Regulatory pathways and virulence inhibition in Listeria...
Transcript of Regulatory pathways and virulence inhibition in Listeria...
Regulatory pathways and virulence inhibition in Listeria monocytogenes
Christopher Andersson
Department of Molecular Biology
Umeå 2016
Responsible publisher under Swedish law: the Dean of the Medical Faculty
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7601-397-7
ISSN: 0346-6612
UMEÅ UNIVERSITY MEDICAL DISSERTATION New Series No: 1772
Elektronisk version tillgänglig på http://umu.diva-portal.org/
Tryck/Printed by: Print och Media, Umeå Universitet
Umeå, Sverige 2016
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Table of Contents Abstract ..................................................................................................................................... ii
Populärvetenskaplig sammanfattning på svenska ................................................................... iii
Papers included in this thesis ................................................................................................... iv
Abbreviations ............................................................................................................................ v
Introduction ............................................................................................................................. 1
General information about Listeria monocytogenes ........................................................... 1
From saprophyte to pathogen............................................................................................. 1
Role in the environment ................................................................................................. 2
Transition to pathogen ................................................................................................... 3
Virulence ............................................................................................................................. 4
Regulation ....................................................................................................................... 4
Virulence factors ............................................................................................................. 8
Pathogenesis ..................................................................................................................... 10
Cells .............................................................................................................................. 10
Homo sapiens sapiens .................................................................................................. 12
Infection models ........................................................................................................... 13
Aims of thesis ......................................................................................................................... 17
Results and Discussion ........................................................................................................... 18
A novel role of the PrfA regulated protein ActA at non-pathogenic conditions ................ 18
L. monocytogenes displays a ring pattern when grown on motility agar ..................... 18
Light exposure increases EPS production ..................................................................... 19
Blue light exposure induces Listeria monocytogenes stress response ......................... 20
Virulence inhibition by regulator inactivation ................................................................... 23
1 and 2 inhibit PrfA:s ability to bind to DNA ................................................................. 24
Chicken embryos as an alternative infection model.......................................................... 25
Conclusions ............................................................................................................................ 28
Acknowledgements ................................................................................................................ 29
References.............................................................................................................................. 30
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Abstract
Listeria monocytogenes is a rod-shaped Gram positive bacterium. It generally
exist ubiquitously in nature, where it lives as a saprophyte. Occasionally it
however enters the food chain, from where it can be ingested by humans
and cause gastro-intestinal distress. In immunocompetent individuals L.
monocytogenes is generally cleared within a couple of weeks, but in
immunocompromised patients it can progress to listeriosis, a potentially life-
threatening infection in the central nervous system. If the infected individual
is pregnant, the bacteria can cross the placental barrier and infect the fetus,
possibly leading to spontaneous abortion.
The infectivity of L. monocytogenes requires a certain set of genes, and the
majority of them is dependent on the transcriptional regulator PrfA. The
expression and activity of PrfA is controlled at several levels, and has
traditionally been viewed to be active at 37 C (virulence conditions) where
it bind as a homodimer to a “PrfA-box” and induces the expression of the
downstream gene.
One of these genes is ActA, which enables intracellular movement by
recruiting an actin polymerizing protein complex. When studying the effects
of a blue light receptor we surprisingly found an effect of ActA at non-virulent
conditions, where it is required for the bacteria to properly react to light
exposure.
To further study the PrfA regulon we tested deletion mutants of several PrfA-
regulated virulence genes in chicken embryo infection studies. Based on
these studies we could conclude that the chicken embryo model is a viable
complement to traditional murine models, especially when investigating
non-traditional internalin pathogenicity pathways. We have also studied the
effects of small molecule virulence inhibitors that, by acting on PrfA, can
inhibit L. monocytogenes infectivity in cell cultures with concentrations in the
low micro-molar range.
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Populärvetenskaplig sammanfattning på svenska
Listeria monocytogenes, även känd som ”Listeriabakterien”, är en
Grampositiv bakterie som i vanliga fall finns i naturen. Ibland kan den dock
sprida sig till mat, och då främst opastöriserade mejeriprodukter. Vad som
gör Listeriabakterien extra farlig är att den kan växa i mat som förvaras i
kylskåp. Om smittad mat därefter äts utan att ha blivit ordentligt upphettad
kommer bakterien i kontakt med människan. För friska individer är det i
allmänhet ingen fara, utan ger oftast bara några dagars magproblem. Om
man däremot har nedsatt immunsystem eller är gravid kan den dock vara
mycket farlig. Om bakterien lyckas sprida sig till hjärnan kan den ge upphov
till sjukdomen ”listerios”, vilket har en dödlighet på ca 20-30 %. Om man som
gravid får i sig bakterien kan den sprida sig till fostret och orsaka missfall.
I denna avhandling beskrivs en ny egenskap hos Listeria där vi visar att
bakterien reagerar på ljus genom att aktivera skyddsmekanismer, vilket kan
vara av vikt för matindustrin. Vi visar även att ett protein som tidigare
framförallt har antagits vara viktigt för bakterien när den orsakar sjukdom
även har en viktig roll utanför människokroppen.
Avhandlingen beskriver även två nya ”små molekyler” som försöker
bekämpa Listeriabakteriens sjukdomsframkallande förmåga, men gör det på
ett sätt som, jämfört med vanlig antibiotika, inte lika lätt ger upphov till
resistans.
