A PrP EGFR signaling axis controls neural stem cell senescence through modulating cellular energy pathways.

Mis-folding of the prion protein (PrP) is known to cause neurodegenerative disease; however, the native function of this protein remains poorly defined. PrP has been linked with many cellular functions, including cellular proliferation and senescence. It is also known to influence epidermal growth factor receptor (EGFR) signaling, a pathway that is itself linked with both cell growth and senescence. Adult neural stem cells (NSCs) persist at low levels in the brain throughout life and retain the ability to proliferate and differentiate into new neural lineage cells. KO of PrP has previously been shown to reduce NSC proliferative capacity. We used PrP KO and WT NSCs from adult mouse brain to examine the influence of PrP on cellular senescence, EGFR signaling, and the downstream cellular processes. PrP KO NSCs showed decreased cell proliferation and increased senescence in in vitro cultures. Expression of EGFR was decreased in PrP KO NSCs compared with WT NSCs and additional supplementation of EGF was sufficient to reduce senescence. RNA-seq analysis confirmed that significant changes were occurring at the mRNA level within the EGFR signaling pathway and these were associated with reduced expression of mitochondrial components and correspondingly reduced mitochondrial function. Metabolomic analysis of cellular energy pathways showed that blockages were occurring at critical sites for production of energy and biomass, including catabolism of pyruvate. We conclude that, in the absence of PrP, NSC growth pathways are downregulated as a consequence of insufficient energy and growth intermediates.

Innate immune responses after stimulation with Toll-like receptor agonists in ex vivo microglial cultures and an in vivo model using mice with reduced microglia.

BACKGROUND: Past experiments studying innate immunity in the central nervous system (CNS) utilized microglia obtained from neonatal mouse brain, which differ developmentally from adult microglia. These differences might impact our current understanding of the role of microglia in CNS development, function, and disease. METHODS: Cytokine protein secretion was compared in ex vivo P3 and adult microglial cultures after exposure to agonists for three different toll-like receptors (TLR4, lipopolysaccharide [LPS]; TLR7, imiquimod [IMQ]; and TLR9, CpG Oligodeoxynucleotide [CpG-ODN] 1585). In addition, changes in inflammatory gene expression in ex vivo adult microglia in response to the TLR agonists was assessed. Furthermore, in vivo experiments evaluated changes in gene expression associated with inflammation and TLR signaling in brains of mice with or without treatment with PLX5622 to reduce microglia. RESULTS: Ex vivo adult and P3 microglia increased cytokine secretion when exposed to TLR4 agonist LPS and to TLR7 agonist IMQ. However, adult microglia decreased expression of numerous genes after exposure to TLR 9 agonist CpG-ODN 1585. In contrast, in vivo studies indicated a core group of inflammatory and TLR signaling genes increased when each of the TLR agonists was introduced into the CNS. Reducing microglia in the brain led to decreased expression of various inflammatory and TLR signaling genes. Mice with reduced microglia showed extreme impairment in upregulation of genes after exposure to TLR7 agonist IMQ. CONCLUSIONS: Cultured adult microglia were more reactive than P3 microglia to LPS or IMQ exposure. In vivo results indicated microglial influences on neuroinflammation were agonist specific, with responses to TLR7 agonist IMQ more dysregulated in mice with reduced microglia. Thus, TLR7-mediated innate immune responses in the CNS appeared more dependent on the presence of microglia. Furthermore, partial responses to TLR4 and TLR9 agonists in mice with reduced microglia suggested other cell types in the CNS can compensate for their absence.

Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported ∼89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (∼78 900 centroids), giving ∼100% coverage, and ∼10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ sLab⟩ ≤ (0.15 ± 0.01) Da (1σx̅). All laboratories achieved ⟨sLab⟩ ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) °C and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories( tHDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort( tHDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.

Microglia Are Critical in Host Defense against Prion Disease.

