Proteome-wide analysis of severe bacterial infections. The battle between host and pathogen.

The pathogenesis of severe infectious diseases is a complex interplay between the host and the pathogen. The development and progression of a disease encompasses a multitude of processes, which balance between host-damage and host-protection. Proteomic analysis provides the necessary tools to interpret the extensive protein networks of host-pathogen interactions underlying the pathogenesis of a particular disease. This thesis focuses on proteome analysis of two blocks; the host-response during disease, as well as the pathogen during colonization and disease. Data-independent acquisition (DIA) mass spectrometry (MS) was utilized to acquire near-to-complete proteome maps of processes involved in the pathogenesis of one severe infectious disease; meningitis, as well as the host-pathogen interaction counterparts of its leading bacterial cause; Streptococcus pneumoniae.High quantitative ability of DIA-MS was used to construct compendiums of digital cerebrospinal fluid (CSF) proteome maps to define the pathogen-specific host response patterns in meningitis. We generated a predictive multiprotein panel of eighteen human proteins with a high sensitivity and specificity, for discrimination of the meningitis-causing pathogens in the CSF during meningitis. The results also showed a large number of neutrophil-associated proteins in the CSF during bacterial meningitis, and these were found to be due to the presence of neutrophil extracellular traps (NETs). The presence of NETs was further confirmed in the CSF in a rat model of pneumococcal meningitis. Treating the animals with DNase resulted in the abolishment of NETs, and led to increased bacterial killing. We further continued to explore the transcriptional landscape and adaptation of S. pneumoniae in human blood plasma by generating a large number of perturbations. A comprehensive pneumococcal proteome repository was constructed to unravel complex protein-protein networks of the bacteria. The results revealed specific regulatory patterns in response to human blood plasma, and pneumococcal transcriptional reorganization regulated by important virulence factors. Furthermore, to describe processes involved in bacterial dissemination in the human nasopharynx, we investigated differences between pneumococcal populations associated with colonization (biofilm bacteria), disease (biofilm-dispersed bacteria) and the conventional broth-grown, planktonic bacteria. The investigated populations showed distinct proteome patterns, especially in regards to metabolic pathways. The virulence of these models was investigated in a murine pneumococcal infection model, where it was showed that virulence of the populations is largely mediated on the investigated pneumococcal serotype. In conclusion, large-scale proteome analyses produced in this thesis generate fundamental knowledge in understanding host-pathogen interactions as a whole. Furthermore, the constructed repositories can be repetitively queried by the scientific community to deepen the understanding in host-pathogen interactions in bacterial infections

Quantitative proteogenomics of human pathogens using DIA-MS.

The increasing number of bacterial genomes in combination with reproducible quantitative proteome measurements provides new opportunities to explore how genetic differences modulate proteome composition and virulence. It is challenging to combine genome and proteome data as the underlying genome influences the proteome. We present a strategy to facilitate the integration of genome data from several genetically similar bacterial strains with data-independent analysis mass spectrometry (DIA-MS) for rapid interrogation of the combined data sets. The strategy relies on the construction of a composite genome combining all genetic data in a compact format, which can accommodate the fusion with quantitative peptide and protein information determined via DIA-MS. We demonstrate the method by combining data sets from whole genome sequencing, shotgun MS and DIA-MS from 34 clinical isolates of Streptococcus pyogenes. The data structure allows for fast exploration of the data showing that undetected proteins are on average more amenable to amino acid substitution than expressed proteins. We identified several significantly differentially expressed proteins between invasive and non-invasive strains. The work underlines how integration of whole genome sequencing with accurately quantified proteomes can further advance the interpretation of the relationship between genomes, proteomes and virulence

Brain inflammation induces post-synaptic changes during early synapse formation in adult-born hippocampal neurons.

