See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/5511543

Immunological, cellular and molecular events in typhoid fever

Article  in  Indian journal of biochemistry & biophysics · November 2007

Source: PubMed

CITATIONS READS
2 1,826

2 authors, including:

Sk Jain
Jamia Hamdard University
168 PUBLICATIONS   2,651 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Defense research and development organization View project

Thalassemia View project

All content following this page was uploaded by Sk Jain on 10 September 2014.

The user has requested enhancement of the downloaded file.

Indian Journal of Biochemistry & Biophysics
Vol. 44, October 2007, pp. 320-330

Minireview

Immunological, Cellular and Molecular Events in Typhoid Fever

Nowsheen Hamid and S K Jain*
Department of Biotechnology, Hamdard University, Hamdard Nagar, New Delhi 110 062, India

Received 02 June 2007; revised 20 September 2007

Salmonella, a facultative intracellular Gram-negative bacterium infects a wide range of hosts causing several
gastrointestinal diseases and enteric fever in humans and certain animal species. Typhoid caused by Salmonella typhi
remains a major health concern in India and worldwide. Also, with emergence of multidrug resistant strains, Salmonella has
acquired increased virulence, communicability and survivability, resulting in increased morbidity and mortality. Though a
number of vaccines for typhoid are available against S. typhi (or also against S. typhimurium), these have certain undesirable
side effects and the search for new immunogens suitable for vaccine formulation is still continuing. The immune response to
primary Salmonella infection involves both humoral and cell-mediated responses. The protective immunity against
Salmonella depends on host- parasite interaction, however; the detailed mechanism of virulence, innate resistance and
susceptibility of host remains unclear. This review focuses on the molecular, immunological and cellular mechanisms of
pathogenesis of Salmonella infection to provide an insight to counteract bacterial infections and allow a better understanding
of its clinical manifestations. It also reviews better technological possibilities combined with increased knowledge in related
fields such as immunology and molecular biology and allow for new vaccination strategies. Some new approaches such as
subunit and nucleic acid vaccines and recombinant antigen which are becoming increasingly important for the development
of potential vaccines have also been discussed. A significant progress has been made in our understanding of Salmonella
pathogenesis. Despite these efforts, however, many challenges exist, especially for investigators who aim to understand how
the pathogenic mechanisms operating in vitro apply to in vivo model systems. However, unyielding work and collaborations
between Salmonella researchers and clinicians worldwide have made significant contributions to understanding the
interaction between virulence determinants and immunity required to stop the spread of this pathogen.

Keywords: Outer membrane proteins, Pathogenesis, Salmonella, Typhoid, Vaccine, Virulence.

Introduction Although many studies have been undertaken to

Typhoid caused by Salmonella typhi, a facultative understand the host response against Salmonella
gram-negative bacterium, remains a public health infection, precise events related to immune response
problem with an annual global burden of about 16 against this microorganism are not fully understood.
million cases, leading to 60,0000 deaths1. Salmonella The development of effective vaccines for protection
species are an important group of pathogens, which against diseases caused by Salmonella spp. continues
infect a wide range of hosts causing a variety of to be an extensively studied field6. Live vaccines can
syndromes. Other Salmonella serovars (S. typhimu- effectively induce both humoral and cell-mediated
rium, S. enteritis) cause infections to domestic immune response and are often preferred over
animals that can be transmitted to humans and also vaccines derived from killed bacteria7. Though the
represent a serious concern for the food industry2. later cannot replicate and are, therefore, non-
Typhoid is endemic in many developing countries, infectious, these are less effective than live vaccines
including areas of Africa, Asia and S. America3 and in inducing protective immunity. The ability to elicit
children in endemic areas, travelers and microbiolo- antibodies, in addition to cell-mediated immunity is
gical laboratory technicians are particularly at risk of important for optimal protection conferred by
contracting the disease. Salmonella vaccines8.
___________
*Author for correspondence: Two new vaccines, a parental capsular
E-mail: skjain@jamiahamdard.ac.in polysaccharide vaccine based on the S. typhi Vi
Tel: +91-11-26059688; Fax: 26059663 antigen9 and a live attenuated oral vaccine containing
Abbreviations: LPS, lipopolysaccharide; MDR, multidrug S. typhi strain Ty21a10 have been licensed for use
resistance; Nramp-1, natural resistance associated macrophage
protein-1; OMP, outer membrane proteins; TTSS, type III against typhoid fever during last 15 years. However,
secretion system. due to adverse reactions and less than desired
 HAMID & JAIN: MOLECULAR BIOLOGY OF TYPHOID 321

