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RESEARCH ARTICLE
Membrane tubules attach Salmonella Typhimurium to eukaryotic
cells and bacteria
Svetlana I. Galkina1, Julia M. Romanova2, Elizaveta E. Bragina1, Irina G. Tiganova2,
Vladimir I. Stadnichuk3, Natalia V. Alekseeva2, Vladimir Y. Polyakov1 & Thomas Klein4
A.N. Belozersky Institute of the Moscow State University, Moscow, Russia; 2N.F. Gamaleya Research Institute of Epidemiology and Microbiology RAMS,
Moscow, Russia; 3Physical Department of the Moscow State University, Moscow, Russia; and 4Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach,
Germany
IMMUNOLOGY & MEDICAL MICROBIOLOGY
1
Correspondence: Svetlana I. Galkina, A.N.
Belozersky Institute of Moscow State
University, Leninskie Gory, MSU, Bldg. 1,
Corp. 40, 119991 Moscow, Russia. Tel.: 17
495 9395 408; fax: 17 495 9393 181; e-mail:
[email protected]
Received 27 May 2010; revised 1 October 2010;
accepted 6 October 2010.
Final version published online 8 November
2010.
DOI:10.1111/j.1574-695X.2010.00754.x
Editor: Eric Oswald
Keywords
membrane tubulovesicular extensions;
membrane tethers; cytonemes; Salmonella
enterica serovar Typhimurium; human
neutrophil; adhesive bacterial tubular
appendages.
Abstract
Using scanning electron microscopy techniques we measured the diameter of
adhesive tubular appendages of Salmonella enterica serovar S. Typhimurium. The
appendages interconnected bacteria in biofilms grown on gallstones or coverslips,
or attached bacteria to host cells (human neutrophils). The tubular appendage
diameter of bacteria of virulent flagellated C53 strain varied between 60 and 70 nm,
thus considerably exceeding in size of flagella or pili. Nonflagellated bacteria of
mutant SJW 880 strain in biofilms grown on gallstones or coverslips were also
interconnected by 60–90-nm tubular appendages. Transmission electron microscopy studies of thin sections of S. Typhimurium biofilms grown on agar or
coverslips revealed numerous fragments of membrane tubular and vesicular
structures between bacteria of both flagellated and nonflagellated strains. The
membrane structures had the same diameter as tubular appendages observed by
scanning electron microscopy, indicating that tubular appendages might represent
membrane tubules (tethers). Previously, we have shown that neutrophils can
contact cells and bacteria over distance via membrane tubulovesicular extensions
(TVE) (cytonemes). The present electron microscopy study revealed the similarities in size and behavior of bacterial tubular appendages and neutrophil TVE.
Our data support the hypothesis that bacteria establish long-range adhesive
interactions via membrane tubules.
Introduction
Recent investigations demonstrate that eukaryotic cells can
communicate with their environment over distance using
nanometer-wide and very long membrane tubular or tubulovesicular cellular extensions (cytonemes, membrane
tethers). These long-range cellular interactions were first
demonstrated for embryonic (Gustafson & Wolpert, 1967)
and blood cells (neutrophils) (Shao et al., 1998; Schmidtke
& Diamond, 2000; Galkina et al., 2001), and more recently
for nerve and other cells as summarized in numerous
comprehensive reviews (Galkina et al., 2006; Gerdes, 2006;
Veranic et al., 2008; Hurtig et al., 2010).
Bacteria communicate with their environment by means
of numerous tubular cellular extensions – flagella, pili,
fimbriae and curli – which differ in size, origin and
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composition. Flagella are 15–20 nm in diameter (Allen &
Baumann, 1971). R-type straight flagella of Salmonella are
5–11 nm (Mimori et al., 1995) and pili do not exceed
6–7 nm (Merz et al., 2000; Zhang et al., 2000), whereas
aggregative fimbriae or curli are far smaller. High-resolution
scanning electron microscopy studies revealed that contact
with cultured epithelial cells results in the formation of
unusually wide (60 nm in diameter) tubular appendages of
S. Typhimurium attaching bacteria to the epithelial cells
(Ginocchio et al., 1994; Reed et al., 1998). The formation of
such appendages did not require de novo protein synthesis
and was transient. These surface structures were shed or
retracted and were not observed on bacteria undergoing
internalization. Helicobacter pylori bacteria were found to
produce bacterial organelles of similar diameter (45–70 nm)
upon interaction with the epithelial cell surface (Rohde
FEMS Immunol Med Microbiol 61 (2011) 114–124
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Salmonella adheres via membrane tubules
et al., 2003). Helicobacter pylori also developed these organelles when grown on agar plates in the absence of eukaryotic cells.
