Malarial EBA-175 region VI crystallographic structure reveals a KIX-like binding interface

The malaria parasite proliferates in the bloodstream of its vertebrate host by invading and replicating within erythrocytes. To achieve successful invasion, a number of discrete and essential events need to take place at the parasite-host cell interface. Erythrocyte-binding antigen 175 (EBA-175) is a member of a family of Plasmodium falciparum erythrocyte-binding proteins involved in the formation of a tight junction, a necessary step in invasion. Here we present the crystal structure of EBA-175 region VI (rVI), a cysteine-rich domain that is highly conserved within the protein family and is essential for EBA-175 trafficking. The structure was solved by selenomethionine single-wavelength anomalous dispersion at 1.8 Å resolution. It reveals a homodimer, containing in each subunit a compact five-α-helix core that is stabilized by four conserved disulfide bridges. rVI adopts a novel fold that is likely conserved across the protein family, indicating a conserved function. It shows no similarity to the Duffy-binding-like domains of EBA-175 involved in erythrocyte binding, indicating a distinct role. Remarkably, rVI possesses structural features related to the KIX-binding domain of the coactivator CREB-binding protein, supporting the binding and trafficking roles that have been ascribed to it and providing a rational basis for further experimental investigation of its function.     

Effects of four Bacillus spp. of soil origin on the Colorado potato beetle Leptinotarsa decemlineata (Say)

Four species of Bacillus were isolated from soil in an effort to find safe, effective and alternative biological control agents against plant pests. These bacteria were identified as Bacillus pumilus, Bacillus sphaericus, Bacillus megaterium and Bacillus cereus on the basis of fatty acid methyl ester analysis and carbon utilization profiles by using Microbial Identification and Biolog Microplate Systems. Laboratory experiments carried out to determine the insecticidal activities of these isolates showed that B. pumilus caused 95.7 and 26.7% mortality and B. sphaericus caused 74.5 and 23.3% mortality of Leptinotarsa decemlineata larvae and adults, respectively. B. cereus and B. megaterium showed 51.1 and 29.7%, respectively, of L. decemlineata larvae. This study presents at least two Turkish isolates from the genus Bacillus showing high insecticidal activity against L. decemlineata.     

Malarial proteases and host cell egress: an 'emerging' cascade

Malaria is a scourge of large swathes of the globe, stressing the need for a continuing effort to better understand the biology of its aetiological agent. Like all pathogens of the phylum Apicomplexa, the malaria parasite spends part of its life inside a host cell or cyst. It eventually needs to escape (egress) from this protective environment to progress through its life cycle. Egress of Plasmodium blood-stage merozoites, liver-stage merozoites and mosquito midgut sporozoites relies on protease activity, so the enzymes involved have potential as antimalarial drug targets. This review examines the role of parasite proteases in egress, in the light of current knowledge of the mechanics of the process. Proteases implicated in egress include the cytoskeleton-degrading malarial proteases falcipain-2 and plasmepsin II, plus a family of putative papain-like proteases called SERA. Recent revelations have shown that activation of the SERA proteases may be triggered by regulated secretion of a subtilisin-like serine protease called SUB1. These findings are discussed in the context of the potential for development of new chemotherapeutics targeting this stage in the parasite's life cycle.     

Structural insights into microneme protein assembly reveal a new mode of EGF domain recognition

The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Adhesive complexes composed of microneme proteins (MICs) are secreted onto the parasite surface from intracellular stores and fulfil crucial roles in host-cell recognition, attachment and penetration. Here, we report the high-resolution solution structure of a complex between two crucial MICs, TgMIC6 and TgMIC1. Furthermore, we identify two analogous interaction sites within separate epidermal growth factor-like (EGF) domains of TgMIC6-EGF2 and EGF3-and confirm that both interactions are functional for the recognition of host cell receptor in the parasite, using immunofluorescence and invasion assays. The nature of this new mode of recognition of the EGF domain and its abundance in apicomplexan surface proteins suggest a more generalized means of constructing functional assemblies by using EGF domains with highly specific receptor-binding properties.     

Karyotype variation in the South American aphid genus Neuquenaphis (Hemiptera,Aphididae, Neuquenaphidinae)

The endemic South American aphid genus Neuquenaphis (Hemiptera, Aphididae, Neuquenaphidinae) forms an important component of the phytophagous insect fauna associated with southern beeches, Nothofagus (Nothofagaceae), but has not previously been studied cytologically. As part of ongoing studies of the taxonomy, evolution and host relationships of this genus, the karyotypes of 12 species are described and illustrated. Species are mostly distinguishable by differences in number and/or relative lengths of chromosomes, with 2n (female) numbers ranging from 6 to 16. The taxonomic and evolutionary significance of the karyotype variation in this group are discussed.     

