Novel approaches to understand the intracellular lifestyle of Salmonella enterica by live cell imaging and ultrastructural studies [Elektronische Ressource] = Neue Ansätze zum Verständnis der Interaktion intrazellulärer Salmonella enterica und Wirtszellen mittels Lebendzell-Mikroskopie und ultrastruktureller Untersuchungen / vorgelegt von Roopa Rajashekar

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• Naturwissenschaften

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2010
• Nombre de lectures: 26
• Poids de l'ouvrage: 6 Mo

Extrait

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




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



Der Naturwissenschaftlichen Facultät
der Friedrich-Alexander-Universität-Nürnberg
zur
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







2


This work is dedicated to my beloved mother






Late Smt. Padma Rajashekar













 
3Table of contents

1 INTRODUCTION 6
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
2 RATIONALE AND AIMS OF THE PROJECT 34
3 RESULTS AND PUBLICATIONS 36
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
4 DICUSSION 116
5 SUMMARY 129
6 LIST OF PUBLICATIONS AND CONTRIBUTION OF CO-
AUTHORS 133
7 REFERENCES 134
8 ABBREVIATIONS 146
9 CURRICULUM VITAE 149
10 ACKNOWLEDGEMENTS 152















51 INTRODUCTION
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 http://www.textbookofbacteriology.net/themicrobialworld/S.enterica.jpeg).

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
excrement.
 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
immunity.
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