Mechanism of infection of a human isolate Salmonella _3_10r-_ in

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					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
                 YASHROY: ULTRASTRUCTURAL MECHANISM OF INFECTION OF SALMONELLA (3,10:r:-)                           559

     Salmonellae are responsible for causing food-                               Material & Methods
poisoning problems and typhoid-like infections in
                                                                    The pathogen, Salmonella 3,10:r:- (a monophasic
humans and animals, resulting in huge monetary losses
                                                              variety of otherwise diphasic serotypes such as S.
due to morbidity-linked reduction in productivity and
                                                              weltvreden and S. simi), originally isolated from human
increased costs of disease treatment/management1,2.
                                                              food poisoning cases, was obtained from National
Therefore, it is important to understand their
                                                              Salmonella Centre at the Indian Veterinary Research
mechanisms of infection at cellular and molecular
levels in order to innovate necessary interventions.          Institute, Bareilly, Uttar Pardesh. This particular strain
Salmonella (3,10:r:-), originally isolated from severe        has been typed distinctly from S. weltvreden3,4 and is
human food poisoning cases3, is capable of infecting          maintained at this Centre. Cultures of the organisms
animals4 and poultry5. Interest in structural aspects of      originally isolated from cases of human food poisoning3
virulence dates back to the year 1992 from this               with 109 cfu were injected into chicken ileum in 24 h
laboratory when it was first demonstrated by                  pre-fasted 3 month old five broiler birds (obtained from
transmission electron microscopy (TEM) that                   Central Avian Research Institute, Bareilly and
Salmonella (3,10:r:-) organisms located in close              specifically maintained under suitable laboratory
proximity of host cells and interacting in vivo with          conditions), using standard ligated ileal loop
microvilli of epithelium in chicken ileum, developed          methodology26,27. The procedures used employ thorough
numerous bacterial outer membrane bound surface               washing-out of the ileum of its contents (including
appendages or periplasmic protrusions filled with             naturally inhabiting organisms). Another saving factor
bacterial secretions 6. Similar surface appendages            in this procedure was that the experimental (injected
(named as invasosomes) were also later observed on            with the dose of organisms) and control ileal loops
Salmonella Typhimurium while closely interacting in           (injected with sterile medium without organisms) were
vitro with cultured MDCK epithelial cells, as studied         located in the same animal(s) so as to allow for excellent
by scanning electron microscopy7. Further TEM studies         control versus experimental sampling. The fluid was
suggested that larger periplasmic protrusions could           found to be exsorbed only in the experimental loops
pinch off as 50- 90 nm diameter sized bacterial outer         after 18 h of injection, and their contents were tested
membrane vesicles (OMVs), which could also be seen            for presence of injected organism to make sure that
in fusion-like membrane-to-membrane contact with              reaction was indeed caused by Salmonella (3,10:r:-)
microvilli of host epithelial cells8,9. Translocation of      pathogens. The fluid exsorbed/accumulated in the ileal
bacterial ‘toxic’ secretions from invading Gram-negative      loops was drained and ileal tissue pieces (size
pathogens into host/target cells was lauded as discovery      approximately 1-2 mm3 ) of the experimental and control
of a novel process viz., vesicular exocytosis in              loops were fixed in 2.5 per cent glutaraldehyde in
prokaryotes 9,10 and was considered to add a new              phosphate buffer (pH 7) at 5o C for 6 h and subsequently
structural dimension of type III secretion system11. Later,   post-fixed/block-stained in aqueous 1 per cent osmium
these OMVs have also been associated with damage              tetroxide for 6 h at room temperature following standard
caused to eukaryotic host cells8-15 and aiding in invasion    methodology28,29. The fixed tissue blocks were made in
of the pathogens16. It is now becoming increasingly clear     arlaldite and ultra-thin sections (approximately 500 Ao
that Gram-negative pathogens employ OMVs for                  in thickness) were cut using glass knife with an ultra-
targeting toxin delivery into mammalian cells17,18 and        microtome (LKB Ultrotome III, Sweden). Ultra-thin
that they contain a variety of bacterial toxins19-21 which    sections obtained on 3 mm diameter copper grids, were
may have some role in virulence22 and pathogenicity21         stained with uranyl acetate and lead citrate stains for
and also in cytokine production by neutrophils23, besides     contrast and examined under JEOL JEM 1200EX
acting as potent inducers of platelet aggregation24.          electron microscope (Japan) working in transmission
Recently, OMV release has also been shown to correlate        mode operated at 100 kilovolts. The electron
directly with level of protein accumulation in the cell       micrographs were interpreted in detail and arranged in
envelope which has been opined to represent a                 an order proposed to be a workable sequence of the
physiological stress response25. The present study            progress of invasion and infection in vivo.
reports a comprehensive ultrastructural mechanism for
invasion and infection of chicken ileum by the human
isolate Salmonella 3,10:r:- organisms, with crucial role           From intensive TEM studies and detailed
assigned to OMV's.                                            interpretation of electron micrographs, a comprehensive
560                                                             INDIAN J MED RES, DECEMBER 2007

