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

Salmonella – the ultimate insider. Salmonella virulence factors that modulate intracellular survival

Salmonella enterica serovar Typhimurium is a common facultative intracellular pathogen that causes food-borne gastroenteritis in millions of people worldwide. Intracellular survival and replication are important virulence determinants and the bacteria can be found in a variety of phagocytic and non-phagocytic cells in vivo. Invasion of host cells and intracellular survival are dependent on two type III secretion systems, T3SS1 and T3SS2, each of which translocates a distinct set of effector proteins. However, other virulence factors including ion transporters, superoxide dismutase, flagella and fimbriae are also involved in accessing and utilizing the intracellular niche

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

Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). DNA-mediated killing occurred within minutes, as a result of perturbation of both the outer and inner membrane (IM) and the release of cytoplasmic contents, including genomic DNA. Sub-inhibitory concentrations of DNA created a cation-limited environment that resulted in induction of the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon PA3552–PA3559 in P. aeruginosa. Furthermore, DNA-induced expression of this operon resulted in up to 2560-fold increased resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides, but had no effect on β-lactam and fluoroquinolone resistance. Thus, the presence of extracellular DNA in the biofilm matrix contributes to cation gradients, genomic DNA release and inducible antibiotic resistance. DNA-rich environments, including biofilms and other infection sites like the CF lung, are likely the in vivo environments where extracellular pathogens such as P. aeruginosa encounter cation limitation

Full Sequence and Comparative Analysis of the Plasmid pAPEC-1 of Avian Pathogenic E. coli χ7122 (O78∶K80∶H9)

(APEC), are very diverse. They cause a complex of diseases in Human, animals, and birds. Even though large plasmids are often associated with the virulence of ExPEC, their characterization is still in its infancy., are also present in the sequence of pAPEC-1. The comparison of the pAPEC-1 sequence with the two available plasmid sequences reveals more gene loss and reorganization than previously appreciated. The presence of pAPEC-1-associated genes is assessed in human ExPEC by PCR. Many patterns of association between genes are found.The pathotype typical of pAPEC-1 was present in some human strains, which indicates a horizontal transfer between strains and the zoonotic risk of APEC strains. ColV plasmids could have common virulence genes that could be acquired by transposition, without sharing genes of plasmid function

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

How cells ensure correct metallation of a given protein and whether a degree of promiscuity in metal binding has evolved are largely unanswered questions. In a classic case, iron- and manganese-dependent superoxide dismutases (SODs) catalyze the disproportionation of superoxide using highly similar protein scaffolds and nearly identical active sites. However, most of these enzymes are active with only one metal, although both metals can bind in vitro and in vivo. Iron(II) and manganese(II) bind weakly to most proteins and possess similar coordination preferences. Their distinct redox properties suggest that they are unlikely to be interchangeable in biological systems except when they function in Lewis acid catalytic roles, yet recent work suggests this is not always the case. This review summarizes the diversity of ways in which iron and manganese are substituted in similar or identical protein frameworks. As models, we discuss (1) enzymes, such as epimerases, thought to use Fe[superscript II] as a Lewis acid under normal growth conditions but which switch to Mn[superscript II] under oxidative stress; (2) extradiol dioxygenases, which have been found to use both Fe[superscript II] and Mn[superscript II], the redox role of which in catalysis remains to be elucidated; (3) SODs, which use redox chemistry and are generally metal-specific; and (4) the class I ribonucleotide reductases (RNRs), which have evolved unique biosynthetic pathways to control metallation. The primary focus is the class Ib RNRs, which can catalyze formation of a stable radical on a tyrosine residue in their β2 subunits using either a di-iron or a recently characterized dimanganese cofactor. The physiological roles of enzymes that can switch between iron and manganese cofactors are discussed, as are insights obtained from the studies of many groups regarding iron and manganese homeostasis and the divergent and convergent strategies organisms use for control of protein metallation. We propose that, in many of the systems discussed, “discrimination” between metals is not performed by the protein itself, but it is instead determined by the environment in which the protein is expressed.National Institutes of Health (U.S.) (Grant GM81393

Host-pathogen interactions : the impact of Nrampl on Salmonella enterica serovar tyuphimurium virulence gene expression

