Search

Immunometabolism and regulation of mitochondrial reactive oxygen species (mROS) control the immune effector phenotype of differentiated macrophages. Mitochondrial function requires dynamic fission and fusion, but whether effector function is coupled to altered dynamics during bacterial responses is unknown. We show that macrophage mitochondria undergo fission after 12 h of progressive ingestion of live Streptococcus pneumoniae (pneumococci), without evidence of Drp-1 phosphorylation at S616. Fission is associated with progressive reduction in oxidative phosphorylation but increased mROS generation. Fission is enhanced by mROS production, PI3Kγ signaling and by cathepsin B, but is independent of inflammasome activation or IL-1β generation. Inhibition of fission reduces bacterial killing. Fission is associated with Parkin recruitment to mitochondria, but not mitophagy. Fission occurs upstream of apoptosis induction and independently of caspase activation. During macrophage innate responses to bacteria mitochondria shift from oxidative phosphorylation and ATP generation to mROS production for microbicidal responses by undergoing fission. Author summary Changes in metabolism regulate function in immune cells, including macrophages which are key cells in pathogen clearance. Mitochondria are cellular organelles that generate energy during metabolism but also mitochondrial reactive oxygen species (mROS) that contribute to bacterial killing. Mitochondria are dynamic organelles that form complex networks with varying degrees of fragmentation or fusion, but the functional consequences of these processes on macrophage function during bacterial infection are unknown. We show that sustained ingestion of live bacteria triggers mitochondrial fragmentation, reducing metabolism but enhancing mROS generation. Mitochondrial fragmentation is not part of a clearance pathway for damaged mitochondria and is initiated before signs of cell death. Macrophage signalling pathways activated during infection, and mROS generation, enhance mitochondrial fragmentation, and inhibition of pathways promoting fragmentation reduces bacterial killing. Overall, these findings suggest that responses to ingested bacteria trigger mitochondrial fragmentation, allowing mitochondria to switch from energy generation during metabolism to organelles facilitating bacterial killing.

This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell.

The generation of immune cells from BM precursors is a carefully regulated process. This is essential to limit the potential for oncogenesis and autoimmunity yet protect against infection. How infection modulates this is unclear. Salmonella can colonize systemic sites including the BM and spleen. This resolving infection has multiple IFN-γ-mediated acute and chronic effects on BM progenitors, and during the first week of infection IFN-γ is produced by myeloid, NK, NKT, CD4+ T cells, and some lineage-negative cells. After infection, the phenotype of BM progenitors rapidly but reversibly alters, with a peak ∼30-fold increase in Sca-1hi progenitors and a corresponding loss of Sca-1lo/int subsets. Most strikingly, the capacity of donor Sca-1hi cells to reconstitute an irradiated host is reduced; the longer donor mice are exposed to infection, and Sca-1hic-kitint cells have an increased potential to generate B1a-like cells. Thus, Salmonella can have a prolonged influence on BM progenitor functionality not directly related to bacterial persistence. These results reflect changes observed in leucopoiesis during aging and suggest that BM functionality can be modulated by life-long, periodic exposure to infection. Better understanding of this process could offer novel therapeutic opportunities to modulate BM functionality and promote healthy aging.

9 páginas, 1 figura, 2 tablas -- PAGS nros. 4854-4862, Streptococcus pneumoniae is an important human pathogen that often carries temperate bacteriophages. As part of a program to characterize the genetic makeup of prophages associated with clinical strains and to assess the potential roles that they play in the biology and pathogenesis in their host, we performed comparative genomic analysis of 10 temperate pneumococcal phages. All of the genomes are organized into five major gene clusters: lysogeny, replication, packaging, morphogenesis, and lysis clusters. All of the phage particles observed showed a Siphoviridae morphology. The only genes that are well conserved in all the genomes studied are those involved in the integration and the lysis of the host in addition to two genes, of unknown function, within the replication module. We observed that a high percentage of the open reading frames contained no similarities to any sequences catalogued in public databases; however, genes that were homologous to known phage virulence genes, including the pblB gene of Streptococcus mitis and the vapE gene of Dichelobacter nodosus, were also identified. Interestingly, bioinformatic tools showed the presence of a toxin-antitoxin system in the phage φSpn_6, and this represents the first time that an addition system in a pneumophage has been identified. Collectively, the temperate pneumophages contain a diverse set of genes with various levels of similarity among them., Streptococcus pneumoniae (the pneumococcus) is an important human pathogen and a major etiological agent of pneumonia, bacteremia, and meningitis in adults and of otitis media in children. The casualties due to the pneumococcus are estimated to be over 1.6 million deaths per year, and most of these deaths are of young children in developing countries (40). S. pneumoniae is also a human commensal that resides in the upper respiratory tract, and it is asymptomatically carried in the nasopharynx of up to 60% of the normal population (48). Bacteriophages of S. pneumoniae (pneumophages) were first identified in 1975 from samples isolated from throat swabs of healthy children by two independent groups (46, 65). Since then, pneumophages have been identified from different sources and a variety of locations (44). The abundance of temperate bacteriophages in S. pneumoniae has been reported in different studies in the past (6, 54). Up to 76% of clinical isolates have been showed to contain prophages (or prophage remnants) when studied with a DNA probe specific for the major autolysin gene, lytA, which hybridizes with many of the endolysin genes of temperate pneumococcal phages (54). Hybridization analyses have identified highly similar prophages among pneumococcal clinical isolates even of different capsular serotypes, a result which indicates the widespread distribution of these mobile genetic elements among virulent strains (26)., Only three S. pneumoniae bacteriophage genomes have been characterized in detail, and their sequences have been determined. Dp-1 and Cp-1 are lytic bacteriophages, whereas MM1 is a temperate pneumophage (45, 50, 52). Genes coding for virulence factors such as toxins or secreted enzymes have been associated with the presence of prophages in both gram-negative (67) and gram-positive bacteria, such as Streptococcus pyogenes (7) and Staphylococcus aureus (23). Because a considerable number of toxin genes are located in prophages, phage dynamics are of apparent importance for bacterial pathogenesis. Unfortunately, the role of temperate bacteriophages in the virulence of S. pneumoniae remains mostly unknown. Recently, the availability of relatively inexpensive next-generation sequencing technologies has permitted the complete genomic analysis of dozens of genomes of pneumococcal clinical isolates. In this report, we present a comparative genomic analysis of 10 pneumophages identified in the genomes of newly sequenced S. pneumoniae strains. The proteome of these phages has been predicted and annotated by comparative sequence analyses by using the available databases at the National Center for Biotechnological Information website (http://www.ncbi.nlm.nih.gov/). This systematic characterization of pneumophage genomes provides for a substantial increase in our knowledge of the global proteome and the overall genetic diversity of this important human pathogen. The comparative analysis of multiple temperate bacteriophages from a single species offers a unique opportunity to study one of the mechanisms of lateral gene transfer that drive prokaryotic genetic diversity, P. Romero is the recipient of a postdoctoral fellowship from the Spanish Ministerio de Educacion y Ciencia (EX-2006-0759). This work was supported by a grant from the Dirección General de Investigación Científica y Técnica (SAF2006-00390) and by grants DC05659, DC04173, and DC02148 from the U.S. NIH NIDCD. CIBER de Enfermedades Respiratorias (CibeRes) is an initiative of ISCIII