Characterisation of SseF, a Salmonella pathogenicity island 2-encoded type three secretion system effector involved in the formation of Salmonella-induced filaments [Elektronische Ressource] = Charakterisierung von SseF, einem auf Salmonella Pathogenitätsinsel-2 kodierten Typ-III Sekretions-System-Effektor, das in die Bildung der durch Salmonellen induzierten Filamente involviert ist / vorgelegt von Petra Müller

De
Characterisation of SseF, a Salmonella pathogenicity island 2-encoded type three secretion system effector involved in the formation of Salmonella-induced filaments Charakterisierung von SseF, einem auf Salmonella Pathogenitätsinsel 2 kodierten Typ III Sekretions System Effektor, das in die Bildung der durch Salmonellen induzierten Filamente involviert ist Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr.rer.nat. vorgelegt von Petra Müller aus Nürnberg Als Dissertation genehmigt von der Naturwissen- schaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 5. Mai 2010 Vorsitzender der Prüfungskommision: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. Michael Hensel Zweitberichterstatter: Prof. Dr. Andreas Burkovski »Messieurs, c`est les microbes qui auront le dernier mot« Louis Pasteurs Table of contents i1 Introduction .........................................................................................................................1 1.1 Salmonella enterica and its pathogenesis .............................................................................. 1 1.1.1 Virulence factors of Salmonella enterica.......................................................................................
Publié le : vendredi 1 janvier 2010
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Source : D-NB.INFO/1003017282/34
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Characterisation of SseF,
a Salmonella pathogenicity island 2-encoded type three
secretion system effector involved in the formation of
Salmonella-induced filaments


Charakterisierung von SseF, einem auf Salmonella
Pathogenitätsinsel 2 kodierten Typ III Sekretions System
Effektor, das in die Bildung der durch Salmonellen
induzierten Filamente involviert ist



Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr.rer.nat.




