The host vasculature is believed to constitute the principal route of dissemination of Neisseria meningitidis (Nm) throughout the body, resulting in septicaemia and meningitis in susceptible humans. In vitro, the Nm outer membrane protein Opc can enhance cellular entry and exit, utilising serum factors to anchor to endothelial integrins; but the mechanisms of binding to serum factors are poorly characterised. This study demonstrates that Nm Opc expressed in acapsulate as well as capsulate bacteria can increase human brain endothelial cell line (HBMEC) adhesion and entry by first binding to serum vitronectin and, to a lesser extent, fibronectin. This study also demonstrates that Opc binds preferentially to the activated form of human vitronectin, but not to native vitronectin unless the latter is treated to relax its closed conformation. The direct binding of vitronectin occurs at its Connecting Region (CR) requiring sulphated tyrosines Y56 and Y59. Accordingly, Opc/vitronectin interaction could be inhibited with a conformation-dependent monoclonal antibody 8E6 that targets the sulphotyrosines, and with synthetic sulphated (but not phosphorylated or unmodified) peptides spanning the vitronectin residues 43–68. Most importantly, the 26-mer sulphated peptide bearing the cell-binding domain 45RGD47 was sufficient for efficient meningococcal invasion of HBMECs. To our knowledge, this is the first study describing the binding of a bacterial adhesin to sulphated tyrosines of the host receptor. Our data also show that a single region of Opc is likely to interact with the sulphated regions of both vitronectin and of heparin. As such, in the absence of heparin, Opc-expressing Nm interact directly at the CR but when precoated with heparin, they bind via heparin to the heparin-binding domain of the activated vitronectin, although with a lower affinity than at the CR. Such redundancy suggests the importance of Opc/vitronectin interaction in meningococcal pathogenesis and may enable the bacterium to harness the benefits of the physiological processes in which the host effector molecule participates.

The development of rapid detection assays for malaria diagnostics is an area of intensive research, as the traditional microscopic analysis of blood smears is cumbersome and requires skilled personnel. Here, we describe a simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures. This homogeneous assay is based on the fluorescence quenching of cyanine 3B (Cy3B)-labeled recombinant Plasmodium falciparum heat shock protein 70 (PfHsp70) upon binding to gold nanoparticles (AuNPs) functionalized with an anti-Hsp70 monoclonal antibody. Upon competition with the free antigen, the Cy3B-labeled recombinant PfHsp70 is released to solution resulting in an increase of fluorescence intensity. Two types of AuNP-antibody conjugates were used as probes, one obtained by electrostatic adsorption of the antibody on AuNPs surface and the other by covalent bonding using protein cross-linking agents. In comparison with cross-linked antibodies, electrostatic adsorption of the antibodies to the AuNPs surfaces generated conjugates with increased activity and linearity of response, within a range of antigen concentration from 8.2 to 23.8 μg.mL(-1). The estimated LOD for the assay is 2.4 μg.mL(-1) and the LOQ is 7.3 μg.mL(-1). The fluorescence immunoassay was successfully applied to the detection of antigen in malaria-infected human blood cultures at a 3% parasitemia level, and is assumed to detect parasite densities as low as 1,000 parasites.μL(-1).

The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Hepatic Plasmodium development occurs inside a specialized membranous compartment termed the parasitophorous vacuole (PV). Here, we show that, during the parasite's hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful Plasmodium liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency, leading to a disease outcome different from that generally induced by infection with wildtype parasites. This study identifies a host-parasite protein interaction during the hepatic stage of infection by Plasmodium parasites. The identification of such vital interactions may hold potential toward the development of novel malaria prevention strategies. malaria | Plasmodium liver stages | host-parasite interaction | ApoH | EXP-1

Cerebral malaria is one of the most severe complications of malaria disease, attributed to a complicated series of immune reactions in the host. The syndrome is marked by inflammatory immune responses, margination of leukocytes, and parasitized erythrocytes in cerebral vessels leading to breakdown of the blood–brain barrier. We show that chemical attenuation of the parasite at the very early, clinically silent liver stage suppresses parasite development, delays the time until parasites establish blood-stage infection, and provokes an altered host immune response, modifying immunopathogenesis and protecting from cerebral disease. The early response is proinflammatory and cell mediated, with increased T cell activation in the liver and spleen, and greater numbers of effector T cells, cytokine-secreting T cells, and proliferating, proinflammatory cytokine-producing T cells. Dendritic cell numbers, T cell activation, and infiltration of CD8+ T cells to the brain are decreased later in infection, possibly mediated by the anti-inflammatory cytokine IL-10. Strikingly, protection can be transferred to naive animals by adoptive transfer of lymphocytes from the spleen at very early times of infection. Our data suggest that a subpopulation belonging to CD8+ T cells as early as day 2 postinfection is responsible for protection. These data indicate that liver stage–directed early immune responses can moderate the overall downstream host immune response and modulate severe malaria outcome.

SummaryNeisseria meningitidis Opc protein is an effective invasin for human endothelial cells. We have investigated novel human endothelial receptors targeted by Opc and observed that Opc-expressing bacteria interacted with a 100 kDa protein in wholecell lysates of human endothelial and epithelial cells. The identity of the protein was established as a-actinin by mass spectrometry. Opc expression was essential for the recognition of a-actinin whether provided in a purified form or in cell extracts. The interaction of the two proteins did not involve intermediate molecules. As there was no demonstrable expression of a-actinin on the surfaces of any of the eight cell lines studied, the likelihood of the interactions after meningococcal internalization was examined. Confocal imaging demonstrated considerable colocalization of N. meningitidis with a-actinin especially after a prolonged period of internalization. This may imply that bacteria and a-actinin initially occur in separate compartments and co-compartmentalization occurs progressively over the 8 h infection period used. In conclusion, these studies have identified a novel and an intracellular target for the N. meningitidis Opc invasin. Since a-actinin is a modulator of a variety of signalling pathways and of cytoskeletal functions, its targeting by Opc may enable bacteria to survive/translocate across endothelial barriers.