David J. P. Ferguson

Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells

The malaria parasite Plasmodium falciparum is one of the most successful human pathogens. Specific virulence factors remain poorly defined, although the adhesion of infected erythrocytes tothe venular endothelium has been associated with some of thesyndromes of severe disease. Immune responses cannot prevent the development of symptomatic infections throughout life, and clinical immunity to the disease develops only slowly during childhood. An understanding of the obstacles to the development of protective immunity is crucial for developing rational approaches to prevent the disease. Here we show that intact malaria-infected erythrocytes adhere to dendritic cells, inhibit the maturation of dendritic cells and subsequently reduce their capacity to stimulate T cells. These data demonstrate both a novel mechanism by which malaria parasites induce immune dysregulation and a functional role beyond endothelial adhesion for the adhesive phenotypes expressed at the surface of infected erythrocytes.

Neurological and behavioral abnormalities, ventricular dilatation, altered cellular functions, inflammation, and neuronal injury in brains of mice due to common, persistent, parasitic infection

BackgroundWorldwide, approximately two billion people are chronically infected with Toxoplasma gondii with largely unknown consequences.MethodsTo better understand long-term effects and pathogenesis of this common, persistent brain infection, mice were infected at a time in human years equivalent to early to mid adulthood and studied 5–12 months later. Appearance, behavior, neurologic function and brain MRIs were studied. Additional analyses of pathogenesis included: correlation of brain weight and neurologic findings; histopathology focusing on brain regions; full genome microarrays; immunohistochemistry characterizing inflammatory cells; determination of presence of tachyzoites and bradyzoites; electron microscopy; and study of markers of inflammation in serum. Histopathology in genetically resistant mice and cytokine and NRAMP knockout mice, effects of inoculation of isolated parasites, and treatment with sulfadiazine or αPD1 ligand were studied.ResultsTwelve months after infection, a time equivalent to middle to early elderly ages, mice had behavioral and neurological deficits, and brain MRIs showed mild to moderate ventricular dilatation. Lower brain weight correlated with greater magnitude of neurologic abnormalities and inflammation. Full genome microarrays of brains reflected inflammation causing neuronal damage (Gfap), effects on host cell protein processing (ubiquitin ligase), synapse remodeling (Complement 1q), and also increased expression of PD-1L (a ligand that allows persistent LCMV brain infection) and CD 36 (a fatty acid translocase and oxidized LDL receptor that mediates innate immune response to beta amyloid which is associated with pro-inflammation in Alzheimers disease). Immunostaining detected no inflammation around intra-neuronal cysts, practically no free tachyzoites, and only rare bradyzoites. Nonetheless, there were perivascular, leptomeningeal inflammatory cells, particularly contiguous to the aqueduct of Sylvius and hippocampus, CD4+ and CD8+ T cells, and activated microglia in perivascular areas and brain parenchyma. Genetically resistant, chronically infected mice had substantially less inflammation.ConclusionIn outbred mice, chronic, adult acquired T. gondii infection causes neurologic and behavioral abnormalities secondary to inflammation and loss of brain parenchyma. Perivascular inflammation is prominent particularly contiguous to the aqueduct of Sylvius and hippocampus. Even resistant mice have perivascular inflammation. This mouse model of chronic T. gondii infection raises questions of whether persistence of this parasite in brain can cause inflammation or neurodegeneration in genetically susceptible hosts.

Novel Type IV Secretion System Involved in Propagation of Genomic Islands

Type IV secretion systems (T4SSs) mediate horizontal gene transfer, thus contributing to genome plasticity, evolution of infectious pathogens, and dissemination of antibiotic resistance and other virulence traits. A gene cluster of the Haemophilus influenzae genomic island ICEHin1056 has been identified as a T4SS involved in the propagation of genomic islands. This T4SS is novel and evolutionarily distant from the previously described systems. Mutation analysis showed that inactivation of key genes of this system resulted in a loss of phenotypic traits provided by a T4SS. Seven of 10 mutants with a mutation in this T4SS did not express the type IV secretion pilus. Correspondingly, disruption of the genes resulted in up to 100,000-fold reductions in conjugation frequencies compared to those of the parent strain. Moreover, the expression of this T4SS was found to be positively regulated by one of its components, the tfc24 gene. We concluded that this gene cluster represents a novel family of T4SSs involved in propagation of genomic islands.

