1. Investigation of mutual utilization of sialic acid by pathogenic microorganisms in bacterial adaptation to the host ...

Although pathogenic bacteria always co-exist with other microbes while colonizing their hosts, little is known about how they interact and compete with each other to successfully survive at mucosal surfaces. Bacteria have evolved to use sialic acid for their own benefit in at least two different ways: they utilise sialic acid as a nutrient, indeed many commensal and pathogenic bacteria use host sialic acids as sources of carbon, nitrogen, energy, and amino sugars for cell wall synthesis, or they coat themselves in sialic acid, providing resistance to components of host innate immune responses. We have recently characterized a carbohydrate-binding protein in Group B streptococcus (GBS), which is important for the uptake of sialic acid from the environment. In addition, we demonstrated that the expression of the protein is necessary for the growth of GBS in a complex medium devoid of glucose, but containing sialic acid. Since this protein is not able to extract sialic acid from host glycoproteins, we hypothesize that GBS rely on the sialidase activity expressed by other bacteria inhabiting the same niche. Indeed, many bacterial and viral pathogens secrete a sialidase that releases sialic acid from a diverse range of host sialo-glycoconjugates that could be scavenged by GBS. Alternatively host sialidases could be activated during the course of inflammatory responses to infection. Therefore we will examine how environmental sialic acid affects the capacity of pathogens sharing the same niche to efficiently colonize the host. This will be achieved both in competition experiments with distinct species and/or taking advantage of mutant strains defective for genes involved in sialic acid catabolism/uptake/transport. These experiments will also be performed in the presence of host epithelial cells to act as a physiologically relevant source of sialic acid. Furthermore, we will examine whether antibodies raised against surface exposed components of the bacterial sialic acid metabolism can impede both bacterial fitness in vitro or effective colonization in vivo.
The collaborative laboratory at the Imperial College in London has a pluriannual experience on bacterial pathogenesis. In particular they have highlighted the importance of metabolic pathways in influencing bacterial serum resistance. Indeed, pathogenic species colonizing the upper respiratory tract, such N. meningitidis, use a limited number of carbon energy sources including glucose and lactate and IC's group has recently elucidated the importance of lactate metabolism for the survival of the bacterium within the host. In addition, they have underlined that the mechanism for attenuation of host response is mediated through the sialic acid biosynthesis pathway, which is directly connected to central carbon metabolism. These findings highlight the intimate relationship between bacterial physiology and resistance to innate immune killing. IC's laboratory has also developed several experimental tools such as an in vitro model of epithelial cells derived from the respiratory tract and serum resistance assays that will be crucial to investigate the role of bacterial carbohydrate metabolism during colonization. Biochemical assays to evaluate carbohydrate uptake and levels of capsule expression are also available in IC's laboratory.

2. Investigation of the biological role of surface proteins in Staphylococcus aureus pathogenesis

The Gram positive pathogen, Staphylococcus aureus is responsible for disorders ranging from skin infections to septic shock. The antibiotic resistant S. aureus strains have been a major medical challenge globally, especially in nosocomial settings. Although S. aureus is generally considered to be an extracellular pathogen, in recent years it has been established that it is capable of leading an intracellular lifestyle in the host. Intracellular bacteria could indeed hinder effective bacterial killing by antibiotics and contribute to persistent staphylococcal infections. The molecular mechanisms involved in S. aureus-mediated host cell invasion or survival are unclear. While some surface proteins such as fibronectin binding proteins have been reported, there is little known about other cell surface proteins in host-pathogen interactions
We propose to use molecular tools to study the role of surface-associated proteins of S. aureus in host cell invasion. We plan to employ a transposon library of mutants in S. aureus to screen for genes that are essential for host cell entry and survival in epithelial cell lines. Genes involved in these processes will be identified by deep sequencing mutant libraries. We will then focus on surface proteins that are important in host cell entry. Using genetic tools we will generate targeted mutants in multiple S. aureus strains. These mutants will be used to further characterize interactions of selected proteins with host cells using microscopic and biochemical methods.
Part of the proposed work will be performed in collaboration with the IC. The lab at the Imperial College London has pioneered research on S .aureus physiology, particularly with regard to surface-associated proteins. IC's group elucidated the pathways involved in the synthesis of lipoteichoic acid, a key component of the staphylococcal cell wall.

