A bistable prokaryotic differentiation system underlying development of conjugative transfer competence

The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that is a model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. Previous work showed bimodal ICE activation, but by using single cell time-lapse microscopy coupled to combinations of chromosomally integrated single copy ICE promoter-driven fluorescence reporters, RNA sequencing and mutant analysis, we now describe the complete regulon leading to the arisal of differentiated dedicated transfer competent cells. The regulon encompasses at least three regulatory nodes and five (possibly six) further conserved gene clusters on the ICE that all become expressed under stationary phase conditions. Time-lapse microscopy indicated expression of two regulatory nodes (i.e., bisR and alpA-bisDC) to precede that of the other clusters. Notably, expression of all clusters except of bisR was confined to the same cell subpopulation, and was dependent on the same key ICE regulatory factors. The ICE thus only transfers from a small fraction of cells in a population, with an estimated proportion of between 1.7–4%, which express various components of a dedicated transfer competence program imposed by the ICE, and form the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements.     

The cytoplasmic trafficking of DNA topoisomerase IIalpha correlates with etoposide resistance in human myeloma cells

In this study we have investigated the role of topoisomerase (topo) IIalpha trafficking in cellular drug resistance. To accomplish this, it was necessary to separate the influence of cell cycle, drug uptake, topo protein levels, and enzyme trafficking on drug sensitivity. Thus, we developed a cell model (called accelerated plateau) using human myeloma H929 cells that reproducibly translocates topo IIalpha to the cytoplasm. Compared to log-phase cells, the cytoplasmic redistribution of topo IIalpha in plateau-phase cells correlated with a 10-fold resistance to VP-16 and a 40-60% reduction in the number of drug-induced double-strand DNA breaks. In addition, 7-fold more VP-16 was necessary to achieve 50% topo IIalpha band depletion, suggesting that there are fewer drug-induced topo-DNA complexes formed in quiescent cells than in log-phase cells. The total cellular amount of topo IIalpha and topo IIbeta protein in log- and plateau-phase cells was similar as determined by Western blot analysis. There was a 25% reduction in S-phase cell number in plateau cells (determined by bromodeoxyuridine (BrdU) incorporation), while there was no significant difference in the equilibrium concentrations of [(3)H]-VP-16 when log cells were compared with plateau cells. Furthermore, the nuclear/cytoplasmic ratio of topo IIalpha is increased 58-fold in accelerated-plateau H929 cells treated with leptomycin B (LMB) when compared to untreated cells. It appears that the nuclear-cytoplasmic shuttling of topo IIalpha, which decreases the amount of nuclear target enzyme, is a major mechanism of drug resistance to topo II inhibitors in plateau-phase myeloma cells.     

Assessment of hepatic glucose metabolism by indirect calorimetry in combination with a non-invasive technique using naturally enriched 13C glucose in healthy children and adolescents

The metabolic fate of hepatic glucose can be best studied using invasive techniques such as tracer infusions and frequent blood sampling which have been revealed to be impractical in the pediatric age group. The aim of this study was to develop a non-invasive method based on indirect calorimetry and expired 13CO2 monitoring in order to gain insight into the mechanisms leading to impaired glucose tolerance in children and teenagers. As a first step, net glucose oxidation (NGO) and energy expenditure (EE) were measured in 47 subjects (range 7.5-17.3 years) of whom 18 were prepubertal (P1), 11 in early puberty (P2-P3) and 18 in late puberty (P4-P5) after 3-hourly loads of 180 mg/kg of oral maize glucose containing naturally enriched 13C. Isotope analysis allowed to calculate exogenous and endogenous glucose oxidation (EXGO, ENGO) and, hence, to derive TGS and NGS, that is glycogen turnover. NGO and EE decreased significantly with pubertal progression, reflecting higher metabolism at younger ages, whereas EXGO remained constant. TGS did not change significantly whereas NGS showed a significant negative correlation with pubertal progression: this can be explained by the fact that glycogenolysis exceeded glycogen synthesis in this experimental setting. This non-invasive method appears to be a promising tool to study the fate of hepatic glucose and therefore glycogen turnover in children at risk of developing glucose intolerance and/or type 2 diabetes.     

Inhibition of natural type I IFN-producing and dendritic cell development by a small molecule receptor tyrosine kinase inhibitor with Flt3 affinity

In vivo steady-state type I natural IFN-producing and dendritic cell (DC) development is largely dependent on Flt3 signaling. Natural IFN-producing and DC progenitors and their respective downstream cell populations express the flt3 receptor, and Flt3 ligand (Flt3L)(-/-) mice have reduced while Flt3L-injected mice develop markedly increased numbers of both cell types. In the present study, we show that SU11657, a small multitargeted receptor tyrosine kinase inhibitor with Flt3 affinity, suppressed in vitro natural IFN-producing and DC development in Flt3L-supplemented mouse whole bone marrow cell cultures in a dose-dependant manner, while DC development in GM-CSF-supplemented cultures was not affected. In vivo SU11657 application led to a significant decrease of both natural IFN-producing and DCs, comparable to the reduction observed in Flt3L(-/-) mice. Conversely, Flt3L plasma levels increased massively in inhibitor-treated animals, likely via a regulatory feedback loop, without being able to compensate for pharmacological Flt3 inhibition. No obvious toxicity was observed, and hemopoietic progenitor cell and stem cell function remained intact as assessed by myeloid colony-forming unit activity and in vivo bone marrow repopulation assays. Furthermore, upon treatment discontinuation, IFN-producing and DCs recovered to normal levels, proving that treatment effects were transient. Given the importance of IFN-producing and DCs in regulation of immune responses, these findings might lead to new pharmacological strategies in prevention and treatment of autoimmune diseases and complications of organ or blood cell transplantation.     