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Papers included in this thesis
Paper I Cycles of light and dark co-ordinate reversible colony differentiation in Listeria monocytogenes. Teresa Tiensuu†, Christopher Andersson†, Patrik Rydén and Jörgen Johansson* Mol Microbiol. 2013 Feb;87(4):909-24. doi: 10.1111/mmi.12140. Epub 2013 Jan 21. Paper II Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA. James A. D. Good†, Christopher Andersson†, Sabine Hansen†, Jessica Wall†, K. Syam Krishnan†, Christin Grundström, Moritz S. Niemiec, Karolis Vaitkevicius, Erik Chorell, Uwe H. Sauer, Pernilla Wittung-Stafshede, A. Elisabeth Sauer–Eriksson*, Fredrik Almqvist*, and Jörgen Johansson* Manuscript Paper III Exploring the chicken embryo as a possible model for studying Listeria monocytogenes pathogenicity. Jonas Gripenland†, Christopher Andersson† and Jörgen Johansson* Front Cell Infect Microbiol. 2014 Dec 10;4:170. doi: 10.3389/fcimb.2014.00170. eCollection 2014. Published but not included in the thesis Using the chicken embryo to assess virulence of Listeria monocytogenes and to model other microbial infections. Christopher Andersson, Jonas Gripenland*, Jörgen Johansson*. Nat Protoc. 2015 Aug;10(8):1155-64. doi: 10.1038/nprot.2015.073. Epub 2015 Jul 2. † indicates equal contribution * indicates corresponding author(s) Articles reproduced with permission
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Abbreviations
L. monocytogenes = Listeria monocytogenes
E. coli = Escherichia coli
LLO = Listeriolysin O
A.a. = Amino acid
Bp = Base pair
RNAP = RNA polymerase
CNS = Central nervous system
TEM = Transmission electron microscopy
EPS = Extracellular polymeric substance
UTR = Untranslated region
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Introduction
General information about Listeria monocytogenes
The Listeria genus consist of 10 different species, but it is mainly Listeria
monocytogenes that cause disease in humans. L. monocytogenes is a rod
shaped Gram-positive facultative anaerobic bacterium, and the causative
agent behind listeriosis, a severe CNS infection (reviewed in Swaminathan
and Gerner-Smidt 2007).
While several different L. monocytogenes strains exist, in this work, unless
otherwise specified, EGDe is used as the wild type strain.
EGDe has a 2.9 Mbp genome that was first sequenced in 2001, and contains
3044 annotated (including putative) genes (Glaser, Frangeul et al. 2001).
Other wild type strains exists, and it has been shown that there are several
important differences between them. The most striking being between the
well-used EGD and EGDe strains, where EGD harbor a point mutation in prfA,
the main virulence regulator, rendering it constitutively active (Becavin,
Bouchier et al. 2014).
From saprophyte to pathogen
L. monocytogenes exists ubiquitously in nature, where it is believed to live as
a saprophyte. In nature it can cause disease in domestic animals, but is
otherwise of little threat to humans. L. monocytogenes can however
occasionally come in contact with our food, either through contaminated
animals or contaminated food processing equipment. The bacterium is
considered a serious concern for the food industry, primarily due to its ability
to not only survive, but also proliferate in a wide range of environmental
conditions. Growth of L. monocytogenes at temperatures from 0 up to 43 C
have been reported (Knabel, Walker et al. 1990, Walker, Archer et al. 1990)
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and add to that a pH range of 4.3-9.6 and high osmotolerance, most of the
common techniques for limiting bacterial growth in food products have been
countered (Cole, Jones et al. 1990). Due to this versatility it can replicate to
high numbers once in contact with food, even if refrigerated. If the food is
then not thoroughly heated before consumption, as is commonly the case
with ready-to-eat food, living bacteria can be ingested.
The presence of L. monocytogenes in the intestine can have different effects,
depending on the infected person’s immune status. If the person is healthy,
no symptoms or some gastrointestinal distress might occur. However, if the
person is immunocompromised the bacteria can cause listeriosis (meningitis)
or, if the person is pregnant, abortion.
Role in the environment
L. monocytogenes has been isolated from a wide range of environmental
sources, e.g. different types of plants and soil, from which the bacteria can
enter livestock. L. monocytogenes has the ability to infect several different
kinds of animals (predominantly ruminants), with studies showing that
approximately 20 % of the investigated manure from cattle harbors Listeria.
Not all of the animals display symptoms of an infection, suggesting that cattle
might be a natural reservoir (Nightingale, Schukken et al. 2004).
While L. monocytogenes is a natural biofilm former, the amount and nature
of the biofilm is heavily dependent on the strain and its environment
(Borucki, Peppin et al. 2003). The L. monocytogenes biofilm consists of EPS
(Extracellular Polymeric Substance), although the exact composition has not
been thoroughly elucidated (see Giaouris, Heir et al. 2015 for a review). A
common factor in the examined biofilms is however the presence of
extracellular DNA. Research have shown that DNA plays a central role in
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formation of L. monocytogenes single species biofilms, and the addition of
DNase is able to disperse already formed biofilm (Harmsen, Lappann et al.
2010, Nguyen and Burrows 2014).
Transition to pathogen
When L. monocytogenes enters a host, the expression of several key
virulence genes is increased. This transition is considered to be facilitated by
the main virulence regulator: PrfA. The level of PrfA is controlled by several
factors, including a thermosensor in its 5’-UTR attenuating translation at
temperatures below 37 C (Johansson, Mandin et al. 2002).
A temperature of 37 C is however not enough to fully activate PrfA; in vitro
experiments have displayed a significant impact of the environmental
conditions, i.e. the growth media. There is also an interplay between
temperature and L. monocytogenes motility. L. monocytogenes can employ
either multi-flagellar based motility or actin based propulsion depending on
its environment. When L. monocytogenes lives outside a host (<30 °C), it is
flagellated and utilizes these flagella for movement. When the bacteria
encounters higher temperatures, like those in a host, the flagellar expression
is halted and the bacteria are non-flagellated (Peel, Donachie et al. 1988,
Williams, Joseph et al. 2005).
The presence of a readily available carbon source, such as glucose and
cellobiose, has been shown to inhibit PrfA activity, possibly through the
phosphotransferase system (Deutscher, Herro et al. 2005, Mertins, Joseph et
al. 2007, Joseph, Mertins et al. 2008).
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Virulence
Regulation
Virulence in L. monocytogenes is regulated in a complex network, but so far
the two main players that have been identified are the transcriptional
regulator PrfA and the alternative sigma factor B.
Sigma Factors
Prokaryotic gene regulation is heavily dependent on the use of sigma factors:
proteins that bind to the RNAP and targets it to specific promoters. Sigma
factors responsible for the baseline expression of genes during normal
conditions are called housekeeping sigma factors, and sigma factors with an
affinity to promoters for genes needed to respond to “non-normal”
conditions are classified as “alternative sigma factors”. The alternative sigma
factors are normally bound to anti-sigma factors, and their release and
activity is modulated in a complex network to yield the desired outcome (see
Browning and Busby 2004 for a review).
