Regulatory pathways and virulence inhibition in Listeria monocytogenes

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Regulatory pathways and

virulence inhibition in

Listeria monocytogenes

Christopher Andersson

Department of Molecular Biology

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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

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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}

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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-anti-sigma factors sequesters the anti-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

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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.

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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

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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

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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.

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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).

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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).

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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

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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

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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 lightbiofilm 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

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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.

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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).

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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

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.

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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

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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.

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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)

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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.

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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.

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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

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Regulatory pathways and virulence inhibition in Listeria monocytogenes