vorgelegt von
Petra Müller

aus Nürnberg




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








Tag der mündlichen Prüfung: 5. Mai 2010

Vorsitzender der
Prüfungskommision: Prof. Dr. Eberhard Bänsch

Erstberichterstatter: Prof. Dr. Michael Hensel

Zweitberichterstatter: Prof. Dr. Andreas Burkovski
























»Messieurs, c`est les microbes qui auront le dernier mot«
Louis Pasteurs

Table of contents i
1 Introduction .........................................................................................................................1
1.1 Salmonella enterica and its pathogenesis .............................................................................. 1
1.1.1 Virulence factors of Salmonella enterica............................................................................................3
1.1.2 Survival strategies of intracellular pathogens......................................................................................5
1.2 Salmonella pathogenicity islands and the T3SS ................................................................... 7
1.2.1 SPI2 and the structure of the T3SS apparatus and its regulation.........................................................8
1.2.2 Secreted and translocated proteins of Salmonella enterica ...............................................................13
1.3 The maturation of the SCV and the formation of Salmonella-induced filaments........... 16
1.4 SseF, a SPI2-encoded effector protein................................................................................. 18
1.5 Approaches to analyse the role of SIF in nutrient acquisition.......................................... 20
2 Aim of the work.................................................................................................................23
3 Materials and Methods .....................................................................................................25
3.1 Chemicals and materials ...................................................................................................... 25
3.2 Bacterial strains and culture conditions ............................................................................. 25
3.3 Molecular and genetic methods ........................................................................................... 31
3.3.1 Electrotransformation........................................................................................................................31
3.3.2 The red recombinase system for gene replacement...........................................................................32
3.3.3 Phage transduction in S.typhimurium................................................................................................32
3.3.4 Construction of mutant alleles of SseF..............................................................................................33
3.3.5 ConstrSseF-HA-Strep ........................................................................................................34
3.3.6 Construction of SseF-fusion proteins.....34
3.3.7 Construction of mutant alleles of sseG::sseF::HA and NsseG::sseF::HA.........................................35
3.3.8 Construction of chromosomal and plasmid-based luciferase reporter strains ...................................36
3.3.9 Construction of a trehalose-inducible gfp reporter plasmid ..............................................................36
3.4 Cell culture ............................................................................................................................ 37
3.4.1 Infection of HeLa cells......................................................................................................................37
3.4.2 Infection of RAW macrophages........................................................................................................37
3.4.3 Intracellular replication in HeLa cells ...............................................................................................37
3.4.4 Pulse chase experiments with Alexa 568 conjugated Dextran ..........................................................38
3.4.5 Transfection of HeLa cells for life cell imaging................................................................................38
3.4.6 Life cell imaging ...............................................................................................................................38
3.5 Biochemical and immunological methods........................................................................... 38
3.5.1 Polyacrylamide electrophoresis.........................................................................................................38
3.5.2 Western Blot and immune detection .................................................................................................40
Table of contents ii
3.5.3 Coomassie staining of proteins .........................................................................................................41
3.5.4 In vitro expression of effector proteins .............................................................................................41
3.5.5 Fluorescence analyses of Salmonella gfp-reporter strains.................................................................42
3.5.6 Immunofluorescence analyses of infected HeLa cells ......................................................................42
3.5.7 Selective permeabilisation of HeLa cells ..........................................................................................42
3.5.8 Subcellular fractionation of infected HeLa cells ...............................................................................42
3.5.9 Fractionation of Salmonella strains...................................................................................................43
3.5.10 Luciferase Assay of bacterial cultures..........................................................................................44
3.5.11 Luciferase Assay of HeLa cells infected with trehalose-inducible reporter strains......................45
3.6 Bioinformatics ....................................................................................................................... 45
4 Results ................................................................................................................................47
4.1 The role of SIF in the nutrient availability of Salmonella ................................................. 47
4.1.1 Recruitment of Alexa568-conjugated dextran to the SCV................................................................48
4.1.2 Construction of trehalose-inducible reporter strains .........................................................................49
4.1.3 In vitro analysis of the trehalose-inducible luciferase reporter strains ..............................................51
4.1.4 Infection of cell culture cells with trehalose-inducible luciferase reporter strains ............................54
4.1.5 In vitro analysis of trehalose-inducible gfp reporter strains ..............................................................56
4.2 SseF is required for intracellular replication ..................................................................... 57
4.3 SseF is an integral membrane protein................................................................................. 58
4.3.1 Translocated SseF is integrated into host cell membranes ................................................................59
4.3.2 SseF is integrated into bacterial membranes .....................................................................................61
4.3.3 Topology analyses revealed that the C-terminus of translocated SseF is accessible from the
cytoplasm.........................................................................................................................................................62
4.4 Translational fusion of SseF and SseG................................................................................ 64
4.4.1 SseF-SseG-HA and SseG-SseF-HA fusion proteins are translocated into the host cell cytoplasm...64
4.4.2 Induction of SIF by SseF-SseG-HA and SseG-SseF-HA..................................................................65
4.4.3 Restoration of SIF formation following co-infection with sseF and sseG strains.............................66
4.5 Functional dissection of translational SseF and SseG-/ N-SseG-HA fusion proteins ..... 69
4.5.1 Expression and translocation of strains with deletions in SseG-SseF-HA ........................................70
4.5.2 ocation of strains NSseG-SseF-HA fusion strains ..........................................70
4.5.3 SIF formation of NSseG-SseF-HA fusion constructs........................................................................71
4.5.4 Expression and translocation of deleted NSseG-SseF-HA................................................................72
4.6 Functional dissection of SseF identified essential residues................................................ 73
4.6.1 The deletions in SseF did not influence the translocation behaviour and the subcellular localisation
of the protein ...................................................................................................................................................74
4.6.2 Several sseF mutants showed a replication defect in HeLa cells ......................................................74
4.6.3 Several sseF deletion mutants showed a reduced ability to form SIF...............................................75
4.6.4 The introduction of deletions in sseF did not have a relevant effect on the topology of SseF..........76
Table of contents iii
4.7 Translocated SseFΔ200-205-HA still integrates into HeLa cell membranes ................... 77
4.8 Effect of amino acid exchanges in the AIGAVL motif ...................................................... 79
4.8.1 Exchange of the hydrophobic amino acids against alanine did not influence the translocation and
subcellular localisation of SseF .......................................................................................................................79
4.8.2 Exchange of all hydrophobic amino acids in “AIGAVL” resulted in reduced SIF formation ..........80
5 Discussion...........................................................................................................................83
5.1 The role of SIF in the nutrient acquisition by Salmonella................................................. 84
5.2 SseF is an integral membrane protein required for intracellular replication ................. 88
5.3 Fusion of SseF and SseG can restore the ΔsseF and ΔsseG phenotype ............................ 94
5.4 The amino acid motif AIGAVL is required for SseF function ......................................... 97
6 Summary/ Zusammenfassung........................................................................................ 103
7 Bibliography ....................................................................................................................106
8 Appendix ..........................................................................................................................119
8.1 Abbreviations ...................................................................................................................... 119
8.2 List of figures....................................................................................................................... 122
8.3 List of tables......................................................................................................................... 123
Introduction 1
1 Introduction
Foodborne diseases are a growing and widespread public health problem in developing, but also
in the developed countries. Foodborne diseases are caused by infectious or toxic agents that
enter the body by ingestion of food. In 2005, 1.8 million people died from diarrhoeal diseases
and the majority of these cases could be ascribed to contamination of food and drinking water.
Diarrhoe is one of the major causes for malnutrition of infants and young children in
developing countries. But even in industrialised countries, the percentage of people suffering
from foodborne diseases per year is up to 30%. In general, foodborne diseases are sporadic and
often not reported, but outbreaks may take on enormous proportions. An outbreak of
Salmonellosis due to contaminated ice cream in the USA in 1994 affected an estimated 224,000
people. Salmonellosis is one of the major foodborne diseases and caused by Salmonella (WHO,
2007). The increased centralisation and industrialisation of our food supply has increased the
distribution of this bacterium. In addition on its impact on public health, Salmonella enterica
serovar Typhimurium is an efficient model for the investigation of elementary mechanisms of
bacterial pathogenesis, because genetic manipulation is easy. In addition, infection of mice
exhibits many of the hallmarks of typhoid fever in humans.
1.1 Salmonella enterica and its pathogenesis
Salmonella enterica is a Gram-negative bacterium belonging to the family of
Enterobacteriaceae. It is a rod-shaped, flagellated and facultative intracellular bacterium (Figure
1-1). Salmonella was first described in 1880 by Eberth and cultured in 1884 by Gaffky
(Burrows, 1959). More than 2,500 serovars have been described which are forming a genus
with the species Salmonella enterica and Salmonella bongori. Salmonella enterica is further
divided into 6 subspecies (I, II, IIIa, IIIb, IV and VI). Nearly all serovars leading to infections in
warm-blooded animals, including humans, are belonging to the subspecies I of S. enterica
(Brenner et al., 2000; RKI, 2009). Salmonella is the causative of diseases in human and also in
a variety of animals with symptoms ranging from enterocolitis to typhoid fever. Typhoid fever
is mainly caused by Salmonella enterica serotype Typhi or Salmonella enterica serotype
Paratyphi whereas the generally mild and self-limiting gastroenterititis is mainly caused by
Salmonella enterica serotype Typhimurium (S.Typhimurium) and serotype Enteritidis. Typhoid
fever is rare in the USA and in Europe but in Asia, South Africa and South America it is
3 6 endemic. The infectious dose for typhoid fever is ranging from 10 -10 bacteria. The bacteria
overcome the acidic gastric barrier and compete with the normal flora resident in intestinal
tract. Once in the small intestine, the bacteria adhere to the mucosal cells in order to penetrate
Introduction 2
the mucosa in the terminal ileum. Although Salmonella are able to invade epithelial cells, the
internalisation is mainly achieved by invading M-cells, specialised epithelial cells of the follicle
associated epithelium of the Peyer`s patches, overlying the gut-associated tissue (Popiel and
Turnbull, 1985; Clark et al., 1994; Kops et al., 1996).