An inflammatory reaction in the brain is primarily characterized by activation of parenchymal microglial cells. Microglia regulate several aspects of adult neurogenesis, i.e. the continuous production of new neurons in the adult brain. Hippocampal neurogenesis is thought to be important for memory formation, but its role in brain diseases is not clear. We have previously shown that brain inflammation modulates the functional integration of newly formed hippocampal neurons. Here, we explored whether there is a defined time period during synaptic development when new neurons are susceptible to brain inflammation. Newly formed hippocampal neurons, born in an intact environment in the adult mouse brain, were exposed to lipopolysaccharide (LPS)-induced inflammation during either early or late phases of excitatory and inhibitory synaptogenesis. We used intra-hippocampal injections of GFP-retroviral vector (RV-GFP) to label the new neurons and ipsilateral LPS injection at either 1 or 4weeks post-RV-GFP injection. A single intra-hippocampal LPS injection induced an inflammatory response for at least 3weeks, including an acute transient pro-inflammatory cytokine release as well as a sub-acute and sustained change in microglial morphology. The general cytoarchitecture of the hippocampal dentate gyrus, including granule cell layer (GCL) volume, and astrocytic glial fibrillary acidic protein expression was not different compared to vehicle controls, and no Fluoro-Jade-positive cell death was observed. New neurons encountering this inflammatory environment exhibited no changes in their gross morphology. However, when inflammation occurred during early stages of synapse formation, we found a region-specific increase in the number of thin dendritic spines and post-synaptic density-95 (PSD-95) cluster formation on spines, suggesting an enhanced excitatory synaptic connectivity in the newborn neurons. No changes were observed in the expression of N-cadherin, an adhesion molecule primarily associated with excitatory synapses. At the inhibitory synapses, alterations due to inflammation were also evident during early but not later stages of synaptic development. Gephyrin, an inhibitory scaffolding protein, was down-regulated in the somatic region, while the adhesion molecules neuroligin-2 (NL-2) and neurofascin were increased in the somatic region and/or on the dendrites. The GABAA receptor-α2 subunit (GABAAR-α2) was increased, while pre/peri-synaptic GABA clustering remained unaltered. The disproportional changes in post-synaptic adhesion molecules and GABAA receptor compared to scaffolding protein expression at the inhibitory synapses during brain inflammation are likely to cause an imbalance in GABAergic transmission. These changes were specific for the newborn neurons and were not observed when estimating the overall expression of gephyrin, NL-2, and GABAAR-α2 in the hippocampal GCL. The expression of interleukin-1-type 1 receptor (IL-1R1) on preferentially the somatic region of new neurons, often in close apposition to NL-2 clusters, may indicate a direct interaction between brain inflammation and synaptic proteins on newborn neurons. In summary, this study provides evidence that adult-born hippocampal neurons alter their inhibitory and excitatory synaptic integration when encountering an LPS-induced brain inflammation during the initial stages of synapse formation. Changes at this critical developmental period are likely to interfere with the physiological functions of new neurons within the hippocampus

Alterations in Brain Inflammation, Synaptic Proteins, and Adult Hippocampal Neurogenesis during Epileptogenesis in Mice Lacking Synapsin2.

Synapsins are pre-synaptic vesicle-associated proteins linked to the pathogenesis of epilepsy through genetic association studies in humans. Deletion of synapsins causes an excitatory/inhibitory imbalance, exemplified by the epileptic phenotype of synapsin knockout mice. These mice develop handling-induced tonic-clonic seizures starting at the age of about 3 months. Hence, they provide an opportunity to study epileptogenic alterations in a temporally controlled manner. Here, we evaluated brain inflammation, synaptic protein expression, and adult hippocampal neurogenesis in the epileptogenic (1 and 2 months of age) and tonic-clonic (3.5-4 months) phase of synapsin 2 knockout mice using immunohistochemical and biochemical assays. In the epileptogenic phase, region-specific microglial activation was evident, accompanied by an increase in the chemokine receptor CX3CR1, interleukin-6, and tumor necrosis factor-α, and a decrease in chemokine keratinocyte chemoattractant/ growth-related oncogene. Both post-synaptic density-95 and gephyrin, scaffolding proteins at excitatory and inhibitory synapses, respectively, showed a significant up-regulation primarily in the cortex. Furthermore, we observed an increase in the inhibitory adhesion molecules neuroligin-2 and neurofascin and potassium chloride co-transporter KCC2. Decreased expression of γ-aminobutyric acid receptor-δ subunit and cholecystokinin was also evident. Surprisingly, hippocampal neurogenesis was reduced in the epileptogenic phase. Taken together, we report molecular alterations in brain inflammation and excitatory/inhibitory balance that could serve as potential targets for therapeutics and diagnostic biomarkers. In addition, the regional differences in brain inflammation and synaptic protein expression indicate an epileptogenic zone from where the generalized seizures in synapsin 2 knockout mice may be initiated or spread

Cerebrospinal fluid proteome maps detect pathogen-specific host response patterns in meningitis

Meningitis is a potentially life-threatening infection characterized by the inflammation of the leptomeningeal membranes. Many different viral and bacterial pathogens can cause meningitis, with differences in mortality rates, risk of developing neurological sequelae and treatment options. Here we constructed a compendium of digital cerebrospinal fluid (CSF) proteome maps to define pathogen-specific host response patterns in meningitis. The results revealed a drastic and pathogen-type specific influx of tissue-, cell- and plasma proteins in the CSF, where in particular a large increase of neutrophil derived proteins in the CSF correlated with acute bacterial meningitis. Additionally, both acute bacterial and viral meningitis result in marked reduction of brain-enriched proteins. Generation of a multi-protein LASSO regression model resulted in an 18-protein panel of cell and tissue associated proteins capable of classifying acute bacterial meningitis and viral meningitis. The same protein panel also enabled classification of tick-borne encephalitis, a subgroup of viral meningitis, with high sensitivity and specificity. The work provides insights into pathogen specific host response patterns in CSF from different disease etiologies to support future classification of pathogen-type based on host response patterns in meningitis

Increased PSD-95 expression during epileptogenesis in Syn2^-/- mice.

