Indian J Med Res 126, December 2007, pp 558-566
Mechanism of infection of a human isolate Salmonella (3,10:r:-) in
chicken ileum: Ultrastructural study
Rakesh Chander YashRoy
Biophysics & Electron Microscopy Section, Indian Veterinary Research Institute, Izatnagar
Received October 9, 2006
Background & objectives: Originally isolated from severe human food-poisoning cases, Salmonella
(3,10:r:-), a monophasic variety of otherwise diphasic serotypes such as S. weltevreden and S. simi,
causes serious infections in man, animals and poultry. Mechanism of infection of this versatile and
deadly organism is important to understand for its control. The objective of this study was to enhance
our understanding of infection of Salmonella (3,10:r:-) in vivo at cellular level.
Methods: Aliquots of 109 cfu of Salmonella (3,10:r:-) organisms were injected intra-ileally in 24 h pre-
fasted 3 month old broiler chickens by standard ligated ileal loop method. After 18 h, the fluid accumulated
in the ileum was drained and small tissue pieces were fixed in 2.5 per cent buffered (pH 7) glutaraldehyde
and subsequently in 1 per cent aqueous osmium tetraoxide. Ultra-thin sections of araldite-embedded
tissue pieces were examined under transmission electron microscope operated at 100 KV after staining
with uranyl acetate and lead citrate.
Results: Over 70 per cent of salmonellae interacting within 300 nm with ileal epithelial cells developed
numerous surface blebs of periplasmic extensions designated “periplasmic organelles” (POs). Large
sized POs were apparently pinched off as outer membrane vesicles (OMVs), 50-90 nm in diameter. Type
III secretion needle complex-like “rivet complexes” (RCs) were viewed to rivet the bacterial outer and
inner membranes together, allowing only pockets of periplasm to expand/inflate in order to liberate
OMVs. Many OMVs were found visibly docked on the plasma membrane of host epithelial cells. The
invading organisms appeared to leave the epithelial cells so as to find entry into the lymphatic vessels,
where, they again appeared to be closely interacting with ileal macrophages, by forming numerous POs
and concomitantly liberating OMVs. Inside the cytoplasm of macrophages, numerous tight phagosomes
were seen, each containing two organisms. The final stage appeared to contain replicated salmonellae,
four in each loose phagosome and, at the same time, macrophages also showed signs of apoptotic
disintegration, culminating in the release of replicated salmonellae.
Interpretation & conclusions: Outer membrane vesicles released from a fiercely virulent human isolate,
Salmonella 3,10:r:- pathogens have been implicated in translocating biochemical signals from the host-
interactive organisms to the eukaryotic cells at both stages of invasion leading to epithelial cell and macrophage
infection in vivo, in the chicken ileal model. A comprehensive cellular mechanism at ultrastructural level is
outlined for typhoid-like Salmonella infections caused by this humans-infecting organism.
Key words Chicken ileum - human isolate - invasion & infection - outer membrane vesicles - Salmonella (3,10:r:-) -
type III secretion - ultrastructure
Salmonellae are responsible for causing food-
poisoning problems and typhoid-like infections in
humans and animals, resulting in huge monetary losses
due to morbidity-linked reduction in productivity and
increased costs of disease treatment/management1,2.
Therefore, it is important to understand their
mechanisms of infection at cellular and molecular
levels in order to innovate necessary interventions.
Salmonella (3,10:r:-), originally isolated from severe
human food poisoning cases3, is capable of infecting
animals4 and poultry5. Interest in structural aspects of
virulence dates back to the year 1992 from this
laboratory when it was first demonstrated by
transmission electron microscopy (TEM) that
Salmonella (3,10:r:-) organisms located in close
proximity of host cells and interacting in vivo with
microvilli of epithelium in chicken ileum, developed
numerous bacterial outer membrane bound surface
appendages or periplasmic protrusions filled with
bacterial secretions6. Similar surface appendages
(named as invasosomes) were also later observed on
Salmonella Typhimurium while closely interacting in
vitro with cultured MDCK epithelial cells, as studied
by scanning electron microscopy7. Further TEM studies
suggested that larger periplasmic protrusions could
pinch off as 50- 90 nm diameter sized bacterial outer
membrane vesicles (OMVs), which could also be seen
in fusion-like membrane-to-membrane contact with
microvilli of host epithelial cells8,9. Translocation of
bacterial ‘toxic’ secretions from invading Gram-negative
pathogens into host/target cells was lauded as discovery
of a novel process viz., vesicular exocytosis in
prokaryotes9,10 and was considered to add a new
structural dimension of type III secretion system11. Later,
these OMVs have also been associated with damage
caused to eukaryotic host cells8-15 and aiding in invasion
of the pathogens16. It is now becoming increasingly clear
that Gram-negative pathogens employ OMVs for
targeting toxin delivery into mammalian cells17,18 and
that they contain a variety of bacterial toxins19-21 which
may have some role in virulence22 and pathogenicity21
and also in cytokine production by neutrophils23, besides
acting as potent inducers of platelet aggregation24.