Figure 1-1 EM micrograph of Salmonella enterica serovar Typhimurium
Depicted is a pseudo-coloured scanning electron micrograph showing S. Typhimurium (red) invading cultured
polarised epithelial cells. A pseudo-sequence of events during invasion is shown. The micrographs are unpublished
data from a collaboration of Roman Gerlach and Manfred Rohde.

M-cells, present under the follicle associated epithelium, are specialised in transporting antigens
from the lumen to the antigen presenting cells (Kraehenbuhl and Neutra, 2000). After crossing
the epithelial cell barrier, Salmonella infect cells found in the subepithelial dome of Peyer's
patches. An alternative mechanism to enter the host is via dendritic cells (DC) present in the
Peyer´s patches or lamina propria that breach the epithelial barrier and sample luminal bacteria
(Rescigno et al., 2001). Bacteria are first found in the lymphoid organs draining the intestine,
Peyer`s patches and mesenteric lymph nodes and later they are also present in the spleen.
Salmonella are able to survive and replicate within the mononuclear phagocytes of the
lymphoid follicles, liver and spleen (House et al., 2001). Intracellular Salmonella can
furthermore activate caspase-1-dependent apoptotic killing of macrophages in the early phase
of infection. Simultaneously, polymorph nuclear monocytes and other components of the
immune system are recruited to the site of infection and may provide a new intracellular niche
for Salmonella (Monack et al., 2000). Dependent on the number of invaded bacteria, their
virulence and the host cell response, later during infection, bacteria can be released into the
blood stream, resulting in bacteraemia and systemic dissemination of the pathogen into the host
(Parry et al., 2002) (Figure 1-2).
Introduction 3