efficacy, a number of new genetically defined such as digestive tract bleeding and intestinal
attenuated strains of S. typhi have been constructed as perforation may also develop. The patients may have
live candidate oral vaccines which have the potential clinical recovery afterwards, however, some patients
as vector for delivery of foreign antigens also11. who are not responsive to the treatment with
Although all clinical and field trials with Salmonella antibiotics against S. typhi may even die after a
as delivery system have been limited to attenuated progressive clinical worsening17.
strains of S. typhi, the use of attenuated S. typhimu- The understanding of typhoid fever pathogenesis,
rium for heterologous antigen delivery is a promising especially the cellular and molecular phenomenon
alternative to S. typhi as vaccine vector12. responsible for clinical manifestations has greatly
increased with several important discoveries: a)
The development of an effective Salmonella Bacterial type III protein secretion system — Many
vaccine is highly relevant and being pursued by a virulence genes are clustered together on
number of workers. A number of well-tolerated pathogenecity islands (PAIs) that encode structurally
attenuated S. typhi strains have been found to be similar but functionally distinct type III secretion
immunogenic in clinical trials13 and show promise as systems (TTSS) which translocate virulence proteins
candidates for new generation of typhoid vaccines. In from bacterial to host cells during the infectious cycle.
addition, recombinant Salmonella vaccines have been b) The five virulence genes of Salmonella spp.
shown to protect against a broad range of pathogens encoding Salmonella invasion proteins (Sips) A, B, C,
in animal models and preliminary results from clinical D and E, which are capable of inducing apoptosis in
trials demonstrate that protective immunity against macrophages. c) The function of Toll receptors R2
heterologous antigens is achievable14. A significant and R4 present in the macrophage surface (originally
development involves the use of Salmonella for discovered in Drosophila) — The Toll family
delivery of DNA vaccines. Use of bivalent receptors are critical in cell signaling mediated
Salmonella strains to deliver DNA vaccines allows through macrophages in association with LBP and
the induction of immunity against the Salmonella CD14. d) The lines of immune defense between
carrier, the heterologous antigen(s) expressed by intestinal lumen and internal organs, and e) The
Salmonella and the antigen(s) encoded by the DNA fundamental role of endothelial cells in inflammatory
vaccines. Further, it should be possible to deviation from bloodstream into infected tissues by
simultaneously express the heterologous antigen bacteria6,17-19.
integrated into Salmonella chromosome as well as
The use of antimicrobials in typhoid therapy needs
cloned into the plasmid(s) carried by the Salmonella.
to be evaluated in light of increased risk of disease
It may even be possible to both ‘prime’ and ‘boost’
due to elevation of cellular concentration of LPS
with a single oral immunization by delivering the
molecules. These molecules are responsible for
DNA vaccine to appropriate antigen presenting cells
mononuclear cell activation that releases cytokines,
(APCs) for expression of antigen and co-delivering an
such as TNFα, IL-1, IL-6, IFNγ etc. They are
expressed antigen. This DNA vaccine delivery system
considered as powerful agent for the inflammatory
is very promising and offers a new and exciting
cell activation and act through CD14 and Toll R2 and
approach to mucosal vaccine delivery15,16. To date,
R4 receptors. The signals are then transmitted to an
however, there have been no reports of human trials
intracellular protein p21 activated kinase18. Other
of recombinant Salmonella for the delivery of DNA
proteins activated by S. typhimurium are the Rho-
vaccines.
GTPases, which stimulate the membrane “ruffling”
Typhoid Fever: Molecular and Immunological (rearrangement of cellular membrane), responsible for
Perspective the entry of Salmonella spp. to the cell. At this stage,
Classically typhoid fever is considered a multiple rearrangement of actin cytoskeleton and nuclear
stage disease. The 1st week is characterized by responses also occur. The contact of cytosol with
progressive elevation of body temperature, followed proteins secreted by invasive bacteria (S. typhi,
by bacteremia, 2nd week with rose spots in the skin, S. typhimurium, S. cholerae-suis etc.) is made through
abdominal pain and splenomegaly and the 3rd week protein channels TTSS that translocate bacterial
with a more intensive intestinal inflammatory process, proteins inside the cells. The genetic acquisition of
particularly in the Peyer’s patches. Complications, TTSS is a major evolutionary leap for gram-negative
322 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, OCTOBER 2007

bacterial pathogens. TTSS allows animal and plant residing within epithelial or macrophage cells
pathogens to inject their own proteins (termed as growing in a medium that mimics the eukaryotic
effectors) directly into host cells that modulate intracellular environment25. The spv-ABCD operon is
specific cellular functions of the host. These controlled by regulatory protein SPVR, encoded by a
‘molecular syringes’ and their effectors are the gene located upstream and in opposite orientation to
essential virulence determinants. The protein Sop E the spv operon. Expression of SPVR, on the other
(the substrate for this secretion system) stimulates the hand, is regulated by the factors such as alternate
cytoskeleton reorganization, followed by the Jun sigma factor (RpoS), integration-host factor and
N-terminal protein activation that are dependent on leucine-responsive regulatory protein26. The expres-
Rac-1 and CDC 4219. The role of mitogen activated sion of spv operon may also be controlled by the
protein kinases in nuclear response and production of PhoP-PhoQ, the two-component regulatory system27,28
cytokines induced by inflammatory cells and cultured and determinants encoded by Salmonella pathogeni-
epithelial cells are other phenomena that are city island 2 (SPI-2)29,30. The bacterial functions
responsible for the symptoms and clinical required for Salmonella proliferation in target organs
manifestation of the disease19. include the cell envelope transporter (Toll B), a
phosphoglycerol-transferase (Mdo B), a methionyl-
Pathogenesis of Typhoid
tRNA-formyl-transferase-homologous protein (Fmt)
Murine salmonellosis has extensively been used as
and a putative malate oxidoreductase (Mdh)31,32.
the model for human typhoid fever. S. typhimurium
causes a systemic infection in mice that is reminiscent Host Controlling Determinants
of human typhoid and hence has been used to study The eukaryotic natural resistance associated
clinically relevant mechanisms of anti-Salmonella macrophage protein-1 (Nramp 1) is a membrane
host defense20,21. This model has also proven useful protein present exclusively in professional phagocytes
for the analysis of genetic basis of Salmonella and essential for resistance to Salmonella33. INF-γ is
virulence and for initial assessment of safety and another host factor that impairs intracellular
immunogenicity of new generation of Salmonella proliferation of Salmonella.
vaccines.
Intracellular Proliferation of Salmonella in
Molecular and Cellular Events in Salmonella
Cultured Cells
Intracellular Proliferation
Infection of cultured phagocytic or epithelial cells
Salmonella proliferates within membrane bound
mimics relevant bacteria-host interaction taking place
vacuoles of eukaryotic cells. Recent work has shown
in in vivo, bacterial invasion and intracellular
that macrophages are the main cells that support
proliferation. In phagocytic cells, diversity of
bacterial growth in vivo. In contrast, tissue culture
phenotypes in cultured macrophages ranges from
models have used epithelial cells as most permissive
active bacterial killing to pathogen proliferation at
cells for bacterial growth22. Intracellular proliferation
rates comparable to those of epithelial cells34.
of Salmonella is fast and almost 100-fold increase in
Salmonella must express and deploy a TTSS located
bacterial load can be seen within 24 h. In liver and
in SPI-2 to survive the host phagocytic vacuoles and
spleen, Salmonella is located in CD18 containing
to cause systemic infection in mouse models of
phagocytes (polymorphonuclear cells and macro-
typhoid fever. The screening of Salmonella genes that
phages) that infiltrate to the infection loci23. If
are transcriptionally co-regulated in vitro with SPI-2
macrophages are immunodepleted, the severity of
genes has been used to identify bacterial loci that
infection increases, resulting in macrophages
might function in a mouse model of systemic disease.
becoming the permissive cells in vivo for proliferation
Strains with mutations in three SPI-2 co-expressed
of Salmonella24.
genes have been constructed and tested for their
Intracellular Proliferation of Salmonella in vivo ability to cause disease in mice35. The SPI-2 encoded
Salmonella Determinants TTSS plays a role in macrophage growth and SPI-2
The products of the virulence plasmid-encoded spv- determinants are involved in inhibition of phagosome-
ABCD operon are the first pathogenic functions lysosome fusion and host NADPH oxidase delivery to
linked to Salmonella proliferation in liver and spleen. the Salmonella containing vacuoles and both these
The spv genes are expressed efficiently in bacteria activities are essential for bacterial growth36. Other
 HAMID & JAIN: MOLECULAR BIOLOGY OF TYPHOID 323