Gram-negative bacteria are also known to develop membrane tubular extensions, referred to as membrane sheaths
of flagella. Electron microscopy studies of flagella of Vibrio
metchnikovii (Follett & Gordon, 1963), Bdellovibrio bacteriovorus (Seidler & Starr, 1968), or H. pylori (Geis et al., 1993)
revealed an internal electron-dense filament and a surrounding flagellar sheath with the typical bilayer structure
of a membrane. These tubular membrane structures
were three to four times thicker than typical bacterial flagella
and had a diameter very similar in size to that of the
adhesive tubular appendages of S. Typhimurium observed
upon interaction with epithelial cells (Ginocchio et al.,
1994). Despite these structures having been observed
many years ago, the function of membrane sheaths of
flagella remains to be elucidated (Sjoblad et al., 1983;
McCarter, 2001). Moreover, the formation of membrane
tubular structures that do not contain flagellar filament
has been observed in some strains of Beneckea. These
tubular membrane projections were often beaded to a
variable degree. Transmission electron microscopy data
revealed that tubular projections were evaginations of the
outer membrane of the cell wall (Allen & Baumann, 1971).
Membranous tubulovesicular protrusions, which spanned
individual cells in colony, were shown to develop in bacteria
of wild-type and mutant Neisseria gonorrhoeae strains
(Wolfgang et al., 2000). Formation of these membrane
structures appears to be associated with type IV pili biogenesis, which includes fiber formation and fiber translocation
to the cell surface. The simultaneous absence of the secretin
family and biogenesis component PilQ and the twitching
motility/pilus retraction protein PilT leads to the expression
of type IV pili, which fail to reach the cell surface and remain
localized inside tubulovesicular membrane protrusions of
bacteria.
We suggest that tubular appendages of bacteria could
represent membrane tubular structures and play the
same role in bacterial adhesive interactions as tubulovesicular extensions (TVE, cytonemes) play in the adhesion of
human neutrophils. Neutrophil TVE consist of membrane
tubules and/or vesicles of the same diameter (150–240 nm
depending on conditions) interconnected in one line.
TVE represent long and rapidly developed (20–100 mm in
length in 20 min) exocytotic neutrophil structures, which
can establish long-range contacts between neutrophils
and substrata or eukaryotic cells, and which can catch and
hold bacteria (Galkina et al., 2001, 2009, 2010). Using
scanning and transmission electron microscopy, we studied
tubular adhesive appendages of S. Typhimurium and compared these in size and behavior with human neutrophil
TVE.
FEMS Immunol Med Microbiol 61 (2011) 114–124
Materials and methods
Bicarbonate-free Hanks solution, 4-bromophenacyl bromide (BPB) and phenylmethylsulfonyl fluoride (PMSF)
were purchased from Sigma (Steinheim, Germany). FicollPaque was obtained from Pharmacia (Uppsala, Sweden) and
fibronectin was from Calbiochem (La Jolla, CA).
Salmonella Typhimurium cells of virulent strain C53 were
a kind gift of Prof. F. Norel (Pasteur Institute, France)
(Kowarz et al., 1994). Salmonella of strain SJW 880 flaR
1656 H1-gt H2-enx, nonmotile nonflagellated mutant
strain, were obtained from S. Kato (Nagoya University,
Japan). Bacteria were grown in Luria–Bertani broth without
NaCl and then washed twice using physiological solution
and centrifuged at 2000 g. Biofilms of bacteria were grown
on coverslips, agar or gallstones. Coverslips were thoroughly
cleaned, washed and placed into Petri dishes. Cholesteroltype gallstones 3–4 mm in diameter were extracted as a
result of surgical intervention from the gallbladder of a
gallstone disease patient, washed, incubated in 70% ethanol
for 12 h, dried in sterile conditions and placed into the vials.
The concentration of bacteria stock suspension was
2 108 CFU mL 1. Bacteria were added in Petri dishes with
coverslips, agar or in vials with gallstones in Luria–Bertani
broth and were grown for 1–3 days at 37 1C with gentle
agitation. Following culture, coverglasses and stones were
taken from the vials and the medium was allowed to flow
down. Coverslips or gallstones were placed into vials containing fixative solution for electron microscopy without
preliminary washing with the buffer solution.
