Background Clostridioides difficile infections (CDI) have emerged over the past decade causing symptoms that range from mild, antibiotic-associated diarrhea (AAD) to life-threatening toxic megacolon. In this study, we describe a multiple and isochronal (mixed) CDI caused by the isolates DSM 27638, DSM 27639 and DSM 27640 that already initially showed different morphotypes on solid media.ResultsThe three isolates belonging to the ribotypes (RT) 012 (DSM 27639) and 027 (DSM 27638 and DSM 27640) were phenotypically characterized and high quality closed genome sequences were generated. The genomes were compared with seven reference strains including three strains of the RT 027, two of the RT 017, and one of the RT 078 as well as a multi-resistant RT 012 strain. The analysis of horizontal gene transfer events revealed gene acquisition incidents that sort the strains within the time line of the spread of their RTs within Germany. We could show as well that horizontal gene transfer between the members of different RTs occurred within this multiple infection. In addition, acquisition and exchange of virulence-related features including antibiotic resistance genes were observed. Analysis of the two genomes assigned to RT 027 revealed three single nucleotide polymorphisms (SNPs) and apparently a regional genome modification within the flagellar switch that regulates the fli operon.ConclusionOur findings show that (i) evolutionary events based on horizontal gene transfer occur within an ongoing CDI and contribute to the adaptation of the species by the introduction of new genes into the genomes, (ii) within a multiple infection of a single patient the exchange of genetic material was responsible for a much higher genome variation than the observed SNPs.

Summary Apicomplexa are unicellular eukaryotic pathogens and cause important diseases including malaria and toxoplasmosis. The discovery of an algal endosymbiont, the apicoplast, provides exciting avenues to develop urgently needed new drugs. However, the physiological function of the apicoplast and its integration into the parasite metabolism remain poorly understood and at times controversial. Using a new approach to genetic engineering in Toxoplasma we show here that the apicoplast phosphate translocator (TgAPT) is an essential link between endosymbiont and cytoplasmic metabolism. Our genetic analyses show that TgAPT is required for fatty acid synthesis in the apicoplast, but indicate also that this might not be its most critical function. Detailed biochemical analyses demonstrate that this transporter has unique properties allowing it to supply the apicoplast with carbon, and indirectly with energy and reduction power. Ablation of the transporter results in remarkably strong and fast inhibition of parasite growth underscoring its merit as a target.

Stage conversion between tachyzoites and bradyzoites of Toxoplasma gondii was investigated in vitro by using murine bone marrow-derived macrophages (BMMs) as host cells. Following infection of untreated BMMs with tachyzoites, spontaneous expression of bradyzoite-specific antigens (Bsa) occurred at low frequency with Toxoplasma strain-dependent ratios from 0.03 to 2%. As previously described for peritoneal macrophages, activation of tachyzoite-infected BMMs with gamma interferon (IFN-gamma) or lipopolysaccharide resulted in the induction of Bsa. When bradyzoites were used for infection, prolonged expression of Bsa could be observed in IFN-gamma-activated BMMs. The induction of Bsa expression seemed to be closely linked to parasite multiplication and increased to maximal values of 50 to 70% in intermediately activated macrophages with nitric oxide (NO) levels that allowed reduced parasite replication. Identical results in stage conversion were obtained when sodium nitroprusside was used as a source of exogenous NO, indicating that NO might be a molecular trigger of stage conversion. NO is reactive with iron-sulfur centers in proteins, thereby inhibiting proteins involved in the mitochondrial respiratory chain. Using oligomycin and antimycin A as inhibitors of mitochondrial function, growth inhibition of parasites and induction of Bsa were obtained. Since microglia are the functional correlates of macrophages in the central nervous system and inhibit T. gondii upon activation with IFN-gamma, a similar mechanism might be involved during cyst development in the brain.

Many apicomplexan parasites, such as Toxoplasma gondii and Plasmodium species, possess a nonphotosynthetic plastid, referred to as the apicoplast, which is essential for the parasites' viability and displays characteristics similar to those of nongreen plastids in plants. In this study, we localized several key enzymes of the carbohydrate metabolism of T. gondii to either the apicoplast or the cytosol by engineering parasites which express epitope-tagged fusion proteins. The cytosol contains a complete set of enzymes for glycolysis, which should enable the parasite to metabolize imported glucose into pyruvate. All the glycolytic enzymes, from phosphofructokinase up to pyruvate kinase, are present in the T. gondii genome, as duplicates and isoforms of triose phosphate isomerase, phosphoglycerate kinase, and pyruvate kinase were found to localize to the apicoplast. The mRNA expression levels of all genes with glycolytic products were compared between tachyzoites and bradyzoites; however, a strict bradyzoite-specific expression pattern was observed only for enolase I. The T. gondii genome encodes a single pyruvate dehydrogenase complex, which was located in the apicoplast and absent in the mitochondrion, as shown by targeting of epitope-tagged fusion proteins and by immunolocalization of the native pyruvate dehydrogenase complex. The exchange of metabolites between the cytosol and the apicoplast is likely to be mediated by a phosphate translocator which was localized to the apicoplast. Based on these localization studies, a model is proposed that explains the supply of the apicoplast with ATP and the reduction power, as well as the exchange of metabolites between the cytosol and the apicoplast.Up to 20 to 30% of the world population is estimated to be chronically infected with the apicomplexan parasite Toxoplasma gondii. The parasite differentiates within the human host between tachyzoites and bradyzoites, which display distinct physiological features. Mature bradyzoites are adapted for lifelong persistence in their hosts and display an extreme reduction in growth rate, up to a complete arrest of the cell cycle (7). In contrast, tachyzoites, which are present during the acute phase of infection, are characterized by a fast duplication time of 6 to 8 h, indicating that this stage possesses effective pathways for nutrient acquisition and energy metabolism.T. gondii possesses, like many other apicomplexan parasites, a nonphotosynthetic plastid, the so-called apicoplast. This organelle contains a 35-kb circular genome with similarities to plastid genomes from algae and is surrounded by four membranes. These features support the secondary endosymbiosis of a photosynthetic alga by the apicomplexan ancestor as the phylogenetic origin of the apicoplast (21,32,43,51). Numerous nucleus-encoded proteins are imported into the apicoplast, aided by a bipartite presequence which is composed of a signal peptide and an adjacent transit peptide (23,24,39,49).The apicoplast is the location of several anabolic pathways, such as type I...