Summary of the project

Pathogenic intracellular bacteria cause many severe forms of disease worldwide with devastating morbidity and high mortality rates. Despite the significance of many facultative intracellular bacteria in animal and human diseases, relatively little is known as to how intracellular parasites use eukaryotic cells as a replication niche to hide from the immune system. Listeria monocytogenes is one of the deadliest food-borne pathogens accounting for up to 10% of community-acquired bacterial meningitis in humans. Following ingestion by a mammalian host, L. monocytogenes has the capacity to cross all bodily barriers such as the intestine, the blood-brain and the feto-placental barrier and to replicate within host cells. The proposal aims at fully exploiting genomicand post genomic data in combination with different in vitro and in vivo infection systems to understand how this microbe subverts cellular functions to favour infection. The bacterial and host cell systems and the signalling, regulatory and effector pathways involved in the different steps of the listerial infectious life cycle will be systematically investigated by transcriptomic profiling, RNAi technology, proteomic analysis, in vivo imaging and complemented with biochemical and pharmacological analyses. The proposal will identify intracellularly expressed surface and secreted proteins, determine the eukaryotic ligands engaged and examine the cellular processes and signalling pathways harnessed by the bacterium. Information deriving from bacterial and host processes will be used to identify potential metabolic pathways that can be manipulated pharmacologically or targeted by new and existing chemotherapeutics. These studies will form the basis to address questions regarding the propensity of particular lineages of L. monocytogenes to either cause epidemics or sporadic infection using previously determined whole genome sequences of these bacteria. The consortium will create a database for integration and in-depth analysis of data with accessibility for all partners and provide platforms for transcriptome, proteome and functional analyses as well as mutant repositories for the wider scientific community.

Background and present state of the art in the field

The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for up to 10% of community-acquired bacterial meningitis in humans. It is the primary cause of central nervous system infection in domestic ruminants. Listeriosis has one of the highest hospitalisation (90%) and mortality rates of all food-borne infections.
L. monocytogenes is also an important model in biomedical research. The concept that T cells are key players in cell-mediated immunity, and that activated macrophages are required for elimination of intracellular pathogens was established by studies using the murine model of Listeria infection. L. monocytogenes has also become one of the best characterised bacterial systems for the molecular analysis of invasion and intracellular parasitism.Molecular epidemiological studies have also made it a model to understand mechanisms of transmission from the environment to the infected host. Much of the existing knowledge on the molecular and cellular mechanisms of Listeriapathogenesis has been contributed by members of the consortium. A brief summary of the interaction of Listeria with the host cell is given here:

(i) Invasion. Entry of Listeria into host cells is mediated by two leucine-rich repeat (LRR)-containing surface proteins, the internalins InlA and InlB that use different entry pathways. The receptor for InlA is E-cadherin, a transmembrane glycoprotein on epithelial cells that is linked to the cortical actin cytoskeleton via ß- and a-catenin. a-catenin is connected by the ARHGAP10 protein to vezatin which in turn anchors it to myosin VIIA. The interaction between myosin VIIA and the actin cytoskeleton induces the tension that is required for uptake by the bacterium. InlB - via its LRR domain- interacts with the hepatocyte growth factor (HGF) receptor Met, a transmembrane heterodimeric protein. Like the growth factor, InlB stimulates the sequential tyrosine-phosphorylation of Met and the adaptor proteins Shc, Gab1 and Cbl, which are recruited to the site of bacterial entry. More recently, purified InlB has been shown to induce mono-ubiquitination of Cbl, inducing endocytosis of the Met receptor. Indeed, all major components ofthe host endocytic machinery are harnessed by the bacterium to promote its own uptake.

(ii) Escape from the phagocytic vacuole. Unlike most other intracellular parasites, Listeriareplicate in the cytosol of mammalian cells. A pore-forming toxin, Hly or listeriolysin O (LLO), which is only active at low pH, is required for the efficient disruption of the phagosomal membrane. LLO has been shown to localize to lipid rafts and has extremely diverse effects on the host cell including facilitating antigen presentation, inducing diacylglycerol and phosphoinositide metabolism, and activating a proinflammatory response. The N-terminally located PEST sequence in LLO appears to control egress of bacteria to the cytosol. Two phospholipases, PlcA and PlcB (and a sphin-gomyelinase in Listeria ivanovii, SmcL), act in concert with LLO to mediate bacterial escape from the phagosome.