For neutrophil preparation, we used the blood of healthy
volunteers who had not had any pharmacological therapy
for the 2 weeks preceding sampling. Blood was taken via
venous puncture and the sampling was approved by the
Ministry of Public Health Service of the Russian Federation.
Blood experimental procedures were approved by the
Institutional Ethics Committee of the A.N. Belozersky
Institute. Neutrophils were isolated from freshly drawn
blood on a bilayer gradient of Ficoll-Paque (at densities of
1.077 and 1.125 g mL 1) (Boyum, 1974). Washed neutrophils were resuspended in bicarbonate-free Hanks solution
containing 10 mM HEPES, pH 7.35. Glass coverslips
were incubated in Hanks solution containing 5 mg mL 1
fibronectin for 2 h at room temperature and were
thoroughly washed with phosphate-buffered saline. Neutrophils (106 cells mL 1) were plated on protein-coated coverslips in corresponding buffer and incubated for 20 min at
37 1C. To induce neutrophil TVE formation, cells were
plated to fibronectin-coated coverslips in the presence of
BPB. BPB was dissolved in dimethyl sulfoxide (DMSO) and
added to the cells before plating. Corresponding amounts of
DMSO (not exceeding 5 mL mL 1) were added to the control
cells.
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116
To study neutrophil interactions with bacteria, neutrophils were incubated over fibronectin-coated coverslips in
control conditions or in the presence of 10 mM BPB for
15 min. Bacteria (bacteria/cell ratio 20 : 1) were then added
and cells further incubated for 5 min. Coverslips with cells
were taken from the dishes and placed into vials with fixative
solution for electron microscopy without preliminary washing with the buffer solution. TVE are very vulnerable
structures and thus easily destroyed during fixation and
drying procedures during preparation for electron microscopy. Interactions with bacteria further increase these
degradative processes. To stabilize the BPB-induced neutrophil TVE in some experiments, we used sulfatide from
bovine brain as described previously (Galkina et al., 2009)
and as is indicated in the figure legends.
For scanning electron microscopy, cells and bacteria were
fixed in 2.5% glutaraldehyde in Hanks buffer without Ca21
and Mg21 ions and in the presence of 5 mM EDTA as
an inhibitor of metalloproteinases, 5 mM PMSF as an
inhibitor of serine proteases, and 10 mM HEPES at pH 7.3.
Cells were postfixed with 1% osmium tetroxide in 0.1 M
sodium cacodylate with 0.1 M sucrose at pH 7.3 (without
washing with a buffer after glutaraldehyde), dehydrated in
acetone series, critical-point-dried with liquid CO2 as
a transitional fluid in a Balzers apparatus, sputter-coated
with gold–palladium and observed at 15 kV with a Camscan
S-2 or JSM-6380 scanning electron microscope. It is
worth noting that air-drying of samples after fixation
completely eliminated thin tubular structures of cells and
bacteria.
For transmission electron microscopy, the pieces of
biofilms of bacteria grown on the coverslips or agar were
transferred in vials with the fixative solution. We used two
types of fixation for the transmission electron microscopy.
One of these was a routinely used fixation in 2.5% solution
of glutaraldehyde in 0.1 M sodium cacodylate pH 7.3,
followed by washing with a buffer and postfixation in 1%
osmium tetroxide in 0.1 M sodium cacodylate pH 7.3. For
the second method, we used the modified-for-scanning
electron microscopy procedure described above. After
fixation, all of the samples were routinely dehydrated
(70% ethanol containing 2% uranyl acetate), and embedded
in Epon 812 (Fluka). Embedded specimens were sliced
into ultrathin sections with a Reichert UltraÑut III,
stained with lead citrate, and examined with a JFM-1011
microscope.
Tubular appendages or membrane vesicles diameters were
measured directly on highly magnified scanning or transmission electron microscopy images and calculated based
on the respective bar’s value. The data were expressed
as mean SEM. Student’s t-test was performed for unpaired observations. Values of P o 0.05 were regarded as
significant.
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S.I. Galkina et al.
Results
Scanning electron microscopy study of adhesive
bacterial appendages in bacterial biofilms
grown on gallstones and coverslips
We studied bacterial connections in biofilms grown on
gallstones, coverslips or agar. Gallstones are recognized as a
natural substrate for persistence of bacteria in chronic
infections. It is suggested that Salmonella form biofilms on
the surface of gallstones, where the bacteria are protected
from high concentrations of bile and antibiotics. The
potential for biofilm formation on the surface of gallstones
in vitro was demonstrated by Prouty et al. (2002). We used
gallstones from a patient undergoing surgical intervention.