                                                              500 nm

                                                                     Periplasmic organelle


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.
                 YASHROY: ULTRASTRUCTURAL MECHANISM OF INFECTION OF SALMONELLA (3,10:r:-)                                    561

cellular mechanism for invasion and infection of chicken      salmonellae located close to the ruffled host cells. The
ileum by a human pathogen, Salmonella 3,10:r:- is             observed loss of surface appendages at time of
proposed (Fig. 1). An amorphous surface ultrastructure        intracytoplasmic entry of salmonellae could be
was observed for most of the organisms located near the       explained as prior liberation of OMVs from inflating
center of the ileal lumen when the organisms were located     periplasmic organellae here as shown in Fig. 1C. It
more that 2000 nm away from the epithelial cell microvilli    appears that the invading Salmonella 3,10:r:- pathogens
(Fig. 1A). Such a surface morphology has therefore, been      use the ileal lining epithelial cells as a safe corridor to
considered to represent a non-interactive (with host cells)   get entry into the inner regions of the ileum. Fig. 2 shows
state of the organisms. Organisms could be observed only      these organisms passing through lymphatic vessels of
in the tissue taken from ileal loops injected with live       chicken epithelium. Here, these organisms were
organisms and not in control loops given placebo. Marked      observed as being abundantly phagocytosed by
surface changes were seen in organisms representing a         macrophages.
majority (over 70%) of organisms located closely (within          A representative salmonella organism is shown to
300 nm) to epithelial cell microvilli in the ilial loops      be located closely face-to-face with a macrophage in
injected live organisms (Fig. 1B). Numerous bacterial         the lymphatic vessel of chicken ileum (Fig. 1E).
outer membrane-bound periplasmic extensions/protusions        Interestingly, once again, the Salmonella 3,10:r:-
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       Fig. 2. Salmonella 3,10:r:- pathogens (arrow-heads) interacting with
the likely macropinocytosis process. Our results also         macrophages while passing through the lumen of lymphatic vessels
showed that the surface appendages, referred to as            (thick bold arrows) in experimentally infected chicken ileum
periplasmic organelles were not observed on the               (P, phagosome; pc, phagocytic cup).
562                                      INDIAN J MED RES, DECEMBER 2007