Recently, OMV release has also been shown to correlate
directly with level of protein accumulation in the cell
envelope which has been opined to represent a
physiological stress response25. The present study
reports a comprehensive ultrastructural mechanism for
invasion and infection of chicken ileum by the human
isolate Salmonella 3,10:r:- organisms, with crucial role
assigned to OMV's.
Material & Methods
The pathogen, Salmonella 3,10:r:- (a monophasic
variety of otherwise diphasic serotypes such as S.
weltvreden and S. simi), originally isolated from human
food poisoning cases, was obtained from National
Salmonella Centre at the Indian Veterinary Research
Institute, Bareilly, Uttar Pardesh. This particular strain
has been typed distinctly from S. weltvreden3,4 and is
maintained at this Centre. Cultures of the organisms
originally isolated from cases of human food poisoning3
with 109 cfu were injected into chicken ileum in 24 h
pre-fasted 3 month old five broiler birds (obtained from
Central Avian Research Institute, Bareilly and
specifically maintained under suitable laboratory
conditions), using standard ligated ileal loop
methodology26,27. The procedures used employ thorough
washing-out of the ileum of its contents (including
naturally inhabiting organisms). Another saving factor
in this procedure was that the experimental (injected
with the dose of organisms) and control ileal loops
(injected with sterile medium without organisms) were
located in the same animal(s) so as to allow for excellent
control versus experimental sampling. The fluid was
found to be exsorbed only in the experimental loops
after 18 h of injection, and their contents were tested
for presence of injected organism to make sure that
reaction was indeed caused by Salmonella (3,10:r:-)
pathogens. The fluid exsorbed/accumulated in the ileal
loops was drained and ileal tissue pieces (size
approximately 1-2 mm3 ) of the experimental and control
loops were fixed in 2.5 per cent glutaraldehyde in
phosphate buffer (pH 7) at 5o C for 6 h and subsequently
post-fixed/block-stained in aqueous 1 per cent osmium
tetroxide for 6 h at room temperature following standard
methodology28,29. The fixed tissue blocks were made in
arlaldite and ultra-thin sections (approximately 500 Ao
in thickness) were cut using glass knife with an ultra-
microtome (LKB Ultrotome III, Sweden). Ultra-thin
sections obtained on 3 mm diameter copper grids, were
stained with uranyl acetate and lead citrate stains for
contrast and examined under JEOL JEM 1200EX
electron microscope (Japan) working in transmission
mode operated at 100 kilovolts. The electron
micrographs were interpreted in detail and arranged in
an order proposed to be a workable sequence of the
progress of invasion and infection in vivo.
From intensive TEM studies and detailed
interpretation of electron micrographs, a comprehensive
YASHROY: ULTRASTRUCTURAL MECHANISM OF INFECTION OF SALMONELLA (3,10:r:-)559
560 INDIAN J MED RES, DECEMBER 2007
Fig. 1. An ultrastuctural mechanism of invasion and infection of a human pathogen Salmonella 3,10:r:- studied in experimental infection of chicken ileum. A proposed sequence of
steps A-I may lead to systemic infection. Inset is the detailed structure of a large-sized periplasmic organelle filled with secretory materials packed within a markedly inflated
periplasmic space (PS), prior to its proposed liberation as an outer membrane vesicle (OMV). Fig. 1 A represents the organisms located near the center of the ileal lumen, which are
not closely apposed to the host epithelial cells and thus considered to be non-interactive organisms. Fig. 1B represents the organisms closely interacting with ileal epithelial cell
microvilli (mv) revealing numerous pockets of protruding periplasm, designated periplasmic organella. Fig. 1C represents salmonellae (sal) liberating bacterial outer membrane-
bounded 50-90 nm diameter vesicles (MV) pinched off from large periplasmic organella. These OMVs appear to dock on the microvillous membrane and a fusion-pore, supposedly
formed at the contact point, is proposed to translocate the biochemical signals of the pathogen into host cytosol. Fig. 1D represents host ileal epithelial cells which have undergone
cytoplasmic reorganization and membrane ruffle (R) formation after focal disruption of microvilli (curved arrow) that allow macro-pinocytosis (straight arrows) of the closely
approaching salmonellae (S). This process is proposed to be signaled by contents of outer membrane vesicles translocated into the host eukaryotic cell cytosol. Fig. 1E represents
salmonellae that pass through 'corridors' created in the ruffled epithelial cells (Fig. 1D) and then travel through lymphatic vessels of infected ileum (see Fig. 2) so as to come in contact
with macrophages. Numerous outer membrane vesicles (MV) once again liberated by pinching off periplasmic organelles (p) are apparently taken up (curved arrows) by macrophages
(M). This uptake is assumed to signal the macrophages to go into a stimulated phagocytosis mode. Fig. 1F represents the process of stimulated multiple-phagocytic cup (p) formation,
helping engulf the approaching salmonellae (sal.). Fig. 1G represents the mode of engulfment of pairs of organisms (A & B) and getting enclosed in single phagosomes (P). Fig. 1H
represents a stage where macrophages may end up getting packed with numerous electron-opaque phagosomes in their cytoplasm, mostly not-fusing with co-incident lysosomes (L).