Microglial cells in the central nervous system play important roles in neurodevelopment and resistance to infection, yet microglia can become neurotoxic under some conditions. An early event during prion infection is the activation of microglia and astrocytes in the brain prior to damage or death of neurons. Previous prion disease studies using two different strategies to manipulate signaling through the microglial receptor CSF-1R reported contrary effects on survival from prion disease. However, in these studies, reductions of microglial numbers and function were variable, thus confounding interpretation of the results. In the present work, we used oral treatment with a potent inhibitor of CSF-1R, PLX5622, to eliminate 78 to 90% of microglia from cortex early during the course of prion infection. Oral drug treatment early after infection with the RML scrapie strain significantly accelerated vacuolation, astrogliosis, and deposition of disease-associated prion protein. Furthermore, drug-treated mice had advanced clinical disease requiring euthanasia 31 days earlier than untreated control mice. Similarly, PLX5622 treatment during the preclinical phase at 80 days postinfection with RML scrapie also accelerated disease and resulted in euthanasia of mice 33 days earlier than infected controls. PLX5622 also accelerated clinical disease after infection with scrapie strains ME7 and 22L. Thus, microglia are critical in host defense during prion disease. The early accumulation of PrPSc in the absence of microglia suggested that microglia may function by clearing PrPSc, resulting in longer survival.IMPORTANCE Microglia contribute to many aspects of health and disease. When activated, microglia can be beneficial by repairing damage in the central nervous system (CNS) or they can turn harmful by becoming neurotoxic. In prion and prionlike diseases, the involvement of microglia in disease is unclear. Previous studies suggest that microglia can either speed up or slow down disease. In this study, we infected mice with prions and depleted microglia from the brains of mice using PLX5622, an effective CSF-1R tyrosine kinase inhibitor. Microglia were markedly reduced in brains, and prion disease was accelerated, so that mice needed to be euthanized 20 to 33 days earlier than infected control mice due to advanced clinical disease. Similar results occurred when mice were treated with PLX5622 at 80 days after infection, which was just prior to the start of clinical signs. Thus, microglia are important for removing prions, and the disease is faster when microglia are depleted.

Prion Strain Differences in Accumulation of PrPSc on Neurons and Glia Are Associated with Similar Expression Profiles of Neuroinflammatory Genes: Comparison of Three Prion Strains.

Misfolding and aggregation of host proteins are important features of the pathogenesis of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, frontotemporal dementia and prion diseases. In all these diseases, the misfolded protein increases in amount by a mechanism involving seeded polymerization. In prion diseases, host prion protein is misfolded to form a pathogenic protease-resistant form, PrPSc, which accumulates in neurons, astroglia and microglia in the CNS. Here using dual-staining immunohistochemistry, we compared the cell specificity of PrPSc accumulation at early preclinical times post-infection using three mouse scrapie strains that differ in brain regional pathology. PrPSc from each strain had a different pattern of cell specificity. Strain 22L was mainly associated with astroglia, whereas strain ME7 was mainly associated with neurons and neuropil. In thalamus and cortex, strain RML was similar to 22L, but in substantia nigra, RML was similar to ME7. Expression of 90 genes involved in neuroinflammation was studied quantitatively using mRNA from thalamus at preclinical times. Surprisingly, despite the cellular differences in PrPSc accumulation, the pattern of upregulated genes was similar for all three strains, and the small differences observed correlated with variations in the early disease tempo. Gene upregulation correlated with activation of both astroglia and microglia detected in early disease prior to vacuolar pathology or clinical signs. Interestingly, the profile of upregulated genes in scrapie differed markedly from that seen in two acute viral CNS diseases (LaCrosse virus and BE polytropic Friend retrovirus) that had reactive gliosis at levels similar to our prion-infected mice.

Statins are ineffective at reducing neuroinflammation or prolonging survival in scrapie-infected mice.

Neuroinflammation is a prominent component of several neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease, Parkinson's disease, tauopathies, amyotrophic lateral sclerosis and prion diseases. In such conditions, the ability to decrease neuroinflammation by drug therapy may influence disease progression. Statins have been used to treat hyperlipidemia as well as reduce neuroinflammation and oxidative stress in various tissues. In previous studies, treatment of scrapie-infected mice with the type 1 statins, simvastatin or pravastatin, showed a small beneficial effect on survival time. In the current study, to increase the effectiveness of statin therapy, we treated infected mice with atorvastatin, a type 2 statin that has improved pharmacokinetics over many type 1 statins. Treatments with either simvastatin or pravastatin were tested for comparison. We evaluated scrapie-infected mice for protease-resistant PrP (PrPres) accumulation, gliosis, neuroinflammation and time until advanced clinical disease requiring euthanasia. All three statin treatments reduced total serum cholesterol ≥40 % in mice. However, gliosis and PrPres deposition were similar in statin-treated and untreated infected mice. Time to euthanasia due to advanced clinical signs was not changed in statin-treated mice relative to untreated mice, a finding at odds with previous reports. Expression of 84 inflammatory genes involved in neuroinflammation was also quantitated. Seven genes were reduced by pravastatin, and one gene was reduced by atorvastatin. In contrast, simvastatin therapy did not reduce any of the tested genes, but did slightly increase the expression of Ccl2 and Cxcl13. Our studies indicate that none of the three statins tested were effective in reducing scrapie-induced neuroinflammation or neuropathogenesis.