Antibody-mediated control of persistent gamma-herpesvirus infection

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, establish life-long latency and can reactivate in immunocompromised individuals. T cells play an important role in controlling persistent EBV infection, whereas a role for humoral immunity is less clear. The murine gamma-herpesvirus-68 has biological and structural similarities to the human gamma-herpesviruses, and provides an important in vivo experimental model for dissecting mechanisms of immune control. In the current studies, CD28(-/-) mice were used to address the role of Abs in control of persistent murine gamma-herpesvirus-68 infection. Lytic infection was controlled in the lungs of CD28(-/-) mice, and latency was maintained in B cells at normal frequencies. Although class-switched virus-specific Abs were initially generated in the absence of germinal centers, titers and viral neutralizing activity rapidly waned. T cell depletion in CD28(-/-) mice with compromised Ab responses, but not in control mice with intact Ab responses, resulted in significant recrudescence from latency, both in the spleen and the lung. Recrudescence could be prevented by passive transfer of immune serum. These data directly demonstrate an important contribution of humoral immunity to control of gamma-herpesvirus latency, and have significant implications for clinical intervention.     

Bacterial superantigen exposure after resolution of influenza virus infection perturbs the virus-specific memory CD8(+)-T-cell repertoire

Heterologous viral infections have been shown to impact the preexisting memory CD8(+)-T-cell repertoire. Bacterial superantigens are products of common human pathogenic bacteria, including staphylococci and streptococci, that are potent T-cell-stimulatory molecules. In this report, we show that exposure to staphylococcal enterotoxin B, a bacterial superantigen, causes a selective functional deletion of cross-reactive influenza virus-specific CD8(+) memory T cells. This perturbation of the memory repertoire can have a significant impact on viral clearance after secondary challenge.     

A new in vitro model to evaluate differential responses of endothelial cells to simulated arterial shear stress waveforms

In the circulation, flow-responsive endothelial cells (ECs) lining the lumen of blood vessels are continuously exposed to complex hemodynamic forces. To increase our understanding of EC response to these dynamic shearing forces, a novel in vitro flow model was developed to simulate pulsatile shear stress waveforms encountered by the endothelium in the arterial circulation. A modified waveform modeled after flow patterns in the human abdominal aorta was used to evaluate the biological responsiveness of human umbilical vein ECs to this new type of stimulus. Arterial pulsatile flow for 24 hours was compared to an equivalent time-average steady laminar shear stress, using no flow (static) culture conditions as a baseline. While both flow stimuli induced comparable changes in cell shape and alignment, distinct patterns of responses were observed in the distribution of actin stress fibers and vinculin-associated adhesion complexes, intrinsic migratory characteristics, and the expression of eNOS mRNA and protein. These results thus reveal a unique responsiveness of ECs to an arterial waveform and begin to elucidate the complex sensing capabilities of the endothelium to the dynamic characteristics of flows throughout the human vascular tree.     

Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate

Activation of the proenzyme form of the malarial protease PfSUB-1 involves the autocatalytic cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this sequence but with the N- and C-terminal residues replaced by cysteines (Ac-CVSADNIDIC-OH) was labeled with 5- or 6-isomers of iodoacetamidotetramethylrhodamine (IATR). The doubly labeled peptides have low fluorescence because of ground-state, noncovalent dimerization of the rhodamines. Cleavage of either peptide by recombinant PfSUB-1 results in dissociation of the rhodamine dimers, which abolishes the self-quenching and consequently leads to an approximately 30-fold increase in the fluorescence. This spectroscopic signal provides a continuous assay of proteolysis, enabling quantitative kinetic measurements to be made, and has also enabled the development of a fluorescence-based assay suitable for use in high-throughput screens for inhibitors of PfSUB-1. The structure of the rhodamine dimer in the 6-IATR-labeled peptide was shown by NMR to be a face-to-face stacking of the xanthene rings. Time-resolved fluorescence measurements suggest that the doubly labeled peptides exist in an equilibrium consisting of rhodamines involved in dimers (closed forms) and rhodamines not involved in dimers (open forms). These data also indicate that the rhodamine dimers fluoresce and that the associated lifetimes are subnanosecond.