A versatile proline/alanine transporter in the unicellular pathogen Leishmania donovani regulates amino acid homoeostasis and osmotic stress responses

Unlike all other organisms, parasitic protozoa of the family Trypanosomatidae maintain a large cellular pool of proline that, together with the alanine pool, serve as alternative carbon sources as well as reservoirs of organic osmolytes. These reflect adaptation to their insect vectors whose haemolymphs are exceptionally rich in the two amino acids. In the present study we identify and characterize a new neutral amino acid transporter, LdAAP24, that translocates proline and alanine across the Leishmania donovani plasma membrane. This transporter fulfils multiple functions: it is the sole supplier for the intracellular pool of proline and contributes to the alanine pool; it is essential for cell volume regulation after osmotic stress; and it regulates the transport and homoeostasis of glutamate and arginine, none of which are its substrates. Notably, we provide evidence that proline and alanine exhibit different roles in the parasitic response to hypotonic shock; alanine affects swelling, whereas proline influences the rate of volume recovery. On the basis of our data we suggest that LdAAP24 plays a key role in parasite adaptation to its varying environments in host and vector, a phenomenon essential for successful parasitism.

Classification of organisms previously reported as the SP and Stewart-Letscher groups, with descriptions of Necropsobacter gen. nov. and of Necropsobacter rosorum sp. nov. for organisms of the SP group

To allow classification of bacteria previously reported as the SP group and the Stewart-Letscher group, 35 isolates from rodents (21), rabbits (eight), a dog and humans (five) were phenotypically and genotypically characterized. Comparison of partial rpoB sequences showed that 34 of the isolates were closely related, demonstrating at least 97.4 % similarity. 16S rRNA gene sequence comparison of 20 selected isolates confirmed the monophyly of the SP group and revealed 98.5 %-100 % similarity between isolates. A blast search using the 16S rRNA gene sequences showed that the highest similarity outside the SP group was 95.5 % to an unclassified rat isolate. The single strain, P625, representing the Stewart-Letscher group showed the highest 16S rRNA gene similarity (94.9-95.5 %) to members of the SP group. recN gene sequence analysis of 11 representative strains resulted in similarities of 97-100 % among the SP group strains, which showed 80 % sequence similarity to the Stewart-Letscher group strain. Sequence similarity values based on the recN gene, indicative for whole genome similarity, showed the SP group being clearly separated from established genera, whereas the Stewart-Letscher group strain was associated with the SP group. A new genus, Necropsobacter gen. nov., with only one species, Necropsobacter rosorum sp. nov., is proposed to include all members of the SP group. The new genus can be separated from existing genera of the family Pasteurellaceae by at least three phenotypic characters. The most characteristic properties of the new genus are that haemolysis is not observed on bovine blood agar, positive reactions are observed in the porphyrin test, acid is produced from (+)-L-arabinose, (+)-D-xylose, dulcitol, (+)-D-galactose, (+)-D-mannose, maltose and melibiose, and negative reactions are observed for symbiotic growth, urease, ornithine decarboxylase and indole. Previous publications have documented that both ubiquinones and demethylmenaquinone were produced by the proposed type strain of the new genus, Michel A/76(T), and that the major polyamine of representative strains (type strain not included) of the genus is 1,3-diaminopropane, spermidine is present in moderate amounts and putrescine and spermine are detectable only in minor amounts. The major fatty acids of strain Michel A/76(T) are C(14 : 0), C(16 : 0), C(16:1)omega7c and summed feature C(14 : 0) 3-OH/iso-C(16 : 1) I. This fatty acid profile is typical for members of the family Pasteurellaceae. The G+C content of DNA of strain Michel A/76(T) was estimated to be 52.5 mol% in a previous investigation. The type strain is P709(T) ( = Michel A/76(T) = CCUG 28028(T) = CIP 110147(T) = CCM 7802(T)).

Safe high-pressure freezing of infectious micro-organisms

We describe how high-pressure freezing of infectious biological material can safely be accomplished with the help of membrane carriers. The method described is easy to perform; however, careful manipulations are required. Existing safety regulations must still be followed. However, the procedure reduces the risk of dissemination of infectious material.

