Differential proteomic analysis of Clostridium perfringens ATCC13124; identification of dominant, surface and structure associated proteins

<p>Abstract</p> <p>Background</p> <p><it>Clostridium perfringens </it>is a medically important clostridial pathogen causing diseases in man and animals. To invade, multiply and colonize tissues of the host, a pathogen must be able to evade host immune system, and obtain nutrients essential for growth. The factors involved in these complex processes are largely unknown and of crucial importance to understanding microbial pathogenesis. Many of the virulence determinants and putative vaccine candidates for bacterial pathogens are known to be surface localized.</p> <p>Results</p> <p>Using 2-DE mass spectrometry strategy, we identified major surface (22) and cell envelope (10) proteins from <it>Clostridium perfringens </it>ATCC13124 and those differentially expressed (11) in cells grown on cooked meat medium (CMM) in comparison with cells grown in reference state (tryptose-yeast extract-glucose medium). Riboflavin biosynthesis protein, ornithine carbamoyltransferase, cystathionine beta-lyase, and threonine dehydratase were the predominant proteins that exhibited 2.19 to 8.5 fold increase in the expression level in cells growing on CMM.</p> <p>Conclusion</p> <p>Ornithine carbamoyltransferase and cystathionine beta-lyase were over-expressed in cells grown on cooked meat medium and also identified in the surface protein fraction and the former was immunogenic; making them potential vaccine candidates. Based upon bioinformatic analysis; choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein were predicted as surface protein markers for specific detection of <it>C. perfringens </it>from the environment and food. Most of the proteins over-expressed in CMM were shown to have putative function in metabolism, of which seven were involved in amino acid transport and metabolism or lipid metabolism.</p

Modulation of neuronal proteome profile in response to Japanese Encephalitis Virus infection

In this study we have reported the in vivo proteomic changes during Japanese Encephalitis Virus (JEV) infection in combination with in vitro studies which will help in the comprehensive characterization of the modifications in the host metabolism in response to JEV infection. We performed a 2-DE based quantitative proteomic study of JEV-infected mouse brain as well as mouse neuroblastoma (Neuro2a) cells to analyze the host response to this lethal virus. 56 host proteins were found to be differentially expressed post JEV infection (defined as exhibiting &#8805;1.5-fold change in protein abundance upon JEV infection). Bioinformatics analyses were used to generate JEV-regulated host response networks which reported that the identified proteins were found to be associated with various cellular processes ranging from intracellular protein transport, cellular metabolism and ER stress associated unfolded protein response. JEV was found to invade the host protein folding machinery to sustain its survival and replication inside the host thereby generating a vigorous unfolded protein response, subsequently triggering a number of pathways responsible for the JEV associated pathologies. The results were also validated using a human cell line to correlate them to the human response to JEV. The present investigation is the first report on JEV-host interactome in in vivo model and will be of potential interest for future antiviral research in this field

Network analysis reveals common host protein/s modulating pathogenesis of neurotropic viruses

Network analysis through graph theory provides a quantitative approach to characterize specific proteins and their constituent assemblies that underlie host-pathogen interactions. In the present study, graph theory was used to analyze the interactome designed out of 50 differentially expressing proteins from proteomic analysis of Chandipura Virus (CHPV, Family: Rhabdoviridae) infected mouse brain tissue to identify the primary candidates for intervention. Using the measure of degree centrality, that quantifies the connectedness of a single protein within a milieu of several other interacting proteins, DJ-1 was selected for further molecular validation. To elucidate the generality of DJ-1’s role in propagating infection its role was also monitored in another RNA virus, Japanese Encephalitis Virus (JEV, Family: Flaviviridae) infection. Concurrently, DJ-1 got over-expressed in response to reactive oxygen species (ROS) generation following viral infection which in the early phase of infection migrated to mitochondria to remove dysfunctional mitochondria through the process of mitophagy. DJ-1 was also observed to modulate the viral replication and interferon responses along with low-density lipoprotein (LDL) receptor expression in neurons. Collectively these evidences reveal a comprehensive role for DJ-1 in neurotropic virus infection in the brain

Japanese encephalitis virus induces human neural stem/progenitor cell death by elevating GRP78, PHB and hnRNPC through ER stress