3. Replication dynamics of Streptococcus pyogenes

IC's group has recently developed a powerful reporter system based on fluorescence dilution that enables direct quantification of the replication dynamics of Salmonella in murine macrophages at both the population and single cell level. Its use has provided the first direct evidence of viable non-replicating Salmonella cells in an infected host. These could represent an important reservoir of persistent bacteria. We now wish to extend the system to other pathogens to study replication dynamics in vivo, and to identify and characterize non-replicating bacteria associated with chronic infections. The group of KI has pioneered the use of multi-photon microscopy and micropuncture techniques to enable visualization of the progression of infection in real-time within the organ of a single live animal, focusing in particular on uropathogenic E. coli-induced pyelonephritis. In this project we will introduce the reporter constructs developed by IC's group into a strain of Streptococcus Pyogenes and examine the dynamics of replication in a live imaging model of chronic pyelonephritis. By combining the expertise of the two groups we expect to gain novel insights into bacterial multiplication rates within the bladders and kidneys of live animals and to learn more about the nature of persistence in this disease.

4. Regulation of Shigella virulence

We have recently discovered two bacterial gene regulators that govern expression of virulence factors during the two key steps of Shigella virulence, cell entry and replication in the cytosol. The transcription factor FNR is known to influence the expression of over 50 genes involved in metabolism that are necessary during the transition from growth of the bacterium in aerobic to anaerobic conditions. We have found that FNR is co-opted by Shigella to also dictate the co-ordinated production of genes on the invasion plasmid that carries the genetic information necessary for host cell entry. We have shown that transcription of two genes, spa32 and spa33, are repressed by FNR in anaerobic growth, and these affect the function of the type three secretion system (T3SS) which is required for entry. Furthermore we have identified a novel gene regulator of the AraC family that contributes to growth in the cytosol, the intra-cytoplasmic regulator ICR. The conditions faced by intracellular Shigella are largely unknown, even though this is a key environment for the bacterium during pathogenesis. Interestingly, this regulator is not present in nonpathogenic E. coli strains.
A: Our initial aim is to define the regulon of both FNR and ICR through microarray analysis and RNA-seq approaches. The bacteria will be grown with and without oxygen (to analyse the FNR regulon), and in the presence or absence of extracts of cytosol for the ICR mutant; the mutant fails to grow in the presence of cytosol derived from infected cells. The data will provide a genome wide catalogue of genes controlled by these key factors. Results for genes of interest (particularly those on the virulence plasmid) will be confirmed by qRT-PCR methods.
B: Next we will define the specific DNA sequences specifically recognised by the transcription factors. Although consensus recognition sequences for FNR have been identified in E. coli, we will determine the sites recognised in Shigella. We have already obtained purified FNR to perform DNA binding experiments, and co-immunoprecipitation experiments.
C: The role of genes in the regulon will be determined by constructing null-mutant strains. These will be analysed in standard assays of virulence, including cell entry, plaque formation, cytosolic replication, and ability to induce pathological changes in the gastrointestinal tract.
By the end of the project we will have a comprehensive understanding of the function of these transcription factors and how they modulate virulence of this important human pathogen in the host.