Eukaryotic translation elongation factor 1A (eEF1A) domain I from S. cerevisiae is required but not sufficient for inter-species complementation

Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisiae.

Extended-spectrum cephalosporin-resistant Gram-negative organisms in livestock: an emerging problem for human health?

Escherichia coli, Salmonella spp. and Acinetobacter spp. are important human pathogens. Serious infections due to these organisms are usually treated with extended-spectrum cephalosporins (ESCs). However, in the past two decades we have faced a rapid increasing of infections and colonization caused by ESC-resistant (ESC-R) isolates due to production of extended-spectrum-β-lactamases (ESBLs), plasmid-mediated AmpCs (pAmpCs) and/or carbapenemase enzymes. This situation limits drastically our therapeutic armamentarium and puts under peril the human health. Animals are considered as potential reservoirs of multidrug-resistant (MDR) Gram-negative organisms. The massive and indiscriminate use of antibiotics in veterinary medicine has contributed to the selection of ESC-R E. coli, ESC-R Salmonella spp. and, to less extent, MDR Acinetobacter spp. among animals, food, and environment. This complex scenario is responsible for the expansion of these MDR organisms which may have life-threatening clinical significance. Nowadays, the prevalence of food-producing animals carrying ESC-R E. coli and ESC-R Salmonella (especially those producing CTX-M-type ESBLs and the CMY-2 pAmpC) has reached worryingly high values. More recently, the appearance of carbapenem-resistant isolates (i.e., VIM-1-producing Enterobacteriaceae and NDM-1 or OXA-23-producing Acinetobacter spp.) in livestock has even drawn greater concerns. In this review, we describe the aspects related to the spread of the above MDR organisms among pigs, cattle, and poultry, focusing on epidemiology, molecular mechanisms of resistance, impact of antibiotic use, and strategies to contain the overall problem. The link and the impact of ESC-R organisms of livestock origin for the human scenario are also discussed.

What a unicellular parasite can tell us about mitochondrial biogenesis?

Most of what we know about mitochondrial biogenesis stems from work in yeast and mammals, which are quite closely related. To understand the conserved features of mitochondria and the evolutionary forces that shaped it, it is important to study a more diverse group of eukaryotes. The parasitic protozoan Trypanosoma brucei and its relatives are excellent systems to do so, since they appear to have diverged from other eukaryotes very early in evolution. This is reflected in a number of unique and extreme features in their mitochondrial biology, including a single continuous mitochondrion that contains a one unit mitochondrial genome that is physically connected across the two membranes with the basal body of the flagellum. Moreover, many mitochondrial transcripts have to be extensively edited in order to become functional mRNAs and organellar translation requires extensive import of cytosolic tRNAs. In my talk I will focus on the discovery and characterization of the elusive mitochondrial protein import system of the mitochondrial outer membrane of trypanosomes. In addition I will present data on a central outer membrane component of the mitochondrial genome inheritance system of T. brucei and compare it to the better characterized system of yeast. - I hope that I can convince you in my talk, that a better understanding of the mitochondrial biology in T. brucei will provide insights into both fundamentally conserved and fundamentally diverged aspects of mitochondrial biogenesis and thus of the evolutionary hstory of mitochondria in general.

The ribosome as novel target for small regulatory RNAs

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. Furthermore it is strongly assumed that the primordial translation system in the ‘RNA world’ most likely received direct regulatory input from ncRNA-like cofactors. The fundamental question that we would like to ask is: Does the ‘RNA world still communicate’ with the ribosome? To address this question, we have analyzed the small ncRNA interactomes of ribosomes of prokaryotic (H. volcanii, S. aureus) and unicellular eukaryotic model organisms. Deep-sequencing and subsequent bioinformatic analyses revealed thousands of putative ribosome-associated ncRNAs. For a subset of these ncRNA candidates we have gathered experimental evidence that they are expressed in a stress-dependent manner and indeed directly target the ribosome. In the archaeon H. volcanii a tRNA-derived fragment was identified to target the small ribosomal subunit upon alkaline stress in vitro and in vivo. As a consequence of ribosome binding, this tRNA-fragment reduces protein synthesis by interfering with the peptidyl transferase activity. Our data reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life. Ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory sRNAs.