Fig. 1I. represents the proposed last stage of the cycle at which replicated salmonellae, four in each loosened phagosome, appear to be released from disintegrating infected
macrophages. Movement of such macrophages may result into systemic spread of the pathogens to lead to typhoid-like salmonella complications.
cellular mechanism for invasion and infection of chicken
ileum by a human pathogen, Salmonella 3,10:r:- is
proposed (Fig. 1). An amorphous surface ultrastructure
was observed for most of the organisms located near the
center of the ileal lumen when the organisms were located
more that 2000 nm away from the epithelial cell microvilli
(Fig. 1A). Such a surface morphology has therefore, been
considered to represent a non-interactive (with host cells)
state of the organisms. Organisms could be observed only
in the tissue taken from ileal loops injected with live
organisms and not in control loops given placebo. Marked
surface changes were seen in organisms representing a
majority (over 70%) of organisms located closely (within
300 nm) to epithelial cell microvilli in the ilial loops
injected live organisms (Fig. 1B). Numerous bacterial
outer membrane-bound periplasmic extensions/protusions
were observed on these organisms, considered to represent
a host cell-interactive state of the organisms. As these
structures were present all around the surface of the
organisms, they might show up as thin and long
appendages or as fat ‘blisters’ which appear to signify a
structural expression of acquisition of a virulent state6,7
during close cross-talk with host eukaryotic cells. These
structures have been designated ‘periplasmic organellae’.
Fig. 1C showed 50-90 Ao diameter size bacterial
outer membrane vesicles (OMVs) apparently pinched
off from large periplasmic organelles. Many of these
OMVs appear to form intimate membrane-to-membrane
contact between vesicular surface and host epithelial
cell microvillous plasma membrane. As the OMV-
membrane should consist mainly of lipopolysaccharide
(LPS) and host cell plasma membrane mainly of
phospholipids, any fusion between them is likely to be
assisted by some specialized proteins/receptors.
Salmonella invasion proteins SipB and SipC have been
shown to be present on OMVs (Hayward RD, University
of Cambridge, England, personal communicaton). This
may help in the generation of pore between OMV and
host cell membranes, thereby, help translocating the
OMV contents into the host cell cytosol &/or direct
endocytosis of OMV's as such18.
A representative Salmonella organism (S) is shown
to be located face-to-face with a ruffled host epithelial
cell membrane (R) (Fig. 1D). A focal disruption of
microvilli and reorganization of host cell cytoskeleton
were markedly clear. The arrows suggest a possible path
for intra-cytoplasmic entry of the organisms explaining
the likely macropinocytosis process. Our results also
showed that the surface appendages, referred to as
periplasmic organelles were not observed on the
salmonellae located close to the ruffled host cells. The
observed loss of surface appendages at time of
intracytoplasmic entry of salmonellae could be
explained as prior liberation of OMVs from inflating
periplasmic organellae here as shown in Fig. 1C. It
appears that the invading Salmonella 3,10:r:- pathogens
use the ileal lining epithelial cells as a safe corridor to
get entry into the inner regions of the ileum. Fig. 2 shows
these organisms passing through lymphatic vessels of
chicken epithelium. Here, these organisms were
observed as being abundantly phagocytosed by
A representative salmonella organism is shown to
be located closely face-to-face with a macrophage in
the lymphatic vessel of chicken ileum (Fig. 1E).
Interestingly, once again, the Salmonella 3,10:r:-
Fig. 2. Salmonella 3,10:r:- pathogens (arrow-heads) interacting with
macrophages while passing through the lumen of lymphatic vessels
(thick bold arrows) in experimentally infected chicken ileum
(P, phagosome; pc, phagocytic cup).
YASHROY: ULTRASTRUCTURAL MECHANISM OF INFECTION OF SALMONELLA (3,10:r:-) 561
organisms located in similar orientations showed
numerous periplasmic organelle on their surface,
coincident with several OMVs liberated therefrom,
coming in close contact with the plasma membrane of
macrophages. Many OMVs were also seen located
inside the peripheral regions of the cytoplasm of these
macrophages on the portions closely interacting with
OMVs. Therefore, a plausible explanation is that these
OMVs are engulfed/endocytosed by the macrophages,
thereby translocating bacterial vesicular contents into
the cytosol of macrophages. Besides the contents of the
OMVs, their membrane lipopolysaccharide and outer
membrane proteins are also taken inside the
macrophages. In case of Salmonella 3,10:r:- infection
of chicken ileum as reported here, the observations
suggest that OMV uptake/engulfment triggered the
macrophages into invigorated activity of phagocytosis
of numerous organisms. This became obvious from
numerous phagocytic cup formations around the closely
approaching salmonellae (Fig.1F) and their entrapment
in pairs in tight phagosomes (Fig.1G). Numerous tight
phagosomes containing these organisms predominated
the scene of such macrophages, which also appeared
not to fuse with close-by located lysosomes in the
macrophage cytoplasm (Fig.1H). Many infected
macrophages also appeared to be undergoing apoptotic
disintegration, concomitantly showing numerous loose
phagosomes containing four organisms each (Fig. 1I).
