Genetic control of chicken heterophil function in advanced intercross lines: associations with novel and with known Salmonella resistance loci and a likely mechanism for cell death in extracellular trap production

Heterophils, the avian polymorphonuclear leukocyte and the counterpart of mammalian neutrophils, generate the primary innate response to pathogens in chickens. Heterophil performance against pathogens is associated with host disease resistance, and heterophil gene expression and function are under genetic control. To characterize the genomic basis of heterophil function, heterophils from F13 advanced intercross chicken lines (broiler × Leghorn and broiler × Fayoumi) were assayed for phagocytosis and killing of Salmonella enteritidis, oxidative burst, and extracellular trap production. A whole-genome association analysis of single nucleotide polymorphisms at 57,636 loci identified genomic locations controlling these functional phenotypes. Genomic analysis revealed a significant association of extracellular trap production with the SAL1 locus and the SLC11A1 gene, which have both been previously associated with resistance to S. enteritidis. Fine mapping supports SIVA1 as a candidate gene controlling SAL1-mediated resistance and indicates that the proposed cell-death mechanism associated with extracellular trap production, ETosis, likely functions through the CD27/Siva-1-mediated apoptotic pathway. The SLC11A1 gene was also associated with phagocytosis of S. enteritidis, suggesting that the Slc11a1 protein may play an additional role in immune response beyond depleting metal ions to inhibit intracellular bacterial growth. A region of chromosome 6 with no characterized genes was also associated with extracellular trap production. Further characterization of these novel genes in chickens and other species is needed to understand their role in polymorphonuclear leukocyte function and host resistance to disease

Adaptative Potential of the Lactococcus Lactis IL594 Strain Encoded in Its 7 Plasmids

The extrachromosomal gene pool plays a significant role both in evolution and in the environmental adaptation of bacteria. The L. lactis subsp. lactis IL594 strain contains seven plasmids, named pIL1 to pIL7, and is the parental strain of the plasmid-free L. lactis IL1403, which is one of the best characterized lactococcal strains of LAB. Complete nucleotide sequences of pIL1 (6,382 bp), pIL2 (8,277 bp), pIL3 (19,244 bp), pIL4 (48,979), pIL5 (23,395), pIL6 (28,435 bp) and pIL7 (28,546) were established and deposited in the generally accessible database (GeneBank). Nine highly homologous repB-containing replicons, belonging to the lactococcal theta-type replicons, have been identified on the seven plasmids. Moreover, a putative region involved in conjugative plasmid mobilization was found on four plasmids, through identification of the presence of mob genes and/or oriT sequences. Detailed bioinformatic analysis of the plasmid nucleotide sequences provided new insight into the repertoire of plasmid-encoded functions in L. lactis, and indicated that plasmid genes from IL594 strain can be important for L. lactis adaptation to specific environmental conditions (e.g. genes coding for proteins involved in DNA repair or cold shock response) as well as for technological processes (e.g. genes encoding citrate and lactose utilization, oligopeptide transport, restriction-modification system). Moreover, global gene analysis indicated cooperation between plasmid- and chromosome-encoded metabolic pathways

Disruption of the Salmonella-Containing Vacuole Leads to Increased Replication of Salmonella enterica Serovar Typhimurium in the Cytosol of Epithelial Cells

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that inhabits a vacuolar compartment, called the Salmonella-containing vacuole (SCV), in infected host cells. Maintenance of the SCV is accomplished by SifA, and mutants of this Salmonella pathogenicity island 2 type III effector replicate more efficiently in epithelial cells. Here we demonstrate that enhanced replication of sifA mutants occurs in the cytosol of these cells. Increased replication of wild-type bacteria was also observed in cells treated with wortmannin or expressing Rab5 Q79L or Rab7 N125I, all of which caused a loss of SCV integrity. Our findings demonstrate the requirement of the host cell endosomal system for maintenance of the SCV and that loss of this compartment allows increased replication of serovar Typhimurium in the cytosol of epithelial cells

The Salmonella enterica Serovar Typhimurium Divalent Cation Transport Systems MntH and SitABCD Are Essential for Virulence in an Nramp1(G169) Murine Typhoid Model

Nramp1 is a transporter that pumps divalent cations from the vacuoles of phagocytic cells and is associated with the innate resistance of mice to diverse intracellular pathogens. We demonstrate that sitA and mntH, genes encoding high-affinity metal ion uptake systems in Salmonella enterica serovar Typhimurium, are upregulated when Salmonella is internalized by Nramp1-expressing macrophages and play an essential role in systemic infection of congenic Nramp1-expressing mice

Host–pathogen interactions: Host resistance factor Nramp1 up-regulates the expression of Salmonella pathogenicity island-2 virulence genes

Nramp1 (Natural resistance-associated macrophage protein-1; also known as Slc11a1) is a host resistance gene that provides protection against several intracellular pathogens, including Salmonella enterica serovar Typhimurium. Little is known about the dynamic interplay that occurs between mammalian host resistance determinants such as Nramp1 and pathogens during infection. To explore these interactions, we examined the effect of Nramp1 on expression of Salmonella typhimurium (STM) virulence factors. We demonstrate that Salmonella pathogenicity island 2 (SPI2) is essential for replication of STM in spleens of infected Nramp1(+/+) mice. Furthermore, the presence of Nramp1 in transfected cell lines and congenic knockout mice resulted in the up-regulation of STM SPI2-associated virulence genes critical for intramacrophage survival. This Nramp1-dependent up-regulation of SPI2 was mimicked in vitro by chelation of iron, demonstrating the iron-responsive nature of expression of STM SPI2-associated virulence genes. We propose that acquisition of SPI2 by S. enterica not only enabled this bacterium to become an effective intracellular pathogen but also allowed the bacterium to withstand the effects of macrophage defense mechanisms such as Nramp1 early in the evolution of its pathogenic character. These dynamic Nramp1–pathogen interactions may be essential for regulating the course of an infection. This study demonstrates the presence of a previously undescribed direct influence of a mammalian innate host resistance locus on a pathogen at the genetic level

pH-Dependent Structures of the Manganese Binding Sites in Oxalate Decarboxylase as Revealed by High-Field Electron Paramagnetic Resonance

A high-field electron paramagnetic resonance (HFEPR) study of oxalate decarboxylase (OxdC) is reported. OxdC breaks down oxalate to carbon dioxide and formate and possesses two distinct manganese(II) binding sites, referred to as site−1 and −2. The Mn(II) zero-field interaction was used to probe the electronic state of the metal ion and to examine chemical/mechanistic roles of each of the Mn(II) centers. High magnetic-fields were exploited not only to resolve the two sites, but also to measure accurately the Mn(II) zero-field parameters of each of the sites. The spectra exhibited surprisingly complex behavior as a function of pH. Six different species were identified based on their zero-field interactions, two corresponding to site-1 and four states to site-2. The assignments were verified using a mutant that only affected site-1. The speciation data determined from the HFEPR spectra for site −2 was consistent with a simple triprotic equilibrium model, while the pH dependence of site-1 could be described by a single pKa. This pH dependence was independent of the presence of the His-tag and of whether the preparations contained 1.2 or 1.6 Mn per subunit. Possible structures of the six species are proposed based on spectroscopic data from model complexes and existing protein crystallographic structures obtained at pH 8 are discussed. Although site-1 has been identified as the active site and no role has been assigned to site-2, the pronounced changes in the electronic structure of the latter and its pH behavior, which also matches the pH-dependent activity of this enzyme, suggests that even if the conversion of oxalate to formate is carried out at site-1, site-2 likely plays a catalytically relevant role