Mycoplasma hyopneumoniae J elicits an antioxidant response and decreases the expression of ciliary genes in infected swine epithelial cells.

Mycoplasma hyopneumoniae is the most costly pathogen for swine production. Although several studies have focused on the host-bacterium association, little is known about the changes in gene expression of swine cells upon infection. To improve our understanding of this interaction, we infected swine epithelial NPTr cells with M. hyopneumoniae strain J to identify differentially expressed mRNAs and miRNAs. The levels of 1,268 genes and 170 miRNAs were significantly modified post-infection. Up-regulated mRNAs were enriched in genes related to redox homeostasis and antioxidant defense, known to be regulated by the transcription factor NRF2 in related species. Down-regulated mRNAs were enriched in genes associated with cytoskeleton and ciliary functions. Bioinformatic analyses suggested a correlation between changes in miRNA and mRNA levels, since we detected down-regulation of miRNAs predicted to target antioxidant genes and up-regulation of miRNAs targeting ciliary and cytoskeleton genes. Interestingly, most down-regulated miRNAs were detected in exosome-like vesicles suggesting that M. hyopneumoniae infection induced a modification of the composition of NPTr-released vesicles. Taken together, our data indicate that M. hyopneumoniae elicits an antioxidant response induced by NRF2 in infected cells. In addition, we propose that ciliostasis caused by this pathogen is partially explained by the down-regulation of ciliary genes.

An integrative, multi-omics approach towards the prioritization of Klebsiella pneumoniae drug targets.

Klebsiella pneumoniae (Kp) is a globally disseminated opportunistic pathogen that can cause life-threatening infections. It has been found as the culprit of many infection outbreaks in hospital environments, being particularly aggressive towards newborns and adults under intensive care. Many Kp strains produce extended-spectrum ß-lactamases, enzymes that promote resistance against antibiotics used to fight these infections. The presence of other resistance determinants leading to multidrug-resistance also limit therapeutic options, and the use of 'last-resort' drugs, such as polymyxins, is not uncommon. The global emergence and spread of resistant strains underline the need for novel antimicrobials against Kp and related bacterial pathogens. To tackle this great challenge, we generated multiple layers of 'omics' data related to Kp and prioritized proteins that could serve as attractive targets for antimicrobial development. Genomics, transcriptomics, structuromic and metabolic information were integrated in order to prioritize candidate targets, and this data compendium is freely available as a web server. Twenty-nine proteins with desirable characteristics from a drug development perspective were shortlisted, which participate in important processes such as lipid synthesis, cofactor production, and core metabolism. Collectively, our results point towards novel targets for the control of Kp and related bacterial pathogens.

Fast-SG: an alignment-free algorithm for hybrid assembly.

Background: Long-read sequencing technologies are the ultimate solution for genome repeats, allowing near reference-level reconstructions of large genomes. However, long-read de novo assembly pipelines are computationally intense and require a considerable amount of coverage, thereby hindering their broad application to the assembly of large genomes. Alternatively, hybrid assembly methods that combine short- and long-read sequencing technologies can reduce the time and cost required to produce de novo assemblies of large genomes. Results: Here, we propose a new method, called Fast-SG, that uses a new ultrafast alignment-free algorithm specifically designed for constructing a scaffolding graph using light-weight data structures. Fast-SG can construct the graph from either short or long reads. This allows the reuse of efficient algorithms designed for short-read data and permits the definition of novel modular hybrid assembly pipelines. Using comprehensive standard datasets and benchmarks, we show how Fast-SG outperforms the state-of-the-art short-read aligners when building the scaffoldinggraph and can be used to extract linking information from either raw or error-corrected long reads. We also show how a hybrid assembly approach using Fast-SG with shallow long-read coverage (5X) and moderate computational resources can produce long-range and accurate reconstructions of the genomes of Arabidopsis thaliana (Ler-0) and human (NA12878). Conclusions: Fast-SG opens a door to achieve accurate hybrid long-range reconstructions of large genomes with low effort, high portability, and low cost.

Mycoplasma non-coding RNA: identification of small RNAs and targets.

