Proteolytic cleavage of factor VIII heavy chain is required to expose the binding-site for low-density lipoprotein receptor-related protein within the A2 domain

Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that contributes to the clearance of coagulation factor (F) VIII from the circulation. Previously, we have demonstrated that region Glu(1811)-Lys(1818) within FVIII light chain constitutes an important binding region for this receptor. We have further found that FVIII light chain and intact FVIII are indistinguishable in their LRP-binding affinities. In apparent contrast to these observations, a second LRP-binding region has been identified within A2 domain region Arg(484)-Phe(509) of FVIII heavy chain. In this study, we addressed the relative contribution of FVIII heavy chain in binding LRP. Surface plasmon resonance analysis unexpectedly showed that FVIII heavy chain poorly associated to the receptor. The binding to LRP was, however, markedly enhanced upon cleavage of the heavy chain by thrombin. The A2 domain, purified from thrombin-activated FVIII, also showed efficient binding to LRP. Competition studies employing a recombinant antibody fragment demonstrated that region Arg(484)-Phe(509) mediates the enhanced LRP binding after thrombin cleavage. We propose that LRP binding of non-activated FVIII is mediated via the FVIII light chain while in activated FVIII both the heavy and light chain contribute to LRP bindin

EspH is a hypervirulence factor for Mycobacterium marinum and essential for the secretion of the ESX-1 substrates EspE and EspF

The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum

Genome-wide transposon mutagenesis indicates that Mycobacterium marinum customizes its virulence mechanisms for survival and replication in different hosts.

The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogen Mycobacterium marinum whether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show that M. marinum contains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore, M. marinum contains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers

Inventories and Mobilization of Unsaturated Zone Sulfate, Fluoride, and Chloride Related to Land Use Change in Semiarid Regions, Southwestern United States and Australia

Unsaturated zone salt reservoirs are potentially mobilized by increased groundwater recharge as semiarid lands are cultivated. This study explores the amounts of pore water sulfate and fluoride relative to chloride in unsaturated zone profiles, evaluates their sources, estimates mobilization due to past land use change, and assesses the impacts on groundwater quality. Inventories of water-extractable chloride, sulfate, and fluoride were determined from borehole samples of soils and sediments collected beneath natural ecosystems (N = 4), nonirrigated ( rain-fed ) croplands (N = 18), and irrigated croplands (N = 6) in the southwestern United States and in the Murray Basin, Australia. Natural ecosystems contain generally large sulfate inventories (7,800-120,000 kg/ha) and lower fluoride inventories (630-3,900 kg/ha) relative to chloride inventories (6,600-41,000 kg/ha). Order-of-magnitude higher chloride concentrations in precipitation and generally longer accumulation times result in much larger chloride inventories in the Murray Basin than in the southwestern United States. Atmospheric deposition during the current dry interglacial climatic regime accounts for most of the measured sulfate in both U.S. and Australian regions. Fluoride inventories are greater than can be accounted for by atmospheric deposition in most cases, suggesting that fluoride may accumulate across glacial/interglacial climatic cycles. Chemical modeling indicates that fluorite controls fluoride mobility and suggests that water-extractable fluoride may include some fluoride from mineral dissolution. Increased groundwater drainage/recharge following land use change readily mobilized chloride. Sulfate displacement fronts matched or lagged chloride fronts by up to 4 m. In contrast, fluoride mobilization was minimal in all regions. Understanding linkages between salt inventories, increased recharge, and groundwater quality is important for quantifying impacts of anthropogenic activities on groundwater quality and is required for remediating salinity problems