factors include PhoP-PhoQ system, ompR/EnvZ and endothelial system, where the bacteria are destroyed
transcription regulators RpoS, SlyA and RpoE37. mainly by phagocytosis through macrophage system.
In cell culture models, epithelial cells are the most However, Salmonella are able to survive and multiply
permissive cells for bacterial growth22 and massive within the mononuclear phagocytic cells40. At a
bacterial growth is observed in cultured epithelial threshold level determined by the number of bacteria,
cells. The onset of bacterial growth correlates to Sif bacterial virulence and host immune response, the
formation though these structures are dispensable for bacteria are released from their sequestered
intra-epithelial growth38. The contribution by both the intracellular habitat into the bloodstream. Hence, this
host and the pathogen for fine adjustment of the bacteremic phase of disease is characterized by
intracellular growth rate has been suggested. dissemination of the organisms to secondary sites of
The severity and outcome of Salmonella infections infection. The most common sites of secondary
in mice depend on several variables, such as virulence infection are the liver, spleen, bone marrow,
of infecting strain, infectious dose, route of infection, gallbladder and Peyer’s patches in the terminal ileum.
genetic background and immunological status of the In the liver, S. typhi provokes Kupffer cell activation
host1. S. typhimurium encounters a diversity of and neutralize the bacteria with oxidative free
environments throughout the course of systemic radicals, nitric oxide as well as enzymes while the
infection. survived bacteria invade hepatocytes resulting into
Intestinal-mucosal Immunity (First line of defense) cellular death.
The infectious dose of S. typhi in volunteers varies
Virulence
widely, ranging between 1,000 to 1,000,000
Salmonella pathogenesis is a complex, multi-
organisms39. The bacteria must survive the gastric
factorial process that results from activity of many
acid barrier to reach the small intestine and the low
bacterial gene products and about 4% of its genome is
gastric pH is an important defense mechanism. In
involved in virulence41. In S. typhimurium, many of
small intestine, S. typhi moves across the intestinal
virulence genes cluster together on pathogenecity
epithelial cell and reaches the M cells, thus
islands (PAIs). SPI-1 and SPI-2 are two PAIs that
penetrating the Peyer’s patches. The M cells are
encode structurally similar, but functionally distinct
specialized epithelial cells overlying Peyer’s patches
TTSS encoded by all serovars of S. enterica, which
(probably originated from intestinal epithelial cells)
translocate virulence proteins from bacteria to host
and are present as small pockets in the mucosal
cells, stimulating cellular functions of the host during
surface. After contact with M cells, the bacteria are
the infectious cycle19. SPI-1 plays an important role in
rapidly internalized and interact with antigen-
invasion of epithelial cells, whereas SPI-2 is required
presenting cells, get partially phagocytized and
for bacterial replication within macrophages and
neutralized22. The infected phagocytes are organized
systemic growth in the mouse42. In addition, a number
in their discrete foci that become pathological lesions
of other genes have also been found to be necessary
surrounded by normal tissue. The formation of these
for S. typhimurium virulence43 and the biochemical
lesions is likely to prevent the uncontrolled spread of
functions of some of these genes have been
bacteria in the body, thus confining the bacteria to
elucidated, though little is known about their
localized foci of infection.
regulation in vivo and mechanism of their interaction
Lesion formation is a dynamic process that requires
during infection. Recently, Salmonella invasion
the presence of adhesion molecules such as ICAM1
proteins coded by a new group of five virulence genes
and the balanced action of cytokines (TNFα, IL12,
Sip A to E have been identified. The Sip B protein is
IL18, IL14, IL15 and IFNγ). Failure to form
involved in protein translocation via TTSS and has
pathological lesions results in abnormal growth and
the potential to induce apoptosis in macrophages
dissemination of bacteria in infected tissue and some
through caspase-1 activation17.
bacteria escaping this barrier reach Peyer’s patches.
The dendritic cells help in antigen presentation that Invasion of Salmonella
provokes T and B lymphocyte activation21. The invasive mechanism of Salmonella has been
Dissemination from Intestinal Mucosa’s Lamina Propria studied extensively in S. typhimurium and several loci
The T and B lymphocytes released from the important for invasion have been identified in various
lymphatic nodules reach liver and spleen via reticulo- Salmonella spp. using molecular genetic approaches.
324 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, OCTOBER 2007

