Refined ab initio gene predictions of Heterorhabditis bacteriophora using RNA-seq.

Interest has recently grown in developing the entomopathogenic nematode Heterorhabditis bacteriophora as a model to genetically dissect the process of parasitic infection. Despite the availability of a full genome assembly, there is substantial variation in gene model accuracy. Here, a methodology is presented for leveraging RNA-seq evidence to generate improved annotations using ab initio gene prediction software. After alignment of reads and subsequent generation of a RNA-seq supported annotation, the new gene prediction models were verified on a selection of genes by comparison with sequenced 5' and 3' rapid amplification of cDNA ends products. By utilising a whole transcriptome for genome annotation, the current reference annotation was enriched, demonstrating the importance of coupling transcriptional data with genome assemblies.

Identification of candidate infection genes from the model entomopathogenic nematode Heterorhabditis bacteriophora.

BACKGROUND: Despite important progress in the field of innate immunity, our understanding of host immune responses to parasitic nematode infections lags behind that of responses to microbes. A limiting factor has been the obligate requirement for a vertebrate host which has hindered investigation of the parasitic nematode infective process. The nematode parasite Heterorhabditis bacteriophora offers great potential as a model to genetically dissect the process of infection. With its mutualistic Photorhabdus luminescens bacteria, H. bacteriophora invades multiple species of insects, which it kills and exploits as a food source for the development of several nematode generations. The ability to culture the life cycle of H. bacteriophora on plates growing the bacterial symbiont makes it a very exciting model of parasitic infection that can be used to unlock the molecular events occurring during infection of a host that are inaccessible using vertebrate hosts. RESULTS: To profile the transcriptional response of an infective nematode during the early stage of infection, we performed next generation RNA sequencing on H. bacteriophora IJs incubated in Manduca sexta hemolymph plasma for 9 h. A subset of up-regulated and down-regulated genes were validated using qRT-PCR. Comparative analysis of the transcriptome with untreated controls found a number of differentially expressed genes (DEGs) which cover a number of different functional categories. A subset of DEGs is conserved across Clade V parasitic nematodes revealing an array of candidate parasitic genes. CONCLUSIONS: Our analysis reveals transcriptional changes in the regulation of a large number of genes, most of which have not been shown previously to play a role in the process of infection. A significant proportion of these genes are unique to parasitic nematodes, suggesting the identification of a group of parasitism factors within nematodes. Future studies using these candidates may provide functional insight into the process of nematode parasitism and also the molecular evolution of parasitism within nematodes.

RNAi-mediated gene knockdown by microinjection in the model entomopathogenic nematode Heterorhabditis bacteriophora.

BACKGROUND: Parasitic nematodes threaten the health of humans and livestock and cause a major financial and socioeconomic burden to modern society. Given the widespread distribution of diseases caused by parasitic nematodes there is an urgent need to develop tools that will elucidate the genetic complexity of host-parasite interactions. Heterorhabditis bacteriophora is a parasitic nematode that allows simultaneous monitoring of nematode infection processes and host immune function, and offers potential as a tractable model for parasitic nematode infections. However, molecular tools to investigate these processes are required prior to its widespread acceptance as a robust model organism. In this paper we describe microinjection in adult H. bacteriophora as a suitable means of dsRNA delivery to knockdown gene transcripts. METHODS: RNA interference was used to knockdown four genes by injecting dsRNA directly into the gonad of adult hermaphrodite nematodes. RNAi phenotypes were scored in the F1 progeny on the fifth day post-injection, and knockdown of gene-specific transcripts was quantified with real-time quantitative RT-PCR (qRT-PCR). RESULTS: RNAi injection in adult hermaphrodites significantly decreased the level of target transcripts to varying degrees when compared with controls. The genes targeted by RNAi via injection included cct-2, nol-5, dpy-7, and dpy-13. In each case, RNAi knockdown was confirmed phenotypically by examining the progeny of injected animals, and also confirmed at the transcriptional level by real-time qRT-PCR. CONCLUSIONS: Here we describe for the first time the successful use of microinjection to knockdown gene transcripts in H. bacteriophora. This technique can be used widely to study the molecular basis of parasitism.

Analysis of the mitochondrial proteome in multiple sclerosis cortex.

Mitochondrial dysfunction has been proposed to play a role in the neuropathology of multiple sclerosis (MS). Previously, we reported significant alterations in the transcription of nuclear-encoded electron transport chain genes in MS and confirmed translational alterations for components of Complexes I and III that resulted in reductions in their activity. To more thoroughly and efficiently elucidate potential alterations in the expression of mitochondrial and related proteins, we have characterized the mitochondrial proteome in postmortem MS and control cortex using Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS). Using principal component analysis (PCA) and hierarchical clustering techniques we were able to analyze the differential patterns of SELDI-TOF spectra to reveal clusters of peaks which distinguished MS from control samples. Four proteins in particular were responsible for distinguishing disease from control. Peptide fingerprint mapping unambiguously identified these differentially expressed proteins. Three proteins identified are involved in respiration including cytochrome c oxidase subunit 5b (COX5b), the brain specific isozyme of creatine kinase, and hemoglobin ß-chain. The fourth protein identified was myelin basic protein (MBP). We then investigated whether these alterations were consistent in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We found that MBP was similarly altered in EAE but the respiratory proteins were not. These data indicate that while the EAE mouse model may mimic aspects of MS neuropathology which result from inflammatory demyelinating events, there is another distinct mechanism involved in mitochondrial dysfunction in gray matter in MS which is not modeled in EAE.

Impaired regulation of electron transport chain subunit genes by nuclear respiratory factor 2 in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease. Recently, decreased expression of nuclear encoded electron transport chain genes was found in neurons in MS cortex. To understand the transcriptional mechanisms responsible for the coordinate down regulation of these genes, we performed electrophoretic mobility shifts with nuclear extracts isolated from gray matter from nonlesion areas of postmortem MS and control cortex. Nine tissue blocks from eight different MS brains and six matched control blocks from five control brains were analyzed. We identified a decrease in a transcription factor complex containing nuclear respiratory factor 2 (NRF-2) in nuclear extracts isolated from MS cortex. This decrease is correlated with decreased expression of electron transport chain subunit genes and increased oxidative damage measured by increased anti-nitrotyrosine immunoreactivity. We conclude that in MS cortex a chronic increase in oxidative stress leads to aberrant regulation of transcription of genes involved in energy metabolism.