Knockout of fractalkine receptor Cx3cr1 does not alter disease or microglial activation in prion-infected mice.

Microglial activation is a hallmark of the neuroimmunological response to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and prion disease. The CX3C chemokine axis consists of fractalkine (CX3CL1) and its receptor (CX3CR1); these are expressed by neurons and microglia respectively, and are known to modulate microglial activation. In prion-infected mice, both Cx3cr1 and Cx3cl1 are altered, suggesting a role in disease. To investigate the influence of CX3C axis signalling on prion disease, we infected Cx3cr1 knockout (Cx3cr1-KO) and control mice with scrapie strains 22L and RML. Deletion of Cx3cr1 had no effect on development of clinical signs or disease incubation period. In addition, comparison of brain tissue from Cx3cr1-KO and control mice revealed no significant differences in cytokine levels, spongiosis, deposition of disease-associated prion protein or microglial activation. Thus, microglial activation during prion infection did not require CX3C axis signalling.

Prion infection of mouse brain reveals multiple new upregulated genes involved in neuroinflammation or signal transduction.

UNLABELLED: Gliosis is often a preclinical pathological finding in neurodegenerative diseases, including prion diseases, but the mechanisms facilitating gliosis and neuronal damage in these diseases are not understood. To expand our knowledge of the neuroinflammatory response in prion diseases, we assessed the expression of key genes and proteins involved in the inflammatory response and signal transduction in mouse brain at various times after scrapie infection. In brains of scrapie-infected mice at pre- and postclinical stages, we identified 15 previously unreported differentially expressed genes related to inflammation or activation of the STAT signal transduction pathway. Levels for the majority of differentially expressed genes increased with time postinfection. In quantitative immunoblotting experiments of STAT proteins, STAT1α, phosphorylated-STAT1α (pSTAT1α), and pSTAT3 were increased between 94 and 131 days postinfection (p.i.) in brains of mice infected with strain 22L. Furthermore, a select group of STAT-associated genes was increased preclinically during scrapie infection, suggesting early activation of the STAT signal transduction pathway. Comparison of inflammatory markers between mice infected with scrapie strains 22L and RML indicated that the inflammatory responses and gene expression profiles in the brains were strikingly similar, even though these scrapie strains infect different brain regions. The endogenous interleukin-1 receptor antagonist (IL-1Ra), an inflammatory marker, was newly identified as increasing preclinically in our model and therefore might influence scrapie pathogenesis in vivo. However, in IL-1Ra-deficient or overexpressor transgenic mice inoculated with scrapie, neither loss nor overexpression of IL-1Ra demonstrated any observable effect on gliosis, protease-resistant prion protein (PrPres) formation, disease tempo, pathology, or expression of the inflammatory genes analyzed. IMPORTANCE: Prion infection leads to PrPres deposition, gliosis, and neuroinflammation in the central nervous system before signs of clinical illness. Using a scrapie mouse model of prion disease to assess various time points postinoculation, we identified 15 unreported genes that were increased in the brains of scrapie-infected mice and were associated with inflammation and/or JAK-STAT activation. Comparison of mice infected with two scrapie strains (22L and RML), which have dissimilar neuropathologies, indicated that the inflammatory responses and gene expression profiles in the brains were similar. Genes that increased prior to clinical signs might be involved in controlling scrapie infection or in facilitating damage to host tissues. We tested the possible role of the endogenous IL-1Ra, which was increased at 70 days p.i. In scrapie-infected mice deficient in or overexpressing IL-1Ra, there was no observable effect on gliosis, PrPres formation, disease tempo, pathology, or expression of inflammatory genes analyzed.

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species.

Patient-centric Comparability Assessment of Biopharmaceuticals.

The comparability assessment of a biological product after implementing a manufacturing process change should involve a risk-based approach. Process changes may occur at any stage of the product lifecycle: early development, clinical manufacture for pivotal trials, or post-approval. The risk of the change to impact product quality varies. The design of the comparability assessment should be adapted accordingly. A working group reviewed and consolidated industry approaches to assess comparability of traditional protein-based biological products during clinical development and post-approval. The insights compiled in this review article encompass topics such as a risk-evaluation strategy, the design of comparability studies, definition of assessment criteria for comparability, holistic evaluation of data, and the regulatory submission strategy. These practices can be leveraged across the industry to help companies in design and execution of comparability assessments, and to inform discussions with global regulators.