5. Deciphering the regulatory mechanisms of transcription of antibacterial factors of the gut epithelium

Our laboratory has demonstrated that the enteroinvasive bacterial pathogen Shigella, in order to cause the rupture, invasion, and inflammatory destruction of the gut mucosa, develops a survival strategy on the mucosal surface which facilitates its colonization. This is based upon the pathogens capacity to inject effector molecules into epithelial cells through a type III secretory system. These effectors (i.e., Osp and IpaH proteins) are regulators of epithelial innate defense mechanisms, such as expression and secretion of antimicrobial peptides (i.e., beta-defensins), and trafficking of immune cells (i.e., PMNs and dendritic cells). Their function is to interfere with the signalling pathways that control these defense processes and dampen them to an extent that allows bacteria to survive and colonize the epithelial surface before invading it, and translocating to sub-epithelial tissues (see references).
These observations led us to hypothesize that the innate defence mechanisms of epithelia, particularly the intestinal epithelium, could be manipulated in the opposite way, in order to strengthen epithelial capacities to resist bacterial colonization and translocation with obvious medical applications such as (i) prevention of recurrent enteric infections in children in the developing world, (ii) prevention of bacterial translocation and bacteraemia/septicaemia in immunocompromised patients, and (iii) better control of the pro-inflammatory capacity of the commensal flora in inflammatory bowel diseases.
The overall project, in which the proposed PhD program is embedded, is to identify molecules of the pharmacopoeia that will be able to strongly stimulate epithelial expression of antimicrobial molecules and mediators of tissue repair, while avoiding induction of bona fide pro-inflammatory genes (i.e., TNFα, IL-1β, IL-6, IL-8, etc.) causing tissue destruction. It will be largely based upon high-throughput approaches combining screening of combinatorial libraries of molecules and siRNA screens.
The proposed PhD program is to elucidate the regulatory mechanisms of the genes encoding the major antimicrobial factors of the colonic epithelium (i.e., HBD2, HBD3 and cathelicidin), and the major repair molecules (i.e.: TGF-β, Trefoil factors, and MUC2), in comparison with those of the bona fide pro-inflammatory cytokines described above. The aim is to find a «breach» in the regulation of transcription that would allow to privilege expression of the genes encoding the microbicidal and repair systems against the expression of the pro-inflammatory genes. This is a new view according to which the innate immune response does not necessarily occur «en bloc», but is susceptible to manipulation, particularly at the transcriptional level, thanks to genetic and epigenetic mechanisms that remain to be identified.
Careful bioinformatics analysis of the respective promoter sequences will be carried out to define possible differential regulatory systems, in combination with a whole-genome siRNA screen aimed at identifying transcriptional and regulatory factors. Stably-transfected cellular reporters will be constructed in order to serve as read-outs for studying the regulation of gene transcription.
In collaboration with the KI group, we will model in the mouse a certain number of situations requiring to strengthen antimicrobial mechanisms of defense of the intestinal epithelium (i.e.: fragilized newborns, patients under chemotherapy, relapsing enteric infections, and senescence). This group offers outstanding experience in analysis of the mechanisms of IBDs, and opportunities to access the germ-free facilities and high-throughput transcriptomics capacities also required by this project.
The IP and KI groups have a long history of interaction, collaboration, and are currently involved in the TORNADO project, a EU-funded program of the 7FP aimed at understanding the role of the gut flora in nutrition.

6. A new secreted protein involved in the virulence of the bacterial pathogen Listeria monocytogenes

Bacteremia, characterized by the presence of viable pathogenic bacteria in the blood, is a major cause of morbidity and mortality worldwide. Bacteremia can lead to sepsis or spread of bacteria to distant sites causing infections such as endocarditis, osteomyelitis and meningitis. Little is known about how pathogenic bacteria adapt to survive and grow in the bloodstream. A model human pathogenic bacterium to address this issue is Listeria monocytogenes, which causes a variety of human diseases, ranging from gastroenteritis to invasive listeriosis leading to life-threatening meningoencephalitis. Bacteremia is required for invasion of the central nervous system by Listeria monocytogenes. We have recently carried out a comprehensive genome-wide transcriptome analysis of Listeria monocytogenes grown in human blood. We identified a gene whose expression was highly induced in human blood and whose deletion strongly attenuated virulence in a murine model of infection. This gene is absent from the genome of non-pathogenic Listeria species and encodes a secreted protein of unknown function. A deletion mutant is attenuated in the mouse model.
The aim of this project is to identify the function of this new virulence factor and to characterize its role in vitro and in vivo. Using a two-hybrid screen, we identified a eukaryotic protein that could interact with this virulence factor. The interaction will be confirmed by immunoprecipitation and fluorescence microscopy and the role of the virulence factor on the activity of its interacting partner will be studied in cells transfected with the factor alone or in cells infected by L. monocytogenes. Its role in the infection process will be then investigated in vitro and in vivo.