This stage of macrophages showed replicated
salmonellae, being let out for re-infection, due to
disintegration of the infected macrophages.
With over 2323 known serotypes, Salmonella
infections which occur commonly in man, animals and
birds the worldover, perpetually take a heavy toll in the
form of morbidity-linked losses besides thrusting heavy
expenditures on management and treatment of the
disease5. The serotype, Salmonella 3,10:r:- is responsible
for causing severe food-poisoning infections in human
beings3 and animals4. In the chicken model of
experimental infection studied in vivo, interesting
ultrastructural changes were observed in both the
interacting pathogens and the eukaryotic host cells. Host-
pathogen interactions encountered in Gram-negative
organisms at close interface with eukaryotic cells have
been recently linked with the bacterial type III secretion
system (T3SS) and OMV-associated export of bacterial
toxins30-32. The Gram-negative pathogens employ T3SS
for translocation of a cocktail of bacterial effector
proteins and virulence determinants from the organisms
to directly into host cell cytosol33-35. Spatio-temporal
regulation of these effectors accomplishes fine-tuned
modulation of host cell machinery36. Such hijacking of
eukaryotic functions is not only accomplished by
intracellular bacterial pathogens37, but a similar
subversion of host cell actin dynamics is also achieved
by extracelluarly infecting enteropathogens like
enteropathogenic and enterohaemorrhagic Escherichia
Our findings showed that periplasm of Salmonella
3,10:r:- organisms located in close proximity (within
300 nm) of host ileal epithelial cells as well as tissue
macrophages played a significant role at the host-
pathogen interface. It was earlier proposed that signals
from host cells like antimicrobial peptides may induce
synthesis of bacterial toxins9. It has recently been
shown that antimicrobial peptides do trigger pathogen
virulence as the two-component regulatory system
PhoP-PhoQ of Salmonella is activated by binding to
antimicrobial peptides, thereby promoting gene
transcription necessary of Salmonella survival within
the host. It was therefore opined that antimicrobial
peptides might act as a double-edged sword, promoting
antibacterial immunity while simultaneously triggering
pathogen virulence39. The tightly packed LPS
molecules in the bacterial outer membrane are the first
barrier to antimicrobial peptides. Further, only the
killer form of antimicrobial peptide penetrates the
lipopolysaccharide layer and induces LPS
micellization40. Virulence proteins and allied
determinants may be quickly synthesized and
transported across the bacterial cell membrane into the
periplasmic space via the general secretory pathway
under influence of suitable inducers like change in
temperature, pH and chemical composition in the
microenvironment around the eukaryotic host cells30.
Indirect evidence on E. coli suggests that H+ -ATPase
machinery uses proton motive force to generate ATP
which, in turn, is essential for protein translocation
via OMVs41. Numerous pockets of protruding
periplasm (designated as periplasmic organelles for
their being physiologically significant structures11)
were observed all around the Salmonella 3,10:r:-
organisms approaching closely and interacting with
host epithelial cells or macrophages. A model for
molecular structure of the periplasmic organelle has
already been proposed and these organelles have been
explained to represent a secretion-active virulent state
of the organisms, ready to secrete the bacterial toxins
and secretory products as OMVs30.
562 INDIAN J MED RES, DECEMBER 2007
OMVs liberated from the secretion-active
Salmonella 3,10:r:-, have been proposed to be released
by pinching off inflated periplasmic organelles filled
with bacterial toxins8 and exoproteins secreted by the
general secretory pathway (GSP)27. It was proposed that
fusion of OMVs with the host epithelial membrane may
result in the translocation of bacterial enterotoxins to
directly inside the host epithelial cells8. A similar process
for the release of heat-labile enterotoxin via general
secretory pathway as OMVs has been observed for E.
coli and further a mechanism of OMV-mediated
receptor-dependent delivery of bacterial toxin into host
cells was implicated42. Similarly, Shiga toxin was also
found to be released as OMVs from periplasmic space
of Shigella dysenteriae and that the secretion was
induced by an antimicrobial compound, mitomycin C43.
On the same pattern, OMVs containing vacuolating
cytotoxin (VacA), which was immuno-localized in the
periplasm and outer membrane of intact Helicobacter
pylori bacteria, appeared to originate from blebs arising
on the bacterial outer membrane. Both soluble secreted
VacA and VacA-containing OMVs were internalized by
MKN28 cells and were detectable in the gastric mucosa
of H. pylori-infected humans12. Likewise, active
cytotoxic necrotizing factor 1 (CNF1) secreted from
uropathogenic E. coli has also been found to be
associated with OMVs thereby suggesting that CNF1
is transported to the environment of the infected tissue
via OMVs44. The salmonella invasion protein SipB of
T3SS was shown to direct heterotypic membrane fusion,
allowing delivery of contents from E. coli-derived
liposomes into cytosol of living mammalian cells45. Such
a mode of translocation of bacterial secretions as OMVs
into another host/recipient cell has, therefore, been
described as “vesicular exocytosis from prokaryotes”
as earlier, the exocytosis process was traditionally
associated only with eukaryotes9,10. OMVs have also
been linked to type I21 and type III30,46 secretory systems
of Gram-nagative organisms.
