identification of new virulence factors in francisella...

Identification of new virulence factors in Francisella tularensis

Anna-Lena Forslund

Department of Molecular Biology 901 87 Umeå Umeå 2009

”Att våga är att förlora fotfästet en stund. Att inte våga är att förlora sig själv”.

Søren Kierkegaard

Copyright©Anna-Lena Forslund

ISBN: 978-91-7264-916-3

Printed by: Print & Media Umeå, Sweden 2009

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Table of Contents

Abstract ________________________________________________ 1

Papers in this thesis ______________________________________ 2

Background _____________________________________________ 3

History _____________________________________________________ 3

Occurance __________________________________________________ 4

Tularemia __________________________________________________ 4

Diagnosis ___________________________________________________ 5

Vaccine ____________________________________________________ 6

Immune response ____________________________________________ 7

Pathogenic lifestyle and general properties _______________________ 8

Virulence factors ____________________________________________ 10 Francisella Pathogenicity Island, FPI ________________________________ 10 Surface structures ______________________________________________ 10 LPS ________________________________________________________ 11 Type IV pili __________________________________________________ 12

Posttranslational modifications ____________________________________ 13 Glycosylation ________________________________________________ 13 Formation of disulphide bridges by DsbA __________________________ 14

Secretion systems ___________________________________________ 15

Regulation _________________________________________________ 15 Fe regulation and iron acquisiton __________________________________ 16 Small non‐coding RNAs __________________________________________ 17 Hfq __________________________________________________________ 18

Aims __________________________________________________ 19

Results and discussion ___________________________________ 20

Hfq has a role in virulence regulation in F. tularensis ______________ 20 Deletion of hfq have pleiotrophic effects on F. tularensis _______________ 21 Identification of genes with altered expression in the hfq mutant _________ 21

DsbA – disulphide oxidoreductase A ____________________________ 22 DsbA has a major impact on virulence in both LVS and FSC200 ___________ 23 Several proteins accumulate or are degraded in the dsbA mutant ________ 23

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Function of Type IV pili genes in F. tularensis ____________________ 25 Pilin proteins in F. tularensis ______________________________________ 26 PilA has a major impact on virulence in mice _________________________ 26 The Tfp biogenesis genes in the virulence of SCHU S4 __________________ 28 Does Francisella express functional Tfp fibres on the surface? ___________ 28 Posttranslational modification of pilin protein ________________________ 29

Conclusions ___________________________________________ 29

Acknowledgements / Tack! _______________________________ 32

Sammanfattning på svenska _____________________________ 34

References ____________________________________________ 35

ABSTRACT

Abstract Francisella tularensis, the causative agent of tularemia, is a highly

virulent bacterium with an infection dose of less than 10 bacteria. The ability of a pathogen to cause infection relies on different virulence mechanisms, but in Francisella tularensis relatively few virulence factors are known. Two F. tularensis subspecies are virulent in humans; the highly virulent subspecies tularensis, also referred to as type A, and the less virulent subspecies holarctica, also called type B. The aim of this thesis has been to improve the knowledge regarding the ability of Francisella to cause disease, with the emphasis on surface located and membrane associated proteins and structures. In addition I have also investigated how virulence is regulated by studying the role of the small RNA chaperone, Hfq.

The genome of Francisella appears to encode few regulatory genes. In my work I found that Hfq has an important role in regulation of virulence associated genes in Francisella. Similar to what has been found in other pathogens, Hfq functions in negative regulation, and this is the first time a negative regulation has been described for genes in the Francisella pathogenicity island. Another protein with a key role in virulence is a homologue to a disulphide oxidoreductase, DsbA, which was identified as an outer membrane lipoprotein in Francisella. A dsbA mutant was found to be severely attenuated for virulence and also induced protection against wild-type infections, thus making it a candidate for exploration as a new live vaccine. Additional genes with homology to known virulence determinants include a type IV pilin system. The pilin homologue, PilA, was identified to be required for full virulence in both type A and type B strains. In addition, genes involved in pili assembly and secretion, pilC and pilQ, were also found to be virulence associated in the type A strain.

In summary, dsbA, hfq and type IV pili associated genes were indentified to be virulence determinants in F. tularensis. DsbA is a potential target for drug development and a dsbA mutant a candidate for a new live vaccine strain. Furthermore the identification of Hfq as a novel regulatory factor opens new insights into the virulence regulatory network in Francisella.

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PAPERS IN THIS THESIS

Papers in this thesis I. Meibom, K., Forslund, A-L., Kuoppa K., Alkhuder, K., Dubail, I., Dupuis,

M., Forsberg, Å., Charbit, A. Hfq, a novel pleiotropic regulator of virulence-associated genes in Francisella tularensis. Infect Immun 2009, 77(5):1866-80

II. Straskova, A., Pavkova, I., Link, M., Forslund, A-L., Kuoppa, K., Noppa, L., Kroca, M., Fucikova, A., Klimentova, J., Krocova, Z., Forsberg, Å., Stulik, J. A dsbA mutant of Francisella tularensis is Highly Attenuated In Vivo and Induces Protective Immunity. J Proteome Res 2009, 8(11):5336-46

III. Forslund, A-L., Kuoppa, K., Svensson, K., Salomonsson, E., Johansson, A., Byström, M., Oyston, P., Michell, S., Titball, R., Noppa, L., Frithz-Lindsten, E., Forsman, M., and Forsberg, Å. Direct Repeat Mediated Deletion of a Type IV Pilin Gene Results in Major Virulence Attenuation of Francisella tularensis. Mol Microbiol 2006, 59:1818-30

IV. Forslund, A-L., Näslund Salomonsson, E., Golovliov, I., Kuoppa, K.,

Michell, S., Titball, R., Oyston, P., Noppa, N., Sjöstedt, A., and Forsberg, Å. Role of Type IV pili in virulence of Francisella tularensis subspecies tularensis. Submitted.

V. Näslund Salomonsson, E., Forslund, A-L., Kuoppa, K., Michell, S., Titball, R., Oyston, P., Noppa, N., and Forsberg, Å. Role of type IV pilin encoding genes in virulence of Francisella tularensis subspecies holarctica. Manuscript

Contribution has also been made by the author to the following studies,

but these are not extensively discussed in the thesis: Salomonsson, E., Kuoppa, K., Forslund, A-L., Golovliov, I., Sjöstedt, A., Noppa, L., and Forsberg, Å. Reintroduction of two deleted virulence loci confers full mouse virulence to the live vaccine strain (LVS) of Francisella tularensis. Infect Immun 2009, 77(8):3424-31 Thelaus, J., Andersson, A., Mathisen, P., Forslund, A-L., Noppa, L., Forsman, M. Influence of nutrient status and grazing pressure on the fate of Francisella tularensis in lake water. Microbiol Ecol. 2009, 67(1):69-80

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BACKGROUND

Background

History

Francisella tularensis is a Gram-negative bacterium causing the disease tularemia. It was first isolated in 1911 in Tulare County, California, and named Bacterium tularence (McCoy, 1911; McCoy et al., 1912). 1947 it was renamed to Francisella tularensis, in the honour of Dr Edward Francis, a pioneer in F. tularensis research in the beginning of the 20th century (Keim et al., 2007). Dr Francis characterized the bacterium and also the disease, for which he proposed the name tularemia. Furthermore, he verified that tularemia could be transmitted to man by blood-sucking insects or by handling infected rabbits or rodents, but not directly between humans (Francis, 1925).

The genus Francisella is divided into three species; Francisella novicida, Francisella tularensis, and Francisella philomiragia (Jensen et al., 1969; Larson et al., 1955). Francisella tularensis is in turn divided into three subspecies; ssp. tularensis, ssp. holarctica, and ssp. mediasiatica (Euzéby, 2007). Since Francisella novicida has been found to be closely related to Francisella tularensis, it is often referred to as a subspecies belonging to Francisella tularensis, making F. novicida a fourth subspecies of Francisella tularensis. This taxonomy is still often used in the literature and will also be used in this thesis. An older nomenclature which is still often used is the two biovars, type A and type B, that corresponds to F. tularensis subspecies tularensis and holarctica respectively (Olsufjev et al., 1982).