It has been suggested that invasion system in S. typhi combat the infection by macrophage activation that
is genetically distinct from S. typhimurium. A large may lead to septic shock. The pathogens have
chromosomal fragment (100 kb) around the antigens such as LPS of gram-negative bacteria,
Salmonella inv locus may be responsible for the cell glycolipids of mycobacteria, lipoteichoic acid of
invasion44. Sip proteins secreted through TTSS gram-positive bacteria, mannans of yeast, RNAs of
apparatus play a key role in the invasion of virus etc that are recognized by the receptors.
S. typhimurium and a similar mechanism is expected Two LPS-binding proteins BPI (bactericide
to work for S. typhi also45. S. typhimurium infection permeability increasing protein) and LBP
results in release of a set of effector proteins that (lipopolysaccharide binding protein) have distinct
induce actin cytoskeleton rearrangement, membrane effects. BPI (mol. mass 55 kD) is present in
ruffling and macropinocytosis. The effector proteins neutrophils, has an antimicrobial role with selective
include an exchange factor for Rho GTPases (Sop E), toxicity against gram-negative bacteria and is more
an inositol phosphate phosphatase (Sop B) and an effective when acting on neutrophil-phagocytosis in
actin-binding protein (Sip A)46. Several factors synergism with defensins (intestinal mucosa’s
including anaerobic growth state, calcium antimicrobial factors). LBP increases the sensitivity to
concentration and osmolarity regulate S. typhimurium LPS, allowing the effector cell activation by
invasiveness, whereas osmolarity and growth phase subpicomolar LPS concentrations. It recognizes lipid
regulate the adherence and invasion of cultured A and also carries out an important role in the
human epithelial cells by S. typhi. bacterial clearance of peripheral blood through CD14.
Some recent studies indicate that Toll family
Apoptosis and Human Toll Receptor Activation in Typhoid receptors are critical in LPS mediated signaling in
Fever association with LBP and CD1423 and discovery
Typhoid is an important example of severe sepsis, of a Toll-like human receptor4 (hTLR4) has
as it causes high degree of cellular death enhanced the understanding of binding of receptors
(necroapoptosis) besides severe toxemia. The innate with LPS-3447-49.
immune system uses Toll family receptors to sign
microbe’s presence and initiate host defense. Bacterial Host Immune Defense
lipoproteins (BLP) expressed in all bacteria species The early growth of Salmonella in mouse tissues is
are potent activators of Toll R2. The innate immune controlled by the innate resistance autosomal
system includes macrophages and natural killer (NK) dominant gene Nramp 1, located on chromosome 1
cells that act directly on pathogens through cytokines and expressed mainly in macrophages and cells of
and other stimulatory molecules and activate the granulocyte lineage50. Nramp1 codes for an integral
adaptive immune responses (cellular and molecular) membrane phosphoglycoprotein that is recruited to
through T and B lymphocytes. It identifies the the bacteria-containing phagosome, where it functions
pathogen by standard recognition receptors, which as a divalent metal ion pump. The main host defense
attach to microbial macromolecules. CD14 and Toll against Salmonella spp. occurs through the
R2 receptors were considered as fundamental in neutrophils, followed by mononuclear cells. These
recognition of LPS/endotoxin on the surface of gram- inflammatory cells produce cytokines such as TNFα
negative bacteria such as Salmonella. These receptors (produced mainly by Kupffer cells in liver), IFNγ,
differ in the composition. CD14 is a GPI (glycosyl- IL-1, IL-2, IL-6 and IL-8. Clearance of bacteria from
phosphatidylinositol) receptor that does not cross tissues requires the CD28-dependent activation of
cellular membranes and does not transmit signals for CD4+ and TCR-α β T cells and is controlled by MHC
cytoplasm or activate protein chains of macrophages, class II genes51-53. Dendritic cells and B-cells are
while Toll R2 molecule crosses cellular membranes involved in the initiation and development of T-cell
and transmits signals for the intracellular protein immunity to Salmonella54,55. Interaction between B
pathways. Activation of these protein pathways and T-cells is needed for development of antibody
spreads to transcriptional factor NF-kB that migrates responses to Salmonella proteins and for isotype
from cytoplasm to nucleus and recognizes and switching of antibody response against LPS
expresses genes encoding the adhesion molecules antigens56. Resistance to reinfection with virulent
TNFα and other Th1 cytokines (IFNγ, IL-2, etc.). The Salmonella microorganisms (secondary infection) in
patients with failure of immune response fail to immunized mice requires the presence of CD4+
 HAMID & JAIN: MOLECULAR BIOLOGY OF TYPHOID 325

dependent Th1 type immunological memory, CD8+ that confer powerful protective effect on host cells
T cells and anti-Salmonella antibodies8. (hepatocytes, CEIs, inflammatory cells etc.) through
Epithelial cells play a key role in the inflammatory partial inhibition of cytokines associated with Th1
response to intestinal pathogens. Their interaction response. The predominant assessment of Th1
with Salmonella spp. leads to the generation of a great immune response can be detected by immunohisto-
number of biochemical signals including the chemistry techniques using specific antibodies,
basolateral release of chemokines (including IL-8) bioassays or immunoassays and detection of cyto-
and apical secretion of `pathogen-elicited epithelial kines in peripheral mononuclear cells by the flow
chemoattractant`. These substances are partially cytometer or ELISPOT17,54.
responsible for guiding the recruitment and traffic of Extensive knowledge about molecular and cellular
polymorphonuclear cells across intestinal epithelial mechanism of pathogenesis of Salmonella infection
cells. After initial localization in resident phagocytes and typhoid has allowed a better understanding of its
(macrophages), the bacteria are associated mainly clinical phases and more rational approaches for
with PMNs in early phase of infection. However, clinical manifestations. In future, it should be
evidence suggests a predominant role of mononuclear possible to decrease the intensity of inflammatory
cells in early resistance to the disease57. In a recent phenomenon using therapeutic maneuvers intended to
study, S. typhimurium infection has been shown to disable inflammatory cells (with cytokines decrease),
induce IL-8 secretion by intestinal epithelium as well as to inhibit the cellular death (hepatocytes,
mediated through increase in intracellular calcium and inflammatory cells, etc.) related to Salmonella
this phenomenon is found to be NF-κ B dependent58. invasion.
The inflammatory deviation on migration of blood
leukocytes across the endothelial cells into hepatic Outer Membrane and Proteins of Salmonella as
and spleen tissues is another important event during Candidate Antigens for Typhoid Vaccine
Salmonella infection that occurs through the action of Salmonella have an inner cell membrane, an outer
adhesion molecules such as integrins present in membrane and in between these a peptidoglycan cell
inflammatory cells and selectins in endothelial cells. wall structure. The inner membrane is the typical
The inflammatory microenvironment is completed by phospholipid bi-layer structure, enclosing cytoplasm
chemokines that are capable of stimulating leukocyte and other cell inclusions. The outer membrane
motility (chemokinesis) and directed movement consisting of two leaflets is constituted by
(chemotaxis) of neutrophils and mononuclear cells. lipopolysaccharides (LPS), proteins, phospholipids
The chemokines help the blood leukocyte migration (PLs) and enterobacterial common antigen (ECA); the
straight for host cells infected by bacteria. TNF-α is inner leaflet is composed of PLs and outer leaflet
produced by macrophages and other mononuclear mainly of LPS59. As the exposed cell wall
cells. Besides the macrophage phagocytosis, TNF-α components of gram-negative bacteria interact
causes neutralization of these invasive bacteria in directly with host cells and antibodies, an immune
association with IFN-γ, IL-2 and other cytokines47. response evoked against these would be able to
Bacteria-infested Peyer's patches produce strong recognize the invading pathogens and confer
inflammatory reaction with the recruitment of protection. Of the surface components, LPS and
leukocytes. The potent inflammatory reaction against protein components of outer membranes have gained
Salmonella spp. provokes host cell death, as well as attention because of the immune response evoked by
apoptosis of both inflammatory and epithelial cells them.
resulting in the appearance of several clinical signs The outer membrane proteins (OMPs) of gram-
such as fever, jaundice (due to hepatocyte death and negative bacteria account for approximately 50% of
cholangiocyte activation) and increase of levels of outer membrane mass and include integral membrane
enzymes AST, ALT, glutamyltranspeptidase, alkaline as well as anchored proteins60. OMPs differ from
phosphatase, etc. The inflammatory response of Th1- other membrane spanning proteins as the spanning
dominant type is destructive for host cells and structure of OMPs is mainly anti-parallel β barrel
bacteria; it attenuates progressively and coincides structure59. Integral OMPs are essential for
with increase of Th2-immune response54. Th2 cells maintaining the integrity and selective permeability of
produce IL-4, IL-10, IL-13 and tissue growth factor bacterial membrane. OMPs represent important
326 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, OCTOBER 2007