Japanese encephalitis virus (JEV), which is a causative agent of sporadic encephalitis, harbours itself inside the neural stem/progenitor cells. It is a well-known fact that JEV infects neural stem/progenitor cells and decreases their proliferation capacity. With mass spectrometry-based quantitative proteomic study, it is possible to reveal the impact of virus on the stem cells at protein level. Our aim was to perceive the stem cell proteomic response upon viral challenge. We performed a two-dimensional gel electrophoresis-based proteomic study of the human neural stem cells (hNS1 cell line) post JEV infection and found that 13 proteins were differentially expressed. The altered proteome profile of hNS1 cell line revealed sustained endoplasmic reticulum stress, which deteriorated normal cellular activities leading to cell apoptosis. The proteomic changes found in hNS1 cell line were validated in vivo in the subventricular zone of JE infected BALB/c mice. Congruent alterations were also witnessed in multipotent neural precursor cells isolated from human foetus and in autopsy samples of human brain clinically diagnosed as cases of JE patients. Endoplasmic reticulum resident chaperone GRP78, mitochondrial protein Prohibitin and heterogeneous nuclear ribonucleoprotein hnRNPC (C1/C2) have been shown to interact with viral RNA. Hence it is proposed that these are the principle candidates governing endoplasmic reticulum stress-induced apoptosis in JEV infection

Cerebrospinal Fluid Biomarkers of Japanese Encephalitis [v1; ref status: indexed, http://f1000r.es/5n3]

Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia. Acute encephalitis syndrome (AES) is a group of central nervous system (CNS) disorders caused by a wide range of viruses, bacteria, fungi, chemicals and toxins. It is important to distinguish between various forms of infectious encephalitis with similar clinical manifestations in order to ensure specific and accurate diagnosis and development of subsequent therapeutic strategies. Cerebrospinal fluid (CSF) is in direct contact with the CNS and hence it is considered to be an excellent source for identifying biomarkers for various neurological disorders. With the recent advancement in proteomic methodologies, the field of biomarker research has received a remarkable boost.  The present study identifies potential biomarkers for JE using a proteomics based approach. The CSF proteomes from ten patients each with JE and Non-JE acute encephalitis were analyzed by 2D gel electrophoresis followed by mass spectrometry. Vitamin D-binding protein (DBP), fibrinogen gamma chain, fibrinogen beta chain, complement C4-B, complement C3 and cytoplasmic actin were found to be significantly elevated in case of JE indicating severe disruption of the blood brain barrier and DBP can be suggested to be an important diagnostic marker

Cerebrospinal Fluid Biomarkers of Japanese Encephalitis [v2; ref status: indexed, http://f1000r.es/5uq]

Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia. Acute encephalitis syndrome (AES) is a group of central nervous system (CNS) disorders caused by a wide range of viruses, bacteria, fungi, chemicals and toxins. It is important to distinguish between various forms of infectious encephalitis with similar clinical manifestations in order to ensure specific and accurate diagnosis and development of subsequent therapeutic strategies. Cerebrospinal fluid (CSF) is in direct contact with the CNS and hence it is considered to be an excellent source for identifying biomarkers for various neurological disorders. With the recent advancement in proteomic methodologies, the field of biomarker research has received a remarkable boost.  The present study identifies potential biomarkers for JE using a proteomics based approach. The CSF proteomes from ten patients each with JE and Non-JE acute encephalitis were analyzed by 2D gel electrophoresis followed by mass spectrometry. Vitamin D-binding protein (DBP), fibrinogen gamma chain, fibrinogen beta chain, complement C4-B, complement C3 and cytoplasmic actin were found to be significantly elevated in case of JE indicating severe disruption of the blood brain barrier and DBP can be suggested to be an important diagnostic marker

Additional file 1: of HSP60 plays a regulatory role in IL-1β-induced microglial inflammation via TLR4-p38 MAPK axis

3 Supplementary figures and 2 Supplementary tables. Table S1. List of proteins showing differential expression after IL-1β treatment in N9 microglia cells, identified by MS/MS analysis. Table S2. List of primers used for quantitative real time PCR (qRT-PCR) analysis. Figure S1. Pie chart showing the molecular functions of differentially expressed proteins in IL-1β treated N9 microglial cells. Figure S2. Protein-protein interaction network of the identified proteins. Figure S3. Effect of IL-1β on phosphorylation of MAPK effector proteins in vitro and in vivo. (DOCX 2526 kb

Temporal expression of selected proteins in mouse brain with disease progression.

<p>BALB/c mice of either sex were injected with 3×10⁵ p.f.u. of JE virus of strain GP78 through tail vein. The animals were then dissected at 1, 3, and 5 day post (JEV) infection (d.p.i) and brain was isolated for protein extraction. (<b>A</b>) Immunoblot showing the protein expression levels during JEV infectionas compared to that of mock-infected control mice. (<b>B</b>) Densitometric analysis of the differential protein expression. Data is representative of three independent experiments.</p

Confirmation of JEV infection in both <i>in vivo</i> and <i>in vitro</i> models using JEV specific antibody.

<p>The onset of JEV infection was confirmed by immunostaining of mouse brain sections and Neuro2a cells using JEV specific antibodies.</p

Validation of proteomic results using Immunostaining.

<p>Differential expression of selected proteins in neuron following JEV-infection in <i>in vivo</i> model. Images are representative of three independent experiments.</p