L. monocytogenes has a total of 5 annotated sigma factors: A, B, C, H and
L (Glaser, Frangeul et al. 2001), with A (RpoD) being the housekeeping
sigma factor, while B, C, H and L are considered alternative sigma factors.
Alternative Sigma Factors
While B is strongly associated with stress response and virulence, the roles
of C, H and L are not as clearly defined, possibly due to limited phenotypic
effects (Mujahid, Orsi et al. 2013).
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B
B (encoded by sigB) is the alternative sigma factor with the largest regulon,
and a sigB deletion mutant have a deficient stress response rendering it more
sensitive to adverse conditions (e.g. acidic environments, oxidative stress,
carbon starvation) (Becker, Cetin et al. 1998, Ferreira, O'Byrne et al. 2001,
Kazmierczak, Mithoe et al. 2003, Mujahid, Orsi et al. 2013). While several
studies have focused on its role in environmental stress survival, many have
also investigated the role of B in virulence, revealing both PrfA (the main
virulence regulator) dependent and independent regulation of virulence
genes (Nadon, Bowen et al. 2002, Ollinger, Bowen et al. 2009). Interestingly,
inlA and inlB belong to a set of genes whose expression is controlled by both
PrfA and by B (McGann, Raengpradub et al. 2008).
Activity modulation
Like all sigma factors, B induces the expression of its target genes by
recruiting the RNAP to an upstream consensus sequence. To facilitate the
ability to quickly change the expression patterns, bacteria usually don’t use
a synthesis/degradation approach for sigma factors, but instead utilize anti-
sigma factors. Anti-sigma factors sequesters the sigma factors, thus
preventing their activity unless certain conditions are met. Often this kind of
regulation exists in several steps, including anti-anti sigma factors.
In L. monocytogenes these factors, including B, are encoded by an operon
consisting of 8 gene. These “regulator of sigma b” genes are, in order: rsbR,
rsbS, rsbT, rsbU, rsbV, rsbW, sigB, rsbX. A lot of what is known about the B
activation pathway in L. monocytogenes is based on its function in Bacillus
subtilis.
In B. subtilis, generally considered a close relative to L. monocytogenes, B
activation is the outcome of a complex signal integration. Assuming the
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function is similar in L. monocytogenes, B is bound to its anti-sigma factor
RsbW in an unstressed cell. When the bacteria encounter stress, RsbT binds
to RsbU. The RsbT-RsbU complex phosphorylates RsbV, enabling it to attack
the RsbW-B complex, releasing B (see Marles-Wright and Lewis 2010 for a
review). Some parts of this pathway have been studied in L. monocytogenes,
and the findings support a similar function in as in B. subtilis. There is
however a significant difference. In B. subtilis environmental and metabolic
stress are divided into integrative, but distinct, pathways, requiring the two
additional genes rsbQ and rsbP, which are not present in the L.
monocytogenes genome (Glaser, Frangeul et al. 2001). L. monocytogenes has
instead been proposed to utilize one pathway for both (Chaturongakul and
Boor 2004).
PrfA
PrfA (Positive regulatory factor A) is a protein consisting of 237 amino acids,
and is a member of the Crp/Fnr family of transcriptional regulators. It
controls the expression of over 30 genes, by binding as a dimer to a “PrfA-
box” located 40.5 bp upstream of the transcriptional start site.
PrfA regulation is in itself regulated at all levels. It is regulated at the
transcriptional level by three promoters: PplcA, P1 and P2 (Fig. 1) (Camilli,
Tilney et al. 1993, Freitag, Rong et al. 1993, Freitag and Portnoy 1994). PplcA
is a PrfA-box dependent promoter (whose transcription results in the
bicistronic operon plcA-prfA), enabling PrfA to upregulate its own expression.
Figure 1. Schematic representation of prfA regulating promoters.
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P1 is A dependent and P2 has an overlapping A-B sequence (Rauch, Luo et
al. 2005). Once it is transcribed, prfA is post-transcriptionally regulated by
both a thermosensor in the 5’ UTR (Johansson, Mandin et al. 2002), and also
by the trans-acting riboswitches SreA and SreB (Loh, Dussurget et al. 2009).
The regulation of PrfA dependent transcription is however not only relying
on the levels of PrfA protein. To induce transcription, members of the
Crp/Fnr family generally bind an activating factor (cAMP in the case of the E.
coli Cap protein), after which the protein bind to the DNA as a homodimer.
In the case of PrfA it has been shown to dimerize and, based on the large
increase of activity during pathogenic conditions, is also believed to require
an activating factor. The activation is proposed to require a conformational
change, stabilizing the DNA binding Helix-Turn-Helix motif. This theory is
supported by findings showing a massive upregulation of PrfA regulated
genes during intracellular growth (Knabel, Walker et al. 1990, Moors, Levitt
et al. 1999), and the existence of prfA mutations rendering PrfA constitutively
active or inactive. The most prominent of these is the strongly activating
G145S mutation (Bockmann, Dickneite et al. 1996, Eiting, Hageluken et al.
2005). A large difference is however that PrfA, unlike Crp, has been found to
be able to bind to DNA even in its inactive state (Freitag, Youngman et al.
1992, Sheehan, Klarsfeld et al. 1995). It has recently been proposed that
glutathione, in its reduced form, is PrfA’s activating factor (Reniere, Whiteley
et al. 2015). When active, PrfA regulates the expression of several genes
important for virulence (Fig. 2), and a mutant lacking functional PrfA is
avirulent. Interestingly, the PrfA-boxes identified does not always follow the
consensus, opening up for another level of regulation. This is also supported
by the relatively high basal expression of hly (perfect consensus), compared
to that of actA (1 mismatch). Mutational experiments indicating that such
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regulation might indeed exist has been conducted, but was not completely
conclusive.
