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Novel approaches to understand the intracellular lifestyle of
Salmonella enterica by live cell imaging and ultrastructural

Neue Ansätze zum Verständnis der Interaktion intrazellulärer Salmonella
enterica und Wirtszellen mittels Lebendzell-Mikroskopie und ultrastruktureller

Der Naturwissenschaftlichen Facultät
der Friedrich-Alexander-Universität-Nürnberg
Erlangung des Doktorgrades Dr. rer.nat.

Vorgelegt von

Roopa Rajashekar
Bangalore, India

Als Dissertation genehmigt von der Naturwissen-
schaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 29.07.2010
Vorsitzender der
Promotionskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Michael Hensel
Zweitberichterstatter: Prof. Dr. Andreas Burkovski


This work is dedicated to my beloved mother

Late Smt. Padma Rajashekar

3Table of contents

1.1 Salmonella and salmonellosis 6
1.2 Epidemiology of Salmonella infection 6
1.2.1 Sources of infection 7
1.2.2 Medically relevant representatives 7
1.2.3 Salmonella pathogenesis and disease outcome 8
1.3 Secretion systems in Salmonella 11
1.3.1 SPI1 and SPI2 Type Three Secretion Systems 11
1.3.2 SPI1 Type Three Secretion Systems 13
1.3.3 SPI2 TSystems 13
1.3.4 Cross-Talk between SPI1 and SPI2 Type Three Secretion Systems 14
1.4 Pathogenicity Islands of Salmonella enterica 14
1.4.1 Salmonella Pathogenicity Island 1 (SPI1) 15
1.4.2 Pathogenicity Island 2 (SPI2) 15
1.5 Endocytosis 17
1.5.1 The Endocytic Pathway 18
1.5.2 Features of Phagolysosome 19
1.5.3 Involvement of SNAREs and Rabs in phagolysosomal biogenesis 19
1.5.4 ent of phosphoinositides in the endocytic pathway 21
1.5.5 The dynamic role of cytoskeleton in phagosome maturation 22
1.6 Intracellular lifestyle of Salmonella 23
1.6.1 Divergent lifestyles of intracellular pathogens 23
1.6.2 Salmonella as a facultative intracellular pathogen 25
1.6.3 SCV- The intracellular habitat of Salmonella 26
1.6.4 Avoidance of host-derived antimicrobial radicals 29
1.6.5 Virulence factors and their molelcular mechanism in controlling the
intracellular fate of Salmonella 29
1.6.6 Effectors proteins of the SPI2-T3SS and their contribution to intracellular
life and Salmonella induced phenotypes 30
43.1 Dynamic Remodeling of the Endosomal System during Formation of
Salmonella-Induced Filametns by Intracellular Salmonella enterica 36
3.2 Novel functions of SPI2 effector proteins during intracellular pathogenesis
of Salmonella enterica revealed by live cell and Ultrastructural analyses 72
3.3 Ultrastructural analysis of SCV and Biogenesis of SIF by Electron
tomography 98

1.1 Salmonella and salmonellosis is a Gram-negative, rod-shaped, peritrichous flagellated and motile
enterobacterium. It is approximately 0.7 to 1.5 µm in diameter and 2 to 5 µm in length (Fig
1). Salmonella are facultative anaerobes, mostly found in contaminated water (sewage water)
and in packed food products like poultry, meat, eggs and milk products which are under-
processed. Salmonella are closely related to the Escherichia genus and widely distributed in
animals and humans. Salmonellacauses food-borne illnesses such as typhoid and paratyphoid
fever which are systemic infections associated with septicemia. However gastroenteritis is a
more self limiting condition.

Salmonella enterica
Peritrichous flagella

Figure 1: Morphology of Salmonella. Salmonella enterica Typhimurium (red) is a rod shape bacteria with
peritrichous flagella. (Adopted from

1.2 Epidemiology of Salmonella infection
Outbreaks of Salmonella infections mainly typhoid fever caused by S. enterica serovar
Typhi and gastroenteritis caused by S. enterica serovar Typhimurium are quite common
globally both in developed and developing countries. There is usually outbreak of
gastroenteritis when humans or animals ingest contaminated food or water. It has been
estimated that there are approximately 20 million cases of human illness every year due to
typhoid resulting in about 200,000 deaths worldwide (Crump et al., 2004).

6In Germany, more than 80% of the human isolates from the cases reported to the Enteric
Reference Centre at the Robert Koch Institute in 1995 were comprised of serovar Enteritidis
(61.3%) and serovar Typhimurium (23.4%) (Rabsch et al., 2001). Intermittent shedding of the
pathogen by domestic animals is thought to provide a constant reservoir for infection and
contamination of food. Industrialization and large scale food distribution, increased
consumption of raw or slightly cooked foods, an increase in immuno-compromised patient
populations, deteriorated public infrastructure and evolution of multi-drug-resistant
Salmonella have been all proposed as possible reasons for the steady increase in the incidence
of infections (Darwin & Miller, 1999). Therefore more stringent quality control
measures are required to prevent these outbreaks.
1.2.1 Sources of infection
Contamination of ground water due to its mixing with sewage water is the common
source of Salmonella where bacteria can survive for several weeks. Aquatic vertebrates,
notably birds and reptiles, are important vectors of Salmonella. Poultry, cattle, and sheep
frequently being agents of contamination, Salmonella can be found in food, particularly meats
and raw eggs.
1.2.2 Medically relevant representatives
 S. enterica serovar Choleraesuis (Bacillus paratyphoid B and C), is an intestinal
commensal in pigs, humans can be infected by ingesting sick animals, the bacteria
causes septicemic Salmonellosis in swine.
 S. enterica serovar Paratyphi
o S. Paratyphi A, solely a human pathogen, causes paratyphoid A, transmission
by contact and contaminated food or water.
o S. Paratyphi B, in central Europe usually a human pathogen, causes
paratyphoid B; transmission by contact and contaminated food, water or fly
 S. enterica serovar Typhi, causes systemic infection called typhoid fever in humans.
The source of infection is usually contaminated food or water. 3–5 % of all patients
remain permanent carriers of the pathogen.