virulence factors and play important role in bacterial The O-antigen of LPS is a virulence factor in
adaptation to host niches, which are usually hostile to enterobacterial infections. S. enterica expresses
invading pathogens. Some of these roles have been β-barrel surface proteases of the omptin family that
enumerated in Table 1. activate human plasminogen. The presence of
The emergence of multi-drug resistance (MDR) O-antigen repeats on wild-type or recombinant
strains of Salmonella has complicated the S. enterica prevents plasminogen activation by PgtE
management of typhoid due to their acquired of S. enterica and does not affect incorporation of
increased virulence, communicability and omptins into bacterial outer membranes. However, its
survivability, leading to increased morbidity and presence prevents PgtE-mediated bacterial adhesion
mortality. Though a number of vaccines for typhoid to basement membranes. Expression of LPS, on the
are currently available, none of these is without side other hand, prevents Pla-mediated adhesion of
effects. Thus, it is pertinent to search for new recombinant E. coli to basement membranes as well
immunogen molecules suitable for vaccine as invasion into human endothelial cells. Pla and PgtE
formulation. The outer membrane proteins of require LPS for their activity and O-antigen sterically
Salmonella have been implicated as possible prevents recognition of large-molecular weight
candidates for conferring protection against typhoid. substrates. Loss of O-antigen facilitates Pla functions
Although significant advances have been made and LPS renders plasminogen activator cryptic in
regarding the structure and function of OMPs, the S. enterica62.
number of OMPs that have been characterized The expression and deployment of TTSS by
represents only a small portion of the total OMPs Salmonella is essential for causing systemic infection
revealed by bacterial genome sequences59. in mouse models of typhoid fever63. A genome-wide
Humoral and cell-mediated immune responses approach for screening of Salmonella genes that are
against OMPs have been studied in control, infected transcriptionally co-regulated in vitro with SPI-2
and immunized-infected mice and the humoral genes has been used to identify bacterial loci that
immune response to crude OMPs of S. typhimurium in might function in a mouse model of systemic disease.
mice has been characterized. The crude OMPs of S. VirK, a homologue of a Shigella virulence
typhimurium (smooth C5 strain) evoke antibody determinant and RcsC, a sensor kinase are important
response to both LPS and proteins. The crude OMPs at late stages of infection. SomA, another Salmonella
from rough mutants (lacking O-specific chain of LPS) gene having homology with VirK is also important
of S. typhimurium produce antibodies to proteins, but for systemic infection in mice. Expression of both
not to LPS61. VirK and SomA requires the transcription factor
Table 1—Various mechanisms of OMP-mediated bacterial adaptive responses to host environment60

Adaptive response(s) Mechanism(s) OMP(s) involved

Iron uptake Expression of OMPs directly binding to host proteins such as Tbp, Lbp, HemR
transferrin, lactoferrin or hemoprotein.
Synthesis of high-affinity iron-siderophores and expression of FhuA, FepA
OMPs and their binding to siderophore complexes.

Antimicrobial peptide Direct degradation of antimicrobial peptides through OmpT, PgtE

resistance production of outer membrane associated proteases.
Modification of bacterial surface through production of OMPs PagP
with enzymatic activities.

Serum resistance Prevent the activation of complement cascades by binding to Por1A, Por1B, OspE
factor H or C4bp, down-regulators of complement activation.
Unknown OmpX

Multi-drug resistance Key roles in multi-drug efflux systems. TolC, OprM

Bile resistance Modulate membrane permeability by regulating the production OmpC, OmpU
of specific porins.
Key roles in multi-drug efflux systems. TolC
 HAMID & JAIN: MOLECULAR BIOLOGY OF TYPHOID 327