Virulence factors
ActA
ActA is a 639 a.a. PrfA regulated protein. It is associated with the bacterial
membrane, protruding out through the cell wall (Kocks, Gouin et al. 1992).
The exact distribution pattern of ActA has however been suggested to vary
depending on the environmental conditions the bacterium lives in.
During intracellular conditions, the expression of ActA is heavily induced,
probably through the increased activity of PrfA (Moors, Levitt et al. 1999,
Shetron-Rama, Marquis et al. 2002). In these conditions ActA display an
“inverse polarized” distribution, resulting in one pole without any ActA
(Kocks, Hellio et al. 1993, Garcia-del Portillo, Calvo et al. 2011). When in the
cytosol, ActA recruits the host ARP2/3-WASP complex. Due to ActA’s
distribution pattern, the ARP2/3-WASP complex can polymerize actin in all
directions but one, thus propelling the bacteria in that direction. ActA is
essential for intracellular movement, which is required for Listeria to
properly spread from cell to cell. L. monocytogenes lacking a functional ActA
is severely dampened in its pathogenicity. This reduction might not only be
Figure 2. Schematic representation over some of the most central PrfA-
dependent promoters.
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due to a lack in intracellular motility however, as recent research have shown
ActA to be involved in aggregation and biofilm formation, possibly affecting
intestinal adherence (Travier, Guadagnini et al. 2013).
Listeriolysin O
Listeriolysin O (LLO, 529 a.a.), transcribed from the hly gene, is one of the
most famous virulence factors of L. monocytogenes, and is essential for its
pathogenicity. LLO is a secreted pore forming toxin that is mainly active in
acidic conditions, such as those found in a L. monocytogenes containing
vacuole (Beauregard, Lee et al. 1997, Glomski, Gedde et al. 2002).
Expression of hly is controlled by PrfA, but basal levels of PrfA is enough to
yield expression. When L. monocytogenes is growing intracellularly LLO is
however not found in any abundance inside the cell cytosol, probably due to
a combination of transcriptional control and a rapid degradation by host
factors (Villanueva, Sijts et al. 1995, Moors, Levitt et al. 1999).
Internalins
The internalin family is characterized by its LLR (Leucin Rich Repeats) and has
over 20 members. Several different proteins in the internalin family has been
shown to be connected to virulence, such as InlC and InlJ (Bergmann,
Raffelsbauer et al. 2002, Sabet, Lecuit et al. 2005), but only the two main
ones, InlA and InlB, have a clearly elucidated role in the actual internalization
of the bacteria.
InlA
InlA is 801 a.a. large protein. It has a LPXTG domain, and its correct (covalent)
anchoring to the cell wall is required for full activity. During the infection
process, InlA bound to the bacterial cell-wall interact with E-cadherin on
mammalian cells (Mengaud, Ohayon et al. 1996). This interaction has been
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shown to induce thyrosine phosphorylation of E-cadherin, leading to
ubiquitination. The ubiquitinated E-cadherin is internalized into the cell, and
it has been suggested that L. monocytogenes hijack this pathway for its own
internalization (Bonazzi, Veiga et al. 2008). While the exact mechanism of
InlA induced internalization remains to be elucidated, its importance for L.
monocytogenes invasion into cells exposing E-cadherin is well documented
(Gaillard, Berche et al. 1991, Lecuit, Ohayon et al. 1997).
inlA is the first gene in a bicistronic operon together with inlB. The expression
of the operon is partially regulated by PrfA, but also B (McGann,
Raengpradub et al. 2008).
InlB
InlB is a 630 a.a. protein belonging to the InlB type family of internalins
(Gaillard, Berche et al. 1991). InlB is, in contrast to InlA, lacking a LPXTG
domain. Instead it is believed to be loosely attached to the cell wall by GW
domains (Jonquieres, Bierne et al. 1999). InlB interacts with the c-Met
receptor and induces bacterial internalization (Braun, Ohayon et al. 1998,
Parida, Domann et al. 1998, Shen, Naujokas et al. 2000).
Expression of inlB is regulated by both PrfA and the B promoter upstream
of inlA, but also by a PrfA dependent promoter in the intragenic region
between inlA and inlB (Wurtzel, Sesto et al. 2012).
Pathogenesis
Cells
When L. monocytogenes comes in contact with human cells it has the
capacity to internalize itself into both professional and non-professional
phagocytes. Depending on the type of cell encountered, the interaction will
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require one or more of a group of L. monocytogenes virulence factors called
internalins. The most famous, and possibly most important, of these are InlA,
which recognizes E-cadherin (Mengaud, Ohayon et al. 1996), and InlB, a c-
Met receptor agonist (Shen, Naujokas et al. 2000). Other virulence factors of
note are P60, which has also been implicated in internalization (Hess,
Gentschev et al. 1995, Pilgrim, Kolb-Maurer et al. 2003) and ActA, which is
needed for maximum uptake into epithelial cells (Suarez, Gonzalez-Zorn et
al. 2001). Several other factors affecting internalization have been identified,
but their mode of action has not been extensively elucidated.
When the bacteria first enter the cell they are confined in a single membrane
vacuole, which is lysed by the actions of the cholesterol dependent cytolysin
LLO together with the action of phospholipases (e.g. PlcA and PlcB) (Gaillard,
Berche et al. 1987, Marquis, Doshi et al. 1995, Grundling, Gonzalez et al.
2003).
In the cytosol the bacterium utilizes Hpt, a hexose phosphate transporter, to
take up glucose-1-phosphate and other hexose phosphates from the cell. Hpt
is not essential for cytosolic replication, but is required for rapid cell division
(generation time of approximately 40 minutes) (Chico-Calero, Suarez et al.
2002).
To spread from an infected cell, L. monocytogenes utilize ActA to recruit
cellular actin through the host factors Arp2/3-WASP, allowing the bacteria to
be propelled through the cytosol. When the bacteria reaches the cell
membrane it extends it into the adjacent cell, finally resulting in a double
membrane enclosed vacuole from which L. monocytogenes escapes to
perpetuate the infection without exposing itself to the extracellular
environment (Fig. 3). It should be noted that prolonged infection will
eventually lead to death of infected cells (data not shown).