Bacteria S. Typhimurium of the C53 strain cultivated for 3
days on gallstones formed biofilms on the surface of the
gallstones. Scanning electron microscopy revealed that the
bacteria in biofilms were interconnected in a network and
anchored to the surface of the gallstones by bacterial tubular
appendages (Fig. 1a and c). The average diameter of these
appendages obtained by scanning electron microscopy was
62 nm (Table 1). The appendages reached 8–10 mm in
length.
We then compared biofilms formed on the surface of
the gallstones by flagellated C53 and nonflagellated
bacteria (mutant strain SJW 880). Bacteria of the mutant
strain SJW 880 carry a mutation in the flaR gene and
therefore do not develop flagella filaments (PattersonDelafield et al., 1973). Mutant bacteria grown on the
gallstones for 3 days formed small colonies (Fig. 1b and d).
Bacteria in these colonies were also interconnected by
multiple tubular appendages with an average diameter of
67 nm (Table 1).
Similar results were obtained with bacteria grown on
coverslips. The Salmonella species of flagellated C53 strain
incubated over fibronectin-coated coverslips for 24 h
formed small colonies. Bacteria were attached to substrata
and to other bacteria by similar tubular appendages (61 nm
in diameter) and developed cell bodies perpendicularly
(Table 1, Fig. 2a). In principle, all bacteria had one to several
tubular appendages. Tubular appendages were also observed
in bacteria of the SJW 880 strain grown on coverslips for
24 h (Fig. 2b), but o 10% of bacteria developed similar
appendages and, where developed, these were slightly wider
in diameter (90 nm) than appendages of the flagellated
strain (Table 1).
Our scanning microscopy data demonstrated that the
formation of 60–90-nm-diameter tubular appendages of
S. Typhimurium appears to be a common feature for
bacterial adhesive interactions with bacteria and substrata.
The diameter of tubular appendages was three times greater
than the diameter of flagella. Moreover, the SJW 880 strain
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Salmonella adheres via membrane tubules
Fig. 1. Scanning electron microscopy images of
Salmonella Typhimurium bacteria of C53 and
SJW 880 strains grown on gallstones. Bacteria of
C53 (a, c) and SJW 880 (b, d) strains were grown
on gallstones for 3 days and fixed and dried for
scanning electron microscopy. Pictures represent
typical images at different amplifications
observed in two independent experiments.
Table 1. Diameter of tubular appendages of Salmonella enterica serovar Typhimurium of C53 and SJW 880 strains in different experimental conditions
obtained by scanning electron microscopy
Object
Diameter of tubular appendages connecting C53 bacteria to the other bacteria and substrata in biofilms grown on
coverslips for 24 h
Diameter of tubular appendages connecting C53 bacteria to other bacteria and substrata in biofilms grown on
gallstones for 3 days
Diameter of tubular appendages connecting C53 bacteria to human neutrophils attached to fibronectin in control
conditions
Diameter of tubular appendages connecting C53 bacteria to human neutrophils attached to fibronectin in the
presence of BPB, 10 mM
Diameter of tubular appendages connecting SJW 880 bacteria to the other bacteria and substrata in biofilms grown
on coverslips
Diameter of tubular appendages connecting SJW 880 bacteria to the other bacteria and substrata in biofilms grown
on gallstones
Diameter of bulges of tubular appendages of bacteria from all experiments
Dmin
Dmax
D SEM
43
87
61 4
42
84
62 3
40
89
66 5
61
101
78 3
81
117
90 4
48
87
67 2
147
292
222 30
Data presented are the mean values of the bacterial appendage diameters (D SEM) obtained from the measurements of appendage diameter in
15–20 bacteria. Dmin and Dmax are the minimal and maximal diameters observed. Results of two to three experiments were summarized.
of S. Typhimurium lacking flagella developed tubular extensions of the same diameter to adhere to bacteria and
substrata.