organisms located in similar orientations showed             to directly into host cell cytosol33-35. Spatio-temporal
numerous periplasmic organelle on their surface,             regulation of these effectors accomplishes fine-tuned
coincident with several OMVs liberated therefrom,            modulation of host cell machinery36. Such hijacking of
coming in close contact with the plasma membrane of          eukaryotic functions is not only accomplished by
macrophages. Many OMVs were also seen located                intracellular bacterial pathogens 37 , but a similar
inside the peripheral regions of the cytoplasm of these      subversion of host cell actin dynamics is also achieved
macrophages on the portions closely interacting with         by extracelluarly infecting enteropathogens like
OMVs. Therefore, a plausible explanation is that these       enteropathogenic and enterohaemorrhagic Escherichia
OMVs are engulfed/endocytosed by the macrophages,            coli38.
thereby translocating bacterial vesicular contents into
the cytosol of macrophages. Besides the contents of the          Our findings showed that periplasm of Salmonella
OMVs, their membrane lipopolysaccharide and outer            3,10:r:- organisms located in close proximity (within
membrane proteins are also taken inside the                  300 nm) of host ileal epithelial cells as well as tissue
macrophages. In case of Salmonella 3,10:r:- infection        macrophages played a significant role at the host-
of chicken ileum as reported here, the observations          pathogen interface. It was earlier proposed that signals
suggest that OMV uptake/engulfment triggered the             from host cells like antimicrobial peptides may induce
macrophages into invigorated activity of phagocytosis        synthesis of bacterial toxins9. It has recently been
of numerous organisms. This became obvious from              shown that antimicrobial peptides do trigger pathogen
numerous phagocytic cup formations around the closely        virulence as the two-component regulatory system
approaching salmonellae (Fig.1F) and their entrapment        PhoP-PhoQ of Salmonella is activated by binding to
in pairs in tight phagosomes (Fig.1G). Numerous tight        antimicrobial peptides, thereby promoting gene
phagosomes containing these organisms predominated           transcription necessary of Salmonella survival within
the scene of such macrophages, which also appeared           the host. It was therefore opined that antimicrobial
not to fuse with close-by located lysosomes in the           peptides might act as a double-edged sword, promoting
macrophage cytoplasm (Fig.1H). Many infected                 antibacterial immunity while simultaneously triggering
macrophages also appeared to be undergoing apoptotic         pathogen virulence 39 . The tightly packed LPS
disintegration, concomitantly showing numerous loose         molecules in the bacterial outer membrane are the first
phagosomes containing four organisms each (Fig. 1I).         barrier to antimicrobial peptides. Further, only the
This stage of macrophages showed replicated                  killer form of antimicrobial peptide penetrates the
salmonellae, being let out for re-infection, due to          lipopolysaccharide layer and induces LPS
disintegration of the infected macrophages.                  micellization 40 . Virulence proteins and allied
                                                             determinants may be quickly synthesized and
                      Discussion                             transported across the bacterial cell membrane into the
     With over 2323 known serotypes, Salmonella              periplasmic space via the general secretory pathway
infections which occur commonly in man, animals and          under influence of suitable inducers like change in
birds the worldover, perpetually take a heavy toll in the    temperature, pH and chemical composition in the
form of morbidity-linked losses besides thrusting heavy      microenvironment around the eukaryotic host cells30.
expenditures on management and treatment of the              Indirect evidence on E. coli suggests that H+ -ATPase
disease5. The serotype, Salmonella 3,10:r:- is responsible   machinery uses proton motive force to generate ATP
for causing severe food-poisoning infections in human        which, in turn, is essential for protein translocation
beings 3 and animals 4 . In the chicken model of             via OMVs 41 . Numerous pockets of protruding
experimental infection studied in vivo, interesting          periplasm (designated as periplasmic organelles for
ultrastructural changes were observed in both the            their being physiologically significant structures11)
interacting pathogens and the eukaryotic host cells. Host-   were observed all around the Salmonella 3,10:r:-
pathogen interactions encountered in Gram-negative           organisms approaching closely and interacting with
organisms at close interface with eukaryotic cells have      host epithelial cells or macrophages. A model for
been recently linked with the bacterial type III secretion   molecular structure of the periplasmic organelle has
system (T3SS) and OMV-associated export of bacterial         already been proposed and these organelles have been
toxins30-32. The Gram-negative pathogens employ T3SS         explained to represent a secretion-active virulent state
for translocation of a cocktail of bacterial effector        of the organisms, ready to secrete the bacterial toxins
proteins and virulence determinants from the organisms       and secretory products as OMVs30.
                 YASHROY: ULTRASTRUCTURAL MECHANISM OF INFECTION OF SALMONELLA (3,10:r:-)                             563