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Homo sapiens sapiens
Infection in humans usually occur through the oral route, and is calculated to
require a relatively high bacterial dose (107-109) for healthy individuals, but
is significantly lower (105-107) for at-risk individuals (elderly, pregnant or
otherwise immunocompromised) (Farber, Ross et al. 1996). Once it has
reached the intestine, it can induce its uptake by mucosal epithelia or
Figure 3. Schematic overview L. monocytogenes cellular infection.
L. monocytogenes: A) comes in contact with a cell and initiates uptake with the
help of (mainly) InlA and InlB. B) is internalized into a vacuole. C) lyse the vacuole
through the actions of LLO, PlcA and PlcB. D) is free in the cytosol, and rapidly
replicate by utilizing host sugars through the hexose transporter Hpt. E) recruits
host factors to polymerize actin, propelling the bacterium forward. F) protrudes
through the cell membrane into an adjacent cell. G) is encapsulated in a new
vacuole, with a double membrane. H) lyse the vacuole, and perpetuate the
infection cycle (I). Adapted from de las Heras, Cain et al. (2011).
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macrophages, from where it can spread to the liver and spleen where it is
primarily taken up by professional phagocytes. In healthy individuals the
infection is generally cleared by this point, but in at-risk persons the bacteria
can spread to the brain or, in the case of pregnant individuals, the fetus. If L.
monocytogenes reaches the brain, it can cause the potentially lethal disease
listeriosis. If the bacteria manages to reach the fetus, it can cause prenatal
infection, possibly leading to abortion/stillbirth.
Immune Response
Most of the knowledge about how the immune system reacts to L.
monocytogenes comes from studies conducted in the murine model. What
has been seen is that the bacteria localize to the liver and spleen, where they
are rapidly internalized by resident macrophages (Conlan 1996). This early
response by the innate immune system is required to combat the infection
until specific CD4+ and CD8+ T-cells can be recruited (see Zenewicz and Shen
2007 for a review).
Infection models
Pre-clinical studies of virulence factors can generally be divided into three
different levels. First is the classical in vitro function, where expression
patterns and protein interactions can be elucidated. To determine what role
virulence factors can have in an actual host, the next level is normally to
employ different kinds of immortalized cell lines. Established cell lines are
easy to work with, but how well they actually represent a normal cell in a
host can definitely be questioned. The final level of pre-clinical research is
usually to utilize an animal model to mimic the conditions in a host.
Several different organisms have been suggested as models for studying L.
monocytogenes infection, each with its distinct advantages and
disadvantages concerning relevance, cost, work load etc.
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Caenorhabditis elegans
The nematode C. elegans is a favored model for research in several fields due
to its very well characterized development and ease of use. It require no
specialized facilities, and its transparent cells makes it ideal for in vivo
studies. The use of C. elegans as an infection model for L. monocytogenes has
been proposed, since L. monocytogenes fed to C. elegans accumulated in the
worm gut, leading to death over the course of several days (Thomsen, Slutz
et al. 2006). The long time to death together with an infection temperature
of 25 °C and an apparent lack of intracellular infection (Thomsen, Slutz et al.
2006) makes to model unsuitable for general infection studies. It should
however be noted that C. elegans has been suggested as a natural reservoir
for L. monocytogenes (Neuhaus, Satorhelyi et al. 2013), possibly making the
model interesting for non-infection studies.
Drosophila melanogaster
The common fruit fly Drosophila melanogaster is widely used in research as
a model organism, and has been proposed as a model for studying Listeria
infections (Mansfield, Dionne et al. 2003). Due to its extensive use in
molecular biology, general methods and mutants are easily available. D.
melanogaster does however have a temperature maximum of 30 °C, limiting
their potential for mimicking a human infection. It has been also shown that
the D. melanogaster model is highly sensitive to the common growth media
“Brain Heart Infusion” (resulting in 30–40 % mortality) as well as bacteria that
are generally not considered pathogenic (Bacillus subtilis and Listeria
innocua) when exposed through the common injection (picking) technique
(Jensen, Pedersen et al. 2007).
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Galleria mellonella
Galleria mellonella (the greater wax moth), or more specifically its larvae, has
recently been promoted as a powerful infection model for bacterial
pathogens. The advantages with the model is its relatively low cost, and its
ability to survive at 37 °C. Experimental procedures for both forced infection
(injection of pathogens into tissue) and forced oral uptake are well
documented (Ramarao, Nielsen-Leroux et al. 2012). Disadvantages with the
model is however a need for routine maintenance of the moths and larvae,
and the method might be more experimentally demanding. It should also be
noted that a relatively specific injection dose is required for injection into the
hemocoel, and data indicate large differences in pathogenicity depending on
the L. monocytogenes strain used (Mukherjee, Altincicek et al. 2010).
Mus musculus
The house mouse (Mus musculus) has for a long time been the “go-to” model
when studying infections in higher organisms. It is very well studied, and
genetic variants are relatively easily available. The model does have its
drawbacks. It requires large specialized facilities, and its relevance for
studying some human infections has been questioned. This is especially true
for L. monocytogenes infection, as a wild type mouse E-cadherin has glutamic
acid instead of proline at position 16 (compared to a human) that renders it
incompatible with wild type InlA, severely reducing L. monocytogenes
infectivity (Lecuit, Dramsi et al. 1999). This limitation can be, and has been,
addressed by mutating either the mouse E-cadherin, or the L.
monocytogenes InlA. Such mutants have been shown to restore receptor
interaction, and increase infectivity (Lecuit, Vandormael-Pournin et al. 2001,
Wollert, Pasche et al. 2007). The need for such action does however highlight
that mice are not a normal host for L. monocytogenes.
16
Meriones unguiculatus
The gerbil (Meriones unguiculatus) has been shown to be a possible model
for studying L. monocytogenes infection. It is suggested as a natural host for
L. monocytogenes, and its E-cadherin and c-Met receptor are compatible
with wild type L. monocytogenes internalins, and can successfully be infected
through the oral route. It does however require specialized facilities similar
to that of the Mus musculus model, and data has shown a discrepancy
between the function of InlA and InlB compared to that of mice carrying the
humanized E-cadherin (Disson, Grayo et al. 2008). The use of gerbils as a
model system is not widespread, and tools for genetic manipulations are not
as mature as those available for other models.