In the ileal epithelial cells showing ruffled
membrane and cytoskeletal reorganization of the
cytoplasm, it was notable that organisms at the surface
of the ruffled site did not reveal any periplasmic
organellae on their exterior. This may be explained as
these organellae have already been pinched off as
OMVs, which in turn have seemingly accomplished their
task of translocating the bacterial virulence determinants
into the interacting ileal epithelial cells. It has been
proposed that SipB (located in/on OMVs) secreted by
the invading Salmonella triggers bacterial entry into
eukaryotic cells and this is blocked by a SipB-derived
polypeptide45,47. Manipulation of the host cell actin
cytoskeleton by Salmonella enterica for entry into
epithelial cells has been extensively studied48. Our study
suggests that Salmonella 3,10:r:- organisms created
corridors via the ruffled locations in order to get access
to inner sites in the ileum, as the organisms were seen
in the lymphatic vessels where they were observed to
closely interact with macrophages (Figs. 1 & 2).
Salmonella 3,10:r:- organisms closely interacting
with macrophages, developed on their surface, large
blebs (periplasmic organelles), which appeared to
liberate numerous OMVs, which in turn, were
apparently taken up by the macrophages. This process
has been assigned the task of translocation of
biochemical signals including LPS from the invading
pathogens to directly into eukaryotic host cells,
macrophages, at this stage. This led to augmented
phagocytic cup formation and consequent engulfment
of organisms into the macrophage cytoplasm (Fig. 1).
Modulation of leukocyte response mediated by other
Gram-negative pathogens has also been reported.
Recently, OMVs of Neisseria menigitidis have been
shown to activate monocytes in an LBP-, CD14-, and
TLR4/MD-2-dependent fashion with pro-inflammatory
effect49. Also, OMV-mediated modulation of leukocyte
adhesion molecule expression and increased reactive
oxygen species (ROS) production is likely to increase
entrapment of leukocytes in the microcirculation and
contribute to untoward inflammatory reactions as
noticed in systemic meningococcal disease50. Another
report 51 shows that OMVs containing cytotoxic
necrotizing factor 1 (CNF-1), but not purified CNF-1,
act in a dose dependent manner, on polymorphonuclear
leukocytes to attenuate their antimicrobial activity. This
study reveals that OMVs provide a means for delivery
of CNF-1 from uropathogenic E. coli to these host cells,
and thus negatively affect the efficacy of acute
inflammatory response to these pathogens. Further, CD+
T cells and toll-like receptors recognize Salmonella
antigens expressed in bacterial surface organelles
including OMVs. Thus, genetically co-ordinated surface
modifications may provide a growth advantage for
Salmonella in host tissues by limiting both innate and
adaptive immune recognition52. OMVs generated by H.
pylori bear serologically recognizable Lewis antigens,
which may contribute to the chronic immune stimulation
of the host. The ability of these OMVs to absorb anti-
Lewis auto-antibodies suggest that they may, in part,
play some role in putative autoimmune aspects of H.
YASHROY: ULTRASTRUCTURAL MECHANISM OF INFECTION OF SALMONELLA (3,10:r:-) 563
Fig.1-G shows that two organisms entering at one
location of the macrophages get enclosed in one tight
phagosome. Numerous tight phagosomes are observed
to occupy the bulk part of the cytoplasm of macrophages,
where they appear to be resistant to fusion with
lysosomes, located nearby (Fig. 1H). Individual
organisms are not really discernible in the tight
phagosomes due highly electron-dense contents.
However, in the spacious phagosomes, four organisms
are clearly visible in each phagosome (Fig. 1I). Thus, it
suggests that two organisms originally entrapped in one
tight phagosome replicate into four organisms coincident
with loosening of the tight phagosomes into spacious
ones. Parallel apoptotic disintegration of infected
macrophages appears to release the replicated pathogens
in body of the host, promoting infection of more host
cells. Circulation of infected macrophages may be
envisaged to lead to systemic infection. Recently, it has
been shown54 that OMVs of H. pylori induce apoptosis
in gastric epithelial cells. Further, this apoptosis is not
mediated by mitochondrial pathway as is demonstrated
by the lack of cytochrome c release with the activation
of caspase 8 and 3.
Overall, this study indicates an important role played
by OMVs released by Salmonella 3,10:r:- pathogens at
both stages of invasion that is of epithelial as well as
macrophage cells. An earlier study revealed that protein
translocation into OMVs required ATP and the proton-
motive force might also contribute but appear not to be
essential in E. coli41. It is plausible to opine that proton-
motive force may generate ATP with the help of H+-
ATPase, and, in turn, ATP may be utilized for transporting
proteins across the cell membrane into the periplasmic
organelles to be eventually released as OMVs. The
specialized T3S assembly of pathogenic and symbiotic
Gram-negative bacteria comprises a multi-protein
transmembrane complex and an ATPase homologous to
F1-ATPase beta-subunit, which forms a double hexameric
ring assembly in the inner membrane as studied for HrcN
of P. syringae55. Earlier, a mechanism was proposed in
which OMVs are formed when the outer membrane
expands faster than the underlying peptidoglycan layer56.