Role of cyclophilin A from brains of prion-infected mice in stimulation of cytokine release by microglia and astroglia in vitro.

Prion diseases or transmissible spongiform encephalopathy diseases are typically characterized by deposition of abnormally folded partially protease-resistant host-derived prion protein (PrPres), which is associated with activated glia and increased release of cytokines. This neuroinflammatory response may play a role in transmissible spongiform encephalopathy pathogenesis. We previously reported that brain homogenates from prion-infected mice induced cytokine protein release in primary astroglial and microglial cell cultures. Here we measured cytokine release by cultured glial cells to determine what factors in infected brain contributed to activation of microglia and astroglia. In assays analyzing IL-12p40 and CCL2 (MCP-1), glial cells were not stimulated in vitro by either PrPres purified from infected mouse brains or prion protein amyloid fibrils produced in vitro. However, significant glial stimulation was induced by clarified scrapie brain homogenates lacking PrPres. This stimulation was greatly reduced both by antibody to cyclophilin A (CyPA), a known mediator of inflammation in peripheral tissues, and by cyclosporine A, a CyPA inhibitor. In biochemical studies, purified truncated CyPA fragments stimulated a pattern of cytokine release by microglia and astroglia similar to that induced by scrapie-infected brain homogenates, whereas purified full-length CyPA was a poor stimulator. This requirement for CyPA truncation was not reported in previous studies of stimulation of peripheral macrophages, endothelial cell cardiomyocytes, and vascular smooth muscle cells. Therefore, truncated CyPA detected in brain following prion infection may have an important role in the activation of brain-derived primary astroglia and microglia in prion disease and perhaps other neurodegenerative or neuroinflammatory diseases.

Borrelia burgdorferi bba66 gene inactivation results in attenuated mouse infection by tick transmission.

The impact of the Borrelia burgdorferi surface-localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, and characterizing the mutant phenotype throughout the natural mouse-tick-mouse cycle. The BBA66-deficient mutant isolate, Bb(ΔA66), remained infectious in mice by needle inoculation of cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. Ixodes scapularis larvae successfully acquired Bb(ΔA66) following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n = 7) demonstrated that the ability of Bb(ΔA66)-infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of Bb(ΔA66) with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. These results suggest a role for BBA66 in facilitating B. burgdorferi dissemination and transmission from the tick vector to the mammalian host as part of the disease process for Lyme borreliosis.

Early cytokine elevation, PrPres deposition, and gliosis in mouse scrapie: no effect on disease by deletion of cytokine genes IL-12p40 and IL-12p35.

Neurodegenerative diseases are typically associated with an activation of glia and an increased level of cytokines. In our previous studies of prion disease, the cytokine response in the brains of clinically sick scrapie-infected mice was restricted to a small group of cytokines, of which IL-12p40, CCL2, and CXCL10 were present at the highest levels. The goal of our current research was to determine the relationship between cytokine responses, gliosis, and neuropathology during prion disease. Here, in time course studies of C57BL/10 mice intracerebrally inoculated with 22L scrapie, abnormal protease-resistant prion protein (PrPres), astrogliosis, and microgliosis were first detected at 40 days after intracerebral scrapie inoculation. In cytokine studies, IL-12p40 was first elevated by 60 days; CCL3, IL-1ß, and CXCL1 were elevated by 80 days; and CCL2 and CCL5 were elevated by 115 days. IL-12p40 showed the most extensive increase throughout disease and was 30-fold above control levels at the terminal stage. Because of the early onset and dramatic elevation of IL-12p40 during scrapie, we investigated whether IL-12p40 contributed to the development of prion disease neuropathogenesis by using three different scrapie strains (22L, RML, 79A) to infect knockout mice in which the gene encoding IL-12p40 was deleted. We also studied knockout mice lacking IL-12p35, which combines with IL-12p40 to form active IL-12 heterodimers. In all instances, knockout mice did not differ from control mice in survival time, clinical tempo, or levels of spongiosis, gliosis, or PrPres in the brain. Thus, neither IL-12p40 nor IL-12p35 molecules were required for prion disease-associated neurodegeneration or neuroinflammation.

The bba64 gene of Borrelia burgdorferi, the Lyme disease agent, is critical for mammalian infection via tick bite transmission.