Gallus gallus
An alternative to the mouse model is the use of chicken (Gallus gallus
domesticus). They have the major advantage of being an occasional host for
natural L. monocytogenes infections (Bailey, Fletcher et al. 1989, Bailey,
Fletcher et al. 1990, Cooper, Charlton et al. 1992), and harbor a compatible
E-cadherin. They are however large, and require specialized large facilities.
The use of chicken embryos is on the other hand easy, requires no specialized
facilities and they are relatively cheap. Previous studies have established that
infection of L. monocytogenes in chicken embryos is possible, but a thorough
study comparing isogenic mutants in the known internalization factors had
not been conducted (Olier, Pierre et al. 2002, Olier, Pierre et al. 2003,
Severino, Dussurget et al. 2007, Yin, Tian et al. 2011).
One should however be aware that, depending on how old the embryos are
at infection, their immunocompetence might vary. Functional macrophages,
for example, have been shown to be present first at day 12-16 (Jeurissen and
Janse 1989).
17
Aims of thesis
The general aim of the thesis was to investigate L. monocytogenes virulence
and virulence associated genes. It began with trying to determine the mode
of action behind a ring formation phenotype, which turned out to be
connected with virulence factors (Paper 1). After seeing that the roles of
virulence factors are far from completely understood, I wanted to find and
characterize small compounds that could act as virulence inhibitors,
preferably by inhibiting PrfA (Paper 2). During the work, a need to find an
easy animal model for screening for virulence deficiency arose (Paper 3).
18
Results and Discussion
A novel role of the PrfA regulated protein ActA at non-pathogenic
conditions
L. monocytogenes displays a ring pattern when grown on motility agar
When L. monocytogenes EGDe was grown on soft (0.3 % w/v agar) BHI-agar
plates at “bench conditions” we observed that the colonies exhibited a ring
forming growth pattern similar to a classical “bulls-eye” (Paper 1: Fig. 1A).
During studies of a lysine riboswitch, located upstream of lmo0798 (encoding
a lysine transporter), a mutant not displaying this bulls-eye pattern was
isolated. This was unexpected, so to determine the cause the region close to
the riboswitch was sequenced, revealing a mutation in lmo0799 resulting in
a premature stop-codon. By sequence comparison (NCBI blastx), Lmo0799
was found to have great similarity to Bacillus proteins annotated as putative
blue light receptors. To test if the difference in colony morphology could be
due to L. monocytogenes light sensing ability, a clean lmo0799 deletion was
constructed and exposed to different light conditions together with wild
type. The experiments revealed a clear correlation, not directly with light
exposure, but rather with the alternation of light levels (Paper 1: Fig. 1D and
S1A). The deletion of lmo0799 rendered the bacterium unable to sense light.
The number of rings in the bulls-eye pattern was directly correlated to the
amount of times the colony had been exposed to a light switch. Upon further
examination it was discovered that the light also restricted L. monocytogenes
motility (Paper 1: Fig. 2). To better elucidate the pathway behind this
mechanism, several different mutants available in the lab were tested.
Interestingly, a ΔprfA mutant was deficient in the ring forming phenotype
(Paper 1: Fig. 5A). In an attempt to identify the cause behind the PrfA
requirement, we tested mutants with deletions in genes known to be directly
19
linked to PrfA. Of those tested only actA exhibited a similar lack of ring
formation as prfA (Paper 1: Fig. 5A). This was exceptionally interesting for
several reasons; first, why would a classical virulence factor be associated
with a distinct colony morphology? Secondly, ActA had, to our knowledge,
never been described to have any function at non-virulence conditions. The
connection to ActA was far from clear, how could a (putative) light receptor
possibly be connected to ActA?
Since PrfA activity is associated with a transfer from flagellar motility to the
use of actin polymerization through the actions of ActA, could the reason for
the decreased motility be a triggering of this change? To test this we used
TEM to determine if there was any difference in the flagellation state
between the different rings. The results did not indicate any such difference
(data not shown), but Northern blots against the antisense RNA that is known
to inhibit motility showed a distinct upregulation in bacteria exposed to light
compared to those in darkness (Paper 1: Fig. 2B).
Light exposure increases EPS production
After identifying that shifts in light levels act as an environmental cue for ring
formation, the next step was to elucidate the contents of the rings and to
determine their connection with virulence.
By visual inspection, it was hypothesized that the ring formation was due to
different amounts of bacteria in the opaque vs translucent rings. This would
fit well into a model where light inhibited motility, but not cell division,
leading to a “build up” of bacteria resulting in the opaque rings. To verify this,
sections of opaque and translucent rings were subjected to viable count. The
results showed, surprisingly, no significant difference in the amount of viable
bacteria in adjacent translucent/opaque rings (Paper 1: Fig. S5A). Since viable
count measurements do not take non-viable bacteria into account, there
20
might still be a difference in the amount of bacteria between the opaque
rings and the translucent rings. Live/dead staining did however not indicate
any differences between adjacent opaque/translucent rings (Paper 1: Fig.
S5B).
Having ruled out a variance in the amount of bacteria as the main difference
between opaque and translucent rings, we hypothesized that the difference
was due to a secreted factor. To investigate this possibility we utilized Congo
red, a stain that preferentially binds to EPS/biofilm. When we supplemented
our plates with Congo red, a striking association of Congo Red to the opaque
rings could be seen (Paper 1: Fig. 3A), strongly indicating that the difference
between the opaque and translucent rings is an increased amount of EPS. It
should however be noted that the exact target(s) of Congo red has not been
determined, so we also conducted classical crystal violet staining to support
the lightbiofilm connection. The results confirmed a deficiency in the
biofilm formation for the actA mutant, but intriguingly not for the
lmo0799 mutant (Paper 1: Fig. 4D). This opened up for the possibility that
the action of ActA was not directly coupled to the light, but rather as a part
of biofilm formation.