PhoP, whereas RcsC expression is independent of number of higher molecular mass proteins (>80 kDa),
PhoP. Further, transcription factor OmpR is needed majority of which appear to be localized in the outer
for the expression of YajN RcsB which is a target of membranes69.
RcsC, even though expression of RcsC itself does not Beside porins, many medium and high molecular
require OmpR. VirK, SomA and RcsC are important mass proteins are present in the outer membrane of
during late stages of enteric fever and probably gram-negative bacteria including Salmonella, some of
contribute to the modulation of bacterial outer which are species-specific70-72. Four non-porin OMPs
membranes in response to the host environment35. (molecular masses 15 kDa, 33 kDa, 37 kDa and 49
Penetration of intestinal epithelial cells is an kDa) have been selected from the OM profile of
important step in the pathogenesis of Salmonella. A S. typhimurium73. These proteins confer varying
gene InvE necessary for Salmonella invasion of degree of protection against bacterial challenge with
cultured epithelial cells has been characterized and its experimentally induced murine salmonellosis and also
predicted amino acid sequence shows significant decrease the number of bacteria reaching liver.
homology to Yersinia outer membrane protein YopN Immunization with 49 kDa protein provides 100%
(LcrE). Unlike wild-type S. typhimurium, InvE protection against Salmonella infection, causing an
mutants fail to change intracellular free calcium levels enhanced phagocytic capacity and index in the
or distribution of polymerized actin in cultured reticulo-endothelial system. However, the role of
epithelial cells or the normal architecture of microvilli these non-porin OMPs in pathogenecity and
of polarized Madin-Darby canine kidney cells. immmunogenicity has not been explored and
Wild-type S. typhimurium can rescue the invasive biological role of majority of bacterial OMPs is still
phenotype of InvE mutants in simultaneous infections unknown.
of cultured epithelial cells. Hence InvE mutants are
deficient in triggering the intracellular events that lead Conclusion
to bacterial internalization. Also, the expression of Salmonellosis (and typhoid fever) continues to be
ShdA, a surface-localized fibronectin-binding protein an important health problem in developing countries.
is induced in vivo in the murine caecum, a tissue The preventive measures against salmonellosis are not
having cognate receptors for this OMP. Expression of adequate and its management is becoming
cloned ShdA gene from T7 promoter in vitro results increasingly difficult due to appearance of new drug
in detection of ShdA in the outer membrane of resistant strains. Presently a number of vaccines
S. typhimurium and binding of fibronectin to the against typhoid are available; however, there is a need
bacterial surface. Deletion of ShdA gene results in for better and improved new generation vaccines
decreased colonization of cecum and Peyer`s patches against Salmonellae. For successful development of a
of terminal ileum to a lesser degree than that of defined vaccine, a clear understanding of both the
mesenteric lymph nodes and spleen 5 days post-oral cellular and humoral components of the immune
inoculation of mice64. responses elicited during infection and identification
Porins are the major class of OMPs that form non- of antigens that trigger protective response is
selective pores for small hydrophobic molecules and essential. Many studies have been undertaken to
have been extensively documented in relation to understand the genetics as well as physiological
immunogenecity and pathogenecity of typhoid fever. responses of Salmonella under different conditions
They are excellent antigens that interact efficiently during infection. Antigens that are currently being
with both arms of the host immune systems and can exploited include the outer membrane proteins
play a role in providing protection against the (OMPs), the heat shock proteins and the Vi capsular
disease61,65. However, they are not specific to antigen.
Salmonella and their efficacy as protective OMPs of Salmonella are being looked upon as new
immunogen depends upon their association with immunizing agents that can confer protection against
LPS66,67. In addition to major OMPs viz. OmpC, typhoid. With the aid of the newly developed
OmpF and OmpA, many minor classes of OMPs have technologies such as functional genomics and DNA
been identified68. Some of these minor species are microarrays, characterization of bacterial OMPs can
expressed only at very low levels. Two-dimensional now be performed at a previously unprecedented large
electrophoresis of S. typhimurium displays a greater scale. These systems in conjunction with other
328 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, OCTOBER 2007

strategies, such as signature-tagged mutagenesis, 9 Hessel L, Debois H, Fletcher M & Dumas R (1999)
subtractive and differential hybridization, in vivo Experience with Salmonella typhi Vi capsular polysaccharide
vaccine. Eur J Clin Microbiol Infect Dis 18, 609-620
expression technology etc would reveal more OMPs 10 Germanier R & Fuer E (1975) Isolation and characterization
that are essential for bacterial virulence and of Gal E mutant Ty21a of Salmonella typhi: A candidate for
adaptation during in vivo infection. These OMPs will a live, oral typhoid vaccine. J Infect Dis 131, 553-558
be promising targets for the design of antimicrobial 11 Garmony H S, Brown K B & Titball R W (2002) Salmonella
vaccines for use in humans: Present and future perspectives.
drugs and vaccines. To circumvent some of the FEMS Microbiol Rev 26, 339-353
adversities, various approaches of immunological 12 Levine M M, Hone D, Tacket C, Ferreccio C & Cryz S
manipulations, either alone or in combination with (1990) Clinical and trials with attenuated Salmonella typhi as
chemotherapy and vaccination have been explored. live oral vaccines and as ‘carrier’ vaccines. Res Microbiol
The development of effective vaccines for 141, 807-816
13 Hone D, Morona R, Attridge S & Hackett J (1987)
protection against diseases caused by Salmonella spp. Construction of defined gal E mutants of Salmonella for use
continues to be an extensively studied field. Use of as vaccines. J Infect Dis 156, 167-174
well-defined recombinant protein in combination with 14 Dunstan S J, Ramsay A J & Strugnell R A (1996) Studies of
appropriate adjuvant is more likely to overcome many immunity and bacterial invasiveness in mice given a
recombinant Salmonella vector encoding murine interleukin-
limitations and provide species or strain specificity. 6. Infect Immun 64, 2730-2736
The nucleic acids have strong potential and may 15 Mollenkopf H J, Groine-Triebkorn D, Anderson P, Hess J &
become the vaccines of future. Better technological Kaufmann S H (2001) Protective efficacy against
possibilities combined with increased knowledge in tuberculosis of ESAT-6 secreted by a live Salmonella
related fields such as immunology and molecular typhimurium vaccine carrier strain and expressed by naked
DNA. Vaccine 19, 4028-4035
biology allow for new vaccination strategies. 16 Niethammer A G, Primus F J, Xiang R, Dolman C S,
Ruehlmann J M, Ba Y, Gillies S D & Riesfeld R A (2001)
Acknowledgement An oral DNA vaccine against human carcinoembryonic
These studies were supported by a CCRUM funded antigen (CEA) prevents growth and dissemination of
short-term research project and a DST sponsored Lewis lung carcinoma in CEA transgenic mice. Vaccine 20,
421-429
research project to SKJ. NH is a UGC (NET) Senior 17 Andrade D R & Andrade D R (2003) Typhoid fever as
Research Fellow. cellular micobiological model. Rev Inst Med Trop S Paulo
45, 185-191
References 18 Chen L M, Bargodia S, Cerione R A & Galan J E (1999)
1 Mastroeni P & Sheppard M (2004) Salmonella infections in Requirement of p21-activated kinase (PAK) for Salmonella
the mouse model: host resistance factors and in vivo typhimurium induced nuclear responses. J Exp Med 189,
dynamics of bacterial spread and distribution in the tissues. 1479-1488
Microb Infect 6, 398-405 19 Hueck C J (1998) Type III protein secretion systems in
2 Mastroeni P (2002) Immunity to systemic Salmonella bacterial pathogens of animals and plants. Microbiol Mol
infections. Curr Mol Med 2, 393-406 Biol Rev 62, 379-433
3 Levine M M (1999) Typhoid fever vaccines. In: Vaccines 20 Conlan J W & North R J (1992) Early pathogenesis of
(Plotkin S A Orenstein W A, eds.), pp 781-814 infection in the liver with the facultative intracellular bacteria
4 Frederico G C A, Silvia M L M & Yara M G (1998) Listeria monocytogenes, Francisella tulacensis and
Vaccines against human parasitic diseases: An overview. Salmonella typhimurium involves lysis of infected
Acta Tropica 71, 237-254 hepatocytes by leukocytes. Immun Infect 60, 5164-5171
5 Doolan D L, Hedstrom R C & Wang R (1997) DNA vaccines 21 Yrlid U, Svensson M, Johansson C & Mary J W (2000)
for malaria: the past the present and the future. Indian J Med Salmonella infection of bone marrow derived macrophages
Res 106, 109-119 and dendritic cells: influence on antigen presentation and
6 Nasser M W, Tariq Hamid & Jain S K (2002) Pathogenesis initiating an immune response. FEMS Immunol Med
of Salmonella: Immunological considerations and vaccines. Microbiol 27, 313-320
Proc Nat Acad Sci India 72 B,135-164 22 Garcia-del Portillo F (2001) Salmonella intracellular
7 Gherardi M M, Gomez M I, Garcia V E, Sordelli D O & proliferation: Where, when and how? Microb Infect 3,
Cerquetti M C (2000) Salmonella enteritidis temperature- 1305-1311
sensitive mutants protect mice against challenge with 23 Richter-Dahlfors A, Buchan A M J & Finlay B B (1997)
virulent Salmonella strains of different serotypes. FEMS Imm Murine salmonellosis studied by confocal microscopy:
Med Microbiol 29, 81-88 Salmonella typhimurium resides intracellularly inside
8 Mastroeni P, Villarreal-Ramos B & Hormaeche, C E (1993) macrophages and exerts a cytotoxic effect on phagocytes
Adoptive transfer of immunity to oral challenge with virulent in vivo. J Exp Med 186, 569-580
salmonellae in innately susceptible BALB/c mice requires 24 Wijburg O L, Simmons C P, van Rooijen N & Strugnell R A
both immune serum and T cells. Infect Immun 61, 3981-3984 (2000) Dual role for macrophages in vivo in pathogenesis
 HAMID & JAIN: MOLECULAR BIOLOGY OF TYPHOID 329

