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Analysis of Salmonella typhi-host cell interaction |
| Principal Investigator : Ayub Qadri
Ph D Students Salmonella
typhi
causes typhoid exclusively in humans. Our current understanding of how this
pathogen interacts with host cells comes largely from studies carried out with
Salmonella typhimurium, which in mice causes an analogous disease
commonly referred to as murine typhoid. However, while these studies have
given us an in-depth understanding of salmonella pathogenesis in general, the
reasons for different manifestations produced in humans by these two closely
related pathogens are not clear. These differences must be produced by
distinct host-pathogen interactions involving unique bacterial determinants
and corresponding receptors on host cells, the identity of which has not been
established. The objective of this project is to decipher Salmonella typhi-specific
host-pathogen interactions and understand the molecular basis of the
cross-talk that ensues during interaction of S.typhi with host cells. Role
of Vi capsular polysaccharide in host-pathogen interaction during infection
with S.typhi We
have previously reported that Vi polysaccharide that is present in S.typhi
but not in S.typhimurium, can interact with host cells through a
surface associated molecular complex containing prohibitin family of
molecules. Prohibitin is a putative tumor suppressor molecule involved in the
regulation of mammlian cell cycle. It binds to the retinoblastoma protein and
represses E2F family of transcription factors, which play a vital role in cell
cycle progression. Although prohibitin binds to the retinoblastoma protein, it
targets a different region on E2F in order to bring about repression.
Moreover, unlike retinoblastoma protein, the repression mediated by prohibitin
is not modulated by cyclin-dependent kinases. On the other hand MAP kinase
pathway is believed to play a crucial role in the regulation of cell cycle by
prohibitin. Considering that this signaling cascade also plays an important
role in inflammation, we asked the question if the interaction between Vi and
prohibitin family of molecules in host cells could modulate inflammatory
responses during infection with S.typhi. The results showed that
engagement of a model human intestinal epithelial cell line Caco-2 with the
polysaccharide could significantly inhibit IL-8 secretion in response to
infection with Vi-negative S.typhi. The inhibitory effect mediated by
the polysaccharide was also revealed by reduced IL-8 secretion from Caco-2
cells infected with Vi-positive S.typhi as compared to those infected
with Vi-negative S.typhi. This reduced response was seen even when
Vi-positive S. typhi produced sufficient amount of flagellin suggesting
that stimulation of cells with Vi might interfere with signaling mediated
through TLR-5, the receptor for flagellin. The latter is believed to be the
major proinflammatory determinant of pathogenic salmonella in the gut. The
results suggested that interaction of Caco-2 with Vi could inhibit early
inflammatory responses during infection with S.typhi. How did Vi bring
about inhibition of IL-8 secretion? Given that MAP-kinase pathway is involved
in the regulation of cell cycle by prohibitin as well as in the induction of
inflammatory responses, we investigated the effect of stimulation with Vi on
the phosphorylation of ERK (extracellular regulated kinase). There was a
significant reduction in ERK phosphorylation when Caco-2 cells were activated
with Vi before being infected with Vi negative S.typhi or stimulated
with PMA indicating that MAP-kinase pathway might be a target for Vi-mediated
suppression of inflammatory responses. The role of this pathway in IL-8
secretion was also ascertained by reduced chemokine secretion from Caco-2
cells in the presence of MEK inhibitor PD98059. We are currently looking at
targets upstream of ERK that might be getting modulated during activation of
cells with Vi. Our findings reveal an important role for Vi in manipulating
host responses during typhoid fever and in turn in establishing infection with
S.typhi. Preliminary data suggests that Vi-mediated inhibition may not
be restricted to IL-8, as secretion of TNF-a
following stimulation of a human monocytic cell line with PMA was also
downregulated in the presence of the polysaccharide indicating that
Vi-mediated inhibtion of inflammatory/innate immune responses may operate not
only during early interaction with intestinal epithelial cells but also during
systemic dissemination of the pathogen. It should be pointed out that Vi is
released by S.typhi in abundance during its growth in vitro and
has also been reported in sera and urine samples of typhoid patients. Our
study also assigns a novel function to prohibitin family of molecules.
Prohibitin and its related members are abundant in the mitochondria but have
also been localized in the membrane and the nucleus. However, while their role
in cell cycle regulation and stabilization of mitochondrial proteins has been
established, very little is know about the regulation of cell signaling by
membrane associated prohibitin. Our studies should therefore also give
insights into how prohibitins regulate intracellular signaling from the
membrane. In
addition to downregulating IL-8 secretion during infection with S.typhi,
we have also seen that stimulation of Caco-2 cells with the polysaccharide
brings about morphological changes indicative of perturbation of tight
junctions. Cells incubated with Vi showed disrupted membrane localization of
ZO-1, one of the key molecules associated with tight junctions. The molecular
mechanism of this deregulation is being investigated. Regulation
of secretion of TLR-5 ligand, flagellin, from salmonella by host stimulus Toll-like
receptors are involved in recognizing pathogen associated molecular patterns (PAMPs)
and in initiating inflammatory responses during infection with microbial
pathogens. Flagellin is one of the PAMPs that is recognized by TLR-5 expressed
by intestinal epithelial cells, dendritic cells and many other cell types. It
constitutes a major proinflammatory determinant of pathogenic salmonella and
can evoke a variety of inflammatory and innate immune responses from cells. In
recent years there is accumulating evidence to suggest that the expression of
many virulence traits of pathogenic bacteria may be induced by signals arising
from host-pathogen cross talk. The question we asked was if secretion of
flagellin was also modulated by a host signal. We have reported earlier that
contact of S.typhi with a human intestinal epithelial cell line Caco-2,
supernatant from these cells or fetal calf serum (FCS) can activate bacteria
to release biologically active flagellin. This activation could be efficiently
reproduced with lysophosphatidic acid (LPA), a bioactive lipid mediator that
is an abundant constituent of serum. We now show that the ability to activate
salmonella to release flagellin is not restricted to LPA but can also be
mediated by other lysophospholipids such as LPC but not by phospholipids such
as phosphatidic acid or phosphatidylcholine. Lysophospholipids are generated
in cells through hydrolysis of existing phospholipids by PLA-2 class of
enzymes. Inhibition of these enzymes in Caco-2 abrogated the ability of these
cells or the supernatant derived from them to induce secretion of flagellin
from pathogenic salmonella. Preliminary data indicates that LPC might be one
of the major lysophospholipids in the culture supernatant derived from Caco-2
cells. Our results suggest that LPC and related lysophospholipids might play a
vital role in regulating inflammatory and innate immune responses during
infection with salmonella by modulating release of a key TLR ligand.
Publications Original peer-reviewed articles 1.
†Huang JC, Han M, Minguela A, Pastor S, Qadri A and Ward ES (2003) T cell
recognition of distinct peptide:I-Au conformers in murine experimental autoimmune encephalomyelitis.J
Immunol 171:2467-2477 (†on
deputation/work done elsewhere). |