Recently, it was shown that OMV production by E. coli
is independent of membrane instability (detergent-
sensitivity, leakiness) but, gene disruption, however, can
cause under or over-production (5 to 200-fold increase)
of OMVs, relative to wild type levels57. Nonetheless, gene
activation leading to synthesis of virulence proteins under
inducement from antimicrobial peptides (present in the
microenvironment or those secreted by eukaryotic host
cells) have been postulated to cause augmented secretion
of OMVs containing secretory exoproteins9. Experimental
proof of this viewpoint has been recently obtained42
confirming that antimicrobial peptides actually trigger
pathogen virulence by binding to Phop-PhoQ regulatory
system of Salmonella. Of late, role T3S needle complex-
like assembly has been implicated in the release of
OMVs in the analogy of blowing off soap bubbles with
the formation of tube-like assembly T3S needle/rivet
complexes30. Although, confirmatory proof for the
existence of a generalized OMV model for T3SS30 is
still awaited, yet it does obviate many unanswered
questions posed to popular injectisome model58 on the
modus operandi of translocation of semi- or folded
proteins through a rather narrow and long conduit of
the T3SS assembly. Interestingly, OMVs have been
recently shown to release type I secreted alpha-
haemolysin21 from E. coli. Also, some T3SS proteins
like SipB & SipC of Salmonella have been shown to be
associated with OMVs. This important ultrastructural
study is hence envisaged to stimulate further work using
monoclonal antibodies and allied techniques to
immunolocalize type I, III and other secretory proteins
in the OMVs of Gram-negative pathogens, under in vitro
and in vivo conditions. To would establish their role in
host-pathogen interactions, inter-species competition of
pathogens, and intercellular communication within
bacterial colonies & inter-kingdom singaling.
The author thanks Prof. B. R. Gupta, Head, Bacteriology and
Mycology Division (retired), Indian Veterinary Research Institute
(IVRI), Bareilly, for providing the organisms and laboratory facilities
for animal experimentation and Director, IVRI for encouragement
and overall support. R.S.I.C., Chandigarh is acknowledged for
making available the transmission electron microscope facility.
1. Guiney DG. Role of host cell death in Salmonella infections.
Curr Top Microbiol Immunol 2005; 289 : 131-50.
Altier C. Genetic and environmental control of Salmonella
invasion. J Microbiol 2005; 43 : 85-92.
Gupta BR, Singh HP, Verma JC, Uppal PK. Isolation of
Salmonella (3,10:r:-) from cases of human food poisoning.
Indian J Med Res 1980; 71 : 175-7.
Kumar AA, Mallick BB, Verma JC, Gupta BR. Isoation of
Salmonella (3,10:r:-) from animals and its public health
importance. Indian J Med Res 1981; 73 : 693-6.
YashRoy RC. Poultry production under Salmonella stress:
Infection mechanisms. In: Moudgal RP, Mohan J, Singh RV,
editors. Poultry production under stress. Izatnagar, India:
Central Avian Research Institute; 2000. p. 292-300.
YashRoy RC. Salmonella 3,10:r:- surface interactions with
intestinal epithelial microvilli. Indian J Anim Sci 1992; 62 :
564 INDIAN J MED RES, DECEMBER 2007
7. Ginocchio CC, Olmsted SB, Wells CL, Galan JE. Contact with
epithelial cells induces the formation of surface appendages on
Salmonella typhimurium. Cell 1994; 76 : 717-24.
YashRoy RC. Electron microscope studies of surface pili and
vesicles of Salmonella 3,10:r:- organisms. Indian J Anim Sci
1993; 63 : 99-102.
YashRoy RC. Discovery of vesicular exocytosis in prokaryotes
and its role in Salmonella invasion. Curr Sci 1998; 75 : 1062-
10. YashRoy RC. Exocytosis from gram-negative bacteria for
Salmonella invasion of chicken ileal epithelium. Indian J Poult
Sci 1998; 33 : 119-23.
11. YashRoy RC. Hijacking of macrophages by Salmonella 3,10:r:-
through “type III” secretion like exocytotic signaling: a
mechanism for infection in chicken ileum. Indian J Poult Sci
2000; 35 : 276-81.
12. Fiocca R, Necchi V, Sommi P, Ricci V, Telford J, Cover TL,
et al. Release of Helicobacter pylori vacuolating cytotoxin by
both a specific secretion pathway and budding of outer
membrane vesicles. Uptake of released toxin and vesicles by
gastric epithelium. J Pathol 1999; 188 : 220-6.
13. Heczko U, Smith VC, Meloche RM, Buchan AMJ, Finlay BB.
Characteristics of Helicobactor pylori attachment to human
primary antral epithelial cells. Microbes Infect. 2000; 2 : 1669-
14. Ismail S, Hampton MB, Keenan JI. Helicobacter pylori outer
membrane vesicles modulate proliferation and interleukin-8
production by gastric epithelial cells. Infect Immun 2003; 71 :
15. Khandelwal P, Banerjee-Bhatnagar N. Insecticidal activity
associated with outer membrane vesicles of Xenorhabdus
nematophilus. Appl Environ Microbiol 2003; 69 : 2032-7.