and control of murine Salmonella enterica var. Typhimurium 40 House D, Bishop A, Parry C M, Dougan G & Wain J (2001)
infections. Eur J Immunol 30, 944-953 Typhoid fever: Pathogenesis and disease. Curr Opin Infect
25 Wilson J A, Doyle T J & Gulig P A (1997) Exponential- Dis 14, 573-578
phase expression of spvA of the Salmonella typhimurium 41 Bowe F, Lipps C J, Tsolis R M, Groisman E, Heffron F &
virulence plasmid: Induction in intracellular salts medium Kusters J G (1998) At least four percent of the Salmonella
and intracellularly in mice and cultured mammalian cells. typhimurium genome is required for fatal infection of mice.
Microbilogy 143, 3827-3839 Infect Immun 66, 3372-3377
26 Marshall D G, Sheehan B J & Dorman C J (1999) A role for 42 Shea J E, Hensel M, Gleeson C & Holden D W (1996)
the leucine-responsive regulatory protein and integration host Identification of a virulence locus encoding a second type III
factor in the regulation of the Salmonella plasmid virulence secretion system in Salmonella typhimurium. Proc Natl Acad
(spv) locus in Salmonella typhimurium. Mol Micobiol 34, Sci (USA) 93, 2539-2547
134-145 43 Groisman E A & Ochman H (1997) How Salmonella became
27 Matsui H, Kawakami T, Ishikawa S, Danbara H & Gulig P A a pathogen. Trends Microbiol 5, 343-349
(2000) Constitutively expressed phoP inhibits mouse- 44 Bliska J B, Galan J E and Falkow S (1993) Signal
virulence of Salmonella typhimurium in an Spv-dependent transduction in the mammalian cell during bacterial
manner. Microbiol Immunol 447, 54-63 attachment and entry. Cell 73, 903-920
28 Heithoff D M, Conner C P, Hentschel U, Govantes F, Hanna 45 Wood M W, Rosqqvist R, Mullan P B, Edwards M H &
P C & Mahan M J (1999) Coordinate intracellular expression Galyov E E (1996) SopE, a secreted protein of Salmonella
of Salmonella genes induced during infection. J Bacteriol dublin, is translocated into the eukaryotic cell via a sip-
181, 799-807 dependent mechanism and promotes bacterial entry. Mol
29 Cirillo D M, Valdivia R H, Monack D M & Falkow S (1998) Microbiol 22, 327-338
Macrophage-dependent induction of the Salmonella
46 Zhou D, Mooseker M S & Galan J E (1999) Role of the
pathogenicity island 2 type III secretion system and its role in
S. typhimurium actinbinding protein Sip A in bacterial
intracellular survival. Mol Microbiol 30, 175-188
internalization. Science 283, 2092-2095
30 Shea J E, Beuzon C R, Gleeson C, Mundy R & Holden D W
47 Andrade Junior D R, Andrade D R, Santos S A & Ori M
(1999) Influence of the Salmonella typhimurium
(2000) Time-dependent progressive production of TNF-alpha
pathogenicity island 2 type III secretion system on bacterial
for rat hepatocytes in a primary culture invaded by
growth in the mouse. Infect Immun 67, 213-219
Salmonella typhimurium. In: International Congress on
31 Bowe F, Lipps C J, Tsolis R M, Groisman E, Heffron F &
Infectious Diseases. Buenos Aires, Anais
Kusters J G (1998) At least four percent of the Salmonella
typhimurium genome is required for fatal infection of mice. 48 Beg A A & Baltimore D (1996) An essential role for NF-
Infect Immun 66, 3372-3377 KappaB in preventing TNF alpha induced cell death. Science
274, 782-784
32 Valentine P J, Devore B P & Heffron F (1998) Identification
of three highly attenuated Salmonella typhimurium mutants 49 Bowie A & O`Neill L A J (2000) The interleukin-1
that are more immunogenic and protective in mice than a receptor/Toll-like receptor superfamily: Signal generators for
prototypical aroA mutant. Infect Immun 66, 3378-3383 pro-inflammatory interleukins and microbial products.
33 Zhang G, Wu H, Ross CR, Minton J E & Blecha F (2000) J Leuk Biol 67, 508-514
Cloning of porcine NRAMP1 and its induction by 50 Vidal S M, Malo D, Vogan K, Skamene E & Gros P (1993)
lipopolysaccharide, tumor necrosis factor alpha, and Natural resistance to infection with intracellular parasites:
interleukin-1beta: Role of CD14 and mitogen-activated isolation of a candidate for Bcg. Cell 469-485
protein kinases. Infect Immun 68, 1086-1093 51 McSorley S J & Jenkins M K (2000) Antibody is required for
34 Monack D M, Navarre W W & Falkow S (2001) Salmonella protection against virulent but not attenuated Salmonella
induced macrophage death: the role of caspase-1 in death and enterica serovar typhimurium. Infect Immun 68, 3344-3348
inflammation. Microb Infect 3, 1201-1212 52 Hess J, Ladel C, Miko D & Kaufmann S H (1996)
35 Detweiler C S, Monack D M, Brodsky I E, Mathew H & Salmonella typhimurium aroA-infection in gene targeted
Falkow S (2003) VirK, SomA and RcsC are important for immunodeficient mice: major role of CD4+ TCR-alpha beta
systemic Salmonella enterica serovar Typhimurium infection cells and IFN-gamma in bacterial clearance independent of
and cationic peptide resistance. Mol Microbiol 48, 385-400 intracellular location. J Immunol 156, 3321-3326
36 Vazquez-Torres A & Fang F C (2001) Salmonella evasion of 53 Hormaeche C E, Harrington K A & Joysey H S (1985)
the NADPH phagocyte oxidase. Microb Infect 3, 1313-1320 Natural resistance to salmonellae in mice: Control by genes
37 Garcia-del Portillo F (1999) In: Microbial Foodborne within the major histocompatibility complex. J Infect Dis
Diseases: Mechanisms of Pathogenesis and Toxin Synthesis. 152, 1050-1056
(Cary J W, Linz J E & Bhatnagar D, eds.), pp 3-49, 54 Mastroeni P, Simmons C, Fowler R, Hormaeche C E &
Technomic Publishing Co. Inc. Lancaster, PA, USA. Dougan G (2000) Igh-6-/- (B-cell deficient) mice fail to
38 Guy R L, Gonias L A & Stein M A (2000) Aggregation of mount solid acquired resistance to oral challenge with
host endosomes by Salmonella requires SP12 translocation virulent Salmonella enterica serovar typhimurium and show
of SseFG and involves SpvR and the fms-aroE intragenic impaired Th1 T-cell responses to Salmonella antigens. Infect
region. Mol Microbiol 37, 1417-1435 Immun 68, 46-53
39 Hornick R B, Greisman S E & Woodward T E (1970) 55 Yrlid U, Svensson M, Johansson C & Wick M J (2000)
Typhoid fever: Pathogenesis and immunologic control. New Salmonella infection of bone marrow-derived macrophages
Engl J Med 283, 686-691 and dendritic cells: influence on antigen presentation and
 330 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, OCTOBER 2007