Transmission electron microscopy study of
adhesive bacterial appendages in bacterial
biofilms grown on agar or coverslips
To demonstrate the membranous nature of bacterial tubular
appendages, we performed a transmission electron microscopy study of thin sections of bacterial biofilms grown on
agar and coverslips. Bacteria of C53 and SJW 880 strains
were grown on agar in Petri dishes and formed biofilms
FEMS Immunol Med Microbiol 61 (2011) 114–124
within 24 h. Very long and thin membrane tubular structures could easily be destroyed during fixation and subsequent preparation steps for electron microscopy studies.
Therefore, it was necessary to apply a fixation procedure to
prevent destruction of membrane tubules. The pieces of
biofilms were fixed in two different ways: (1) by the
procedure modified for scanning electron microscopy; and
(2) by the routinely used procedure described in Materials
and methods. When bacterial biofilms of C53 strain grown
on agar were fixed by this modified procedure (Fig. 3a and
c–f), transmission electron microscopy evaluation revealed
multiple flagella (arrows) and multiple membrane tubular
structures (arrowheads) originating from bacteria (Fig. 3a).
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118
Fig. 2. Scanning electron microscopy images of Salmonella Typhimurium bacteria of C53 and mutant SJW 880 strains grown on coverslips.
Bacteria of C53 (a) and SJW 880 (b) strains were grown on coverslips for
24 h. Pictures represent typical images observed in two independent
experiments.
In samples fixed by the routinely used procedure, we
observed only bacterial flagella (Fig. 3b, arrows).
Transmission electron microscopy studies of bacterial
biofilms of the C53 strain demonstrated that tubular appendages branched from bacteria (Fig. 3a, c and d) appeared to
be the extensions of the outer bacterial membrane (Fig. 3c).
The diameter of these tubular appendages varied from
60–90 nm and strongly differed in diameter from flagella
(15–20 nm), but coincided with the diameter of S. Typhimurium tubular appendages obtained by scanning electron
microscopy (Table 1). The relationship between flagella and
membrane tubular structures remains to be further elucidated. In size, membrane tubules corresponded to ‘membrane sheaths’ of flagella. We did not observe membrane
tubules containing bacterial flagellar filaments inside, as was
demonstrated for H. pylori (Geis et al., 1993) or B. bacter2010 Federation of European Microbiological Societies
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S.I. Galkina et al.
iovorus (Seidler & Starr, 1968). Only in a few cases did we
observe images showing the flagellar filament extending
from membrane tubules (Fig. 3d). Mostly, we observed
membrane tubular structures that did not interact with
flagella. Examination of nonflagellated SJW 880 strain
biofilms also revealed fragments of 50–100 nm diameter
membrane tubular extensions of bacterial membrane (Fig.
3e and f).
In a further experiment, S. Typhimurium (C53 strain)
bacteria were grown on coverslips for 3 days and formed
a biofilm. Subsequent fixation did not cause disaggregation
of the biofilm layer, indicating tight interconnection of
bacteria within a biofilm-structured organization. In
contrast, biofilms of SJW 880 bacteria grown under identical
conditions demonstrated transformation in suspension
upon fixation. We investigated part of the C53 biofilm by
means of transmission electron microscopy with a modified
method for scanning electron microscopy fixation.
According to our hypothesis, in biofilms the bacterial
tubular appendages interconnecting bacteria represent flexible membrane tethers. Sections of such structures can
represent tubules, ovals or circles of the same diameter.
Transmission electron microscopy analysis of the C53 biofilm
revealed some membrane tubular (Fig. 4a–c) and numerous
oval (Fig. 4c and d) and circular (Fig. 4d and e) structures
between bacteria. Membrane circles were often organized in
series, originating from the same location on the bacteria
surface (Fig. 4g). These membrane tubular, oval and circular
structures differ distinctly in size from flagella (Fig. 4e and h,
black arrows). We further studied the distribution of membrane structures in relation to their diameters. Those types of
membranes were most commonly 60-70 nm in diameter
(Fig. 5). This strongly correlates to the diameter of
S. Typhimurium tubular appendages revealed by scanning
electron microscopy (Table 1).
Describing bacterial appendages connecting
S. Typhimurium bacteria to human neutrophils
by means of scanning electron microscopy
Using scanning electron microscopy, we studied S. Typhimurium interactions with neutrophils plated to fibronectincoated substrata in control conditions or in the presence of
BPB. BPB is known to induce TVE formation in human
neutrophils in 4 90% of cells in preparation (Galkina et al.,
2001). BPB-induced TVE did not differ from TVE formed in
the presence of nitric oxide, the natural causative factor for
TVE formation (Galkina et al., 2005; Galkina et al., 2009).