Between 1932 and 1945, Japanese examined the potential of F. tularensis as a biological weapon, and similar programs also existed in the former Soviet Union and the United States. USA ended the offensive research program in 1972 when they signed the “Convention against development of Biological weapons”, and officially the former Soviet Union also ended this type of research in 1990. Francisella has, due to its high infectivity and potential for aerosol transmission, been classified as a category A of biological weapon by the American CDC, Center for Disease control and Prevention (Dennis et al., 2001). During the 20th century the numbers of publications regarding Francisella research were limited, but after the events of 11th September 2001, and the following anthrax letters, more funding was given to research on potentially dangerous pathogens like Francisella. This

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has lead to a dramatic increase in the field of Francisella research, and as a consequence, new genetic tools have been developed, and the genomes of several different Francisella species have been sequenced. As a result our knowledge of this important pathogen has improved significantly the last decade.

Occurance

Francisella species are distributed all over the northern hemisphere and the distribution is based on clinical reports in medical literature. In brief, Francisella type A only occurs in North America, while type B strains are found in Europe and northern areas of Asia, as well as in North America. The natural reservoir of F. tularensis has still not been conclusively identified, but it is known that this bacterium can survive in water and soil for several weeks, and also in dry environments in association with lagomorphs (Keim et al., 2007). It has also been demonstrated that F. tularensis can persist in natural ecosystems by surviving within protozoa, like amoeba (Abd et al., 2003). The occurrence of tularemia cases has undergone a major change over the years. USA and Russia have reported much fewer cases in recent years compared to the peak in the 1940s (Sjostedt, 2007). On the other hand, some countries like Kosovo, have reported new major outbreaks in recent years (Allue et al., 2008; Eliasson et al., 2002; Reintjes et al., 2002). A well studied area with endemic tularemia is Martha´s Vineyard, Massachusetts, USA, where the transmission was found to be a result of human activities - in this case farming (Feldman et al., 2003). It is likely that human activities are important factors that could trigger tularemia outbreaks. For instance, recent outbreaks in Europe, like that in Kosovo, are probably caused by the war disturbances. Other human activities, resulting in ecological and climate changes, may also have influence on the occurrence of tularemia outbreaks (Sjostedt, 2007).

Tularemia

There are different types of Francisella infections, depending on the route of infection. One of the most common forms is ulceroglandular tularemia which is a consequence of a local infection in the skin, via tick bite or direct contact with infected animals, and a following glandular infection. Glandular tularemia is similar to ulceroglandular but without a local infection site and

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the pneumonic form occurs after inhalation of F. tularensis. In addition oculoglandular, gastrointestinal or oropharyngeal forms occur, as a result of infection of the eye or ingestion of contaminated food or water (Fig. 1). A typhoidal form of tularemia is a systemic Francisella infection without any other typical signs. The incubation time varies between 3-6 days, and the clinical symptoms are flue like, with fever, headache, and swollen lymph nodes (Fig. 2) (Dienst, 1963; Evans et al., 1985; Tarnvik et al., 1996). The severity of infection depends both on the infection route and what type of Francisella species that causes the infection. Infections with type A strains are usually severe, and if untreated the mortality rate can be up to 30%-60%, while type B infections are milder and cases of mortality are rare (Dennis et al., 2001). Tularemia is, if diagnosed in time, relatively easy to treat, and ciprofloxacin is recommended as a first choice of treatment (Johansson et al., 2001).

Oropharyngeal

Ulcero-glandularGlandular

Typhoidal

Gastrointes�nalPneumonic

Oculoglandular

Fig 2. A swollen lymph node in a patient suffering from oculoglandular tularemia. With permission from: Dr Henrik Eliasson, Örebro University Hospital, Sweden.

Fig 1. The different forms of tularemia depend on the route of infection. The most common variants are; glandular, ulceroglandular and pneumonic tularemia.

Diagnosis

It is possible to diagnose tularemia by direct isolation of bacteria from patients and cultivate them on blood agar plates supplemented with cystein, but since F. tularensis is relatively difficult to cultivate this is not a fully reliable method. Serological tests are to prefer, with agglutination or ELISA

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(Labayru et al., 1999; Meshcheriakova et al., 1988), but here the disadvantage is the delay of the antibody response, which occurs about two weeks after the first symptoms (Koskela et al., 1985). Therefore, today PCR is often used as a diagnostic method, and the range of different PCR methods for diagnosis are increasing, and many are highly specific and sensitive (Bystrom et al., 2005; Sjostedt et al., 1997; Tomaso et al., 2007). The advantage with PCR is that it is rapid and sensitive, and it also makes it possible to distinguish between different Francisella species and subspecies.

Vaccine

One of the long term goals with Tularemia research has been to develop a vaccine against the disease. Today the only used vaccine is the live vaccine strain, LVS. LVS was originally developed in the former Soviet Union and attenuated by several in vitro passages of a type B strain. LVS was transferred to the US in 1956, and further characterised with separation of the two colony types “blue” and “grey” (page 11) and passages through mice, before it was found to provide protection against aerosolized SCHU S4 (Eigelsbach et al., 1961; Saslaw et al., 1961). This vaccine was initially produced by the National Drug Company in 1959, and over the years the efficacy of LVS has been monitored (Burke, 1977).

Since LVS it is not licensed, it is mainly available for investigative studies and for vaccination of laboratory and military personnel. LVS is administered in humans by scarification, and induces a high level of protection against type B infections, but protection against aerosolized SCHU S4 is more limited (Eigelsbach et al., 1961). One reason that LVS has not been licensed is that the mechanism behind the induced protection is not fully understood, even if it is believed to be T-cell mediated (Tarnvik et al., 1985). Additionally, the molecular basis of the attenuation has not yet been completely evaluated, but recently it was shown that the lack of two specific regions encoding virulence determinants is the cause of the attenuation (Salomonsson et al., 2009b). There is also a naturally attenuated strain of SCHU S4 lacking one of these virulence determinants that induces protective immunity in animal infection models (Twine et al., 2005a). Still, using an attenuated type A strain is regarded to pose a high risk, especially since it potentially could revert to higher virulence. The aromatic amino acid and the purine biosynthesis pathways have also been identified as suitable

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targets for construction of defined attenuated live vaccine strains (Karlsson et al., 2000)

Subunit vaccines are attractive alternatives to live vaccines, although it is a challenge to generate high levels of cell mediated immunity (Fulop et al., 1995; Robinson et al., 2005). Therefore there is a strong focus in ongoing studies to identify new targets for subunit vaccines. Novel approaches to deliver subunit vaccines to the host have also been evaluated, and in a recent study Listeria monocytogenes was successfully used as a delivery vehicle to express F. tularensis antigens into the host (Jia et al., 2009).

Immune response

To establish infections, microorganisms have to penetrate different barriers and avoid killing by the innate immune system of the host. The host humoral immune response is triggered upon contact with microorganisms, and if an infection is established the adaptive immune response will be activated. The immune response induced during F. tularensis infections has mainly been studied in mice and primarily by using LVS, even though other infection models have been studied, as well as infections with other Francisella subspecies. The general opinion is that cell mediated immunity is required for protection against intracellular bacteria and is more important than humoral immunity with antibody response. Still, for F. tularensis infections, antibody responses are generated in humans against LPS (Cole et al., 2006; Koskela et al., 1982; Sundaresh et al., 2007). This is not surprising since F. tularensis has been shown to exist in an extracellular phase in mice (Forestal et al., 2007). However, due to the mainly intracellular nature of F. tularensis infections, the cell mediated immune response is thought to be the most important for protective immunity against tularemia (Sjostedt et al., 1989).

Macrophages are considered to be the primary host cell for F. tularensis, but several other cell types in the immune system, like neutrophils, dendritic cells, hepatocytes and alveolar epithelial cells, also serve as host cells for F. tularensis (Clemens et al., 2007). During early stages of infection, a wide range of cytokines, like tumor necrosis factor-alfa (TNF-α), Interleukin 10 and 12 (IL-10, IL-12) and interferon-gamma (IFN-γ) are produced by different immune cells like keratinocytes and NK-cells (Elkins et al., 1993). The expression of cytokines induces proliferation and differentiation of different immune cells like B- and T-cells. Later during infection, CD4+ T-

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cells and CD8+ T-cells play a major role in infection response (Conlan et al., 1994; Fulop et al., 2001; Sjostedt et al., 1991). αβ-T-cells have been found to be required for protection, and in addition γδ-T-cells are also predicted to play a role in protection since these cells are found in patient sera after infection (Kroca et al., 2000; Yee et al., 1996).

It is likely that there is a synergy between humoral and cell mediated immune responses in protection against F. tularensis. Still, further investigations are needed to fully elucidate the protective immune response against tularemia.

