in flow cell
chambers . At the times indicated, biofilms were observed from a top-down
perspective by using phase-contrast microscopy . In images from 4 and 8
h, light regions represent bacterial macrocolonies (indicated by the
white arrows) and dark areas are the surface of the flow cell chamber .
The magnification is x675 for images
from 4 and 8 h . At 24 h, the images were recorded at an original
magnification of x230 . (They are
shown at a magnification of x173.)
In these images from 24 h, the very dense macrocolonies formed by the
wild type appear as dark regions and the light areas define macrocolony
borders or channels between the macrocolonies . Small clusters of cells,
but no macrocolonies, were observed for the hla mutant at all
time points.
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In this study, we show a role for alpha-hemolysin in S . aureus
biofilm formation, and in particular, this toxin appears to be
required for cell-to-cell interactions . We were initially surprised
to find that a secreted toxin had such a dramatic impact on biofilm
formation; however, other examples exist in which secreted toxins may
play a role in biofilm formation (12,
13, 22) . The fact that cells carrying a
mutant allele of hla are capable of initially colonizing a
surface but never organize into multicellular macrocolonies indicates
a defect in cell-to-cell interactions . Based on the data presented in
this study, we propose that alpha-hemolysin plays a role primarily in
cell-to-cell interactions during biofilm formation .
Alpha-hemolysin is, in part, controlled by the agr system . It
has been shown that an agr mutant produces less alpha-hemolysin
but is a hyper-biofilm-forming strain (22) . However,
the agr system regulates a wide array of virulence factors,
including those involved in surface binding and surface-associated
virulence . Thus, even though alpha-hemolysin production is reduced in
an agr mutant, other surface-associated virulence factors may
be overexpressed, functionally compensating for the lack of
alpha-hemolysin . Furthermore, in vivo studies of device-related
infections have shown that alpha-hemolysin is not regulated by agr
but that its expression is predominately controlled by the
two-component regulator sae (7) . Therefore,
alpha-hemolysin may be produced in an agr-independent fashion
when S . aureus colonizes in-dwelling devices in the biofilm
mode of growth .
We thank A . Chueng and M . Palma for helpful advice and Richard J .
O'Callaghan for sending strains DU1090 and DU1090/pDU1212 . We also
thank Jerry Pier for providing the PIA/PNAG antibodies and Cuong
Vuong and Michael Otto for providing clinical S . aureus
isolates .
This work was supported by grants from Microbia, Inc., by the
American Cancer Society institutional research grant #IRG-82-003-17,
and by the Pew Charitable Trusts to G.A.O . G.A.O . is a Pew Scholar in
the Biomedical Sciences .
* Corresponding author . Mailing address: Department of
Microbiology & Immunology, Rm . 202 Vail Building, Dartmouth Medical School,
Hanover, NH 03755 . Phone: (603) 650-1248 . Fax: (603) 650-1318 . E-mail: georgeo@Dartmouth.edu.
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