The spirochetal agent of Lyme disease, Borrelia burgdorferi, is transmitted by bites of Ixodes ticks to mammalian reservoir hosts and humans. The mechanism(s) by which the organism is trafficked from vector to host is poorly understood. In this study, we demonstrate that a B. burgdorferi mutant strain deficient in the synthesis of the bba64 gene product was incapable of infecting mice via tick bite even though the mutant was (i) infectious in mice when introduced by needle inoculation, (ii) acquired by larval ticks feeding on infected mice, and (iii) able to persist through tick molting stages. This finding of a B. burgdorferi gene required for pathogen transfer and/or survival from the tick to the susceptible host represents an important breakthrough toward understanding transmission mechanisms involved for the Lyme disease agent.

CD11c is not required by microglia to convey neuroprotection after prion infection.

Prion diseases are caused by the misfolding of a normal host protein that leads to gliosis, neuroinflammation, neurodegeneration, and death. Microglia have been shown to be critical for neuroprotection during prion infection of the central nervous system (CNS), and their presence extends survival in mice. How microglia impart these benefits to the infected host are unknown. Previous transcriptomics and bioinformatics studies suggested that signaling through the heterodimeric integrin receptor CD11c/CD18, expressed by microglia in the brain, might be important to microglial function during prion disease. Herein, we intracerebrally challenged CD11c-/- mice with prion strain RML and compared them to similarly infected C57BL/6 mice as controls. We initially assessed changes in the brain that are associated with disease such as astrogliosis, microgliosis, prion accumulation, and survival. Targeted qRT-PCR arrays were used to determine alterations in transcription in mice in response to prion infection. We demonstrate that expression of Itgax (CD11c) and Itgb2 (CD18) increases in the CNS in correlation with advancing prion infection. Gliosis, neuropathology, prion deposition, and disease progression in prion infected CD11c deficient mice were comparable to infected C57BL/6 mice. Additionally, both CD11c deficient and C57BL/6 prion-infected mouse cohorts had a similar consortium of inflammatory- and phagocytosis-associated genes that increased as disease progressed to clinical stages. Ingenuity Pathway Analysis of upregulated genes in infected C57BL/6 mice suggested numerous cell-surface transmembrane receptors signal through Spleen Tyrosine Kinase, a potential key regulator of phagocytosis and innate immune activation in the prion infected brain. Ultimately, the deletion of CD11c did not influence prion pathogenesis in mice and CD11c signaling is not involved in the neuroprotection provided by microglia, but our analysis identified a conspicuous phagocytosis pathway in the CNS of infected mice that appeared to be activated during prion pathogenesis.

Examining the role of actin-plasma membrane association in Pseudomonas aeruginosa infection and type III secretion translocation in migratory T24 epithelial cells.

The opportunistic pathogen Pseudomonas aeruginosa targets wounded epithelial barriers, but the cellular alteration that increases susceptibility to P. aeruginosa infection remains unclear. This study examined how cell migration contributes to the establishment of P. aeruginosa infections using (i) highly migratory T24 epithelial cells as a cell culture model, (ii) mutations in the type III secretion (T3S) effector ExoS to manipulate P. aeruginosa infection, and (iii) high-resolution immunofluorescent microscopy to monitor ExoS translocation. ExoS includes both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities, and P. aeruginosa cells expressing wild-type ExoS preferentially bound to the leading edge of T24 cells, where ExoS altered leading-edge architecture and actin anchoring in conjunction with interrupting T3S translocation. Inactivation of ExoS GAP activity allowed P. aeruginosa to be internalized and secrete ExoS within T24 cells, but as with wild-type ExoS, translocation was limited in association with disruption of actin anchoring. Inactivation of ExoS ADPRT activity resulted in significantly enhanced T3S translocation by P. aeruginosa cells that remained extracellular and in conjunction with maintenance of actin-plasma membrane association. Infection with P. aeruginosa expressing ExoS lacking both GAP and ADPRT activities resulted in the highest level of T3S translocation, and this occurred in conjunction with the entry and alignment of P. aeruginosa and ExoS along actin filaments. Collectively, in using ExoS mutants to modulate and visualize T3S translocation, we were able to (i) confirm effector secretion by internalized P. aeruginosa, (ii) differentiate the mechanisms underlying the effects of ExoS GAP and ADPRT activities on P. aeruginosa internalization and T3S translocation, (iii) confirm that ExoS ADPRT activity targeted a cellular substrate that interrupted T3S translocation, (iv) visualize the ability of P. aeruginosa and ExoS to align with actin filaments, and (v) demonstrate an association between actin anchoring at the leading edge of T24 cells and the establishment of P. aeruginosa infection. Our studies also highlight the contribution of ExoS to the opportunistic nature of P. aeruginosa infection through its ability to exert cytotoxic effects that interrupt T3S translocation and P. aeruginosa internalization, which in turn limit the P. aeruginosa infectious process.