Pathogenic lifestyle and general properties

F. tularensis is able to multiply in several types of cells but the most important and relevant host cells for F. tularensis are phagocytic cells like macrophages (Hall et al., 2007; McCaffrey et al., 2006). The intracellular uptake involves a mechanism termed “looping phagocytosis”. With this mechanism, the bacteria are ingested by the host cell via formation of a pseudopod loop, resulting in enclosure of the bacterium inside a phagosome. Subsequently, the phagosome undergoes a processing pathway where it shrinks quickly and moves towards the centre of the cell. A phagosome matures and later fuses with a lysosome, to form a phagolysosome, where many microbes are degraded. During the phagosomal maturation, different markers are expressed at different stages, making it possible to follow the progress with microscopic methods (Clemens et al., 2009; Ghigo et al., 2002; Hackstadt, 2000; Nagai et al., 2002). Intracellular pathogens have evolved different strategies to avoid degradation like preventing fusion with the lysosome, modulating the phagosome biogenesis in the endosomal-lysosomal degradation pathway, adapting to the acid environment, or by escaping from the phagosome. It has been established that Francisella is able to survive in the phagosome during the phagosomal maturation, and then the bacterium somehow escapes from the late phagosome to initiate replication in the cytosol (Checroun et al., 2006; Chong et al., 2008; Clemens et al., 2004; Golovliov et al., 1997). Phagocytosis is a receptor mediated process that requires a variety of receptors and associated signalling pathways. In F. tularensis, the mechanisms that trigger the bacterial uptake are not fully understood, but it is known that it involves host cell receptors like complement receptor 3 (Cr3), class A scavenger and

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mannose receptors (Balagopal et al., 2006; Clemens et al., 2005; Pierini, 2006; Schulert et al., 2006).

F. tularensis is a small Gram-negative bacterium with a relatively small genome size of 1.89 Mbp, a coding percentage of 79 %, and a low G+C content of 33% (Larsson et al., 2005). It is a slow growing bacterium that is routinely grown in 5% CO2 and requires blood agar supplemented with cystein for growth. Recent development of new powerful genetic tools, together with access to complete genome sequences of several strains, have opened new possibilities to characterise the function of specific genes in F. tularensis. Established methods for transformation of DNA, like chemical transformation, electroporation, cryotransformation, and conjugation have been modified to fit manipulation of Francisella. Several vectors for genetic modification and expression have been constructed (Golovliov et al., 2003; Kuoppa et al., 2001; LoVullo et al., 2006; Maier et al., 2004). Also the ability to utilise transposon mutagenes has been demonstrated to be a useful tool for genetic manipulation of F. tularensis (Kawula et al., 2004; Reznikoff et al., 2004).

To be able to examine a particular virulence mechanism during bacterial infection, there is a need to develop an animal model relevant for the human infection. Even though animal models may have some limitations, they usually provide more information than cell infection studies. Historically both humans and nonhuman primates have been used in tularaemia infection studies, as well as rabbits and guinea pigs. However, data are so far not consistent enough regarding infections in rabbits and guinea pigs to fully evaluate if they are useful models for tularemia infection (Bell et al., 1955; Cross et al., 2007; Owen et al., 1964). Also rats have previously been tested in infection studies, and recent studies indicate that a rat model might be useful as an infection model (Jemski, 1981; Rick Lyons et al., 2007; Wu et al., 2009). However, mice are most commonly used in tularemia infection studies and regarded by many as an appropriate model, even though it clearly has limitations. One is that both type B strains and type A strains are highly virulent irrespective of infection route in mice, while in humans type A strains are significantly more virulent. During the years LVS has been frequently used as a model strain for tularemia infections due to the lower virulence, which allows studies outside high containment laboratories. In addition it also lowers the risks for acquired infections among laboratory personnel. LVS is highly virulent via the intraperitoneal route and via inhalation in mice, while via the subcutaneous route the infection dose is

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quite high with an infection dose of about 106 bacteria. However, the advantages with mice as a model are, besides the fact that mice are highly susceptible to tularemia, that they are easy to handle and that inbred and transgenic mice for genetic analysis of host factors are available.

Virulence factors

Francisella Pathogenicity Island, FPI

Francisella pathogenicity island, FPI, is a ~30 kb locus identified within the genome of F. tularensis. One region of FPI has a significantly lower GC content compared to the average GC content of the F. tularensis genome, indicating that this DNA region was transferred from another microbe (Nano et al., 2004). The intracellular growth locus, iglABCD, as well as pdpABCD, the latter not organized in an operone, have all been verified to be required for virulence. Functions associated to genes encoded by the FPI include intracellular growth and phagosomal escape (Golovliov et al., 2003; Gray et al., 2002; Santic et al., 2005). Genomic studies have also revealed that FPI encodes genes with homology to those encoding type VI secretion systems in other pathogens (Broms et al., 2009; Ludu et al., 2008). Interestingly, the FPI exists in two copies in ssp. tularensis, ssp. holarctica, and ssp. mediasiatica, while there is only one copy in ssp. novicida and Francisella philomiragia (Golovliov et al., 2003; Larsson et al., 2009). The biological significance of the duplication of FPI remains to be evaluated.

Surface structures

Gram negative bacteria are surrounded by an outer membrane that includes a broad variety of proteins – outer membrane proteins (OMPs). OMPs are crucial for several bacterial functions like virulence, immune evasion and adaption to environmental conditions (Buchanan, 1999; Lin et al., 2002). In addition, OMPs could also serve as targets for vaccine development and provide drug targets, since they are the most accessible bacterial proteins (Pal et al., 2005). In F. tularensis, the OMPs that are studied more in detail are the 43-kDa protein, FopA and the 17 kDa protein, TUL4 (Nano, 1988; Sjostedt et al., 1991). Recently, in part due to new proteomic methods, an increasing number of OMPs have been identified in

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F. tularensis (Fulop et al., 1995; Huntley et al., 2007; Pavkova et al., 2006; Twine et al., 2005b; Wehrly et al., 2009).

LPS

The outer membrane of Gram-negative bacteria is decorated with Toll-like receptor (TLR) ligands like lipopolysaccarides (LPS), lipoproteins and peptidoglycans. LPS consists of three main structures, the lipid part, the core and the O-antigen. The lipid A component is the conserved biologically active component of LPS that is anchored in the OM, while the variable O-antigen is exposed on the bacterial surface, and the core is the structure that link lipid A and O-antigen (Trent et al., 2006). LPS are large molecules that usually are endotoxic and thus involved in immune response in response to infection, and LPS also serve to stabilize the outer membrane structure.

The LPS of F. tularensis is unusual compared to LPS from most other Gram-negatives, since it is not recognised by host cells and therefore lacks endotoxic activity (Cole et al., 2006; Hajjar et al., 2006; Sandstrom et al., 1992). It has been established that the O-antigen is identical between type A and type B strains, while it differs in ssp. novicida. In contrast, the core and lipid A are identical between all F. tularensis subspecies. Probably, as a consequence of these differences, only LPS derived from ssp. novicida can induce production of proinflammatory cytokines in the host (Kay et al., 2006; Kieffer et al., 2003; Thomas et al., 2007).

F. tularensis strain LVS has for a long time been known to spontaneously produce different phenotypes. In brief; gray or blue colonies as well as rough or smooth colonies have been identified, but among these characteristics there are several alterations, and therefore it may be difficult to distinguish between them. (Cowley et al., 1996; Eigelsbach et al., 1951; Hartley et al., 2006). The blue variants are usually more virulent and immunogenic, and it is clear that LPS plays a role in this phase variation since it seems that the grey variants lack O-antigen (Hartley et al., 2006).

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Type IV pili

Type IV pili (Tfp), are important for host colonization and virulence in many bacterial pathogens like Pseudomonas aeruginosa, Neisseria ssp, Vibrio cholerae, Moraxella catarrhalis (Fullner et al., 1999; Luke et al., 2004; Mattick et al., 1996; Tønjum et al., 1997). The function of Tfp is to promote adhesion, twitching motility, biofilm formation, and DNA transfer (Aas et al., 2002; O'Toole et al., 1998). Tfp are flexible extracellular filaments, usually 5-7 nm in diameter, and the biogenesis includes polymerisation of a single protein subunit, in Pseudomonas aeruginosa named PilA. The pilin proteins have conserved features with a positively charged leader sequence, a conserved cleavage site followed by a highly hydrophobic domain that forms the core of the pilus fiber, and the C-terminal domain often contains two conserved cysteins. In addition, there are so called minor pilins that are similar to the structural pilin proteins since they also have a prepilin like N-terminal sequence. The exact function of minor pilins is not known, but they may play a role in the Tfp biogenesis or function (Helaine et al., 2007; Koomey, 1995). A peptidase, PilD, cleaves the signal peptide from the prepilin before the mature pilin is secreted. The secretion occurs via the PilQ secretin pore in the outer membrane. Another main component, PilC, is an inner membrane protein needed for Tfp biogenesis. In the Tfp system there are also two ATPases, PilB and PilT, which are involved in extension and retraction of the pilus respectively (Fig. 3). The PilT dependent ability to retract the pilus is also required for motility on solid surfaces, which is also denoted twitching motility (Jakovljevic et al., 2008; O'Toole et al., 1998; Strom et al., 1993a).