(iii) Growth in the cytosol. Whole genome profiling has revealed that ~17% of the total genome (484 genes)1 was mobilised to enable adaptation to intracellulargrowth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria with decrease in mRNAs encoding enzymes in central metabolism and temporal induction of genes involved in alternative-carbon-source utilization pathwaysand their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, transport, the general stress response, cell division, changes in cell-wall structure, and included many genes with unknown functions.

(iv) Actin-based intracellular motility and direct cell-to-cell spread. After replicating in the cytosol, Listeria spread from cell to cell by actin-based propulsion mediated by ActA. This surface protein activates the Arp2/3 complex by mimicking the natural function of the Cdc42 downstream effector WASP, to which sections of the N-terminal domain of ActA are structurally related. Cell-to-cell spread ensures that Listeria avoid humoral effectors of the immune system, and allow escape from the cellular immune response typically directed against infected cells. ActA exhibits “structural mimicry” of host cell components and studies on its mechanism of action have been instrumental in understanding how eukaryotic cells control the actin cytoskeleton 2;3. Five years ago, publication of the whole genome sequences of a pathogenic L. monocytogenes serotype 1/2a strain as well as that of a non-pathogenic L. innocuaspecies by members of the consortium ushered in the era of Listeria genomics. This data initiated whole genome-based approaches to studying gene expression using transcriptomics, proteomics, comparative genomics, as well as bioinformatics approaches to understanding metabolism and physiology of the bacterium. In the past three years members of the consortium have:

(i) used comparative whole genome hybridization approaches to determine the population
    structure of L. monocytogenes (IP)4
(ii) used comparative genomics to identify novel virulence factors, such as a bile salt
    hydrolase, Bsh, and surface proteins Vip, Auto, InlJ (IP)5-8 and InlH (GI,GBF)9
(iii) determined the genome sequences of six strains representative for all species of the
    genus Listeria (L. ivanovii, L. welshimeri, L. seeligeri, L. grayi) as well as for strains
     representing specific lineages of L. monocytogenes (L. monocytogenes serotype 4b and 4a)
     (GI, IP,WÜ,GBF, LE) (http://www.genomik.uni-wuerzburg.de)10
(iv) used whole genome transcription profiling to delineate all members of the PrfA regulon,
     the master regulator of virulence, for serotype 1 strains (IP,GI, LE)1;11
(v) used whole genome transcription profiling to describe all listerial genes required for
    intracellular survival in epithelial and macrophage cell lines and identify pathovar specific
    gene expression (GI,WÜ)1;12
(vi) used proteome analysis to determine the repertoire of secreted cell wall and membrane
    proteins expressed during extracellular growth (GBF,MA,IP)13-15
(vii) used metabolomics to enable reconstruction of amino acid biosynthesis pathways and
    document the influence of PrfA on branched amino acid production (WÜ)16
(viii) pioneered studies on host cell interactions that revealed hijacking of the
     clathrin-dependent endocytic pathway by Listeria during invasion (IP)17
(ix) described a novel interaction between a listerial protein and a receptor on the
    endoplasmic reticulum (Vip-Gp96) (IP,PT,MA)5
(x) identified a novel regulon modifying cell wall components during intracellular survival using
    transcriptomics (IP)18
(xi) shown that Listeria monocytogenes produces GSH by a unique multifunctional protein
    (GshF) that combines both catalytic steps in a single enzyme (ISR, WÜ)19
(xii) identified a new species-specific pathogenicity island, LIPI2, which is believed to play
    a key role in the pathogenic tropism of L. ivanovii (LE, WÜ)20
(xiii) used fundamental knowledge gained through Listeria genomics research to identify
    a new potential treatment for listeriosis (LE)21
(xiv) created bioinformatic databases with tools for comparative genomics and proteomics
    of Listeria species (GI, GBF)22-26

Transcriptome-based analysis of the host responses to listerial infection have identified an "early/persistent" gene expression program consistent with NF-kB-dependent responses downstream of TLRs, and a subsequent "late response" cluster largely composed of IFN-responsive genes (IRGs) that was dependent on the presence of the bacterium in the host cytosol27. Studies of whole genome-based RNAi knockdowns in Drosophilacells have identified host cell components involved in endocytosis and vesicular transport as well as in the generation of signalling molecules such as phophoinositides, sphingosine and ceramide28;29. More recently, the insulin-like growth factor II receptor has been implicated in the adhesion and uptake of Listeria30.

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