We performed experiments with BPB to compare size and
behavior of the neutrophil TVE and the adhesive tubular
appendages of bacteria.
Under control conditions, neutrophils attached and spread
(flattened) on fibronectin-coated coverslips (Fig. 6a). When
FEMS Immunol Med Microbiol 61 (2011) 114–124
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Salmonella adheres via membrane tubules
Fig. 3. Transmission electron microscopy images
of thin sections of biofilms of Salmonella
Typhimurium bacteria of the C53 and mutant
SJW 880 strains grown on agar. Bacteria of C53
(a–d) and SJW 880 (e, f) strains were grown in
Petri dishes on agar for 24 h and formed biofilms.
Biofilms of C53 and SJW 880 strains were fixed
by the procedure modified for scanning electron
microscopy (a, c–f) and by the routinely used
procedure (b) described in Materials and
methods. Black arrowheads indicate membrane
tubules and black arrows indicate flagella.
Pictures represent typical images observed in two
independent experiments.
bacteria S. Typhimurium were added to neutrophils, these
were either ingested by neutrophils or remained on the
neutrophil surface. In contrast to the control cells (Fig. 6a),
specific membrane ruffles were observed on the surface of
neutrophils exposed to bacteria (Fig. 6b, arrowheads).
Similar ruffles were observed on the surface of enterocytes,
M-cells or macrophages infected with S. Typhimurium.
Thin-section electron microscopy revealed that these ruffles
indicated the sites of bacteria entry into the cell (Bliska et al.,
1993). Based on these studies, we consider that ruffles on the
neutrophil surface point to the sites of bacteria ingestion.
Bacteria that remained on the cell surface (Fig. 6b) were
attached to the neutrophils by tubular appendages with an
average diameter of 66 nm (Table 1). Similar appendages
attached the bacteria to substrata in the area between
attached neutrophils (Figs 6f and 7b).
Neutrophils attached to substrata in the presence of BPB
did not spread and developed membrane TVE on their
surface, which anchored the cells to substrata and interconnected the neutrophils (Fig. 6c). When bacteria were added
to BPB-treated neutrophils, the bacteria were attached to
FEMS Immunol Med Microbiol 61 (2011) 114–124
neutrophil cell bodies, to TVE (Fig. 6d and e), substrata
between neutrophils (Fig. 6g), and other bacteria (Fig. 6d
and e) by means of their own tubular extensions, which had
an average diameter of 78 nm (Table 1).
Initial studies with tubular appendages of S. Typhimurium (60 nm in diameter) and epithelial MDCK cells or
murine Peyer’s patch follicle-associated epithelia (Ginocchio
et al., 1994; Reed et al., 1998) described these as specific
eukaryotic cell-induced structures (Ginocchio et al., 1994;
Reed et al., 1998). In our experiments, tubular appendages
of the same diameter connected bacteria to substrata or to
bacteria in biofilms in the absence of eukaryotic cells.
Formation of bacterial biofilms on the different surfaces
required an incubation period of 1–3 days. However, in
interactions with epithelial cells, bacteria developed appendages within 15 min (Ginocchio et al., 1994). Thus, it
appears that eukaryotic cells accelerate the development of
tubular appendages in bacteria.
To reveal the effect of neutrophils on S. Typhimurium
adhesion and formation of tubular appendages, we compared attachment of bacteria to fibronectin-coated substrata
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S.I. Galkina et al.
Fig. 4. Transmission electron microscopy images
of tubular and vesicular membrane structures
inside biofilm of Salmonella Typhimurium of C53
strain grown on coverslips. Transmission electron
microscopy images of tubular (a–c), oval (d, e)
and vesicular (f, g) membrane structure found
between bacteria in thin sections of C53 biofilm.
Flagellar filaments are presented to compare
tubular appendages and bacterial flagella (e and
h, black arrows). Bacteria were grown on
coverslips for 3 days (a, b) and fixed by a
procedure modified for scanning electron
microscopy. Pictures represent typical images
observed in two independent experiments.
under control conditions and in the presence of attached
neutrophils (Fig. 7). No tubular appendages were formed
under control conditions (Fig. 7a), but in the presence of
neutrophils, bacteria developed tubular appendages which
connected bacteria to substrata and to other bacteria (Fig.