7 S. enterica serovar Typhimurium (also referred to as S. typhimurium), causes a wide
range of infections in birds and mammals ranging from self limiting gastroenteritis to
severe systemic paratyphoid diseases; conveyed by contaminated food.

1.2.3 Salmonella pathogenesis and disease outcome
Entry of Salmonella to its animal host is a huge challenge as it encounters a series of
unique environments, such as temperature, pH, osmolarity, and nutrient availability. Bacteria
also encounters host innate immunity like phagocytes which could engulf and kill the
bacteria. Pathogens sense these changes and adapts to the environment by coordinated
programs of gene expression that provides an adaptive advantage in each new host
environment. In order to adapt to such conditions pathogens activates specific virulence
mechanisms that allow them to resist, evade, or even systematically manipulate the innate
As Salmonella is a Gram-negative bacterium it possess antigen repertoire such as
lipopolysaccharide and lipoproteins of the outer membrane and the host innate immune
system detects the presence of microbial pathogens using receptors that recognize these
structures (Medzhitov & Janeway, 2000). A host response is stimulated by interaction of these
microbial signature molecules with specific host receptors at the intestinal mucosa. Bacterial
pathogens that survive the innate immune effectors may persist in the host, which allows
recognition of microbial signature molecules which in turn activate cytokine production and
inflammation. All these persistent host responses lead to disease outcome.
The clinical symptoms associated with Salmonella infection are enteric (typhoid) fever
and gastroenteritis (Miller & Pegues, 2000). Enteric fever is a systemic illness that results
from infection with the exclusively human pathogens, Salmonella enterica serovar Typhi and
Paratyphi. Clinical symptoms include pain in abdomen, diarrhea, headache, fever etc. The
pathological hallmark of enteric fever is mononuclear cell infiltration in the intestinal Peyer's
patches, mesenteric lymph nodes, spleen, and bone marrow. Many non-typhoidal Salmonella
strains, such as S. enteritidis and S. typhimurium, cause self-limited enteritis in humans. As
the stomach environment is acidic due to gastric juices with high pH, Salmonellae exhibit an
adaptive acid-tolerance response on exposure to low pH (Garcia-del Portillo et al., 1993). In
the stomach, the bacterium comes in contact with the intestinal mucosal layer before
encountering and adhering to cells of the intestinal epithelium.
8Salmonellae express several fimbriae that contribute to their ability to adhere to intestinal
epithelial cells (Baumler et al., 1996).
Microscopic studies have revealed that Salmonellae invade epithelial cells by a
morphologically distinct process termed as bacterial-mediated endocytosis (Francis et al.,
1992). This unique process is characterized by membrane ruffles formation and is different
from receptor-mediated endocytosis. Shortly after bacteria adhere to the apical epithelial
surface, profound cytoskeletal rearrangements occur in the host cell, disrupting the normal
epithelial brush border and inducing the subsequent formation of membrane ruffles that reach
out and enclose adherent bacteria in large vesicles (Fig 2). This process is different from the
receptor-mediated endocytosis. Following bacterial internalization, Salmonella resides in a
host-derived vacuole called as Salmonella-containing vacuole (SCV) which transacts to the
basolateral membrane and the apical epithelial brush border reconstitutes.

Fig 2: Invasion of Salmonella and membrane ruffle formation in epithelial cells: Scanning electron micrograph
showing Salmonella.typhimurium entering a Hep-2 cell through bacterial mediated endocytosis. Membrane
ruffles extend from the cell surface, enclosing and internalizing adherent bacteria. (Adopted from Ohl & Miller,

9As studied in mice models, Salmonellaappear to preferentially adhere to and enter the
microfold cell (M cells) of the intestinal epithelium, although invasion of normally
nonphagocytic enterocytes also occurs (Jones et al, 1994). M cells (Fig 3) are specialized
epithelial cells that sample intestinal antigens through pinocytosis and transport these antigens
to lymphoid cells that underlie the epithelium in Peyer's patches (Brandtzaeg et al., 1989).
This activity is important in the priming of mucosal immunity. In bovine epithelium,
however, Salmonella do not appear to interact preferentially with M cells and the relative role
of M cells and enterocyte invasion in different animal hosts is not well understood.
Salmonellamay also passively cross the intestinal epithelial barrier following phagocytosis by
migrating CD18-positive phagocytes (Vazquez-Torres et al., 1999). Furthermore, many SPI1
encoded genes have also been implicated in mediating macrophage apoptosis in vitro (Chen et
al., 1996) and loss of electrolytes and fluid secretion, contributing to enteritis and intestinal
inflammation (Wallis & Galyov et al., 2000).

Fig 3: Diagrammatic representation of pathogenesis of Salmonella in the human gut: Orally ingested
Salmonellae survive at the low pH of the stomach and evade the multiple defenses of the small intestine in order
to gain access to the epithelium. Salmonellae preferentially enter M cells, which transport them to the lymphoid
cells (T and B) in the underlying Peyer's patches. Once across the epithelium, Salmonella serotypes that are
associated with systemic illness enter intestinal macrophages and disseminate throughout the reticuloendothelial
system. By contrast, non-typhoidal Salmonella strains induce an early local inflammatory response, which
results in the infiltration of PMNs (polymorphonuclear leukocytes) into the intestinal lumen and causes diarrhea.
(Adopted from Haraga et al., 2008).