Blue light exposure induces Listeria monocytogenes stress response
To further our understanding of the mechanism(s) involved in the ring
formation, a mariner transposon library was constructed and screened for
phenotypic absence of ring formation. Identified mutants were sequenced,
revealing 36 genes/operons essential for ring formation (Paper 1: Table S1).
When mapping the identified genes to their predicted function, a large group
became apparent: “Adaptations to atypical conditions”. This was interesting,
since genes classified as belonging to “Adaptation to atypical conditions”
generally are connected to the stress response. Finding that disruption of
21
stress response genes knocked out the ring formation phenotype was not so
surprising, since a deletion of central regulators could affect the whole
bacterium. What made it so interesting was that this group made up 30 % of
the hits, indicating that it probably was not only unspecific effects (Fig. 4).
Most of the identified mutants in this group had a transposon insertion into
genes coding for proteins involved in B activation/repression.
This fitted well with the data showing an increased production of EPS, which
is known to be dependent on the stress response, but the connection to ActA
was still far from obvious. Could it be that ActA was involved in the stress
response? To verify that light indeed induced a stress response, we used the
expression of lmo2230, a highly B dependent gene, as a readout for the
9%5%
7%2%
3%2%
5%
30%5%
19%
2%11%
Transport/binding proteins and lipoproteins Cell surface proteins
Specific pathways Metabolism of amino acids and related molecules
DNA restriction/modification and repair DNA recombination
Regulation Adaptation to atypical conditions
From Listeria From other organisms
No similarity Intergenic region
Figure 4. Pie chart of functional categories of identified transposon mutants
lacking ring formation.
From Listeria
22
stress response. Bacteria exposed to light had a high amount of lmo2230
mRNA, which was almost completely absent in a ΔactA background and in
the examined transposon mutants (Paper 1: Fig. 4A and 5D). To examine if
this deficiency was specific to light exposure, we triggered the stress
response by exposing bacteria to oxidative stress through the use of
hydrogen peroxide. The results showed the same deficiency for ΔactA and all
except two of the transposon mutants. Western blots established that the
amount of B protein was not significantly downregulated for ΔactA and all
but one transposon mutant (Paper 1: Fig. 4B-C and 5B-C). The effect of the
ΔactA mutant must thus be due to a decreased B activity, and the different
effects of the transposon mutants could indicate that separate stress
pathways are involved in the ring formation.
When considering all the data, we came up with a model (Paper 1: Fig. 7)
where light exposure activates lmo0799 which acts as an early warning
system alerting the bacterium for an impending increase in ROS. This
activates a B dependent pathway, leading to a general induction of the
stress response, increasing prfA expression causing ActA upregulation. While
the details regarding the feedback from ActA back to the stress response still
remains to be elucidated, it does present a very interesting role for this
classical virulence factor.
Just after the paper was published, another work describing a role for ActA
in aggregation through direct ActA::ActA interactions was published, further
showing that we do in fact know very little about the actual roles of bacterial
proteins (Travier, Guadagnini et al. 2013). During the work with Paper 1,
another article was published characterizing the light-receptor. In the paper
they saw an effect on B and possibly also on virulence, where light exposure
was proposed to increase virulence in a light receptor dependent manner
(Ondrusch and Kreft 2011).
23
Virulence inhibition by regulator inactivation
Small 2-pyridones had previously been identified to have an effect in E. coli
(Svensson, Larsson et al. 2001). To test if similar compounds could have an
effect in L. monocytogenes, we screened several compounds for virulence
attenuation in cell cultures by flow cytometry. The identified hits were
verified by classical cell infection experiments, resulting in two positive hits
(Paper 2: Fig. 1a). When testing the compounds another, similar, compound
was predicated to also be effective. The compound did however not show
any significant virulence inhibition in cell based assays, allowing us to use it
as a negative control (Paper 2: Fig. 1a).
Once we had established the compounds effects on virulence, we wanted to
examine if they affected PrfA or another of the known virulence genes. To do
this we conducted Northern blot experiments against inlAB, hly, actA and
plcA-prfA. The results showed a clear down-regulation of hly, actA and plcA-
prfA when the bacteria were treated with compound 1 and 2, but not 3
(Paper 2: Fig. S4). The compounds inability to down-regulate inlAB, the only
genes not completely requiring PrfA, did indeed indicate that the compounds
might act on PrfA directly. To see if the effect could also be seen on the
protein level, Western blots were performed, revealing a decrease of ActA
and LLO, but remarkably not of PrfA (Paper 2: Fig. 2A-C). This suggested that
the compounds acted directly on PrfA’s activity, possibly by direct binding.
To investigate if the compounds bound to PrfA, we used ITC (Isothermal
titration calorimetry), confirming a 1:1 binding of the compounds to PrfA.
Interestingly, 3 also bound to PrfA with similar affinity as 1 and 2 (Paper 2:
Fig. 4a and S6), opening up for the possibility of the compound binding in
different locations or having different functional interactions.
24
1 and 2 inhibit PrfA:s ability to bind to DNA
The ITC data combined with the previous Western blot results strongly
indicated that the compounds somehow impacted PrfA’s activity. One
possible mode of action for the compounds would be that they interfere with
the conformational change necessary for PrfA’s ability to bind to DNA. To
verify this we conducted SPR (Surface Plasmon Resonance) experiments,
where we analyzed the amount of PrfA binding to target DNA in the presence
or absence of compound. The data verified our conclusion, showing reduced
PrfA binding to the hly promoter/”PrfA box” in the presence of 1 or 2, but
only slightly with 3 (Paper 2: Fig. 4b). The effect of 3 increases dramatically
with higher concentrations of compound, with no significant difference
between 1, 2 or 3 at 100 µM. The relevance of such high concentrations in in
vitro experiments can however be questioned, especially in the case of the
SPR since it results in a protein::compound ratio of 1:5000.