7b). Quantification of attached bacteria resulted in 6 2
(mean SEM) S. Typhimurium per 0.01 mm2 under control conditions and 25 5 per 0.01 mm2 in the presence of
neutrophils (P o 0.05). Thus, neutrophils facilitated the
development of tubular bacterial appendages and induced
bacterial adhesion to fibronectin-coated substrata.
Discussion
We studied tubular appendages formed by flagellated and
nonflagellated S. Typhimurium bacteria, which attached
bacteria to substrata and interconnected bacteria in biofilms. These appendages varied in diameter from 60 to
90 nm and reached 8–10 mm in length. Tubular appendages
do not represent flagellar filaments or flagellar hooks. The
bacterial flagella consist of the basal body, the filament and
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the hook (a flexible tube which connects the basal body and
the filament). In the flagellated C53 strain, the flagellar
filaments correspond to tubular appendages in length;
however, the diameter of these filaments does not exceed
20 nm. Bacteria of the SJW 880 strain (polyhook mutant)
have no flagella, but hooks of polyhook mutants can reach
900 nm in length (Hirano et al., 1994), which is comparable
to the length of tubular appendages. In diameter, hooks do
not exceed flagella (Aizawa et al., 1985; Hirano et al., 1994;
Cornelis, 2006), thus differing strongly from the 60-nm
tubular appendages.
Our transmission electron microscopy data confirmed
our suggestion that bacterial adhesive tubular appendages
(diameters of 60–90 nm) represent membrane tubules
(membrane tethers), which are formed as extensions of the
outer membrane. Whether membrane tubular structures are
related to flagella or pili, and serve as ‘membrane sheaths’
for flagella or pili remains to be further elucidated. Our
electron microscopy data demonstrated mainly empty
membrane tubules without filaments inside. Membrane
tubules and flagellar filaments had different sensitivities to
FEMS Immunol Med Microbiol 61 (2011) 114–124
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Salmonella adheres via membrane tubules
25
Frequency
20
15
10
5
0
50
60
70
80
90
100
Diameter (nm)
110
120
Fig. 5. Distribution of the vesicular structures observed between bacteria inside the biofilm of Salmonella Typhimurium of C53 strain according
to the diameters measured on transmission electron microscopy images.
The diameters of tubular and vesicular membrane structures observed in
bacteria inside the biofilm of S. Typhimurium of C53 strain grown on
coverslips were measured on transmission electron images. For oval
structures, the minimum diameter was measured. To summarize the
distribution pattern, data (n = 62) from two independent experiments
were collected.
fixation and other preparation procedures. Therefore, we
cannot exclude that the preparation techniques we used,
preserved membrane tubules more effectively than flagellar
filaments. Nonetheless, the membrane tubules may represent structures independent of other cell protrusions, and
which are destined for cell adhesion and communication.
Further, our data revealed the prominent similarity
between bacteria tubular appendages and TVE of neutrophils. TVE represent membrane tubules and vesicles containing neutrophil cytoplasm (Galkina et al., 2001, 2009,
2010). Neutrophil TVE have a strictly uniform diameter
along the entire length (150–240 nm, depending on conditions) and can reach 80–100 mm in length, equivalent to
several neutrophil diameters, within 20 min. Bacterial adhesive appendages had a strong tubular form and their average
diameter varied from 60 to 90 nm, depending on the
conditions (Table 1). In our experiments, bacterial appendages reached 8–10 mm in length (Fig. 6e), which is several
times the bacterial length. Like neutrophil TVE (Fig. 6c),
bacterial appendages were highly flexible and were able to
coil several times around bacteria (Fig. 6f). Neutrophil TVE
are believed to be exocytotic structures and are capable of
being shed from the neutrophil surface (Galkina et al.,
2009). Bacterial appendages of S. Typhimurium also
Fig. 6. Scanning electron microscopy images of
tubular appendages of Salmonella Typhimurium
and tubulovesicular extensions of human
neutrophils. Neutrophils were plated to
fibronectin-coated substrata for 15 min at 37 1C
in control conditions (a, b, f); in the presence of
10 mM BPB (c, g); in the presence of 10 mM BPB
and 25 mg bovine brain sulfatide (d, e). Bacteria
S. Typhimurium of virulent C53 strain were then
added (bacteria/cell ratio 20 : 1) and cells were
further incubated for 5 min at 37 1C (b–g). White
arrowheads indicate ruffles on the neutrophil
surface. Small arrows indicate bulges on
neutrophil TVE and on bacterial tubular
appendages. Large arrows indicate tubular
appendages shed from bacteria. Pictures
represent typical images observed in three
independent experiments.