     OMVs liberated from the secretion-active                   eukaryotic cells and this is blocked by a SipB-derived
Salmonella 3,10:r:-, have been proposed to be released          polypeptide45,47. Manipulation of the host cell actin
by pinching off inflated periplasmic organelles filled          cytoskeleton by Salmonella enterica for entry into
with bacterial toxins8 and exoproteins secreted by the          epithelial cells has been extensively studied48. Our study
general secretory pathway (GSP)27. It was proposed that         suggests that Salmonella 3,10:r:- organisms created
fusion of OMVs with the host epithelial membrane may            corridors via the ruffled locations in order to get access
result in the translocation of bacterial enterotoxins to        to inner sites in the ileum, as the organisms were seen
directly inside the host epithelial cells8. A similar process   in the lymphatic vessels where they were observed to
for the release of heat-labile enterotoxin via general          closely interact with macrophages (Figs. 1 & 2).
secretory pathway as OMVs has been observed for E.                   Salmonella 3,10:r:- organisms closely interacting
coli and further a mechanism of OMV-mediated                    with macrophages, developed on their surface, large
receptor-dependent delivery of bacterial toxin into host        blebs (periplasmic organelles), which appeared to
cells was implicated42. Similarly, Shiga toxin was also         liberate numerous OMVs, which in turn, were
found to be released as OMVs from periplasmic space             apparently taken up by the macrophages. This process
of Shigella dysenteriae and that the secretion was              has been assigned the task of translocation of
induced by an antimicrobial compound, mitomycin C43.            biochemical signals including LPS from the invading
On the same pattern, OMVs containing vacuolating                pathogens to directly into eukaryotic host cells,
cytotoxin (VacA), which was immuno-localized in the             macrophages, at this stage. This led to augmented
periplasm and outer membrane of intact Helicobacter             phagocytic cup formation and consequent engulfment
pylori bacteria, appeared to originate from blebs arising       of organisms into the macrophage cytoplasm (Fig. 1).
on the bacterial outer membrane. Both soluble secreted          Modulation of leukocyte response mediated by other
VacA and VacA-containing OMVs were internalized by              Gram-negative pathogens has also been reported.
MKN28 cells and were detectable in the gastric mucosa           Recently, OMVs of Neisseria menigitidis have been
of H. pylori-infected humans 12 . Likewise, active              shown to activate monocytes in an LBP-, CD14-, and
cytotoxic necrotizing factor 1 (CNF1) secreted from             TLR4/MD-2-dependent fashion with pro-inflammatory
uropathogenic E. coli has also been found to be                 effect49. Also, OMV-mediated modulation of leukocyte
associated with OMVs thereby suggesting that CNF1               adhesion molecule expression and increased reactive
is transported to the environment of the infected tissue        oxygen species (ROS) production is likely to increase
via OMVs44. The salmonella invasion protein SipB of             entrapment of leukocytes in the microcirculation and
T3SS was shown to direct heterotypic membrane fusion,           contribute to untoward inflammatory reactions as
allowing delivery of contents from E. coli-derived              noticed in systemic meningococcal disease50. Another
liposomes into cytosol of living mammalian cells45. Such        report 51 shows that OMVs containing cytotoxic
a mode of translocation of bacterial secretions as OMVs         necrotizing factor 1 (CNF-1), but not purified CNF-1,
into another host/recipient cell has, therefore, been           act in a dose dependent manner, on polymorphonuclear
described as “vesicular exocytosis from prokaryotes”            leukocytes to attenuate their antimicrobial activity. This
as earlier, the exocytosis process was traditionally            study reveals that OMVs provide a means for delivery
associated only with eukaryotes9,10. OMVs have also             of CNF-1 from uropathogenic E. coli to these host cells,
been linked to type I21 and type III30,46 secretory systems     and thus negatively affect the efficacy of acute
of Gram-nagative organisms.                                     inflammatory response to these pathogens. Further, CD+
                                                                T cells and toll-like receptors recognize Salmonella
    In the ileal epithelial cells showing ruffled               antigens expressed in bacterial surface organelles
membrane and cytoskeletal reorganization of the                 including OMVs. Thus, genetically co-ordinated surface
cytoplasm, it was notable that organisms at the surface         modifications may provide a growth advantage for
of the ruffled site did not reveal any periplasmic              Salmonella in host tissues by limiting both innate and
organellae on their exterior. This may be explained as          adaptive immune recognition52. OMVs generated by H.
these organellae have already been pinched off as               pylori bear serologically recognizable Lewis antigens,
OMVs, which in turn have seemingly accomplished their           which may contribute to the chronic immune stimulation
task of translocating the bacterial virulence determinants      of the host. The ability of these OMVs to absorb anti-
into the interacting ileal epithelial cells. It has been        Lewis auto-antibodies suggest that they may, in part,
proposed that SipB (located in/on OMVs) secreted by             play some role in putative autoimmune aspects of H.
the invading Salmonella triggers bacterial entry into           pylori pathogenisis53.
564                                        INDIAN J MED RES, DECEMBER 2007

