Gabriele Margos

Apical Membrane Antigen 1, a Major Malaria Vaccine Candidate, Mediates the Close Attachment of Invasive Merozoites to Host Red Blood Cells

ABSTRACT Apical membrane antigen 1 (AMA-1) of Plasmodium merozoites is established as a candidate molecule for inclusion in a human malaria vaccine and is strongly conserved in the genus. We have investigated its function in merozoite invasion by incubating Plasmodium knowlesi merozoites with red cells in the presence of a previously described rat monoclonal antibody (MAb R31C2) raised against an invasion-inhibitory epitope of P. knowlesi AMA-1 and then fixing the material for ultrastructural analysis. We have found that the random, initial, long-range (12 nm) contact between merozoites and red cells occurs normally in the presence of the antibody, showing that AMA-1 plays no part in this stage of attachment. Instead, inhibited merozoites fail to reorientate, so they do not bring their apices to bear on the red cell surface and do not make close junctional apical contact. We conclude that AMA-1 may be directly responsible for reorientation or that the molecule may initiate the junctional contact, which is then presumably dependent on Duffy binding proteins for its completion.

A new Borrelia species defined by multilocus sequence analysis of housekeeping genes.

ABSTRACT Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.

Plasmodium falciparum apical membrane antigen 1 (PfAMA-1) is translocated within micronemes along subpellicular microtubules during merozoite development

During the assembly of Plasmodium falciparum merozoites within the schizont stage, the parasite synthesizes and positions three sets of secretory vesicles (rhoptries, micronemes and dense granules) that are active during red cell invasion. There are up to 40 micronemes per merozoite, shaped like long-necked bottles, about 160 nm long and 65 nm at their widest diameter. On their external surfaces, they bear bristle-like filaments, each 3-4 nm thick and 25 nm long. Micronemes are translocated from a single Golgi-like cisterna near the nucleus along a band of two or three subpellicular microtubules to the merozoite apex, where they dock with the rhoptry tips. Dense granules are also formed around the periphery of the Golgi cisternae but their distribution is unrelated to microtubules. Three polyclonal antibodies raised against the recombinant PfAMA-1 ectodomain sequence recognizing both the 83 kDa and processed 66 kDa molecules label the peripheries of translocating and mature micronemes but do not label rhoptries significantly at any stage of merozoite development within schizonts. This result confirms that PfAMA-1 is a micronemal protein, and indicates that within the microneme it is located near or inserted into this organelles boundary membrane.

Active and Passive Surveillance and Phylogenetic Analysis of Borrelia burgdorferi Elucidate the Process of Lyme Disease Risk Emergence in Canada

Background Northward expansion of the tick Ixodes scapularis is driving Lyme disease (LD) emergence in Canada. Information on mechanisms involved is needed to enhance surveillance and identify where LD risk is emerging. Objectives We used passive and active surveillance and phylogeographic analysis of Borrelia burgdorferi to investigate LD risk emergence in Quebec. Methods In active surveillance, we collected ticks from the environment and from captured rodents. B. burgdorferi transmission was detected by serological analysis of rodents and by polymerase chain reaction assays of ticks. Spatiotemporal trends in passive surveillance data assisted interpretation of active surveillance. Multilocus sequence typing (MLST) of B. burgdorferi in ticks identified likely source locations of B. burgdorferi. Results In active surveillance, we found I. scapularis at 55% of sites, and we were more likely to find them at sites with a warmer climate. B. burgdorferi was identified at 13 I. scapularis–positive sites, but infection prevalence in ticks and animal hosts was low. Low infection prevalence in ticks submitted in passive surveillance after 2004—from the tick-positive regions identified in active surveillance—coincided with an exponential increase in tick submissions during this time. MLST analysis suggested recent introduction of B. burgdorferi from the northeastern United States. Conclusions These data are consistent with I. scapularis ticks dispersed from the United States by migratory birds, founding populations where the climate is warmest, and then establishment of B. burgdorferi from the United States several years after I. scapularis have established. These observations provide vital information for public health to minimize the impact of LD in Canada.

Formation of the Food Vacuole in Plasmodium falciparum: A Potential Role for the 19 kDa Fragment of Merozoite Surface Protein 1 (MSP119)

Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP119), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP119 during the parasites subsequent intracellular development using immunochemical analysis of metabolically labeled MSP119, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP119 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP119 and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP119 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.