Figure 1-2 Pathogenesis of Salmonella
A model of the possible routes used by Salmonella to cross the intestinal epithelial cells is depicted. The main
entry is via M-cells, although epithelial cells can also be invaded. Another route is the sampling of luminal bacteria
by DCs. Invaded Salmonella infect cells of the subepithelial dome and can induce caspase-1-dependent cell death
of macrophages in the early phase of infection, resulting in release of bacteria and inflammation. Additionally,
Salmonella can replicate and survive within macrophages. The figure is adapted and modified from Sansonetti
(2002). SPI: Salmonella pathogenicity island (see 1.2)

1.1.1 Virulence factors of Salmonella enterica
Salmonella have evolved remarkable mechanisms to persist within their hosts allowing the
survival and replication within the host as well as transmission between different hosts.
Salmonella invading the host encounter a variety of hazards: nutrient limitations, low
osmolarity and extreme changes in pH. Furthermore intracellular Salmonella have to overcome
several levels of mammalian host responses. First, Salmonella have to cope with the low pH in
the stomach. In the intestine the bacteria are encountered by low oxygen levels, increased
Introduction 4
osmolarity and the normal gut flora competing for nutrition. Later during infection, within
macrophages, Salmonella reside within phagolysosomes and are threatened by low pH,
oxidative stress, various antimicrobial peptides and nutrient limitations. If the bacteria are
released into the blood stream, they additionally have to survive attack by the complement
system. Different virulence factors have been identified that help the bacteria to cope with the
host defence mechanisms they are exposed to during the infection process.
After ingestion, Salmonella have to overcome the harsh acidic conditions in the stomach and
later during infection the bacteria are taken up in the acidic phagolysosomes of macrophages.
Salmonella respond to the acidic environment by a complex adaptive system called the acidic
tolerance response (ATR) (Foster and Hall, 1990). Acid stress is a combination of low pH
(inorganic acid) and the concentration of weak organic acids, present in the microbial
environment. The ATR requires the synthesis of over 50 acid shock response proteins (ASP)
that are thought to aid cell survival by preventing or repairing macromolecular damages caused
by stress and can be grouped into different systems. The first system includes a subset of ASP
induced by the major iron regulatory protein Fur, which is dependent on the alternative sigma
factor RpoS. The second system is controlled by the two-component regulatory system PhoPQ.
The RpoS system is mainly required for the protection against organic acid stress, whereas the
PhoPQ system is mostly activated during inorganic acid stress (Foster, 1991; Bearson et al.,
1998).
Phagocyte-derived reactive oxygen species generated by the NADPH phagocyte oxidase as
well as the reactive nitrogen species produced by the inducible NO synthase (iNOS) play an
important role in innate immunity to Salmonella (Vazquez-Torres et al., 2000a; MacMicking et
al., 1997). Reactive oxygen as well as reactive nitrogen species can mediate damages of the
cellular components including oxidation of cellular membranes and enzymes, DNA damage and
mutagenesis and the inhibition of membrane transport processes (Weiss, 1986). Salmonella
evolved different antioxidant strategies to resist against reactive oxygen species such as
scavengers, detoxifying enzymes, and repair systems. During oxidative stress a subset of
proteins is induced including catalases, superoxide dismutases or peroxinitrase (Kagaya et al.,
1992; De Groote et al., 1997; Sly et al., 2002; Bryk et al., 2000; Piddington et al., 2001).
Epithelial cells of the gut lumen are protected by antimicrobial agents including cationic
antimicrobial peptides (CAMP). In Gram-negative organisms they interact with Lipid A of LPS
on the outer membrane of the bacteria and induce leakage in the bacterial membrane.
Salmonella avoid damage by CAMP through modification of the Lipid A structure or CAMP
cleavage (Guo et al., 1997; Guo et al., 1998). The outer membrane protease PgtE protects

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