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The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.tRNA contains modified nucleosides that are posttranscriptionally generated by the activity of specific enzymes (http: //modomics.genesilico.pl/sequence?seqtypeϭtRNA; 2). Many of these modifications frequently appear in the anticodon wobble position and are pivotal in the decoding process by stabilizing correct codon-anticodon interactions (1, 9). In Escherichia coli, the enzymes MnmE and MnmG carry out the GTPand flavin adenine dinucleotide (FAD)-dependent incorporation of the cmnm (CH 2 -NH-CH 2 -COOH) group at position 5 of the wobble uridine in several tRNAs, a reaction whose specific steps remain to be elucidated (4,7,8). MnmG and MnmE form a heterotetrameric ␣ 2 ␤ 2 complex in vitro (8). MnmG is a highly conserved FAD-binding protein (8) with 49% sequence identity to human MTO1.We cloned E. coli MnmG (MnmG Ec ; NCBI gi:2367273), MnmG , and MnmE (gi:12518545) into a modified pET15b vector (Novagen). The proteins were expressed in E. coli BL21(DE3) (Novagen). Cells were grown in LB medium, induced with 100 M isopropyl-1-thio-␤-D-galactopyranoside, and incubated for ϳ16 h at 16°C. Proteins were purified using nickelnitrilotriacetic acid (Ni-NTA) resin (Qiagen, Mississauga, Ontario, Canada) and concentrated to ϳ8 mg/ml in 20 mM Tris-HCl (pH 8.0), 0.8 M NaCl, 5% (vol/vol) glycerol, 5 mM dithiothreitol (DTT). MnmG Ec was crystallized by the hanging-drop method, equilibrating the protein drop against reservoir solution containing 100 mM Tris-HCl (pH 7.5), 100 mM sodium formate, 6.5% (wt/vol) polyethylene glycolPEG 8000, 6% (vol/vol) ethylene glycol. The crystals belong to space group P2 1 , with a ϭ 85.9 Å, b ϭ 144.1 Å, c ϭ 147.6 Å, and ␤ ϭ 106.8°, with four molecules in the asymmetric unit (V m ϭ 3.03 Å 3 Da Ϫ1 ) (6). They differ from the previously reported crystals and diffract to significantly higher resolution (7). Selenomethionine-labeled protein [expressed in strain DL41(DE3)] crystallized under the same conditions. Crystals of MnmG were obtained at 20°C by equilibrating protein (7 mg/ml) supplemented with the anticodon stem-loop fragment of tRNA Glu (Dharmacon) (molar ratio, 1:1.2) against a reservoir solution of 1.3 M Li 2 SO4, 0.1 M Tris-HCl (pH 8.0). They belong to space group P3 1 21, with a ϭ 144 Å.6 and c ϭ 271.0 Å, with two molecules in the asymmetric unit (V m ϭ 6.39 Å 3

BackgroundFew, if any, protozoan parasites are reported to exhibit extreme organ tropism like the flagellate Tritrichomonas foetus. In cattle, T. foetus infects the reproductive system causing abortion, whereas the infection in cats results in chronic large bowel diarrhoea. In the absence of a T. foetus genome, we utilized a de novo approach to assemble the transcriptome of the bovine and feline genotype to identify host-specific adaptations and virulence factors specific to each genotype. Furthermore, a subset of orthologs was used to characterize putative druggable targets and expose complications of in silico drug target mining in species with indefinite host-ranges.ResultsIllumina RNA-seq reads were assembled into two representative bovine and feline transcriptomes containing 42,363 and 36,559 contigs, respectively. Coding and non-coding regions of the genome libraries revealed striking similarities, with 24,620 shared homolog pairs reduced down to 7,547 coding orthologs between the two genotypes. The transcriptomes were near identical in functional category distribution; with no indication of selective pressure acting on orthologs despite differences in parasite origins/host. Orthologs formed a large proportion of highly expressed transcripts in both genotypes (bovine genotype: 76%, feline genotype: 56%). Mining the libraries for protease virulence factors revealed the cysteine proteases (CP) to be the most common. In total, 483 and 445 bovine and feline T. foetus transcripts were identified as putative proteases based on MEROPS database, with 9 hits to putative protease inhibitors. In bovine T. foetus, CP8 is the preferentially transcribed CP while in the feline genotype, transcription of CP7 showed higher abundance. In silico druggability analysis of the two genotypes revealed that when host sequences are taken into account, drug targets are genotype-specific.ConclusionGene discovery analysis based on RNA-seq data analysis revealed prominent similarities between the bovine and feline T. foetus, suggesting recent adaptation to their respective host/niche. T. foetus represents a unique case of a mammalian protozoan expanding its parasitic grasp across distantly related host lineages. Consequences of the host-range for in silico drug targeting are exposed here, demonstrating that targets of the parasite in one host are not necessarily ideal for the same parasite in another host.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-955) contains supplementary material, which is available to authorized users.