    Fig.1-G shows that two organisms entering at one            cells) have been postulated to cause augmented secretion
location of the macrophages get enclosed in one tight           of OMVs containing secretory exoproteins9. Experimental
phagosome. Numerous tight phagosomes are observed               proof of this viewpoint has been recently obtained42
to occupy the bulk part of the cytoplasm of macrophages,        confirming that antimicrobial peptides actually trigger
where they appear to be resistant to fusion with                pathogen virulence by binding to Phop-PhoQ regulatory
lysosomes, located nearby (Fig. 1H). Individual                 system of Salmonella. Of late, role T3S needle complex-
organisms are not really discernible in the tight               like assembly has been implicated in the release of
phagosomes due highly electron-dense contents.                  OMVs in the analogy of blowing off soap bubbles with
However, in the spacious phagosomes, four organisms             the formation of tube-like assembly T3S needle/rivet
are clearly visible in each phagosome (Fig. 1I). Thus, it       complexes 30. Although, confirmatory proof for the
suggests that two organisms originally entrapped in one         existence of a generalized OMV model for T3SS30 is
tight phagosome replicate into four organisms coincident        still awaited, yet it does obviate many unanswered
with loosening of the tight phagosomes into spacious            questions posed to popular injectisome model58 on the
ones. Parallel apoptotic disintegration of infected             modus operandi of translocation of semi- or folded
                                                                proteins through a rather narrow and long conduit of
macrophages appears to release the replicated pathogens
                                                                the T3SS assembly. Interestingly, OMVs have been
in body of the host, promoting infection of more host
                                                                recently shown to release type I secreted alpha-
cells. Circulation of infected macrophages may be
                                                                haemolysin21 from E. coli. Also, some T3SS proteins
envisaged to lead to systemic infection. Recently, it has       like SipB & SipC of Salmonella have been shown to be
been shown54 that OMVs of H. pylori induce apoptosis            associated with OMVs. This important ultrastructural
in gastric epithelial cells. Further, this apoptosis is not     study is hence envisaged to stimulate further work using
mediated by mitochondrial pathway as is demonstrated            monoclonal antibodies and allied techniques to
by the lack of cytochrome c release with the activation         immunolocalize type I, III and other secretory proteins
of caspase 8 and 3.                                             in the OMVs of Gram-negative pathogens, under in vitro
     Overall, this study indicates an important role played     and in vivo conditions. To would establish their role in
by OMVs released by Salmonella 3,10:r:- pathogens at            host-pathogen interactions, inter-species competition of
both stages of invasion that is of epithelial as well as        pathogens, and intercellular communication within
macrophage cells. An earlier study revealed that protein        bacterial colonies & inter-kingdom singaling.
translocation into OMVs required ATP and the proton-                                   Acknowledgment
motive force might also contribute but appear not to be
                                                                     The author thanks Prof. B. R. Gupta, Head, Bacteriology and
essential in E. coli41. It is plausible to opine that proton-   Mycology Division (retired), Indian Veterinary Research Institute
motive force may generate ATP with the help of H +-             (IVRI), Bareilly, for providing the organisms and laboratory facilities
ATPase, and, in turn, ATP may be utilized for transporting      for animal experimentation and Director, IVRI for encouragement
proteins across the cell membrane into the periplasmic          and overall support. R.S.I.C., Chandigarh is acknowledged for
                                                                making available the transmission electron microscope facility.
organelles to be eventually released as OMVs. The
specialized T3S assembly of pathogenic and symbiotic                                       References
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Reprint requests: Prof. R.C. YashRoy, Principal Scientist, O/C Biophysics & Electron Microscopy Section, Indian Veterinary
                  Research Institute, Izatnagar, Bareilly 243122, India

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