initiating an immune response. FEMS Immunol Med infections: Entiritis versus typhoid fever. Microbes Infect 3,
Microbiol 27, 313-320 1335-1344
56 Sinha K, Mastroeni P, Harrison J, de Hormaeche R D& 65 Sharma P, Sharma B K & Sharma S (1990) Mechanism of
Hormaeche C E (1997) Salmonella typhimurium aroA, htrA, protection provided by active immunization with porins in
and aroD htrA mutants cause progressive infections in mice challenged with Salmonella typhi. J Exp Med 60,
athymic (nu/nu) BALB/c mice. Infect Immunol 65, 1566- 247-252
1599 66 Nakae T (1976) Outer membranes of Salmonella. Isolation of
57 Hormaeche C E, Mastroeni P, Arena A, Uddin J & Joysey H protein complex that produces transmembrane channels.
S (1990) T-cells do not mediate the initial suppression of a J Boil Chem 251, 2170-2178
Salmonella infection in the RES. Immunology 70, 247-250 67 Muthukkumar S & Muthukkaruppan V R (1993) Mechanism
58 Gewirtz A T, Reed K A, Merlin D, Hobert M, Neish A S & of protective immunity induced by Porin-lipopolysaccharide
James L (2002) 21 Modeling microbial-epithelial interactions against murine salmonellosis. Infect Immun 61, 3017-3025
in the intestine. Met Micobiol 31, 377-396 68 Fernandez-Mora M, Oropeza R, Puente J L & Calva E
59 Samuelson P, Gunneriusson E, Per-Ake Nygren, Stahl S (1995) Isolation and characterization of ompS1, a novel
(2002) Display of proteins on bacteria. J Biotech 96, 129-154 Salmonella typhi OMP-encoding gene. Gene 58, 67-72
69 Molloy P M (2000) Two-Dimensional Electrophoresis of
60 Lin J, Huang S & Zhang Q (2002) Outer membrane proteins:
Membrane Proteins Using Immobilized pH Gradients. Anal
Key players for bacterial adaptation in host niches. Microb
Biochem 280, 1-10
Infect 4, 325-331
70 Ames G F L (1974) Resolution of bacterial proteins by
61 Natarajan M, Udhayakumar V, Krishnaraju K & polyacrylamide gel electrophoresis on slab gels. J Biol Chem
Muthukkaruppan V (1985) Role of outer membrane proteins 249, 634-644
in immunity against murine salmonellosis-1. Antibody 71 Ortiz V, Isibasi A, Garcia-Ortigoza E & Kumate J (1989)
response to crude outer membrane proteins of Salmonella Immunoblot detection of class specific humoral immune
typhimurium. Comp Immun Microbiol Infect Dis 8, 9-16 response to outer membrane protein isolated from
62 Kukkonen M, Timo T K & Korhonen K (2004) The omptin Salmonella typhi in humans with typhoid fever. J Clin
family of enterobacterial surface proteases/adhesions: from Microbiol 27 1640-1645
housekeeping in Escherichia coli to systemic spread of 72 Blanco F, Isibasi A, Gonzalez C R, Ortiz V, Paniagua J,
Yersenia pestis. Int J Med Microbiol 7-14 Arregnin C et al (1993) Human cell mediated immunity to
63 Lawhon S D, Frye J G, Soyemoto M, Porwollik S, McClel- porins from Salmonella typhi. Scand J Infect Dis 25, 73-80
land & Altier C (2003) Global regulation by CsrA in 73 Hamid T (2001) Biological characterization of the outer
Salmonella typhimurium. Mol Micobiol 48, 1633-1645 membrane proteins of S. typhi and S. typhimurium and
64 Kingsley R A, Santos R L, Zhang S, Tsolis R M, Adams L G studies on their role in protection against typhoid, Ph. D.
& Baumler A J (2001) Animal models of Salmonella thesis, Jamia Hamdard, New Delhi

View publication stats