Once we had established that 1 and 2 were effective in inhibiting PrfA::DNA
binding, we wanted to test the hypothesis that they interfered with PrfA:s
ability to bind to DNA by keeping the HTH in a non-active confirmation. We
reasoned that a mutant with the HTH locked in an active confirmation should
be unaffected by the compounds, and for that reason utilized a PrfA with the
G145S amino acid substitution, which has been shown to be constitutively
active through stabilization of the HTH (Eiting, Hageluken et al. 2005). When
including this mutant in the SPR experiments, we could show that 1 and 2
were indeed unable to prevent PrfA-binding (Paper 2: Fig. 4c). Interestingly
the PrfAG145S mutant did not display the same level of binding reduction at
higher concentrations as the wild type PrfA, possibly due to a general greater
stability of the PrfAG145S protein::DNA interaction. This was also confirmed by
cell infection assays, where a PrfAG145S strain was unaffected by 1 or 2 (Paper
2: Fig. S2A).
25
Chicken embryos as an alternative infection model
During the work with identifying inhibitory compounds for PrfA, it became
apparent that we had a need to screen for virulence inhibition in vivo. For L.
monocytogenes several model systems have been proposed, most famous is
probably the Meriones unguiculatus (gerbil) and the Mus musculus model.
Both of these were immediately excluded due to their need for large
specialized facilities. Our search lead us to the chicken embryo model. The
model has previously been used for L. monocytogenes, but has not been
extensively characterized. The model has several advantages: the
temperature requirement (37.5 °C) closely resembles that of a human
infection, it is easily handled in a normal lab environment and it is relatively
cheap.
Several different methods of infecting chicken embryos with both virus and
bacteria exists, so we started by determining what would be most
appropriate for our needs. Injection into the egg yolk or onto the
chorioallantoic membrane proved too technically challenging. Instead we
decided to inject the bacteria through a small perforation in the eggshell
directly through the chorioallantoic membrane into the allantoic cavity (Fig.
5). The advantage with this type of infection is its relative ease and the high
certainty of the amount of injected bacteria; the drawback being that
bacteria are artificially forced through several natural barriers.
26
To evaluate its effectiveness as a model we started by comparing the
pathogenicity of wild type EGDe to its isogenic ΔprfA mutant. A successful
model should have complete mortality when infected with wild type
bacteria, while a prfA mutant should be avirulent. Death of embryos
infected with a prfA strain would indicate that the model was sensitive to
general bacterial growth, and death might not be due to specific virulence.
The results fit the criteria of a relevant model well, showing complete
embryo mortality after approximately 40 hours post infection for wild type,
Figure 5. Schematic representation of a chicken egg after 9 days and the
point of inoculation.
The needle is injected through the chorioallantoic membrane into the allantoic
cavity. Adapted from Andersson, Gripenland et al. (2015)
27
while ΔprfA did not display any significant mortality at the conclusion of the
experiment (72 h) (Paper 3: Fig. 1A). During the work with the chicken
embryos, the need for high quality eggs quickly became apparent. A low
quality of the eggs resulted in a high unspecific death, not related to
infection.
To further validate the model we also tested a Δhly mutant which, as has
been shown previously in other models, also was avirulent (Paper 3: Fig. 2)
(Freitag, Youngman et al. 1992). Once we had established the validity of the
model we wanted to determine if the pathogenicity was dependent on the
internalins InlA and/or InlB. When infecting chicken embryos with deletion
mutants of inlA or inlB we could, surprisingly, not see any attenuation of the
pathogenicity (Paper 3: Fig. 2). Taken together, the data indicated that the
chicken embryo model can be used as a method to detect virulence deficient
L. monocytogenes mutants. It is of special interest for studying pathways not
dependent on InlA or InlB, for example when studying effects of other
internalins or internalization associated factors whose effects might be
obscured by the actions of InlA and/or InlB.
Once it had been established that chicken embryos was a reliable model for
L. monocytogenes infection, we wanted to employ the model for use when
screening new virulence inhibitory compounds. Unfortunately this proved to
be impossible with the current compounds, mainly due to the combination
of a relatively large target volume (a chicken egg contains approximately 70
ml), a small injection volume and a low water solubility. This resulted in the
formation of precipitates, leading to an inability to reach the minimum
inhibitory concentration.
28
Conclusions
L. monocytogenes utilizes a blue light receptor to modulate its
colony behavior at a morphological level in response to shifting
levels of blue light.
L. monocytogenes utilize light sensing to initiate the stress
response.
The classical virulence factor ActA is closely tied to the stress
response, and is required for ring formation.
Small 2-pyridone compounds can bind to and inhibit PrfA activity.
The inhibition is probably due to a stabilization of PrfA, preventing
the HTH from entering an active confirmation.
Chicken embryos fulfill the requirements for use as a model system for L. monocytogenes infection.
Pathogenicity in chicken embryos is independent of InlA and InlB.
29
Acknowledgements
Jag skulle vilja tacka mina föräldrar, mor/far-föräldrar samt Lennart och Berit
för allt stöd och uppmuntran ni har gett mig under min studietid (trots att
den aldrig verkar ta slut…).
I would of course also like to thank my fantastic supervisor (yes, I’m talking
about you Jörgen; and no, I won’t repeat it ). Thank you for all the support
and giving me both guidance and freedom, even though my instinctive
response to any suggestion is “NO!”…
My group, both old and new members: Without you, life at the department
would not have been the same.
The Friday fika group: I don’t know if I would have survived without you (or
at least not without the fika…). Keep fighting oh Lord of the Cookies!
Johnny: Utan dig vet jag inte om institutionen hade stått kvar.
Även våra administratörer och media-personal ska ha tack för allt de gör.
Enormt stort tack till alla mina vänner utanför labbet, utan ert stöd hade det
nog inte blivit någon avhandling:
Lisa: Den bästa vän jag någonsin haft. Marcus och David: Tack för underbara
spelkvällar och härligt sällskap. Helena: Tack för allt stöd och vänskap.
Marta: It all started with squash, but quickly progressed to wine, beer and
vodka. Thank you for all the fun times!
Rickard och Lars: Tack för allt från klättersällskap till vetenskapliga
diskussioner.
Monika: Tack för alla enastående middagar och underbart sällskap. Tycker
fortfarande att du borde stannat här uppe =)
30
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