BACKGROUND: Bacterial non-coding RNAs act by base-pairing as regulatory elements in crucial biological processes. We performed the identification of trans-encoded small RNAs (sRNA) from the genomes of Mycoplama hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis, which are Mycoplasma species that have been identified in the porcine respiratory system. RESULTS: A total of 47, 15 and 11 putative sRNAs were predicted in M. hyopneumoniae, M. flocculare and M. hyorhinis, respectively. A comparative genomic analysis revealed the presence of species or lineage specific sRNA candidates. Furthermore, the expression profile of some M. hyopneumoniae sRNAs was determined by a reverse transcription amplification approach, in three different culture conditions. All tested sRNAs were transcribed in at least one condition. A detailed investigation revealed a differential expression profile for two M. hyopneumoniae sRNAs in response to oxidative and heat shock stress conditions, suggesting that their expression is influenced by environmental signals. Moreover, we analyzed sRNA-mRNA hybrids and accessed putative target genes for the novel sRNA candidates. The majority of the sRNAs showed interaction with multiple target genes, some of which could be linked to pathogenesis and cell homeostasis activity. CONCLUSION: This study contributes to our knowledge of Mycoplasma sRNAs and their response to environmental changes. Furthermore, the mRNA target prediction provides a perspective for the characterization and comprehension of the function of the sRNA regulatory mechanisms.

Biosynthesis of vitamins and cofactors in bacterium-harbouring trypanosomatids depends on the symbiotic association as revealed by genomic analyses.

Some non-pathogenic trypanosomatids maintain a mutualistic relationship with a betaproteobacterium of the Alcaligenaceae family. Intensive nutritional exchanges have been reported between the two partners, indicating that these protozoa are excellent biological models to study metabolic co-evolution. We previously sequenced and herein investigate the entire genomes of five trypanosomatids which harbor a symbiotic bacterium (SHTs for Symbiont-Haboring Trypanosomatids) and the respective bacteria (TPEs for Trypanosomatid Proteobacterial Endosymbiont), as well as two trypanosomatids without symbionts (RTs for Regular Trypanosomatids), for the presence of genes of the classical pathways for vitamin biosynthesis. Our data show that genes for the biosynthetic pathways of thiamine, biotin, and nicotinic acid are absent from all trypanosomatid genomes. This is in agreement with the absolute growth requirement for these vitamins in all protozoa of the family. Also absent from the genomes of RTs are the genes for the synthesis of pantothenic acid, folic acid, riboflavin, and vitamin B6. This is also in agreement with the available data showing that RTs are auxotrophic for these essential vitamins. On the other hand, SHTs are autotrophic for such vitamins. Indeed, all the genes of the corresponding biosynthetic pathways were identified, most of them in the symbiont genomes, while a few genes, mostly of eukaryotic origin, were found in the host genomes. The only exceptions to the latter are: the gene coding for the enzyme ketopantoate reductase (EC:1.1.1.169) which is related instead to the Firmicutes bacteria; and two other genes, one involved in the salvage pathway of pantothenic acid and the other in the synthesis of ubiquinone, that are related to Gammaproteobacteria. Their presence in trypanosomatids may result from lateral gene transfer. Taken together, our results reinforce the idea that the low nutritional requirement of SHTs is associated with the presence of the symbiotic bacterium, which contains most genes for vitamin production.

Predicting the proteins of Angomonas deanei, Strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family.

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

Efficient representation and P-value computation for high-order Markov motifs.

MOTIVATION: Position weight matrices (PWMs) have become a standard for representing biological sequence motifs. Their relative simplicity has favoured the development of efficient algorithms for diverse tasks such as motif identification, sequence scanning and statistical significance evaluation. Markov chainbased models generalize the PWM model by allowing for interposition dependencies to be considered, at the cost of substantial computational overhead, which may limit their application. RESULTS: In this article, we consider two aspects regarding the use of higher order Markov models for biological sequence motifs, namely, the representation and the computation of P-values for motifs described by a set of occurrences. We propose an efficient representation based on the use of tries, from which empirical position-specific conditional base probabilities can be computed, and extend state-of-the-art PWM-based algorithms to allow for the computation of exact P-values for high-order Markov motif models. AVAILABILITY: The software is available in the form of a Java objectoriented library from http://www.cin.ufpe.br/approxiamtely paguso/kmarkov.