Fig 3. Schematic overview of a type IV pili system. The nomenclature of the Tfp components is the same as in P. aeruginosa.

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The Type IV pilins can be further divided into two subgroups, Type IVa and Type IVb pilins which show distinct differences. Type IVa pilin has a shorter signal peptide and the mature protein is also shorter than Type IVb pilins. Furthermore, they could also be distinguished by the different amino acids that flank the PilD cleavage site (Craig et al., 2004; Kachlany et al., 2001; Strom et al., 1993a). Type IVb pili are usually associated with enteropathogenic bacteria like Vibrio cholerae, Salmonella enterica and enteropathogenic E. coli (Donnenberg et al., 1992; Shaw et al., 1990; Zhang et al., 2000).

In the genome of F. tularensis several homologues to genes encoding Tfp system have been identified. There are six putative pilin genes; pilA, pilE, pilV, FTT0861, FTT0230 and FTT1314, several minor pilins and/or pseudopilins and additional genes encoding components postulated to be involved in function and biogenesis of Tfp, like; pilB, pilC, pilD, pilT, and pilQ (Larsson et al., 2005).

Posttranslational modifications

Many proteins undergo posttranslational modification (PTM). There are different types of PTMs such as enzymatically mediated attachment of functional groups like phosphate, acetate, lipids and carbohydrates, but modification can also involve changes of the protein structure involving formation of disulfide bridges. These modifications can be critical for proper function of the protein, and the nature of modifications can often be detected by Western blotting using specific antibodies and/or by mass spectrometry.

Glycosylation

One of the most common PTM is glycosylation, which is an enzymatic process where sugars (mono- or oligosaccarides) are added to proteins building flagella, pili or other adhesins, or LPS (Spiro, 2002). N-glycosylation and O-glycosylation are the two main types of protein glycosylation, where the O-linked is most common in prokaryotes. N-linked glycosylation is a mechanism where oligosaccarides are attached to an aspargine side chain residue. N-linked glycosylation is uncommon in prokaryotes, and has so far only been documented to occur in Campylobacter (Szymanski et al., 2003; Young et al., 2002). In the O-linked glycosylation pathway oligosaccarides are attached to the hydroxyl group of

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serine or threonine (Aas et al., 2007; Castric, 1995; Faridmoayer et al., 2007).

Formation of disulphide bridges by DsbA

Oxidative folding processes are needed for proper function of a broad range of bacterial proteins. Interestingly, these proteins include virulence factors, like proteins involved in Type IV pili system, Type II secretion, capsule biogenesis and toxin expression. For instance, type III secretion and DNA uptake systems are dependent upon proper disulphide bond formation for function (Jackson et al., 1999; Pugsley et al., 2001; Stenson et al., 2002; Tinsley et al., 2004; Yu, 1998; Zav'yalov et al., 1997; Zhang et al., 1996). The folding process is catalysed by a group of enzymes named disulfide bond, Dsb, proteins. This oxidative folding machinery has been extensively studied in E. coli where it involves the periplasmic oxidative protein, DsbA,

and the membrane-bound protein, DsbB (Fig. 4). The two cysteins in DsbA are reduced when the disulphide bond is formed, and DsbB catalyses a redox reaction which restores DsbA to an active oxidised form, ready to catalyse new reactions (Inaba et al., 2006; Zhou et al., 2008). Loss of components in the Dsb pathway usually results in impaired virulence and altered ability to cause infection (Jackson et al., 1999; Pugsley et al., 2001; Yu, 1998).

Fig 4. Model of the oxidative disulphide folding mechanism in E. coli, where the process occurs in the periplasmic space and is mediated by DsbA and DsbB.

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In F. tularensis FTT1103 has been postulated to encode a disulfide oxidoreductase, DsbA. FTT1103, like the 17-kDa protein TUL4, has also been shown to stimulate the cellular receptor TLR2 (Thakran et al., 2008).

Secretion systems

Microbes have evolved different strategies to invade human cells and/or blood circulation, but commonly they have to defeat different barriers like epithelial cells, cell membranes, and not least, the immune response of the host. The strategies employed are highly diverse, but one common strategy is based on the ability to secrete proteins across bacterial membrane, and in some cases also across the host cell membrane. In Gram negative bacteria six different secretion systems have been identified, type I-VI secretion (Backert et al., 2006; Delepelaire, 2004; Filloux, 2004; Kaniga et al., 1995; Schell et al., 2007).

In F. tularensis, genes with homology to the T1SS, T2SS, and T6SS have been identified, even if functional studies of these systems are still limited (Broms et al., 2009; Champion et al., 2009; Gil et al., 2006; Hager et al., 2006; Hansen et al., 2006). The limited evidence for functional secretion systems in Francisella is partially due to the difficulties to analyse secretion, since human pathogenic F. tularensis do not secrete detectable levels of proteins during in vitro growth. In contrast, F. novicida secretes higher levels of proteins, and is therefore often used as a model for secretion studies.

Regulation

Bacteria are able to sense environmental changes and adapt to these changes by altering expression of different enzymes, structural proteins, toxins etc. In many bacterial species, a broad range of regulatory genes have been identified, and many of those are so called two-component systems (TCS). TCS are composed of a membrane situated sensor, that senses changes in the environment, and a transcriptional response regulator located in the cytoplasm (Beier et al., 2006).

In F. tularensis relatively few regulatory genes have been identified. Some of these have been shown to regulate genes in the Francisella Pathogenicity Island (FPI) (page 10). Many FPI genes are regulated by MglA, including several genes required for intracellular growth in macrophages as well as in

15

BACKGROUND

amoebas (Baron et al., 1998; Brotcke et al., 2006; Lauriano et al., 2004). In addition, an MglA like protein, SspA, interacts with MglA, and this SspA-MglA complex associates with the RNA polymerase to regulate transcription (Charity et al., 2007). The genes regulated by MglA-SspA are also regulated by FevR, which in turn is regulated by MglA-SspA (Brotcke and Monack 2008). PmrA is another regulatory protein that regulates a broad range of genes, including many that are under the control of MglA. Still, PmrA does not regulate MglA expression and vice versa (Mohapatra et al., 2007). MigR is another, recently identified regulator found to regulate the iglABCD operone encoded by the FPI (Buchan et al., 2009). Taken together the regulation of virulence genes in F. tularensis appears to be complex, where several regulatory proteins regulate the same group of genes, and the significance and consequences of this co-regulation in vivo remains to be resolved.

Besides identification of the specific regulatory proteins, it is important to bear in mind that several environmental signals, and other factors that influence a regulation of virulence gene expression, often act in concert with the regulators. For Francisella iron limitation, temperature and intracellular growth, are factors that have been shown to be of major importance for virulence gene expression (Deng et al., 2006; Golovliov et al., 1997; Horzempa et al., 2008).

Fe regulation and iron acquisiton

A key requirement for bacteria to be able to establish infection in the host is the acquisition of essential nutrients and metal ions that are required for the activity of many key enzymes. Iron is one of the most limited nutrients in mammalians, and free iron is not present in the blood or tissues, instead it is sequestered by specific binding proteins or in ferritin storage complexes (Weinberg, 1993). To be able to acquire ferric ions, despite the very low iron levels, high-affinity iron uptake systems have evolved in many bacteria. These systems are well characterised in pathogens like Yersinia pestis and Legionella pneumophila (Perry et al., 1999; Robey et al., 2002; Wandersman et al., 2004).