<p>Representative immunoblots and quantification of PSD-95 (95 kDa) (A-C) and neuroligin-1 (NL-1) (~101 kDa) (D-F) in cortex, hippocampus, and sub-cortex at 1 month (A, D), 2 months (B, E), and 3.5 months (C, F) relative to WT and normalized to β-actin in cortex and hippocampus for PSD-95 and in all three regions for NL-1 and to GAPDH in sub-cortex for PSD-95. Data are presented as mean ± SEM, n = 4 WT and 9 Syn2^<b>-/-</b> for 1 month, n = 4 WT and 6 Syn2^<b>-/-</b> for 2 months, and n = 5 WT and 6 Syn2^<b>-/-</b> for 3.5 months group. *, <i>p</i> ≤ 0.05, unpaired <i>t</i> test. Ctx = cortex, HPC = hippocampus, SC = sub-cortex.</p

Reduced hippocampal neuroblast production in epileptogenic 2-months old Syn2^-/- mice.

<p>Microphotographs of doublecortin (DCX) immunostaining of migrating neuroblasts in 2-months old WT (A) and Syn2^<b>-/-</b> (KO) (B) mice and 3.5-months old WT (C) and Syn2^<b>-/-</b> (D) mice. Quantification of mean number of DCX^<b>+</b> cells per brain section in the granule cell layer/sub granular zone (GCL/SGZ) of the dentate gyrus at 1, 2, and 3.5 months (E). Images showing Ki67^<b>+</b> proliferating cells in 2-months old WT (F) and Syn2^<b>-/-</b> (G) mice and 3.5-months old WT (H) and Syn2^<b>-/-</b> (I) mice. Quantification of mean number of Ki67^<b>+</b> cells per brain section in the GCL/SGZ at 1, 2, and 3.5 months (J). Data are presented as mean ± SEM, n = 7 WT and 8 Syn2^<b>-/-</b> for 1 month, n = 8 WT and 8 Syn2^<b>-/-</b> for 2 months, and n = 10 WT and 7 Syn2^<b>-/-</b> for 3.5 months group. *, <i>p</i> ≤ 0.05, unpaired <i>t</i> test. Scale bar is 40 μm (in A for A-D and F-I).</p

Increased gephyrin, neuroligin-2, neurofascin, and KCC2 expression during epileptogenesis in Syn2^-/- mice.

<p>Representative immunoblots and quantification of gephyrin (93 kDa) (A-C), NL-2 (93 kDa) (D-F) neurofascin (NF) (~186 kDa) (G-I), and potassium chloride co-transporter (KCC2) (~140 kDa) (J-L) in cortex, hippocampus, and sub-cortex at 1 month (A, D, G, J), 2 months (B, E, H, K) and 3.5 months (C, F, I, L) relative to WT and normalized to β-actin in cortex and hippocampus for gephyrin and NL-2 and to GAPDH in sub-cortex for gephyrin and NL-2 and to GAPDH for all three regions for NF and KCC2. Data are presented as mean ± SEM, n = 4 WT and 9 Syn2^<b>-/-</b> for 1 month, n = 4 WT and 6 Syn2^<b>-/-</b> for 2 months, and n = 5 WT and 6 Syn2^<b>-/-</b> for 3.5 months group. *, <i>p</i> ≤ 0.05, unpaired <i>t</i> test. Ctx = cortex, HPC = hippocampus, SC = sub-cortex.</p

Cytokine and chemokine expression in Syn2^-/- mice.

<p>A panel of pro- and anti-inflammatory cytokines was measured in cortical, hippocampal and sub-cortical homogenates using mesoscale multiplex ELISA. Quantifications of the cytokine expression in cortex (A), hippocampus (B), and sub-cortex (C) in 1-month old Syn2^<b>-/-</b> mice, and KC/GRO expression in cortex, hippocampus, and sub-cortex at 1 month (D), 2 months (E), and 3.5 months (F). Representative immunoblots and quantification of CX3CR1 (~50 kDa) (G) in cortex and hippocampus of 1-month old Syn2^<b>-/-</b> mice relative to WT and normalized to β-actin (42 kDa). Representative confocal images of Iba1^<b>+</b> microglial cells (H-J) including an orthogonal projection of an Iba1-labelled cell (blue), iNOS (red) and arginase-1 (green), with a defined iNOS immunostaining on an Iba1-labelled cell (cross) (H), double-labeled Iba1/iNOS cell in the entorhinal cortex from 1 month old WT (I) and Syn2^<b>-/-</b> mice (J). Note the prominent expression of iNOS in morphologically activated microglial cell in J as compared to iNOS expression in a ramified microglial cell in I. Data are presented as mean ± SEM, n = 4 WT and 9 Syn2^<b>-/-</b> for 1 month, n = 4 WT and 6 Syn2^<b>-/-</b> for 2 months, and n = 5 WT and 6 Syn2^<b>-/-</b> for 3.5 months group. *, <i>p</i> ≤ 0.05, #, <i>p</i> = 0.07, unpaired <i>t</i> test. Ctx = cortex, HPC = hippocampus, SC = sub-cortex. Scale bar is 8.5 μm (in H), 10 μm (in I for I and J).</p