Three-dimensional ultrastructure of the ring stage of Plasmodium falciparum: Evidence for export pathways

The three-dimensional structure of the Plasmodium falciparum ring stage has been explored by reconstruction from serial sections and stereoscopic examination of tilted sections. The ring-like light microscopic appearance is related to the shape and contents of the biconcave discoidal parasite at this stage, its thick perimeter containing most of the ribosomes and its thin center containing smooth membrane organelles. The shapes of rings vary between flat and curved cuplike forms. The rough endoplasmic reticulum is a branched network continuous with the nuclear envelope. Evidence for a simple Golgi complex is seen in the presence on the outer nuclear envelope of a locus of coated vesicle budding associated with a single membranous cisterna or cluster of smooth vesicles. In middle and late stage rings this complex migrates along an extension of the nuclear envelope continuous with the rough endoplasmic reticulum. Evidence is also presented for a mechanism of exporting membrane from the parasite into the parasitophorous vacuole membrane and beyond into the red blood cell, by means of double-membraned vesicle-based exocytosis.

Interaction between Host Complement and Mosquito-Midgut-Stage Plasmodium berghei

ABSTRACT After ingestion by mosquitoes, gametocytes of malaria parasites become activated and form extracellular gametes that are no longer protected by the red blood cell membrane against immune effectors of host blood. We have studied the action of complement onPlasmodium developmental stages in the mosquito blood meal using the rodent malaria parasite Plasmodium berghei and rat complement as a model. We have shown that in the mosquito midgut, rat complement components necessary to initiate the alternative pathway (factor B, factor D, and C3) as well as C5 are present for several hours following ingestion of P. berghei-infected rat blood. In culture, 30 to 50% of mosquito midgut stages of P. berghei survived complement exposure during the first 3 h of development. Subsequently, parasites became increasingly sensitive to complement lysis. To investigate the mechanisms involved in their protection, we tested for C3 deposition on parasite surfaces and whether host CD59 (a potent inhibitor of the complement membrane attack complex present on red blood cells) was taken up by gametes while emerging from the host cell. Between 0.5 and 22 h, 90% of Pbs21-positive parasites were positive for C3. While rat red and white blood cells stained positive for CD59, Pbs21-positive parasites were negative for CD59. In addition, exposure of parasites to rat complement in the presence of anti-rat CD59 antibodies did not increase lysis. These data suggest that parasite or host molecules other than CD59 are responsible for the protection of malaria parasites against complement-mediated lysis. Ongoing research aims to identify these molecules.

Louse-borne relapsing fever (Borrelia recurrentis) diagnosed in 15 refugees from northeast Africa: epidemiology and preventive control measures, Bavaria, Germany, July to October 2015

We report 15 imported louse-borne relapsing fever (LBRF) cases in refugees in Bavaria, Germany. One patient died. Epidemiological findings confirmed that all were young males from the Horn of Africa (12 from Somalia), who had similar migration routes converging in Sudan continuing through Libya and Italy. The majority likely acquired their infection during migration. Healthcare workers should be aware of LBRF in refugees passing through north Africa to ensure correct treatment and preventive measures.

Correlation of structural development and differential expression of invasion-related molecules in schizonts of Plasmodium falciparum

During asexual development Plasmodium schizonts undergo a series of complex biochemical and structural changes. Using tightly synchronized cultures of 2 P. falciparum lines (clone C10 and strain ITO4) for light microscopy and fluorescence imaging we monitored the timing and sequence of expression of proteins associated with invasion-related organelles. Antibodies to rhoptry, micronemal and dense granule proteins (Rhoptry Associated Protein 1, Apical Membrane Antigen 1, Erythrocyte Binding Antigen 175, Ring-infected Erythrocyte Surface Antigen) and to pellicle-associated proteins (Merozoite Surface Protein 1, PfMyosin-A) were used. Clone C10 developed faster than ITO4; this difference was also found in the timing of protein expression seen by immunofluorescence. Light microscopic data were combined with transmission electron microscopic analysis using serial sectioning of ITO4 schizonts to determine nuclear number and organellar development. Thus a timetable of schizont structural maturation was established. Generally, the timing of organelle-specific antigen expression correlates well with the ultrastructural data. Rhoptries are formed mainly between second and fourth nuclear divisions, micronemes between the end of the fourth nuclear division and merozoite separation from the residual body, while dense granules are generated mainly after the micronemes. PfAMA-1 appears in micronemes before EBA-175, suggesting micronemal heterogeneity.

Borrelia miyamotoi-Associated Neuroborreliosis in Immunocompromised Person.

Borrelia miyamotoi is a newly recognized human pathogen in the relapsing fever group of spirochetes. We investigated a case of B. miyamotoi infection of the central nervous system resembling B. burgdorferi–induced Lyme neuroborreliosis and determined that this emergent agent of central nervous system infection can be diagnosed with existing methods.