It has been known for a long time that F. tularensis requires iron for growth, and there are also several genes where expression is affected by iron limitation, such as iglC and pdpB, encoded by the FPI (Deng et al., 2006). In addition, it has been shown that F. tularensis expresses siderophores with

16

BACKGROUND

very high binding affinity for iron. Siderophore expression requires at least fslA in the fslABCDEF gene cluster (Sullivan et al., 2006). No homologue to a gene encoding a siderophore receptor for iron uptake has been identified (Larsson et al., 2005), but fslE has been proposed to encode a novel siderophore receptor since it is needed for siderophore mediated iron acquisition in F. tularensis (Ramakrishnan et al., 2008). In addition, a recent study postulated that the membrane protein, encoded by FTT0918 and designated the Fe utilisation protein A, FupA, is required for siderophore-independent iron acquisition and proper Fe uptake in F. tularensis (Lindgren et al., 2009). Since F. tularensis is able to survive in many niches in nature, like in water, protozoa, and in mammals, it is likely that this bacterium has evolved effective systems also for iron acquisition in order to adapt to different growth conditions.

Small non-coding RNAs

Small non-coding RNAs (sRNAs) exist in a wide variety of organisms, where they usually act as post-transcriptional repressors by base paring with mRNA to inhibit translation. The mechanisms to achieve this regulation, is by changing the RNA conformation and stability. Some of the advantages of these regulators are that they are less energy consuming, since they are shorter (50-250 nucleotides) compared to protein encoding mRNA (on average about 1000 nucleotides), and does not require the translation step. Overall, these features allow a very quick response to environmental signals (Majdalani et al., 2005).

The base paring by sRNAs can occur via cis-encoded or trans-encoded pathways. Cis-encoded sRNAs are often 75 nucleotides or longer, and are highly complementary to the target RNA since they are encoded on the DNA strand opposite the target RNA. In contrast to cis-encoded sRNAs, the trans-encoded RNAs share only limited complementary sequences with their target mRNAs, and upon binding they usually negatively regulate the translation and/or stability of target mRNAs. To facilitate the RNA-RNA interaction between trans-encoded sRNA and mRNA, the RNA chaperone Hfq is often required since the complementarity is relatively low and the sRNA molecules can be quite unstable. Many of the cis-encoded sRNAs are more or less constitutively expressed, but in contrast, the trans-encoded sRNAs are synthesized under very specific growth conditions, for example

17

BACKGROUND

18

during oxidative stress, and under altered glucose concentration or iron levels (Brantl, 2002; Waters et al., 2009).

Hfq

Hfq is a RNA binding protein that was originally identified as a host factor required for replication of the RNA phage Qβ in E.coli. Hfq is also similar both structurally and functionally to the eukaryotic Sm protein, which has been identified as a posttranscriptional regulator. In recent years RNA-based regulation, mediated by Hfq, has been an area of increasing interest and the number of sRNAs that require Hfq to exert its regulatory function are rapidly growing (Valentin-Hansen et al., 2004). In addition to facilitate base pairing, Hfq contributes to sRNA regulation by modulating RNA levels. This modulation by Hfq can either be by acting as a chaperone to stabilise the sRNA structure before binding to the target mRNA, or by stabilising the already existing sRNA-mRNA complex. The binding by Hfq to RNA usually results in degradation of the target RNA by RNAse E, and therefore the regulatory function of Hfq is usually negative. The RNAse E mediated degradation is somehow facilitated by Hfq but the mechanism is not fully understood (Aiba, 2007). Even if Hfq usually has a negative effect on transcription, it can also promote transcription by stabilising sRNA that binds to RNA to prevent formation of an inhibitory structure and thereby promote transcription (Waters et al., 2009).

In several bacterial pathogens, like in Salmonella, the function of Hfq has been extensively studied, where the transcription of at least 20% of all genes have been shown to be directly or indirectly controlled by Hfq. A deletion of hfq in Salmonella impairs the expression of the general stress sigma factors, sigma S and sigma E. Therefore, it is not surprising that deletion of hfq results in pleiotrophic effects in many bacteria, like decreased ability to invade host cells and impaired survival in macrophages, reduced secretion of virulence factors and virulence attenuation in mice (Aiba, 2007; Pannekoek et al., 2009; Vogel, 2009). F. tularensis encodes an Hfq homologue and it has also been verified to be expressed (Havlasova et al., 2005).

AIMS

Aims The objective of this thesis was to increase the understanding of the

molecular mechanisms that govern the ability of Francisella tularensis to cause disease.

The main focus has been to identify and study the biological role and function in virulence of surface located and membrane associated proteins and structures. In addition, I also wanted to address how virulence genes are regulated and specifically the significance of Hfq in the regulatory process.

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RESULTS AND DISCUSSION

Results and discussion So far, the number of virulence factors identified in F. tularensis is

relatively limited, but it is clear that one of the most important virulence strategies is the ability to survive and replicate inside host cells. The specific mechanisms of uptake and intracellular survival are under intensive exploration, and a few host cells receptors that are involved in the initiation of bacterial uptake have been identified (Balagopal et al., 2006; Clemens et al., 2005; Pierini, 2006; Schulert et al., 2006). On the other hand less is known about the bacterial adhesins, even though genomic and proteomic analysis has identified several outer membrane proteins and other potential surface organelles or structures (Huntley et al., 2007; Thakran et al., 2008). Bacterial surface proteins and structures may be of fundamental importance for several processes during infection and immune evasion. Therefore, the focus on membrane associated proteins in this thesis, and their role in virulence, could provide new important insights into a so far understudied area.

Hfq has a role in virulence regulation in F. tularensis

The key to understanding how a specific pathogen overcomes the host defence to cause disease, is to identify the virulence factors required to promote infection. Equally important is to understand how these virulence factors are regulated in response to infection. Disclosing such regulatory mechanisms could provide targets for development of new strategies for antimicrobial compounds, and could also be a way for subsequent identification of new virulence factors. Overall the Francisella genome contains few regulatory genes (Brotcke et al., 2008; Buchan et al., 2009; Charity et al., 2007; Mohapatra et al., 2007)(page 15-16), and therefore, regulation mediated by small non coding RNA molecules (sRNAs) could be an important regulatory mechanism in F. tularensis. In many bacteria, most sRNAs require the chaperon like protein, Hfq, for stabilisation and proper function in regulation (Brown et al., 1996; Sittka et al., 2007)(page 17-18). In E.coli, an hfq mutant has a pleiotrophic phenotype involving altered growth and stress sensitivity, and as a consequence the deletion also has an impact on virulence (Tsui et al., 1994).

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Deletion of hfq have pleiotrophic effects on F. tularensis

A previous study identified an hfq homologue in the genome of F. tularensis (Havlasova et al., 2005), and here we decided to mutate hfq by in-frame deletion of the gene in both LVS and in the virulent type B strain, FSC200 (Paper I). The hfq mutant showed a decreased growth in vitro, as well as higher sensitivity to osmotic stress, heat stress and membrane stress. The altered phenotype was in general the same for LVS and the FSC 200 strain, with the exception that the FSC200 hfq mutant was more resistant against heat stress compared to the LVS mutant. Interestingly, for both strains, the ability to multiply in macrophages did not require Hfq expression, as the growth of the two mutant strains were comparable to the growth of the respective isogenic wild-type strains. This actually suggests that the phenotype of the mutant was more pronounced during in vitro conditions. To further validate if Hfq had a role in virulence, mice were infected via subcutaneous and/or intraperitoneal route, and thereby we could verify that Hfq was required for full virulence of both LVS and FSC200. In addition we also studied the infection kinetics of LVS in liver and spleen and found that the hfq mutant was delayed and impaired in its ability to multiply inside host tissue (Paper I).

In other pathogens like S. typhimurium, P. aeruginosa and E. coli, expression of σS and σE, two sigma factors involved in different stress responses, are known to be dependent on Hfq for expression (Brown et al., 1996; Guisbert et al., 2007; Sonnleitner et al., 2006). Since neither σS nor σE have been identified in the genome of Francisella, the effect of an hfq deletion was assumed to be altered compared to what is seen in other pathogens. However, our results verifies that also in F. tularensis, hfq mutants are defective in stress responses, similar to what has been observed in E. coli hfq mutants. These findings suggest that there may be some yet unidentified targets involved in stress responses of F. tularensis that are part of the Hfq regulatory network.