Virulent Toxoplasma gondii Evade Immunity-Related GTPase-Mediated Parasite Vacuole Disruption within Primed Macrophages

Cytokine-activated macrophages restrain the replication of intracellular parasites and disrupt the integrity of vacuolar pathogens. In this study, we show that inducible nitric oxide synthase and the immunity-related GTPase (IRG) family member Irgm3, respectively, are required for the ability of in vivo primed macrophages to restrain the growth of Toxoplasma gondii and to destroy the parasite’s intracellular niche. Remarkably, virulent Type I strains of T. gondii evade IRG-dependent vacuolar disruption, while remaining susceptible to iNOS-dependent restriction. The ability of virulent T. gondii to escape killing by macrophages is controlled at the level of the individual vacuole and is associated with differential permissiveness for association of the IRG proteins Irga6 (IIGP1) and Irgb6 (TGTP) to the vacuolar membrane. Surprisingly, expression of the Type I ROP-18 virulence determinant in an avirulent strain did not confer the evasive phenotype. These results pinpoint evasion of vacuolar disruption by IRG proteins as a new determinant of pathogen virulence.

Fatal Plasmodium falciparum Malaria Causes Specific Patterns of Splenic Architectural Disorganization

ABSTRACT The spleen is critical for host defense against pathogens, including Plasmodium falciparum. It has a dual role, not only removing aged or antigenically altered erythrocytes from the blood but also as the major lymphoid organ for blood-borne or systemic infections. The human malaria parasite P. falciparum replicates within erythrocytes during asexual blood stages and causes repeated infections that can be associated with severe disease. In spite of the crucial role of the spleen in the innate and acquired immune response to malaria, there is little information on the pathology of the spleen in human malaria. We performed a histological and quantitative immunohistochemical study of spleen sections from Vietnamese adults dying from severe falciparum malaria and compared the findings with the findings for spleen sections from control patients and patients dying from systemic bacterial sepsis. Here we report that the white pulp in the spleens of patients dying from malaria showed a marked architectural disorganization. We observed a marked dissolution of the marginal zones with relative loss of B cells. Furthermore, we found strong HLA-DR expression on sinusoidal lining cells but downregulation on cordal macrophages. P. falciparum infection results in alterations in splenic leukocytes, many of which are not seen in sepsis.

Developmental Expression of a Tandemly Repeated, Glycine- and Serine-Rich Spore Wall Protein in the Microsporidian Pathogen Encephalitozoon cuniculi

ABSTRACT Microsporidia are intracellular organisms of increasing importance as opportunistic pathogens in immunocompromised patients. Host cells are infected by the extrusion and injection of polar tubes located within spores. The spore is surrounded by a rigid spore wall which, in addition to providing mechanical resistance, might be involved in host cell recognition and initiation of the infection process. A 51-kDa outer spore wall protein was identified in Encephalitozoon cuniculi with the aid of a monoclonal antibody, and the corresponding gene, SWP1, was cloned by immunoscreening of a cDNA expression library. The cDNA encodes a protein of 450 amino acids which displays no significant similarities to known proteins in databases. The carboxy-terminal region consists of five tandemly arranged glycine- and serine-rich repetitive elements. SWP1is a single-copy gene that is also present in the genomes ofEncephalitozoon intestinalis and Encephalitozoon hellem as demonstrated by Southern analysis. Indirect immunofluorescence and immunoelectron microscopy revealed that SWP1 is differentially expressed during the infection cycle. The protein is absent in replicative meronts until 24 h postinfection, and its expression is first induced in early sporonts at a time when organisms translocate from the periphery to the center of the parasitophorous vacuole. Expression of SWP1 appears to be regulated at the mRNA level, as was shown by reverse transcriptase PCR analysis. Further identification and characterization of stage-specific genes might help to unravel the complex intracellular differentiation process of microsporidia.