Overview and phylogeny of Mycobacterium tuberculosis complex organisms: Implications for diagnostics and legislation of bovine tuberculosis

Members of the Mycobacterium tuberculosis complex (MTBC) cause a serious disease with similar pathology, tuberculosis; in this review, bovine tuberculosis will be considered as disease caused by any member of the MTBC in bovids. Bovine tuberculosis is responsible for significant economic loss due to costly eradication programs and trade limitations and poses a threat to both endangered and protected species as well as to public health. We here give an overview on all members of the MTBC, focusing on their isolation from different animal hosts. We also review the recent advances made in elucidating the evolutionary and phylogenetic relationships of members of the MTBC. Because the nomenclature of the MTBC is controversial, its members have been considered species, subspecies or ecotypes, this review discusses the possible implications for diagnostics and the legal consequences of naming of new species.

What can a unicellular parasite tell us about mitochondrial biogenesis?

Most of what we know about mitochondrial biogenesis stems from work in yeast and mammals, which are quite closely related. To understand the conserved features of mitochondria and the evolutionary forces that shaped it, it is important to study a more diverse group of eukaryotes. The parasitic protozoan Trypanosoma brucei and its relatives are excellent systems to do so, since they appear to have diverged from other eukaryotes very early in evolution. This is reflected in a number of unique and extreme features in their mitochondrial biology, including a single continuous mitochondrion that contains a one unit mitochondrial genome that is physically connected across the two membranes with the basal body of the flagellum. Moreover, many mitochondrial transcripts have to be extensively edited in order to become functional mRNAs and organellar translation requires extensive import of cytosolic tRNAs. In my talk I will focus on the discovery and characterization of the elusive mitochondrial protein import system of the mitochondrial outer membrane of trypanosomes. In addition I will present data on a central outer membrane component of the mitochondrial genome inheritance system of T. brucei and compare it to the better characterized system of yeast. - I hope that I can convince you in my talk, that a better understanding of the mitochondrial biology in T. brucei will provide insights into both fundamentally conserved and fundamentally diverged aspects of mitochondrial biogenesis and thus of the evolutionary history of mitochondria in general.

What can a unicellular parasite tell us about mitochondrial biogenesis?

In the unicellular parasite Trypanosoma brucei, as in other eukaryotes, more than 95% of all mitochondrial proteins are imported from the cytosol. The recently characterized multisubunit ATOM complex, the functional analogue of the TOM complex of yeast, mediates import of essentially all proteins across the outer mitochondrial membrane in T. brucei. Moreover, an additional protein termed pATOM36, which is loosely associated with the ATOM complex, has been implicated in the import of only a subset of mitochondrial proteins. Here we have investigated more precisely which role pATOM36 plays in mitochondrial protein import. RNAi mediated ablation of pATOM36 specifically depletes a subset of outer mitochondrial membrane proteins including ATOM complex subunits and as a consequence results in the collapse of the ATOM complex as shown by Blue native PAGE. In addition, a SILAC-based global proteomic analysis of uninduced and induced pATOM36 RNAi cells together with in vitro import experiments suggest that pATOM36 might be a novel protein import factor acting on a subset of alpha-helically anchored mitochondrial outer membrane proteins. Identification of pATOM36 interaction partners by co-immunoprecipitation together with immunofluorescence analysis shows that unexpectedly a fraction of the protein is associated with the tripartite attachment complex (TAC). This complex is essential for proper inheritance of the mitochondrial DNA in T. brucei. It forms a physical connection between the single unit mitochondrial DNA and the basal body of the flagellum that is stable throughout the cell cycle. Thus, pATOM36 simultaneously mediates ATOM assembly, and thus protein import, as well as mitochondrial DNA inheritance since it is an essential component of the TAC.