Tma108, a putative M1 aminopeptidase,is a specific nascent chain-associated protein in Saccharomyces cerevisiae

The discovery of novel specific ribosome-associated factors challenges the assumption that translation relies on standardized molecular machinery. In this work, we demonstrate that Tma108, an uncharacterized translation machinery-associated factor in yeast, defines a subpopulation of cellular ribosomes specifically involved in the translation of less than 200 mRNAs encoding proteins with ATP or Zinc binding domains. Using ribonucleoparticle dissociation experiments we established that Tma108 directly interacts with the nascent protein chain. Additionally, we have shown that translation of the first 35 amino acids of Asn1, one of the Tma108 targets, is necessary and sufficient to recruit Tma108, suggesting that it is loaded early during translation. Comparative genomic analyses, molecular modeling and directed mutagenesis point to Tma108 as an original M1 metallopeptidase, which uses its putative catalytic peptide-binding pocket to bind the N-terminus of its targets. The involvement of Tma108 in co-translational regulation is attested by a drastic change in the subcellular localization of ATP2 mRNA upon Tma108 inactivation. Tma108 is a unique example of a nascent chain-associated factor with high selectivity and its study illustrates the existence of other specific translation-associated factors besides RNA binding proteins.     

Characterization of the universal stress protein F from atypical enteropathogenic Escherichia coli and its prevalence in Enterobacteriaceae

Atypical enteropathogenic Escherichia coli (aEPEC) are heterogeneous strains in terms of serotypes, adherence patterns and the presence of novel virulence factors. This heterogeneity is intriguing, promoting studies trying to characterize these novel proteins and to better comprehend this pathotype group. In a previous study analyzing low‐molecular mass proteomes of four representative aEPEC strains of three different adhesion phenotypes, we classified proteins according to their annotated function, with most of them being involved in metabolism and transport; while some of them were classified as hypothetical proteins. The majority of the hypothetical proteins were homologue products of genes identified in the genome of enterohemorrhagic E. coli. One of the hypothetical proteins was annotated as Z2335, with orthologue in EPEC, and by bioinformatics analysis, this protein was revealed to be the universal stress protein F (UspF). Thus, herein we successfully obtained a recombinant UspF protein from aEPEC, which is a α/β, ATP‐binding protein involved in stress response, with comparable protein production among the four studied strains, but showing noteworthy differences when cultivated in different stress conditions, also present in other enterobacterial species, such as Shigella sonnei and Citrobacter freundii. Furthermore, our results confirm that the Usp protein superfamily encompasses a conserved group of proteins involved in stress resistance in aEPEC and other Enterobacteriaceae.     

Alteration of sphingolipid metabolism as a putative mechanism underlying LPS‐induced BBB disruption

Septic encephalopathy with confusion and agitation occurs early during sepsis and contributes to the severity of the disease. A decrease in the sphingosine‐1‐phosphate (S1P) blood levels has been shown in patients and in animal models of sepsis. The lipid mediator S1P is known to be involved in endothelial barrier function in a context‐dependent manner. We utilized lipopolysaccharide (LPS )‐injected mice as a model for septic encephalopathy and first performed tracer permeability assays to assess the blood–brain barrier (BBB ) breakdown in vivo. At time points corresponding to the BBB breakdown post LPS injection, we aimed to characterize the regulation of the sphingolipid signaling pathway at the BBB during sepsis. We measured sphingolipid concentrations in blood, in mouse brain microvessels (MBMV s), and brain tissue. We also analyzed the expression of S1P receptors, transporters, and metabolizing enzymes in MBMV s and brain tissue. Primary mouse brain microvascular endothelial cells (MBMEC s) were isolated to evaluate the effects of LPS on transendothelial electrical resistance (TEER ) as a measure of permeability in vitro . We observed a relevant decrease in S1P levels after LPS injection in all three compartments (blood, MBMV s, brain tissue) that was accompanied by an increased expression of the S1P receptor type 1 and of sphingosine kinase 1 on one hand and of the S1P degrading enzymes lipid phosphate phosphatase 1 (LPP 1) and S1P phosphatase 1 on the other hand, as well as a down‐regulation of sphingosine kinase 2. Application of LPS to a monolayer of primary MBMEC s did not alter TEER , but serum from LPS ‐treated mice lead to a breakdown of the barrier compared to serum from vehicle‐treated mice. We observed profound alterations of the sphingolipid metabolism at the BBB after LPS injection that point toward a therapeutic potential of drugs interfering with this pathway as novel approach for the detrimental overwhelming immune response in sepsis.

Read the Editorial Highlight for this article on page 115 . Cover Image for this Issue: doi. 10.1111/jnc.14161 .

     

Pathogenicity in the tubercle bacillus: molecular and evolutionary determinants

In contrast to the great majority of mycobacterial species that are harmless saprophytes, Mycobacterium tuberculosis and other closely related tubercle bacilli have evolved to be among the most important human and animal pathogens. The need to develop new strategies in the fight against tuberculosis (TB) and related diseases has fuelled research into the evolutionary success of the M. tuberculosis complex members. Amongst the various disciplines, genomics and functional genomics have been instrumental in improving our understanding of these organisms. In this review we will present some of the recent key findings on molecular determinants of mycobacterial pathogenicity and attenuation, the evolution of M. tuberculosis, genome dynamics, antigen mining for improved diagnostic and subunit antigens, and finally the identification of novel drug targets. The genomics revolution has changed the landscape of TB research, and now underpins our renewed efforts to defeat this deadly pathogen.