Current status of antisense RNA-mediated gene regulation in Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive human-pathogen bacterium that served as an experimental model for investigating fundamental processes of adaptive immunity and virulence. Recent novel technologies allowed the identification of several hundred non-coding RNAs (ncRNAs) in the Listeria genome and provided insight into an unexpected complex transcriptional machinery. In this review, we discuss ncRNAs that are encoded on the opposite strand of the target gene and are therefore termed antisense RNAs (asRNAs). We highlight mechanistic and functional concepts of asRNAs in L. monocytogenes and put these in context of asRNAs in other bacteria. Understanding asRNAs will further broaden our knowledge of RNA-mediated gene regulation and may provide targets for diagnostic and antimicrobial development.

Detection of very long antisense transcripts by whole transcriptome RNA-Seq analysis of Listeria monocytogenes by semiconductor sequencing technology.

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 community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30%) of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs). We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected eukaryotic host.

Serodiagnosis of Schistosoma japonicum infection: genome-wide identification of a protein marker, and assessment of its diagnostic validity in a field study in China.

BACKGROUND: Schistosomiasis remains a highly prevalent and serious parasitic disease. A major factor preventing its effective management is the scarcity of effective diagnostic tools. We did a genome-wide identification of diagnostic protein markers for schistosome infection and assessed their diagnostic validity in a field study. METHODS: We predicted putative secreted proteins of Schistosoma japonicum (SjSPs) and expressed them as glutathione S-transferase (GST)-fusion proteins. The fusion proteins were arrayed on glutathione (GSH)-immobilised microplates and screened with serum samples from patients with schistosomiasis diagnosed by the Kato-Katz method. We further assessed an identified protein marker for sensitivity and specificity, first in infected serum samples collected from Jiangxi and Hunan Provinces, China, and then through a field study, done in two villages located in a high schistosomiasis-endemic area of the southeast of China. FINDINGS: Of 204 recombinant proteins, 35 yielded seropositive reactions, eight showed strong immunoreactivity, and only one (SjSP-13) reacted to the entire panel of 14 archived samples. The reactivity of SjSP-13 to 476 serum samples showed 90·4% (95% CI 86·5-93·5) sensitivity and 98·9% (95% CI 95·9-99·9) specificity. Of 1371 residents enrolled in a field study from Dec 6, 2010, to June 23, 2011, only 74 individuals were identified as being egg-positive, whereas 465 were diagnosed as positive by the SjSP-13-based ELISA kit (rSP13-ELISA). Of the 394 individuals found egg-negative but rSP13-ELISA-positive, 363 (92·4%) were confirmed to be positive for schistosome infection by PCR detection of S japonicum SjR2 retrotransposon. INTERPRETATION: The application of this sensitive, specific, and affordable rSP13-ELISA method should help reduce schistosomiasis transmission through targeted treatment of individuals, particularly with low intensity infections, and therefore support schistosomiasis control and elimination strategies. FUNDING: National 973 project in China.

Ultra deep sequencing of Listeria monocytogenes sRNA transcriptome revealed new antisense RNAs.

Listeria monocytogenes, a gram-positive pathogen, and causative agent of listeriosis, has become a widely used model organism for intracellular infections. Recent studies have identified small non-coding RNAs (sRNAs) as important factors for regulating gene expression and pathogenicity of L. monocytogenes. Increased speed and reduced costs of high throughput sequencing (HTS) techniques have made RNA sequencing (RNA-Seq) the state-of-the-art method to study bacterial transcriptomes. We created a large transcriptome dataset of L. monocytogenes containing a total of 21 million reads, using the SOLiD sequencing technology. The dataset contained cDNA sequences generated from L. monocytogenes RNA collected under intracellular and extracellular condition and additionally was size fractioned into three different size ranges from <40 nt, 40-150 nt and >150 nt. We report here, the identification of nine new sRNAs candidates of L. monocytogenes and a reevaluation of known sRNAs of L. monocytogenes EGD-e. Automatic comparison to known sRNAs revealed a high recovery rate of 55%, which was increased to 90% by manual revision of the data. Moreover, thorough classification of known sRNAs shed further light on their possible biological functions. Interestingly among the newly identified sRNA candidates are antisense RNAs (asRNAs) associated to the housekeeping genes purA, fumC and pgi and potentially their regulation, emphasizing the significance of sRNAs for metabolic adaptation in L. monocytogenes.

Having a pair: the key to immune evasion for the diploid pathogen Schistosoma japonicum.

Schistosomes, unlike malaria parasites, are in their diploid stage when targeted by the human immune system. Diploids can be either homozygous or heterozygous. The difference has profound significance for developing immunity and yet has not previously been addressed. We examined the implications of zygosity on immunity to a diploid pathogen, Schistosoma japonicum and showed that the diploid state, and its associated heterozygous advantage, significantly affects the outcome of attack by the immune system and the accumulation of antigenic diversity in the parasite population. We demonstrate here that diploidy provides a novel means of immune evasion for diploid pathogens.

Identification and characterization of six novel tetraspanins from Schistosoma japonicum.

BACKGROUND: Tetraspanins (TSPs), also known as members of the trans-membrane 4 super-family (TM4SF), comprise an assemblage of surface antigens reported in eukaryotic organisms. In the work presented here, six novel TSP proteins from the human blood fluke Schistosoma japonicum (S. japonicum) were produced and analyzed through a combination of bioinformatics and experimental approaches. RESULTS: Six novel TSP proteins of Schistosoma japonicum (designated as Sj-TSP-#1~6) contained four trans-membrane regions and one large extracellular loop (LEL) with a conserved CCG motif. Size of the proteins varied from 227 to 291 amino acid residues. All the six proteins were produced in E.coli and immune sera to each protein were prepared. Analysis of transcription profiles of the proteins by RT-PCR showed that Sj-TSP-#4 was transcribed only in the egg stage while transcription of the Sj-TSP-#2 was detected in female worms but not in males. The similar results were obtained by Western blot. Immunolocalization of the TSP proteins by immunofluorescence assay showed that the Sj-TSP-#2, Sj-TSP-#5 and Sj-TSP-#6 were located in the tegument of worms. CONCLUSIONS: This study provided six novel TSP members of S. japonicum including their sequences and recombinant proteins. Availability of the novel proteins and information on their expression profile and location provided a basis for further investigation of the TSP proteins for their biological functions and as vaccine candidates.