7. The role of immune cells in pneumococcal infections

Streptococcus pneumoniae (pneumococcus) is a common colonizer in healthy pre-school children, yet capable of causing pneumonia and invasive disease. The mechanisms how pneumococci can traverse biological barriers remains largely unknown.
Project aim: the project aims to investigate the interaction between inflammatory cells, such as macrophages/monocytes, neutrophils, dendritic cells and T-cells, and pneumococci in pneumococcal infections Preliminary data: our group in collaboration with the NVD group has discovered a pneumococcal pilus structure that promotes adhesion as well as pneumonia and invasive disease. The pilus consists of one major subunit protein (RrgB) and two minor pilus subunits (RrgA, and RrgC) of which RrgA act as an adhesion for epithelial cells. RrgA has recently been shown also to mediate binding to the CD11b phagocytic receptor on murine macrophages. Pneumococci expressing RrgA are taken up better by macrophages as compared to isogenic mutants lacking the CD11b binding RrgA adhesin. The rate of intracellular killing was not affected, meaning that a higher number of live intracellular bacteria were present up to 10 hours after infection when RrgA was expressed as compared to an isogenic mutant. In vivo imaging and transwell assays revealed that wildtype but not CD11b deficient macrophages infected with RrgA expressing pneumococci exhibited an increase migratory behaviour. Parallel experiments revealed that RrgA expressing pneumococci reached the blood stream faster as compared to bacteria lacking the adhesin, after intraperitoneal infection suggesting that entry into the blood stream is due to migratory immune cells carrying viable pneumococci. Furthermore, we have preliminary data suggesting that pneumococcal cell wall fragments are important in the interaction with T-cells and monocytes to elicit IL17 production
Work plan: in vitro imaging techniques will be used to study the interaction between pneumococci and immune cells.  In vitro assays will be set up to study the migratory behaviour of macrophages and dendritic cells infected with wild type and mutant pneumococci. Depletion of macrophages and its effect on passage across biological barriers will be followed. Live imaging techniques will be used by introducing in vitro infected immune cells carrying pneumococci to directly follow migration of infected immune cells across biological barriers. An artificial lung model established at Karolinska Institutet will be used in infection studies with wild type and mutant pneumococci as well as with immune cells infected with pneumococci.

8. Comparative genomics and functional characterization of pneumococci and interactions with host cells ...

Streptococcus pneumoniae is a highly diverse organism consisting of a large number of capsular serotypes and clonal types. Epidemiological studies reveal that isolates belonging to certain serotypes and likely also clonal types have a much higher likelihood of causing invasive disease in humans. A problem in using comparative genomics comparing isolates of different invasive disease potential has been the large number of differences that exist between strains of different clonal types potentially affecting disease outcome.
Project Aim: to study pneumococcal virulence attributes that have been identified using whole genome sequencing of pneumococcal isolates as well as their interactions with host cells and innate immune responses
Preliminary data: in a large Swedish collection of carrier and invasive disease isolates from small children, molecular characterization was performed both by multi-locus sequence typing (MLST), and by the more discriminatory techniques pulse-field gel electrophoresis (PFGE). Within one single clonal lineage of the common serotype 6B, strains with different PFGE patterns were found. Interestingly, strains with these PFGE patterns did differ significantly from one another in their odds ratio of causing invasive disease. Whole genome sequencing performed on four of these related isolates identified a number of genetic differences in surface proteins associated with invasive disease in mice models.
Workplan: Pneumococcal isolates belonging to different clonal types will be subjected to whole genome sequencing and functional characterization with respect to virulence attributes by creating defined mutants and performing infection experiments in vitro and in mice. In vitro experiments such as testing parameters such as adhesion to host cells, resistance to phagocytosis, complement mediated killing as well as studies on the involvement of the inflammasome will be included in the study.

