SummaryThe Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signaturetag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.

a b s t r a c tAmphipods of the superfamily Lysianassoidea are ubiquitous at hadal depths ( 46000 m) and therefore are an ideal model group for investigating levels of endemism and the drivers of speciation in deep ocean trenches. The taxonomic classification of hadal amphipods is typically based on conventional morphological traits but it has been suggested that convergent evolution, phenotypic plasticity, intra-specific variability and ontogenetic variation may obscure the ability to robustly diagnose taxa and define species. Here we use phylogenetic analysis of DNA sequence variation at two mitochondrial (COI and 16S rDNA) and one nuclear (18S rDNA) regions at to examine the evolutionary relationships among 25 putative amphipod species representing 14 genera and 11 families that were sampled from across seven hadal trenches. We identify several instances where species, genera and families do not resolve monophyletic clades, highlighting incongruence between the current taxonomic classification and the molecular phylogeny for this group. Our data also help extend and resolve the known biogeographic distributions for the different species, such as identifying the co-occurrence of Hirondellea dubia and Hirondellea gigas in the Mariana trench.

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In this study, we identified lysine residues in the fibrinogen A␣ chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with ␣ 2 -antiplasmin (␣ 2 -AP), which is known to cross-link to lysine 303 of the A␣ chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to ␣ 2 -AP but not for crosslinking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-␣C fibrinogens, which lack 100 and 390 amino acids from the C terminus of the A␣ chain, respectively. PAI-2 or ␣ 2 -AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the A␣ chain. ␣ 2 -AP was crosslinked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with ␣ 2 -AP, and the two proteins utilized different lysines in the A␣ chain. Therefore, PAI-2 and ␣ 2 -AP can cross-link simultaneously to the ␣ polymers of a fibrin clot and promote resistance to lysis.

Fibrin deposition is characteristic of inflammatory diseases. The monocytes is central to the inflammatory response and can affect fibrinolysis by expression of urokinase (u-PA) and plasminogen activator inhibitor types 1 and 2 (PAI-1 and PAI-2, respectively). This study examines whether thrombin, which promotes fibrin deposition, can contribute to fibrin persistence by modulating expression of proteins of the fibrinolytic system. Monocytes were isolated from human peripheral blood and analyzed for PAI-2, PAI-1, and u-PA antigens by enzyme-linked immunosorbent assay (ELISA). Monocytes responded to thrombin by increased expression of PAI-2 in a dose- and time-dependent manner, with maximal synthesis at a concentration of 1 U/mL to 10 U/mL. This trend was also evident for PAI-1, which was present at much lower levels. Thrombin and lipopolysaccharide (LPS) stimulated comparable levels of PAI-2, studied at the antigen and mRNA level. The dose effet of LPS on PAI-2 and PAI-1 was found to differ from that of thrombin. The level of u-PA was undetectable by ELISA and zymography in all samples. Thrombin stimulates PAI-2 synthesis by human monocytes, therefore creating an imbalance in the fibrinolytic system. This may contribute to persistence of fibrin, deposited during inflammation.

During the screening of a Staphylococcus aureus signature-tagged mutagenesis library, it was noted that nonhemolytic bacteria became more abundant as time passed in murine abscess and wound models, but not within organ tissues associated with systemic infections. To examine this further, a mixed population of hyperhemolytic, hemolytic, and nonhemolytic S. aureus strain RN6390 cells were inoculated into mice using abscess, wound, and systemic models of infection. After 7 days in the abscess, the hyperhemolytic group markedly declined, whereas the nonhemolytic population increased significantly. A similar phenomenon occurred in murine wounds, but not during the systemic infection. Sequencing of several of the signature-tagged mutants indicated mutations in the agrC gene or within the agrA-agrC intergenic region. Both alpha-hemolysin and delta-hemolysin activity was curtailed in these mutants, but beta-hemolysin activity was unaffected. Single strain comparisons between wild-type strain 8325-4 and strain DU1090 (hla-) as well as between strain RN6911 (agr) and wild-type strain RN6390 were performed using the same three animal models of infection. The agr mutant strain and the hla mutant strain showed no difference in bacterial counts in murine wounds compared to their respective parent strains. The same held true in murine abscesses at day 4, but strain RN6911 counts then declined at day 7. Considerable clearing of the hla mutant strain and the agr mutant strain occurred in the systemic model of infection. Mixed infections with the DU1090 and 8325-4 strains in the abscess model showed a slight advantage given to the DU1090 population, but a distinct selection for the parental 8325-4 strain in the liver. These results suggest that agr mutations cause reductions in the expression of several secreted proteins, including alpha- and delta-hemolysin, which in turn contribute to a growth advantage of this agr mutant group within a mixed population of S. aureus cells residing in abscesses and wounds.

Staphylococcus aureus is an important pathogen of humans and other animals, causing bacteremia, abscesses, endocarditis, and other infectious syndromes. A signature-tagged mutagenesis (STM) system was adapted for use in studying the genes required for in vivo survival of S. aureus. An STM library was ultimately created in S. aureus RN6390, with Tn917 being used to create the transposon mutations. Pools of S. aureusRN6390 mutants were screened in mouse abscess, bacteremia, and wound infection models for growth attenuation after in vivo passage. One of the mutants that was identified displayed marked attenuation following large-pool screening in all three animal models, which was confirmed in bacteremia and endocarditis models of infection with a smaller pool of mutants. Sequence analysis of the entire open reading frame showed a 99% identity to the high-affinity proline permease (putP) gene characterized in another strain of S. aureus. In wound and murine abscess infection models, the putP mutant was approximately 10-fold more attenuated than was wild-type strain RN6390. Another S. aureus strain transduced with theputP mutation also displayed an attenuated phenotype after passage in the wound model. A [3H]proline uptake assay showed that less proline was specifically transported into theputP mutant than into strain RN6390. The reduced viability of the bacteria possessing the mutation in the S. aureushigh-affinity proline permease suggests that proline scavenging by the bacteria is important for in vivo growth and proliferation and that analogs of proline may serve as potential antistaphylococcal therapeutic agents.

Newcastle University ePrints -eprint.ncl.ac.uk Ritchie H, Jamieson AJ, Piertney SB. Population genetic structure of two congeneric deep-sea amphipod species from geographically isolated hadal trenches in the Pacific Ocean. Abstract 12The deep ocean trenches that comprise the hadal zone have traditionally been perceived as a series 13 of geographically isolated and demographically independent features likely to promote local species 14 endemism through potent natural selection and restricted dispersal. Here we provide the first 15 descriptions of intraspecific population genetic structure among trenches from which the levels of 16 genetic connectivity can be examined explicitly. A total of 109 individuals across two species of 17Paralicella amphipods (Lysianassoidea: Alicellidae) were genotyped at 16 microsatellite DNA loci. An 18 analysis of molecular variance identified that 22% of the overall genetic variance was attributable to 19 differences between the species and a further 7% was attributable to differences between 20 populations. The two species showed different patterns of genetic structure, with the levels of 21 genetic differentiation between trenches explained by geographical proximity, the geological ages of 22 the trenches, contemporary bottom current patterns and seabed topography around the Pacific 23Ocean. Overall, the inferred levels of gene flow among trenches was sufficient to reject the 24 hypothesis that they are evolutionarily independent units. 25 Highlights 27 Hadal trenches do not represent demographically independent entities in Paralicella 28  Patterns of connectivity between trenches differ for two Paralicella species 29  Trench geological ages, deep water currents and topography explain connectivity 30