FEMS Immunol Med Microbiol 61 (2011) 114–124
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122
Fig. 7. Scanning electron microscopy images of Salmonella Typhimurium bacteria plated to fibronectin-coated substrata in control conditions
and in the presence of neutrophils. Neutrophils were attached to
fibronectin-coated substrata for 15 min at 37 1C. Bacteria S. Typhimurium of virulent C53 strain were then added (bacteria/cell ratio 20 : 1) for
5 min (a). Bacteria were plated to fibronectin-coated substrata without
neutrophils for 5 min (b).
underwent shedding from bacteria (Fig. 6e and f, large
arrows). In addition, TVE are capable of transporting
neutrophil mediators as bulges along TVE (Fig. 6c, small
arrows). Similar bulges were observed on bacterial appendages (Fig. 6g, small arrows). Whether these bulges move
inside appendages or slide along the appendages remains to
be elucidated.
Bacteria reach 2 mm in length and the diameter of tubular
appendages varies from 60 to 90 nm. Nonspread human
neutrophils are 6–7 mm in diameter and the TVE diameter
varies from 160 to 240 nm. Consequently, the ratio between
the diameters of cell and tubular appendages in neutrophils
and bacteria is comparable.
In neutrophils, membrane tethers, similar to BPB-induced neutrophil TVE in size and behavior, can be pulled
from the neutrophil cell bodies by micropipette manipulation (Shao et al., 1998; Marcus & Hochmuth, 2002).
Diamond and colleagues observed the pulling of extremely
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c
S.I. Galkina et al.
long membrane tethers from flowing neutrophils as a result
of neutrophil attachment to platelets, P-selectin or endothelial cells under physiological flow conditions (Schmidtke &
Diamond, 2000; Oh & Diamond, 2008; Oh et al., 2009).
Recent investigations also demonstrate that membrane
tethers can be extracted from the bacteria Escherichia coli
by optical tweezer manipulation (Jauffred et al., 2007). Like
neutrophil tethers, bacterial tethers are extremely long and
are primarily composed of the asymmetric lipopolysaccharide-containing bilayer of the outer membrane.
The membrane circles observed in thin sections of C53
biofilms may represent either sections of membrane tubules
or sections of membrane vesicles of the same diameter
(Fig. 4e–g). It is known that virtually all gram-negative
bacteria, including Salmonella, produce membrane vesicles
50–250 nm in diameter, commonly filled with components
considered to be secretion products (Li et al., 1998; Beveridge, 1999; Schooling & Beveridge, 2006). These vesicles are
shown to have the capacity of fusing with the outer
membrane of the other gram-negative bacteria. In other
words, the vesicles can serve as secretory carriers between
bacteria. In eukaryotic cells, membrane tubules, along with
membrane vesicles, serve as secretory carriers. In this
capacity, membrane vesicles and tubules are interconvertable. Large GTPase dynamin and dynamin-like proteins
mediate the membrane tubulation and vesicle scission
that occurs during intracellular trafficking, endocytosis
and exocytosis (Sweitzer & Hinshaw, 1998; Praefcke &
McMahon, 2004).
Recently, a dynamin-like protein of cyanobacteria capable
of tubulation of E. coli lipid liposomes was prepared and its
crystal structure was resolved (Low & Lowe, 2006; Low et al.,
2009). Given the presence of large GTPases with predicted
dynamin-like domain organization in many members of
Eubacteria (van der Bliek, 1999), it is likely that bacterial
dynamins or dynamin-like proteins are common features of
bacteria and play an identical role in membrane tubulation/
vesiculation to that of eukaryotic dynamins.
In conclusion, we have demonstrated that pathogenic
bacteria S. Typhimurium use 60–90 nm diameter tubular
appendages to establish contacts with substrata, bacteria and
eukaryotic cells. Scanning and transmission electron microscopy data revealed that bacterial tubular appendages represent membrane tubular extensions. Our work supports the
hypothesis that bacteria-like eukaryotic cells can establish
long-range contact with cells and bacteria via membrane
tubules.
Acknowledgements
This work was supported by grants from the Russian
Foundation of Basic Research 09-04-00367. The authors
FEMS Immunol Med Microbiol 61 (2011) 114–124
123
Salmonella adheres via membrane tubules
very much appreciate the support from Galina Sud’ina for
neutrophil preparations and fruitful discussion of the work.
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