9. The human B cell response to factor H binding protein of Neisseria meningitidis

Meningococci (Neisseria meningitidis) are commensals of the human nasopharynx, which reside asymptomatically in 10-40% of the human population. However, encapsulated bacteria can cross the nasopharyngeal epithelial barrier and cause severe bacterial meningitis and septicemia with frequently fatal outcome.
Immunity to N. meningitidis is associated with the development of protective serum bactericidal activity that involves the alternative and classical pathways of complement activation. However, antibody-mediated opsonisation may contribute significantly to bacteria killing. Naturally acquired serum antibodies are predominantly directed against capsular polysaccharide (PS) antigens and are indirectly induced by exposure to nonpathogenic bacteria, which elicit cross-reactive antibodies that recognise meningococcal PS antigens. However, PS antigens do not elicit long-lasting memory responses and fail to elicit universal humoral protection due to strain variation. Therefore, protein antigens, which are able to elicit long-lasting immunity even in infants who are at high risk for meningococcal disease, may induce more potent protective antibodies against N. meningitidis. Factor H binding protein (fHBP) is expressed on all strains of N. meningitidis and contributes to avoidance of complement mediated killing and induces protective antibodies thus making it a prime vaccine candidate antigen. The binding sites of anti-fHBP antibodies have been characterized in mice but the humoral immune response to fHBP in humans has not been characterized.
To obtain information on human anti-fHBP antibodies we will use fluorescently labeled fHBP to isolate single fHBP-reactive CD27-positive memory B cells from healthy individuals by fluorescence activated cell sorting (FACS). Immunoglobulin (Ig) heavy and light chain genes will be amplified by RT-PCR and sequenced. Cloning and in vitro expression of matching IgH and IgL chain genes will allow us to generate fully human recombinant monoclonal antibodies, which can be tested for i) fHBP reactivity in vitro, ii) bactericidal activity in vitro and in vivo, and iii) their ability to block fH:fHBP interactions in vitro. We will determine which antibodies are able to recognize the extensive fH binding site on fHBP, by examining their reactivity with fH:fHBP complexes. Furthermore, the antibodies will enable us to precisely map fHBP epitopes that are relevant for antibody mediated protection and thus must be included in future vaccine candidates.
In summary, the experiments will help characterize the Ig gene features, reactivity properties and bactericidal activity of anti-fHBP antibodies in humans.

10. Identification and analysis of host cell functions essential for viral replication

Viruses constitute a major threat to human health with respect to the natural occurrence of infections, their misuse as biological weapons and also concerning animal health. Currently, two main strategies for combating viral infections - vaccination and standard antiviral therapy - are being pursued with variable success. A new third strategy aimed at targeting host cell factors, dispensable for the host but essential for virus propagation, promises to be a powerful alternative., It is now possible to scan the entire human genome for critical determinants of infection using high-throughput RNA interference technology . Identification of these determinants has the potential to offer therapeutic solutions for the most important (widespread?) human viruses.
We have previously performed a genome-wide RNAi screen for factors influencing influenza virus replication (Karlas et al. Nature 2010). This work has yielded a plethora of host cell factors either stimulating or repressing viral replication. Consequently, we have identified promising host cell targets which proved in vitro efficacy for a variety of influenza A virus isolates (including the new pandemic H1N1 swine flu and the H5N1 chicken flu). Furthermore, our target identification provided insights into the exploitation of cellular reaction pathways by this virus .  These will  be studied in further detail. The respective host cell processes include uptake of viral particles, nuclear import of viral DNA, replication, RNA splicing, autophagy, and viral release.
Here we wish to extend our existing knowledge to a comparative analysis of flu virus variants and to other important RNA viruses e.g. West Nile Fever, Chikungunya virus and Yellow fever. Moreover, we aim at assessing the significance of the so identified host factors to other host species, including chicken and swine. Having established suitable cellular assays (using e.g. microscopic read outs) we wish to assess the role of these host cell processes for the replication cycles of several of the above mentioned clinically important RNA virus species.
While the expertise at MPIIB is centered aroung the development and use of cell biological assays, RNA interference loss-of-function analyses and microscopical screening, the collaboration with IP will be essential with regard to its expertise in handling and characterisation of specific viral agents.