16. Rolhion N, Barnich N, Claret L, Darfeuille-Michaud A. Strong
decrease in invasive ability and outer membrane vesicle release
in Crohn’s disease-associated adherent-invasive Escherichia
coli strain LF82 with the yfgL gene deleted. J Bacteriol 2005;
187 : 2286-96.
17. Kesty NC, Mason KM, Reedy M, Miller SE, Kuehn MJ.
Enterotoxigenic Escherichia coli vesicles target toxin delivery
into mammalian cells. EMBO J 2004; 23 : 4538-49.
18. Kuehn MJ, Kesty NC. Bacterial outer membrane vesicles and
the host-pathogen interaction. Genes Dev 2005; 19 : 2645-55.
19. Horstman AL, Kuehn MJ. Enterotoxigenic Escherichia coli
secretes active heat-labile enterotoxin via outer membrane
vesicles. J Biol Chem 2000; 275 : 12489-96.
20. Kato S, Kowashi Y, Demuth DR. Outer membrane-like vesicles
secreted by Actinobacillus actinomycetemcomitans are enriched
in leukotoxin. Microb Pathol 2002; 32 : 1-13.
21. Balsalobre C, Silvan JM, Berglund S, Mizunoe Y, Uhlin BE,
Wai SN. Release of type I secreted alpha-haemolysin via outer
membrane vesicles from Escherichia coli. Mol Microbiol. 2006;
59 : 99-112.
22. O’Hagan, Patrick S. McKenna JPS, Dermott E. A comparison
of haemagglutinating and enzymatic activities of Bacteroides
fragilis whole cells and outer membrane vesicles. Microb
Pathog 1996; 20 : 191-202.
23. Lapinet JA, Scapini P, Calzetti F, Perez O, Cassatella MA. Gene
expression and production of tumor necrosis factor alpha,
interleukin-1 beta (IL-1 beta), IL-8, macrophage inflammatory
protein 1 alpha (MIP-1 alpha), MIP-1 beta, and gamma
interferon-inducible protein 10 by human neutrophils stimulated
with group B meningococaal outer membrane vesicles. Infect
Immun 2000; 68 : 6917-23.
24. Sharma A, Novak EK, Sojar HT, Swank RT. Kuramitzu HK,
Genco RJ. Porphyromonas gingivalis platlet aggregation
activity: outer membrane vesicles are potent activators of murine
platelets. Oral Microbiol Immunol 2000; 15 : 393-6.
25. McBroom AJ, Kuehn MJ. Release of outer membrane vesicles
by Gram-negative bacteria is a novel envelope stress response.
Mol Microbiol 2007; 63 : 545-58.
26. Giannella RA, Formal SB, Dommins DJ, Collins H.
Pathogenesis of salmonellosis, studies of fluid secretion,
mucosal invasion and morphological reaction in rabbit ileum.
J Clin Invest 1973; 52 : 441-53.
27. Daniels JJD, Autenrieth IB, Ludwig A, Goebel W. The gene
slyA of Salmonella typhimurium is required for destruction of
M cells and intracellular survival but not for invasion or
colonization of the murine small intestine. Infect Immun 1996;
64 : 5075-84.
28. Hunter PE. Practical electron microscopy. A beginner’s guide.
Praeger Special Studies. New York: Praeger Scientific; 1984.
29. YashRoy RC. Sample preparation techniques for transmission
and scanning electron microscopy. Indian Veterinary Research
Institute, Bareilly, UP (India): Suneet Offset Printers for
Biophysics Section; 1993. p. 1-32.
30. YashRoy RC. Eucaryotic cell intoxication by gram-negative
pathogens: a novel bacterial outermembrane-bound
nanovesicular exocytosis model for type-III secretion system.
Toxicol Int 2003; 10 : 1-9.
31. Hayward RD, Cain RJ, McGhie EJ, Phillips S, Garner MJ,
Koronakis V. Cholesterol binding by the bacterial type III
translocon is essential for virulence effector delivery into
mammalian cells. Mol Microbiol 2005; 56 : 590-603.
32. Lafont F, Goot FG. Oiling the key hole. Mol Microbiol 2005;
56 : 575-7.
33. Wai SN, Lindmark B, Soderblom T, Takade A, Westermark M,
Oscarsson J, et al. Vesicle-mediated export and assembly of
pore-forming oligomers of enterobacterial ClyA cytotoxin. Cell
2003; 115 : 25-35.
34. Foster JW, Spector MP. How Salmonella survive against the
odds. Ann Rev Microbiol 1995; 49 : 145-74.
35. Lee VT, Schneewind O. Protein secretion and the pathogenesis
of bacterial infections. Genes Dev 2001; 15 : 1725-52.
36. Schlumberger MC, Hardt WD. Salmonella type III secretion
effectors: pulling the host cell’s strings. Curr Opin Microbiol
2006; 9 : 46-54.
37. Alonso A, Garcia-del Portillo F. Hijacking of eukaryotic
functions by intracellular bacterial pathogens. Int Microbiol
2004; 7 : 181-91.