Identification of genes with altered expression in the hfq mutant

As Hfq is needed for virulence in mice, Hfq could be postulated to affect expression of virulence associated genes. Therefore we decided to compare the transcriptomes between the LVS hfq mutant and the LVS wild-type by microarray analysis. We found that Hfq mainly functions in negative regulation, since only 16 genes were expressed at lower levels and 88 genes

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at higher levels in the mutant, when compared to the wild-type (Paper I). Among the repressed genes, several were found to be involved in metabolism, and in addition, genes encoding Type IV pili proteins, ribosomal proteins and nucleases were also expressed at lower levels in the hfq mutant. The most significant finding was that ten genes encoded in the pdp-operon of the Francisella Pathogenicity Island, FPI, were negatively regulated by Hfq. The ability to survive and replicate intracellularly is connected with virulence of F. tularensis, and this replication rely on many genes encoded by FPI (Nano et al., 2004). This is the first example of negative regulation of FPI genes, since all previously identified regulators of these genes have been shown to be positive regulators (Brotcke et al., 2008; Charity et al., 2007; Mohapatra et al., 2007). These findings may, at least partly, explain why hfq mutants were not impaired for intracellular growth in macrophages.

Taken together, we have verified that Hfq is indeed involved in virulence gene regulation in F. tularensis, and we would predict that Hfq exerts its role in regulation by interacting with different regulatory sRNAs. So far, no homologues to sRNAs identified in other bacteria have been identified in the genome of F. tularensis. Therefore, it is possible that F. tularensis harbours unique regulatory properties in the context of sRNAs. At this moment it has not been verified if the altered gene expression seen in the hfq mutant is caused by Hfq directly and/or if it is via an indirect effect. This question can only be resolved once sRNAs of Francisella have been identified. Furthermore, since we only identified affected genes by studying the transcriptome, we will also need to evaluate what influence Hfq have on fully translated genes. Therefore a comparative proteomic analysis of the hfq mutant and the isogenic wild-type strain will reveal if Hfq affects expression of additional genes.

DsbA – disulphide oxidoreductase A In the study of Hfq above, we identified Hfq as a key player in regulation

of F. tularensis, and in addition we could also identify genes postulated to have a more direct role in virulence. By targeting proteins with potential roles in regulation that are required for function of several other proteins, could therefore be a successful strategy to identify new virulence factors. Another example of proteins with a key role in virulence are proteins belonging to the Dsb family of disulphide oxidoreductases. There are several

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examples of bacterial proteins from different pathogens, including some with roles in virulence, that are dependent on DsbA-mediated disulphide bond formations for proper folding and function, and as a consequence dsbA mutants are often attenuated for virulence (page 14). For instance, DsbA is known to be necessary for pilus-mediated adhesion in E. coli, Vibrio cholerae and Neisseria (Peek et al., 1992; Tinsley et al., 2004; Zhang et al., 1996). A protein with homology to DsbA was earlier identified in a comparative proteomic study of different Francisella strains, where it was postulated to be a lipoprotein and a potential virulence factor due to its putative function as a disulphide oxidoreductase (Pavkova et al., 2006).

DsbA has a major impact on virulence in both LVS and FSC200

In order to verify if the DsbA homologue in Francisella could play a role in virulence an in-frame deletion mutant of dsbA was generated, both in LVS and the virulent type B strain, FSC200 (Paper II). The dsbA mutants were found to be severely impaired in their ability to infect and multiply intracellularly in macrophages. The dsbA mutants were also highly attenuated in their capacity to infect mice compared to the corresponding isogenic wild-type strains, and interestingly, the mutant strains were able to induce protection against challenge with the virulent wt strain. Thus, DsbA was indeed required for virulence in Francisella which indicated that DsbA might have an important role in folding of proteins that are directly involved in virulence.

Several proteins accumulate or are degraded in the dsbA mutant

The oxidative process mediated by DsbA occurs in the periplasmic space, and the target proteins are either periplasmic, destined to localize to the outer membrane or to be secreted. Therefore, when dsbA is deleted, some proteins may be misfolded and could therefore accumulate or alternatively be degraded. When analysing proteins in the membrane fraction of the dsbA mutant we found that seven proteins accumulated while one appeared to be degraded when compared to membrane proteins of the wild-type strain (Paper II). These proteins are possible DsbA substrates and candidates for causing the virulence attenuation of the dsbA mutant. Interestingly, in all these identified proteins, there was a consensus sequence for a signal peptide, indicating that these proteins are either outer membrane,

23


periplasmic or secreted proteins. Among the proteins that were found to accumulate was a serine-type D-Ala-D-Ala carboxypeptidase, which could be involved in cell wall biosynthesis (Kikuchi et al., 2006). Additionally, proteins belonging to the chitinase family 18 proteins were also among those identified, and these enzymes are known to be important for arthropod invasion and could thereby contribute to natural dissemination of pathogens (Tsai et al., 2001). The protein that was found in lower amount in the membrane fraction was a macrophage infectivity potentiator (MIP), which is a virulence factor in the intracellular pathogen Legionella pneumophila, and might, at least partly, explain why the dsbA mutant was impaired for intracellular growth (Helbig et al., 2003). Still, it is possible that there are additional proteins that depend on DsbA for function, since in this first approach we only studied membrane associated proteins. A complete comparative proteomic analysis with bacterial as well as secreted proteins will be required to get a more complete pattern of DsbA dependent proteins.

In addition, we verified that F. tularensis DsbA in fact has disulphide oxidoreductase activity and that it is a lipoprotein and therefore predicted to locate to the outer membrane (Paper II). The outer membrane localization was also recently confirmed (Qin A., 2009), and one portion of DsbA was found to be exposed on the bacterial surface – a finding that explains how DsbA in SCHU S4 can induce TLR2 mediated signalling (Thakran et al., 2008). The oxidative folding mechanisms can vary between different bacteria, but one common model for the DsbA-DsbB mechanism is that DsbB is localised in the cytoplasmic membrane, while DsbA localises to the periplasmic space (Fig. 4, page 14). Since there are only one homologue to each of dsbA and dsbB in the F. tularensis genome, the membrane localisation of the DsbA homologue is somewhat difficult to explain as the functional enzymatic domain is supposed to localise in the periplasmic space. In contrast, in N. meningitidis there are three dsbA homologues where two are membrane associated lipoproteins (Tinsley et al., 2004). Even if dsbA in F. tularensis and N. meningitidis are not highly homologous, these findings might still argue for a unique oxidative folding mechanism in F. tularensis with dsbB located in the inner membrane and dsbA in the outer membrane. For these reasons we propose a model for the localisation of DsbA, with the enzymatic domain exposed in the periplasmic space and a surface exposed domain as well (Fig. 5).

Taken together, DsbA could serve as a target for development of a novel attenuated F. tularensis live vaccine strain, as the mutant was found to

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Fig 5. A proposed model of the oxidative disulphide folding mechanism in F. tularensis, where DsbA is localised in the outer membrane but still has the enzymatic activity in the periplasmic space and surface exposed domains for activation of TLR2.

induce robust immunity in mice when challenged with a virulent strain of the same subspecies. The mechanism behind this ability to induce protection is

still an issue, since a deletion mutant of the dsbA partner in the oxidative folding process, dsbB, in SCHU S4 did not induce protection even though the level of attenuation was similar to that we found for dsbA (Qin et al., 2008; Qin et al., 2009). Nevertheless, one very important advantage with a dsbA mutant as a live vaccine candidate is that a dsbA mutant probably is required for the function of several virulence determinants and therefore reversion to higher virulence is unlikely. Moreover, DsbA could also serve as a target for drug development for several reasons. By blocking DsbA function, the virulence of F. tularensis would be attenuated, but drugs that block the function of DsbA would probably not be lethal for the bacterium which reduces the risk of resistance development. In addition - since DsbA localizes to the outer membrane, it should also be accessible to a drug that block the function.

Function of Type IV pili genes in F. tularensis

As described in the background section (page 12-13), Type IV pili (Tfp) are highly specialized bacterial adhesins that are found in many important Gram-negative pathogens. The different Francisella tularensis subspecies; ssp. tularensis, ssp. holarctica and ssp. novicida, all encode homologues to genes required for biogenesis and expression of functional Tfp. I wanted to address the function and potential role in virulence of genes encoding Tfp in F. tularensis (Paper III, IV, and V). As seen in the previous section

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(Paper I), Hfq was found to play a role in the regulation of genes associated with Tfp biogenesis which further highlights the potential relevance of these genes in the pathogenesis of tularemia (Helaine et al., 2007; Mattick et al., 1996; Strom et al., 1993b; Wolfgang et al., 1998).