Effective growth and replication of obligate intracellular pathogens depend on host cell metabolism. How this is connected to host cell mitochondrial function has not been studied so far. Recent studies suggest that growth of intracellular bacteria such as Chlamydia pneumoniae is enhanced in a low oxygen environment, arguing for a particular mechanistic role of the mitochondrial respiration in controlling intracellular progeny. Metabolic changes in C. pneumoniae infected epithelial cells were analyzed under normoxic (O2 ≈ 20%) and hypoxic conditions (O2 < 3%). We observed that infection of epithelial cells with C. pneumoniae under normoxia impaired mitochondrial function characterized by an enhanced mitochondrial membrane potential and ROS generation. Knockdown and mutation of the host cell ATP synthase resulted in an increased chlamydial replication already under normoxic conditions. As expected, mitochondrial hyperpolarization was observed in non-infected control cells cultured under hypoxic conditions, which was beneficial for C. pneumoniae growth. Taken together, functional and genetically encoded mitochondrial dysfunction strongly promotes intracellular growth of C. pneumoniae.

Two clinical isolates of the opportunist pathogen Pseudomonas aeruginosa named PAO1 and PA14 are commonly studied in research laboratories. Despite the isolates being closely related, PA14 exhibits increased virulence compared to that of PAO1 in various models. To determine which players are responsible for the hypervirulence phenotype of the PA14 strain, we elected a transcriptomic approach through RNA sequencing. We found 2,029 genes that are differentially expressed between the two strains, including several genes that are involved with or regulated by quorum sensing (QS), known to control most of the virulence factors in P. aeruginosa. Among them, we chose to focus our study on QslA, an antiactivator of QS whose expression was barely detectable in the PA14 strain according our data. We hypothesized that lack of expression of qslA in PA14 could be responsible for higher QS expression in the PA14 strain, possibly explaining its hypervirulence phenotype. After confirming that QslA protein was highly produced in PAO1 but not in the PA14 strain, we obtained evidence showing that a PAO1 deletion strain of qslA has faster QS gene expression kinetics than PA14. Moreover, known virulence factors activated by QS, such as (i) pyocyanin production, (ii) H2-T6SS (type VI secretion system) gene expression, and (iii) Xcp-T2SS (type II secretion system) machinery production and secretion, were all lower in PAO1 than in PA14, due to higher qslA expression. However, biofilm formation and cytotoxicity toward macrophages, although increased in PA14 compared to PAO1, were independent of QslA control. Together, our findings implicated differential qslA expression as a major determinant of virulence factor expression in P. aeruginosa strains PAO1 and PA14. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen responsible for acute nosocomial infections and chronic pulmonary infections. P. aeruginosa strain PA14 is known to be hypervirulent in different hosts. Despite several studies in the field, the underlining molecular mechanisms sustaining this phenotype remain enigmatic. Here we provide evidence that the PA14 strain has faster quorum sensing (QS) kinetics than the PAO1 strain, due to the lack of QslA expression, an antiactivator of QS. QS is a major regulator of virulence factors in P. aeruginosa; therefore, we propose that the hypervirulent phenotype of the PA14 strain is, at least partially, due to the lack of QslA expression. This mechanism could be of great importance, as it could be conserved among other P. aeruginosa isolates.