Mammalian Rif1 contributes to replication stress survival and homology-directed repair

Multifunctional protein Rif1 accumulates at stalled replication forks to facilitate DNA repair during S phase.

Melting during late-stage rifting in Afar is hot and deep

Investigations of a variety of continental rifts and margins worldwide have revealed that a considerable volume of melt can intrude into the crust during continental breakup, modifying its composition and thermal structure. However, it is unclear whether the cause of voluminous melt production at volcanic rifts is primarily increased mantle temperature or plate thinning. Also disputed is the extent to which plate stretching or thinning is uniform or varies with depth with the entire continental lithospheric mantle potentially being removed before plate rupture. Here we show that the extensive magmatism during rifting along the southern Red Sea rift in Afar, a unique region of sub-aerial transition from continental to oceanic rifting, is driven by deep melting of hotter-than-normal asthenosphere. Petrogenetic modelling shows that melts are predominantly generated at depths greater than 80 kilometres, implying the existence of a thick upper thermo-mechanical boundary layer in a rift system approaching the point of plate rupture. Numerical modelling of rift development shows that when breakup occurs at the slow extension rates observed in Afar, the survival of a thick plate is an inevitable consequence of conductive cooling of the lithosphere, even when the underlying asthenosphere is hot. Sustained magmatic activity during rifting in Afar thus requires persistently high mantle temperatures, which would allow melting at high pressure beneath the thick plate. If extensive plate thinning does occur during breakup it must do so abruptly at a late stage, immediately before the formation of the new ocean basin.

PfEMP1 expression is reduced on the surface of knobless Plasmodium falciparum infected erythrocytes

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a key virulence factor for this species of human malarial parasite. PfEMP1 is expressed on the surface of infected erythrocytes (IEs) and directly mediates adhesion to a variety of host cells. A number of other parasite-encoded proteins are similarly exported to the IE plasma membrane and play an indirect role in this adhesion process through the modification of the erythrocyte cytoskeleton and the formation of electron dense knobs into which PfEMP1 is anchored. Analysis of the specific contribution of knob-associated proteins to adhesion is difficult due to rapid PfEMP1 switching during in vitro culture. Furthermore, these studies typically assume that the level and distribution of PfEMP1 exposed in knobby (K+) and knobless (K–) IEs is unaltered, an assumption not yet supported with data. We describe here the preparation and characterisation of a panel of isogenic K+ and K– parasite clones that express one of two defined PfEMP1 variants. Analysis of the cytoadhesive properties of these clones shows that both static and flow adhesion is reduced in all the K– clones and, further, that this correlates with an approximately 50% reduction in PfEMP1 displayed on the IE surface. However, despite this reduction, the gross distribution of PfEMP1 in K– IEs appears unaltered. These data impact on our current interpretation of the role of knobs in adhesion and the mechanism of trafficking PfEMP1 to the IE surface.

The Toxoplasma Acto-MyoA Motor Complex Is Important but Not Essential for Gliding Motility and Host Cell Invasion

Apicomplexan parasites are thought to actively invade the host cell by gliding motility. This movement is powered by the parasites own actomyosin system, and depends on the regulated polymerisation and depolymerisation of actin to generate the force for gliding and host cell penetration. Recent studies demonstrated that Toxoplasma gondii can invade the host cell in the absence of several core components of the invasion machinery, such as the motor protein myosin A (MyoA), the microneme proteins MIC2 and AMA1 and actin, indicating the presence of alternative invasion mechanisms. Here the roles of MyoA, MLC1, GAP45 and Act1, core components of the gliding machinery, are re-dissected in detail. Although important roles of these components for gliding motility and host cell invasion are verified, mutant parasites remain invasive and do not show a block of gliding motility, suggesting that other mechanisms must be in place to enable the parasite to move and invade the host cell. A novel, hypothetical model for parasite gliding motility and invasion is presented based on osmotic forces generated in the cytosol of the parasite that are converted into motility.