Pilin proteins in F. tularensis

Six potential pilin subunit genes; pilA, pilE, pilV, FTT0861, FTT0230, and FTT1314 have been identified in the genome of F. tularensis, and overall these genes are almost identical between the different subspecies, but there are some distinct differences (Paper V). In type B strains, pilE and pilV are non functional due to nonsense mutations within the genes, and in addition FTT0861 harbours a mutation in the stop codon which results in longer open reading frame, and in some type B strains there is also an additional frame-shift mutation, but still FTT0861 might well be functional in all these strains. All six pilin genes are intact and functional in type A strains, as well as in F. novicida, but interestingly, the pilA gene in F. novicida differs in the C-terminal while the N-terminal is almost identical to pilA in type A and type B strains (Larsson et al., 2005). Another very interesting finding is that several regions in the F. tularensis genome are flanked by direct repeats, and as a consequence these regions have been deleted in some strains. One of these regions encodes pilA, where the deletion can occur due to the flanking direct repeats located in the 5´- part of pilA and pilE. This deletion has been verified in LVS and also in a type B strain isolated from a hare (Svensson et al., 2005).

PilA has a major impact on virulence in mice

In one of our studies we evaluated the function of pilA by using a type B strain where pilA has been naturally deleted. When restoring pilA in cis, by genetic recombination, we could establish that PilA was absolutely needed for virulence via the subcutaneous route of infection in mice, and also for optimal spread from the initial site of infection to cause a systemic infection involving the spleen. In contrast, PilA was found not to be needed for intracellular growth or adhesion to cells (Paper III). With this knowledge we decided to expand the work to study the function of PilA, as well as the additional identified pilin genes in the virulent type B strain, FSC200 (paper V). In frame deletions of the four functional pilin genes; pilA, FTT0861,

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FTT0230 and FTT1314 were constructed, and four pseudopilin genes, FTT1621-22 and FTT1496-97 were mutated by polar insertion mutation and in frame deletion respectively. The ability of these mutant strains to induce lethal infection in mice was compared to the isogenic wild-type strain. Strain FSC200 is highly virulent in mice, where less than five bacteria are required to cause a lethal infection. With the exception of the pilA mutant none of the other pilin mutants appeared to be attenuated in single strain infection experiments. Therefore competitive infection experiments were performed, where the ability of each pilin mutant to compete with the wild-type strain in mixed infections was assessed. We found that only pilA and the polar insertion mutant FTT1621-22 were attenuated for virulence in mice, while the other pilin or pseudopilin genes did not have any impact on virulence in the mouse infection model (Paper V). When the function of PilA in the type A strain SCHU S4 was analysed, with the same methods as above, we could establish that the virulence of SCHU S4 is also dependent on a functional pilA gene (Paper IV).

Overall, the expression of PilA is central for virulence in mice of type B strains, and appears to have a role also in the highly virulent type A strain, SCHU S4 (Paper III, IV, and V). On the other hand, when comparing these results to the function of PilA in F. novicida, there are conflicting results. One study established, similar to my findings here, that PilA of F. novicida was necessary for full virulence in mice (Zogaj et al., 2008), while another study showed that a F. novicida pilA mutant was even more virulent than the wild-type (Hager et al., 2006). In the latter study, the authors speculated that this enhanced virulence might be due to abolished secretion of PepO, a protease involved in vasoconstriction, and therefore could limit the spread of F. novicida. Since PepO is lacking in the other F. tularensis subspecies, it is not possible to compare the different subspecies regarding these defects. However, in both these studies they speculated that the function of Tfp genes in F. novicida is connected to a type II secretion system, T2SS. The ability to secrete detectable levels of proteins seems to be unique to F. novicida, but still this does not mean that there is no secretion in the other subspecies of Francisella. It is possible that we have not yet found the specific growth conditions needed for secretion of detectable protein levels by ssp. holarctica and ssp. tularensis.

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The Tfp biogenesis genes in the virulence of SCHU S4

In order to investigate the role and function of the Tfp biogenesis genes, pilA, pilC, pilQ, and pilT, were individually deleted in the type A strain SCHU S4, and the resulting mutants were evaluated for virulence in mice via the subcutaneous route of infection (Paper IV). Mutation of pilA, pilC and pilQ were all expected to result in lack of, or non-functional, pili and result in an attenuated virulence phenotype. Also here, as in the FSC200 infection studies (Paper V), single strain infections failed to reveal any differences and therefore mixed infections were performed. The CI values (Table 1, Paper IV) revealed that PilC and PilQ contributed to virulence in mice to a similar extent as PilA. However, the pilT mutant was somewhat surprisingly found to be even more virulent compared to the wild-type strain. This was unexpected as in other pathogens like P. aeruginosa, deletion of pilT results in a hyperpiliated and attenuated phenotype (Comolli et al., 1999). Another circumstance that argue for a significant role for PilT in type A strains, is that PilT is functional only in type A strains, while there is a stop codon within the gene in type B strains, rendering pilT non functional. Still, in a recent study results were presented where deletion of the truncated, presumably non-functional, pilT gene in LVS resulted in fewer surface fibres as well as attenuation in mice. These data are contradictory to our studies of PilT, where we have established that pilT is not expressed in type B strains, i.e. there is no suppression of the stop codon. As seen in Fig. 3, Paper V, only the intact PilT is expressed, which supports that pilT is a pseudogene.

Does Francisella express functional Tfp fibres on the surface?

Our studies regarding Tfp genes in F. tularensis have linked PilA to virulence. In paper III (Fig. 5B) we also verified that PilA is exported to the surface of the bacterium, but so far, we have been unable to prove any existing surface located filaments composed of PilA. However, in paper III, the pilA gene was tagged with a FLAG epitope, and it could not been excluded that this epitope somehow interfere with filament formation. One argument that the pilin proteins actually are involved in pili formation come from work by Salomonsson et al. where PilA from a type A/B strain and F. novicida both were able to express pili when expressed in the heterologous system in Neisseria gonorrhoeae (Salomonsson et al., 2009a). There is also evidence indicating that both LVS and F. novicida express pili

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like structures, and here the authors proposed that FTT0861 was the structural component of the pili (Chakraborty et al., 2008; Gil et al., 2004). However, to date no one has been able to actually verify that the expressed filaments are Tfp and/or what protein-/s these filaments are composed of.

Posttranslational modification of pilin protein

Many proteins undergo posttranslational modifications, and one of the most common modification is glycosylation, a process where sugars are added to proteins in flagella, pili, other adhesins or LPS (Spiro, 2002). Posttranslational glycosylation has been extensively studied in bacteria like Neisseria gonnorhoeae and Psudomonas aeruginosa (Comer et al., 2002; Miller et al., 2008; Vik et al., 2009) (page 13).

In F. tularensis a homologue to a glycosyltransferase, pilO, was targeted and evaluated for its ability to modify the identified pilin proteins (Paper V).Our result verifies that PilA, as well as proteins encoded by FTT0230 and FTT1314, are posttranslationally modified and that this modification requires PilO. These interesting findings provide at least some support for the idea that F. tularensis actually expresses a Tfp or at least that these proteins are surface located, as surface proteins often are modified by glycosylation. The nature of the modification is currently under investigation.

Conclusions F. tularensis can be isolated from different hosts, ranging from mammals

to insects and protozoa. Type A strains are mainly found in dry environments and probably occupies lagomorphs and rodents, while type B strains are more associated with water, where protozoa could act as a reservoir. In addition, F. tularensis is able to enter the “viable but not culturable” state when grown in lake water, which could be one strategy for the bacterium to adapt to different natural environmental stress conditions (Thelaus et al., 2009). Furthermore, it has been established that Francisella can alter between an intracellular and extracellular lifestyle which implicate that it is able to adapt to multiple cellular microenvironments. This broad spectrum of hosts and lifestyle properties indicate that this bacterium has a very high potential for adaptation to different environmental conditions. The

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challenge to adapt to specific environments can partly be met by genetic adaptation to a specific niche, and partly by modulating gene expression to sense and rapidly respond to environmental signals (Chong et al., 2008). Since F. tularensis has a relatively small genome and so far only a few known regulatory genes including no two-component system, it is likely that there is a tight and possibly also overlapping regulatory network in F. tularensis that maintain these diverse functions.

In my studies different subspecies and strains were included, the avirulent live vaccine strain, LVS, a natural isolate from a hare, the virulent type B strain, FSC200, and the highly virulent type A strain, SCHU S4. The reason for including these different strains was to evaluate if the distinct genomic differences seen at the level of Tfp related genes, also was reflected in virulence properties. These differences include two pilin genes, pilE and pilV, and the predicted ATPase, pilT, that are all non functional in type B strains but are intact in type A strains. We were however unable to establish any significant differences in the phenotype regarding growth and virulence between the two subspecies. On the other hand, our studies were for the most part limited to virulence in mice and growth in macrophages, so therefore there might be additional differences that could have been revealed if other infection models and growth conditions had been included. Alternatively, it is also possible that the observed distinct differences seen between the subspecies rather reflect adaptation to specific environmental conditions and transmission, than having a direct role in animal and human infection. The only pilin gene that strongly correlated to virulence in all the strains that I investigated was pilA. There appears to be a selection to maintain pilA as it has not been deleted in pathogenic type A and type B strains, even if the direct repeats are present and flank the pilA gene also in these two subspecies.

