Growth of Chlamydia pneumoniae Is Enhanced in Cells with Impaired Mitochondrial Function.

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.

Comparative transcriptomics reveals striking similarities between the bovine and feline isolates of Tritrichomonas foetus: consequences for in silico drug-target identification.

BACKGROUND: Few, 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. RESULTS: Illumina 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. CONCLUSION: Gene 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.

Enzymology of tRNA modification in the bacterial MnmEG pathway.

Among all RNAs, tRNA exhibits the largest number and the widest variety of post-transcriptional modifications. Modifications within the anticodon stem loop, mainly at the wobble position and purine-37, collectively contribute to stabilize the codon-anticodon pairing, maintain the translational reading frame, facilitate the engagement of the ribosomal decoding site and enable translocation of tRNA from the A-site to the P-site of the ribosome. Modifications at the wobble uridine (U34) of tRNAs reading two degenerate codons ending in purine are complex and result from the activity of two multi-enzyme pathways, the IscS-MnmA and MnmEG pathways, which independently work on positions 2 and 5 of the U34 pyrimidine ring, respectively, and from a third pathway, controlled by TrmL (YibK), that modifies the 2'-hydroxyl group of the ribose. MnmEG is the only common pathway to all the mentioned tRNAs, and involves the GTP- and FAD-dependent activity of the MnmEG complex and, in some cases, the activity of the bifunctional enzyme MnmC. The Escherichia coli MnmEG complex catalyzes the incorporation of an aminomethyl group into the C5 atom of U34 using methylene-tetrahydrofolate and glycine or ammonium as donors. The reaction requires GTP hydrolysis, probably to assemble the active site of the enzyme or to carry out substrate recognition. Inactivation of the evolutionarily conserved MnmEG pathway produces a pleiotropic phenotype in bacteria and mitochondrial dysfunction in human cell lines. While the IscS-MnmA pathway and the MnmA-mediated thiouridylation reaction are relatively well understood, we have limited information on the reactions mediated by the MnmEG, MnmC and TrmL enzymes and on the precise role of proteins MnmE and MnmG in the MnmEG complex activity. This review summarizes the present state of knowledge on these pathways and what we still need to know, with special emphasis on the MnmEG pathway.

Structure-function analysis of Escherichia coli MnmG (GidA), a highly conserved tRNA-modifying enzyme.

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-A 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.