Impact of Continuous Axenic Cultivation in Leishmania infantum Virulence

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species

Mobilization of genomic islands of Staphylococcus aureus by temperate bacteriophage

The virulence of Staphylococcus aureus, in both human and animal hosts, is largely influenced by the acquisition of mobile genetic elements (MGEs). Most S. aureus strains carry a variety of MGEs, including three genomic islands (νSaα, νSaβ, νSaγ) that are diverse in virulence gene content but conserved within strain lineages. Although the mobilization of pathogenicity islands, phages and plasmids has been well studied, the mobilization of genomic islands is poorly understood. We previously demonstrated the mobilization of νSaβ by the adjacent temperate bacteriophage ϕSaBov from strain RF122. In this study, we demonstrate that ϕSaBov mediates the mobilization of νSaα and νSaγ, which are located remotely from ϕSaBov, mostly to recipient strains belonging to ST151. Phage DNA sequence analysis revealed that chromosomal DNA excision events from RF122 were highly specific to MGEs, suggesting sequence-specific DNA excision and packaging events rather than generalized transduction by a temperate phage. Disruption of the int gene in ϕSaBov did not affect phage DNA excision, packaging, and integration events. However, disruption of the terL gene completely abolished phage DNA packing events, suggesting that the primary function of temperate phage in the transfer of genomic islands is to allow for phage DNA packaging by TerL and that transducing phage particles are the actual vehicle for transfer. These results extend our understanding of the important role of bacteriophage in the horizontal transfer and evolution of genomic islands in S. aureus

Impact of multi-metals (Cd, Pb and Zn) exposure on the physiology of the yeast Pichia kudriavzevii

Metal contamination of the environment is frequently associated to the presence of two or more metals. This work aimed to study the impact of a mixture of metals (Cd, Pb and Zn) on the physiology of the non-conventional yeast Pichia kudriavzevii. The incubation of yeast cells with 5 mg/l Cd, 10 mg/l Pb and 5 mg/l Zn, for 6 h, induced a loss of metabolic activity (assessed by FUN-1 staining) and proliferation capacity (evaluated by a clonogenic assay), with a small loss of membrane integrity (measured by trypan blue exclusion assay). The staining of yeast cells with calcofluor white revealed that no modification of chitin deposition pattern occurred during the exposure to metal mixture. Extending for 24 h, the exposure of yeast cells to metal mixture provoked a loss of membrane integrity, which was accompanied by the leakage of intracellular components. A marked loss of the metabolic activity and the loss of proliferation capacity were also observed. The analysis of the impact of a single metal has shown that, under the conditions studied, Pb was the metal responsible for the toxic effect observed in the metal mixture. Intracellular accumulation of Pb seems to be correlated with the metals toxic effects observed.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "BioInd-Biotechnology and Bioengineering for improved Industrial and Agro-Food processes" (NORTE-07-0124-FEDER-000028), Co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. Manuela D. Machado gratefully acknowledges the post-doctoral grant from FCT (SFRH/BPD/72816/2010). Vanessa A. Mesquita gratefully acknowledges the grant from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES). The authors also thank to Doctor Rosane Freitas Schwan to offer the yeast strain and to Doctor Helena M.V.M. Soares, from the Faculty of Engineering of Porto University, for the use of analytical facilities (AAS with flame atomization and AAS with electrothermal atomization)

Intra- and Interspecies Genomic Transfer of the Enterococcus faecalis Pathogenicity Island

Enterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibiotic resistance and pathogenicity traits. Enterococcus faecalis harbours a pathogenicity island (PAI) of 153 kb containing several virulence factors including the enterococcal surface protein (esp). Until now only internal fragments of the PAI or larger chromosomal regions containing it have been transfered. Here we demonstrate precise excision, circularization and horizontal transfer of the entire PAI element from the chromosome of E. faecalis strain UW3114. This PAI (ca. 200 kb) contained some deletions and insertions as compared to the PAI of the reference strain MMH594, transferred precisely and integrated site-specifically into the chromosome of E. faecalis (intergenic region) and Enterococcus faecium (tRNAlys). The internal PAI structure was maintained after transfer. We assessed phenotypic changes accompanying acquisition of the PAI and expression of some of its determinants. The esp gene is expressed on the surface of donor and both transconjugants. Biofilm formation and cytolytic activity were enhanced in E. faecalis transconjugants after acquisition of the PAI. No differences in pathogenicity of E. faecalis were detected using a mouse bacteraemia and a mouse peritonitis models (tail vein and intraperitoneal injection). A 66 kb conjugative pheromone-responsive plasmid encoding erm(B) (pLG2) that was transferred in parallel with the PAI was sequenced. pLG2 is a pheromone responsive plasmid that probably promotes the PAI horizontal transfer, encodes antibiotic resistance features and contains complete replication and conjugation modules of enterococcal origin in a mosaic-like composition. The E. faecalis PAI can undergo precise intra- and interspecies transfer probably with the help of conjugative elements like conjugative resistance plasmids, supporting the role of horizontal gene transfer and antibiotic selective pressure in the successful establishment of certain enterococci as nosocomial pathogens

The importance of imprinting in the human placenta.

As a field of study, genomic imprinting has grown rapidly in the last 20 years, with a growing figure of around 100 imprinted genes known in the mouse and approximately 50 in the human. The imprinted expression of genes may be transient and highly tissue-specific, and there are potentially hundreds of other, as yet undiscovered, imprinted transcripts. The placenta is notable amongst mammalian organs for its high and prolific expression of imprinted genes. This review discusses the development of the human placenta and focuses on the function of imprinting in this organ. Imprinting is potentially a mechanism to balance parental resource allocation and it plays an important role in growth. The placenta, as the interface between mother and fetus, is central to prenatal growth control. The expression of genes subject to parental allelic expression bias has, over the years, been shown to be essential for the normal development and physiology of the placenta. In this review we also discuss the significance of genes that lack conservation of imprinting between mice and humans, genes whose imprinted expression is often placental-specific. Finally, we illustrate the importance of imprinting in the postnatal human in terms of several human imprinting disorders, with consideration of the brain as a key organ for imprinted gene expression after birth

Study of production and cold nuclear matter effects in pPb collisions at=5 TeV

Production of mesons in proton-lead collisions at a nucleon-nucleon centre-of-mass energy = 5 TeV is studied with the LHCb detector. The analysis is based on a data sample corresponding to an integrated luminosity of 1.6 nb(-1). The mesons of transverse momenta up to 15 GeV/c are reconstructed in the dimuon decay mode. The rapidity coverage in the centre-of-mass system is 1.5 < y < 4.0 (forward region) and -5.0 < y < -2.5 (backward region). The forward-backward production ratio and the nuclear modification factor for (1S) mesons are determined. The data are compatible with the predictions for a suppression of (1S) production with respect to proton-proton collisions in the forward region, and an enhancement in the backward region. The suppression is found to be smaller than in the case of prompt J/psi mesons

Rapid fucosylation of intestinal epithelium sustains host–commensal symbiosis in sickness

Systemic infection induces conserved physiological responses that include both resistance and ‘tolerance of infection’ mechanisms. Temporary anorexia associated with an infection is often beneficial, reallocating energy from food foraging towards resistance to infection or depriving pathogens of nutrients. However, it imposes a stress on intestinal commensals, as they also experience reduced substrate availability; this affects host fitness owing to the loss of caloric intake and colonization resistance (protection from additional infections). We hypothesized that the host might utilize internal resources to support the gut microbiota during the acute phase of the disease. Here we show that systemic exposure to Toll-like receptor (TLR) ligands causes rapid α(1,2)-fucosylation of small intestine epithelial cells (IECs) in mice, which requires the sensing of TLR agonists, as well as the production of interleukin (IL)-23 by dendritic cells, activation of innate lymphoid cells and expression of fucosyltransferase 2 (Fut2) by IL-22-stimulated IECs. Fucosylated proteins are shed into the lumen and fucose is liberated and metabolized by the gut microbiota, as shown by reporter bacteria and community-wide analysis of microbial gene expression. Fucose affects the expression of microbial metabolic pathways and reduces the expression of bacterial virulence genes. It also improves host tolerance of the mild pathogen Citrobacter rodentium. Thus, rapid IEC fucosylation appears to be a protective mechanism that utilizes the host’s resources to maintain host–microbial interactions during pathogen-induced stress