38. Caron E, Crepin VF, Simpson N, Knutton S, Garmendia J,
Frankel G. Subversion of actin dynamics by EPEC and EHEC.
Curr Opin Microbiol 2006; 9 : 40-5.
39. Bishop JL, Finlay BB. Friend or foe? Antimicrobial peptides
trigger pathogen virulence. Trends Mol Med 2006; 12 : 3-6.
40. Papo N, Shai Y. A molecular mechanism for lipopolysaccharide
protection of Gram-negative bacteria from antimicrobial
peptides. J Biol Chem 2005; 280 : 10378-87.
YASH ROY: ULTRASTRUCTURAL MECHANISM OF INFECTION OF SALMONELLA (3,10:r:-)565
Reprint requests: Prof. R.C. YashRoy, Principal Scientist, O/C Biophysics & Electron Microscopy Section, Indian Veterinary Download full-text
Research Institute, Izatnagar, Bareilly 243122, India
41. Chen L, Tai PC. ATP is essential for protein translocation into
Escherichia coli membrane vesicles. Proc Natl Acad Sci USA
1985; 82 : 4384-8.
42. Horstman AL, Kuehn MJ. Bacterial surface association of heat-
labile enterotoxin through lipopolysaccharide after secretion
via the general secretory pathway. J Biol Chem 2002; 277 :
43. Dutta S, Iida K, Takade A, Meno Y, Nair GB,Yoshida S. Release
of Shiga toxin by membrane vesicles in Shigella dysenteriae
serotype 1 strains and in vitro effects of antimicrobials on toxin
production and release. Microbiol Immunol 2004; 48 : 965-9.
44. Kouokam JC, Wai SN, Fallman M, Dobrindt U, Hacker J, Uhlin
BE. Active cytotoxic necrotizing factor 1 associated with outer
membrane vesicles from uropathogenic Escherichia coli. Infect
Immun 2006; 74 : 2022-30.
45. Hayward RD, McGhie EJ, Koronakis V. Membrane fusion
activity of purified SipB, a Salmonella surface protein essential
for mammalian cell invasion. Mol Microbiol 2000; 37 : 727-39.
46. Deakin WJ, Marie C, Saad MM, Krishnan HB, Broughton J.
NopA is associated with cell surface appendages produced by
the type III secretion system of Rhizobium sp. Strain NGR234.
Mol Plant Micobe Interact 2005; 18 : 499-507.
47. Hayward RD, Hume PJ, McGhie EJ, Koronakis V. A Salmonella
SipB-derived polypeptide blocks the ‘trigger’ mechanism of
bacterial entry into eukaryotic cells. Mol Microbiol 2002; 45 :
48. Patel JC, Galan JE. Manipulation of the host actin cytoskeleton
by Salmonella -all in the name of entry. Curr Opin Microbiol
2005; 8 : 12-5.
49. Post DM, Zhamg D. Eastvoild JS, Teghanemt A, Gibson BW,
Weiss JP. Biochemical and functional characterization of
membrane blebs purified from Neisseria meniningitidis
serogroup B. J Biol Chem 2005; 280 : 38383-94.
50. Mirlashari MR, Hoiby EA, Holst J, Lyberg T. Outer membrane
vesicles from Neisseria meningitides. APMIS 2002; 110 : 193-
51. Davis JM, Carvalho HM, Rasmussen SB, O’brien AD.
Cytotoxic necrotizing factor type 1 delivered by outer membrane
vesicles of uropathognic Escherichia coli attenuates
polymorphonuclear leukocyte antimicrobial activity and
chemotaxis. Infect Immun 2006; 74 : 4401-8.
52. Bergman MA, Cummings LA, Barrett SLR, Smith KD, Lara
JC, Aderem A, et al. CD+ T cells and toll-like receptors recognize
Salmonella antigens expressed in bacterial surface organelles.
Infect Immun 2005; 73 : 1350-6.
53. Hynes SO, Keenan JL, Ferris JA, Annuk H, Moran AP. Lewis
epitopes on outer membrane vesicles of relevance to
Helicobacter pylori pathogenesis. Helicobacter 2005; 10 : 146-
54. Ayala G, Torres L, Espinosa M, Fierres-Zarate G, Maldonado
V, Melendez-Zajgla J. External membrane vesicles from
Helicobacter pylori induce apoptosis in gastric epithelial cells.
FEMS Microbiol Lett 2006; 260 : 178-85.
55. Muller SA, Pozidis C, Stone R, Meesters C, Chami M, Engel
A, et al. Double hexameric ring assembly of the type III protein
translocase ATPase HrcN. Mol Microbiol 2006; 61 : 119-25.
56. Wensink J, Witholt B. Outer-membrane vesicles released by
normally growing Escherichia coli contain very little
lipoprotein. Eur J Biochem 1981; 116 : 331-5.
57. McBroom AJ, Johnson AP, Vemulpalli S, Kuehn MJ. Outer
membrane vesicle production by Escherichia coli is
independent of membrane instability. J Bacteriol 2006; 188 :
58. Yip CK, Strynadka NCJ. New structural insights into bacterial
type III secretion system. Trends Biochem Sci 2006; 31 : 223-30.
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