Nrampl (natural resistance-associated macrophage protein 1) 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 Nrampl and pathogens during infection. To explore these interactions we examined the effect of Nrampl on selected S. Typhimurium (STM) genes. Nrampl is believed to be a divalent cation transport system responsible for transport of Fe2+ and Mn2+from the phagolysosome to the cytoplasm of macrophages. We chose to investigate the role of the bacterial Mn2+ transporters MntH and SitABCD in STM pathogenesis. Nrampl-transfected cell lines infected with STM harboring mntH and sitA transcriptional fusions were used to investigate the intracellular patterns of expression of these STM genes. Both genes are expressed in the intracellular environment and are further upregulated in the presence of Nrampl. Analyses using axenic bacterial cultures demonstrated that expression of mntH and sitA responds to levels of Fe2+ and Mn2+ through the regulators Fur and MntR, while expression of mntH also responds to hydrogen peroxide through the regulator OxyR. Studies using strains deleted for oxyR or an expression plasmid deleted for the OxyR box indicated that hydrogen peroxide is important for initial expression of mntH inside macrophages both in the presence and absence of Nrampl. Similar studies investigating the role of Fur and MntR on mntH expression in cultured cells were inconclusive. Virulence studies using congenic Nrampl knockout mice demonstrated that both mntH and sitA are essential for virulence of STM in Nrampl+/+ animals. An mntH sitA double knockout strain was more attenuated than either single knockout strain, indicating that mntH and sitA contribute independently to the pathogenic nature of STM. The effect of Nrampl was also studied on selected STM virulence genes associated with the Type III Secretion System encoded within Salmonella pathogenicity island-2 (SPI2). In vivo studies demonstrated that SPI2 is essential for the ability of STM to replicate in the spleen of Nrampl+/+ mice. To investigate a potential interaction between SPI2 and Nrampl, Nrampl-transfected cell lines were used to identify that the SPI2-associated genes ssrA, sseA and sseJ, but not phoP or the SPI1 regulator hilA, are upregulated in the presence of Nrampl. Studies using axenic cultures indicate that these SPI2-associated genes are responding to extracellular levels of Fe2+ via a novel iron-responsive regulatory system. Further, these genes are differentially regulated by the global regulatory systems PhoPQ and OmpR, suggesting a previously uncharacterized complexity of SPI2 gene regulation. Overall STM is directly influenced on the genetic level by the presence of Nrampl. This impact on STM virulence gene expression correlates with the proposed function of Nrampl as a divalent cation transport system. We propose that upregulation of STM virulence genes in response to limiting levels of divalent cations is a pathogenic strategy developed by the bacterium to maintain some level of replication in naturally resistant (i.e. Nrampl+/+) hosts. We discuss how this may be involved in the development of chronic disease states by Salmonellae.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Slc11a1 limits intracellular growth of Salmonella enterica sv. Typhimurium by promoting macrophage immune effector functions and impairing bacterial iron acquisition

The natural resistance-associated macrophage protein 1, Slc11a1, is a phagolysosomal transporter for protons and divalent ions including iron that confers host protection against diverse intracellular pathogens including Salmonella. We investigated and compared the regulation of iron homeostasis and immune function in RAW264.7 murine phagocytes stably transfected with non-functional Slc11a1 and functional Slc11a1 controls in response to an infection with Salmonella enterica serovar Typhimurium. We report that macrophages lacking functional Slc11a1 displayed an increased expression of transferrin receptor 1, resulting in enhanced acquisition of transferrin-bound iron. In contrast, cellular iron release mediated via ferroportin 1 was significantly lower in Salmonella-infected Slc11a1-negative macrophages in comparison with phagocytes bearing Slc11a1. Lack of Slc11a1 led to intracellular persistence of S.?enterica serovar Typhimurium within macrophages, which was paralleled by a reduced formation of nitric oxide, tumour necrosis factor-alpha and interleukin-6 in Slc11a1-negative macrophages following Salmonella infection, whereas interleukin-10 production was increased. Moreover, Slc11a1-negative phagocytes exhibited higher cellular iron content, resulting in increased iron acquisition by intracellular Salmonella. Our observations indicate a bifunctional role for Slc11a1 within phagocytes. Slc11a restricts iron availability, which first augments pro-inflammatory macrophage effector functions and second concomitantly limits microbial iron access. <br/