I have used different strategies to identify and evaluate the function of virulence genes, in order to reveal some aspects of F. tularensis virulence mechanisms. My studies of Hfq, lead me to the statement that Hfq is required for resistance against different stress conditions via its role in regulation of a broad spectrum of genes. Future studies are needed to confirm the genes that are responsible for stress resistance and virulence. The recognition that DsbA has a major role in virulence, and the ability of a dsbA mutant strain to induce protective immunity are interesting and important findings since this mutant have potential to be explored as a new live vaccine strain. The proposed model for both a periplasmic activity and a

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surface located portion of DsbA is likely but still not fully explained. Maybe this is a unique dual function possessed by a protein in F. tularensis, and perhaps only one of many not yet identified secrets of F. tularensis that in part explain how this bacterium can demonstrate such broad functions despite its relatively small genome. The role of DsbA in virulence has not been fully explored and further analysis could reveal and identify novel genes involved in virulence.

Taken together – this thesis summarises the results from the different strategies I have used to identify and characterise virulence genes in F. tularensis. I have specifically targeted membrane associated proteins like DsbA and genes postulated to encode a Tfp. I have also studied regulation of virulence genes where I focused on analysing the role and function of Hfq. This is still a young research field and F. tularensis is a highly adaptable bacterium. My studies may raise as many new questions as they may answer, but this is also how basic research works. Therefore I feel that my work has significance and contributes to the understanding of the complex interactions between the pathogen Francisella, its various hosts and the different environmental niches that it successfully colonises.

ACKNOWLEDGEMENTS / TACK!

Acknowledgements / Tack! Till er som nu uppenbarligen hunnit fram till denna sida: Jag hoppas att ni

har haft en trevlig resa genom avhandlingen, för nu är vi då framme vid raderna där jag får möjlighet att framföra mina tack och även lite reflektioner över tillvaron.

Jag har haft förmånen att få byta inriktning i mitt yrkesliv för drygt 10 år sedan. I min förra yrkesroll trivdes jag också väldigt bra, men att sedan få möjlighet att studera i vuxen ålder var något nytt och enormt stimulerande. Jag kom sedan till FOI och fick börja jobba med forskning och upptäcka nöjet med detta. Ända sedan 2003, då jag började studera harpestbakterien och fram till nu, ja då det mesta av det praktiska arbetet till denna avhandling tog form, har varit både intressant, inspirerande och tålamodsprövande. Det sista året har jag till stor del ägnat åt att sammanställa allt arbete och sammantaget kan jag säga att den största känslan från detta år är att det har varit väldigt roligt.

Först av allt vill jag framföra mitt stora tack till Åke – min fd projektledare, ”coach” och numera även handledare. Jag imponeras av din förmåga att ha närvaro, trots att du har, inte bara några bollar utan ett helt bollhav i luften. Åke, jag är väldigt tacksam över att du lyckades ingjuta mod i mig att sammanställa de senaste årens arbete i en doktorsavhandling. Utan din support vet både du och jag att jag aldrig vågat ta det här steget. De förutsättningar och det förtroende jag har fått har jag försökt förvalta på mitt bästa sätt. Till er i ”Francisella gruppen” Kerstin, Emelie och Laila, vill jag också rikta ett stort tack, och även till den tidigare gruppmedlemmen Elisabet F. Utan er tillgång i fråga om diskussioner, visioner, idéer, händer och tid så hade detta arbete inte varit möjligt att genomföra. Ett stort tack även till Solveig, min skrivrumskamrat och min stora ”djurstudie-partner”. Thanks also to collaborators in Czechia, France, Umeå University and Great Britain. Under sista året har jag fått vara på institutionen för Molekylärbiologi, tack även till er för era åsikter samt alla gruppmöten och journal clubs.

Vidare vill jag tacka alla som förgyller tillvaron och bidrar till att jag får komma till en arbetsplats där jag trivs. Alla ni i tidigare ”analysgruppen” för sakkunnighet och support. Alla som jag lunchat och fikat med i ”gamla” fikarummet, ”Syd” eller matsalen, och fått dela inspirerande diskussioner i allt från triviala ämnen till att lösa världsproblemen. Tack också till er som

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ACKNOWLEDGEMENTS / TACK!

33

tagit er tid till att vara ett stöd de gånger jag haft behov av det – ovärderligt med sådana arbetskamrater. En del av er som tillhör kategorin ”alla” har slutat, men ni har i högsta grad också tillhört ovanstående kategorier.

Sen till mina ”privata” vänner som jag understundom försummar, framförallt det senaste året. Tack för att ni finns även om jag gjort mig svårtillgänglig. Jag har, som jag tidigare sagt, svårligen lidit av social abstinens, men jag hoppas att det inte gett obotliga defekter utan att jag nu ska kunna reparera skadan. Att bara prata med två katter kan verkligen sätta spår i mentaliteten, eller också kanske det är katterna som hållit mig någorlunda normal.

Avslutningsvis så har jag min familj som är grunden i min tillvaro. Mina syskon, Nisse och Karin med familjer, som finns där när det blåser och när lillsyrran behöver. Mamma Kerstin som alltid är mån om att vi har det bra, och min pappa Sixten, som inte finns kvar hos oss idag, men som var min allra största supporter i många av de beslut man tar i livet. Pappa var även med i detta beslut att doktorera och han hade varit omåttligt stolt om han hade fått vara med nu. Sist och allra mest – mina bästa, kloka, självständiga, älskade döttrar Ida och Malin – jag är så tacksam att ni finns.

SAMMANFATTNING PÅ SVENSKA

Sammanfattning på svenska

Francisella tularensis, är en Gram negativ bakterie som orsakar sjukdomen harpest, även kallad tularemi. Francisella är en mycket virulent bakterie där en infektionsdos på mindre än 10 bakterier kan orsaka sjukdom. Förmågan att orsaka infektioner baseras på olika virulensmekanismer, men hos F. tularensis är inte många av dessa faktorer kända. Det finns i huvudsak två underarter av Francisella tularensis som orsakar sjukdom hos människor, den högvirulenta underarten tularensis, även kallad typ A, och den något mindre virulenta underarten holarctica, kallad typ B. Jag har fokuserat mina studier på att analysera några olika stammar av typ A och typ B. Syftet med denna avhandling har varit att öka kunskapen om den förmåga som Francisella har att orsaka sjukdom, med tonvikt på yt- och membranassocierade proteiner och strukturer. Dessutom har jag undersökt den regulatoriska rollen hos ett chaperone liknande protein, Hfq.

Hittills är endast ett fåtal regulatoriska proteiner är kända hos F. tularensis. Jag har visat att Hfq spelar en roll i regleringen av gener associerade till virulens i Francisella. Detta är första gången en reglulator identifieras som negativt reglerar gener lokaliserade i Francisella patogenicitets ön. Ett annat protein identifierades i yttermembranet på Francisella-bakterien. Detta protein är en homolog till ett disulfid oxidoreduktas, DsbA, som krävs för att ett protein ska inta rätt funktionell konformation. Det visade sig att DsbA bidrar till virulens, och dessutom att en dsbA mutant har förmåga att inducera skydd mot Francisella infektioner. Det finns också andra gener med homologi till kända virulensmekanismer som är identifierade i Francisella genomet, där en sådan mekanism är typ IV pili systemet. I detta system ingår många komponenter, däribland ett pilin, PilA, som konstaterades bidra till virulens i både typ A- och typ B-stammar. Dessutom är två gener, pilC och pilQ, vilka kodar för proteiner som är involverade i pilits uppbyggande och sekretion, också betydelsefulla för virulensen hos en typ A stam.

Sammanfattningsvis så bidrar dsbA, hfq och typ IV pili associerade gener till virulens i F. tularensis. Vidare är DsbA ett potentiellt objekt för läkemedelsutveckling och en dsbA mutant en kandidat för utveckling av en ny levande vaccinstam. Förutom detta så har identifieringen av Hfq som en ny regulator gett ny kunskap vad gäller det regulatoriska nätverket i Francisella tularensis.

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