Characterization of the Bat proteins in the oxidative stress response of Leptospira biflexa.

BACKGROUND: Leptospires lack many of the homologs for oxidative defense present in other bacteria, but do encode homologs of the Bacteriodes aerotolerance (Bat) proteins, which have been proposed to fulfill this function. Bat homologs have been identified in all families of the phylum Spirochaetes, yet a specific function for these proteins has not been experimentally demonstrated. RESULTS: We investigated the contribution of the Bat proteins in the model organism Leptospira biflexa for their potential contributions to growth rate, morphology and protection against oxidative challenges. A genetically engineered mutant strain in which all bat ORFs were deleted did not exhibit altered growth rate or morphology, relative to the wild-type strain. Nor could we demonstrate a protective role for the Bat proteins in coping with various oxidative stresses. Further, pre-exposing L. biflexa to sublethal levels of reactive oxygen species did not appear to induce a general oxidative stress response, in contrast to what has been shown in other bacterial species. Differential proteomic analysis of the wild-type and mutant strains detected changes in the abundance of a single protein only - HtpG, which is encoded by the gene immediately downstream of the bat loci. CONCLUSION: The data presented here do not support a protective role for the Leptospira Bat proteins in directly coping with oxidative stress as previously proposed. L. biflexa is relatively sensitive to reactive oxygen species such as superoxide and H2O2, suggesting that this spirochete lacks a strong, protective defense against oxidative damage despite being a strict aerobe.

Microglia have limited influence on early prion pathogenesis, clearance, or replication.

Microglia (MG) are critical to host defense during prion infection, but the mechanism(s) of this neuroprotection are poorly understood. To better examine the influence of MG during prion infection, we reduced MG in the brains of C57BL/10 mice using PLX5622 and assessed prion clearance and replication using multiple approaches that included bioassay, immunohistochemistry, and Real-Time Quaking Inducted Conversion (RT-QuIC). We also utilized a strategy of intermittent PLX5622 treatments to reduce MG and allow MG repopulation to test whether new MG could alter prion disease progress. Lastly, we investigated the influence of MG using tga20 mice, a rapid prion model that accumulates fewer pathological features and less PrPres in the infected brain. In C57BL/10 mice we found that MG were excluded from the inoculation site early after infection, but Iba1 positive infiltrating monocytes/macrophage were present. Reducing MG in the brain prior to prion inoculation did not increase susceptibility to prion infection. Short intermittent treatments with PLX5622 in prion infected C57BL/10 mice after 80 dpi were unsuccessful at altering the MG population, gliosis, or survival. Additionally, MG depletion using PLX5622 in tga20 mice had only a minor impact on prion pathogenesis, indicating that the presence of MG might be less important in this fast model with less prion accumulation. In contrast to the benefits of MG against prion disease in late stages of disease, our current experiments suggest MG do not play a role in early prion pathogenesis, clearance, or replication.

Increased excitatory amino acid transport into murine prion protein knockout astrocytes cultured in vitro.

Prion protein (PrP) is expressed on a wide variety of cells and plays an important role in the pathogenesis of transmissible spongiform encephalopathies. However, its normal function remains unclear. Mice that do not express PrP exhibit deficits in spatial memory and abnormalities in excitatory neurotransmission suggestive that PrP may function in the glutamatergic synapse. Here, we show that transport of D-aspartate, a nonmetabolized L-glutamate analog, through excitatory amino acid transporters (EAATs) was faster in astrocytes from PrP knockout (PrPKO) mice than in astrocytes from C57BL/10SnJ wild-type (WT) mice. Experiments using EAAT subtype-specific inhibitors demonstrated that in both WT and PrPKO astrocytes, the majority of transport was mediated by EAAT1. Furthermore, PrPKO astrocytes were more effective than WT astrocytes at alleviating L-glutamate-mediated excitotoxic damage in both WT and PrPKO neuronal cultures. Thus, in this in vitro model, PrPKO astrocytes exerted a functional influence on neuronal survival and may therefore influence regulation of glutamatergic neurotransmission in vivo.