Characteristics of intracellular iron transport in erythroid cells

Transferrin mediated uptake of iron is the physiological and most efficient way for introduction of iron into vertebrate cells. Though not accepted by all investigators, receptor-mediated endocytosis of ferric-transferrin is thought to be the normal mechanism of iron uptake. However, the concept of this pathway does not answer many elementary questions about the internalization of iron, its release from transferrin, its transfer to the cytosol, and the possible existence of a transport intermediate. To contribute to the knowledge of intracellular iron metabolism we aimed to obtain answers to the following questions: What is the nature of the low molecular weight iron binding fraction found in reticulocytes? At what point in the transferrin cell cycle does iron leave the endosome; and what are the conditions necessary for endosomal iron release? In chapter 3 the composition of the low molecular weight iron binding fraction is analyzed to determine the existence of physiologically important low molecular weight iron transporting species in the cytosol. Chapter 4 discusses in the phase of the transferrin cycle at which iron is removed from endosomes. In chapter 5 the experimental work is described which explores the presence of endogenous acceptor molecules for iron released from isolated endosome

Cooperative virulence can emerge via horizontal gene transfer but is stabilized by transmission

Intestinal inflammation fuels Salmonella Typhimurium ( S .Tm) transmission despite a fitness cost associated with the expression of virulence. Cheater mutants can emerge that profit from inflammation without enduring this cost. Intestinal virulence in S .Tm is therefore a cooperative trait, and its evolution a conundrum. Horizontal gene transfer (HGT) of cooperative alleles may facilitate the emergence of cooperative virulence, despite its instability. To test this hypothesis, we cloned hilD , coding for a master regulator of virulence, into a conjugative plasmid that is highly transferrable during intestinal colonization. We demonstrate that virulence can emerge by hilD transfer between avirulent strains in vivo . However, this was indeed unstable and hilD mutant cheaters arose within a few days. The timing of cheater emergence depended on the cost. We further show that stabilization of cooperative virulence in S .Tm is dependent on transmission dynamics, strengthened by population bottlenecks, leading cheaters to extinction and allowing cooperators to thrive

Genetics and resistance/Génétique et résistance Development of EST-derived simple sequence repeat markers for wheat leaf rust fungus, Puccinia triticina Eriks Molecular markers for wheat leaf rust

Abstract: Gene-associated simple sequence repeat (SSR) markers were developed for Puccinia triticina through the data mining of existing EST libraries. Analysis of 7134 expressed sequence tags (ESTs) from cDNA libraries of P. triticina detected 204 EST-SSRs with a minimum of 12 repeating nucleotides. The majority of EST-SSRs contained short di-or tri-nucleotide repeats. These EST-SSRs were evaluated on 35 P. triticina isolates collected in Canada and 21 EST-SSRs were polymorphic and informative in determining intraspecific genetic diversity. A comparison of virulence and EST-SSR genotypes showed a strong correlation between virulence to Lr2a, Lr2c and Lr17a and EST-SSRs genotypes. The differentiation of the P. triticina population based on EST-SSR genotypes was comparable to that obtained with genomic SSRs, despite differences between two types of SSR markers. Eight of the 21 EST-SSRs produced the cross amplification in Puccinia coronata and Puccinia graminis, suggesting that EST-SSRs are more applicable than genomic SSRs for interspecific analysis. In summary, our study suggests that the data mining of EST databases is a feasible way to generate informative molecular markers for genetic studies of P. triticina. Keywords: population genetics, Puccinia triticina, simple sequence repeat, virulence Résumé: Des marqueurs microsatellites (SSR) associés à des gènes ont été conçus pour Puccinia triticina à partir l'exploration de données contenues dans des banques d'étiquettes de séquences exprimées (EST). L'analyse de 7134 EST issues de banques d'ADNc de P. triticina ont permis de détecter 204 EST-SSR avec un minimum de 12 répétitions de nucléotides. La majorité des EST-SSR contenaient de courtes répétitions di-ou tri-nucléotidiques. Ces EST-SSR ont été évaluées sur 35 isolats de P. triticina collectés au Canada. Parmi celles-ci, 21 étaient polymorphiques et ont fourni de l'information servant à établir la diversité génétique intraspécifique. Une comparaison de la virulence et des génotypes EST-SSR a montré une forte corrélation entre la virulence à l'égard de Lr2a, Lr2c et Lr17a ainsi qu'à l'égard des génotypes EST-SSR. La différenciation de la population de P. triticina, basée sur les génotypes EST-SSR, était comparable à celle obtenue avec les SSR génomiques, malgré les différences entre les deux types de marqueurs. Huit des 21 EST-SSR ont engendré la transférabilité chez Puccinia coronata et Puccinia graminis, ce qui suggère que les EST-SSR sont plus adaptables à l'analyse interspécifique que les SSR génomiques. En résumé, notre étude suggère que l'exploration de données des banques d'EST permet de générer des marqueurs moléculaires informatifs pour les études génétiques portant sur P. triticina

Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo

Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data