RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum

Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of Plasmodium falciparum. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in P.falciparum blood-stage parasites to disrupt the RAP1 gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.

Host cell remodelling and protein trafficking

Inside their respective vertebrate hosts, Plasmodium spp spend most of their life residing within hepatocytes and erythrocytes, with large-scale infection of the latter responsible for the clinical symptoms associated with malaria. These parasites extensively remodel these host cells for a variety of purposes relating to both pathogenesis and maintaining growth. Remodelling of the erythrocytic stage has been most intensively studied in P. falciparum and is the subject of this chapter. To help remodel their hosts these parasites export hundreds of proteins into the erythrocytic compartment. This principally alters the architecture of the erythrocyte, rendering the host membrane more permeable to solutes and nutrients, and also increasing the rigidity and adhesiveness of the infected erythrocyte. Moreover, because erythrocytes lack a secretory apparatus, the parasite must also export many additional proteins to help traffic other proteins to their correct destination within the host cell. The functions of some of these exported proteins will be discussed as will recent progress that has been made in unravelling how exported proteins gain access to the host compartment.

Helicobacter pylori-associated hypochlorhydria in children, and development of iron deficiency

Aims Acute Helicobacter pylori infection is associated with transient hypochlorhydria. In H pylori-associated atrophy, hypochlorhydria has a role in iron deficiency (ID) through changes in the physiology of iron-complex absorption. The aims were to evaluate the association between H pylori-associated hypochlorhydria and ID in children. Methods Symptomatic children (n=123) were prospectively enrolled. Blood, gastric juice and gastric biopsies were taken, respectively, for haematological analyses, pH assessment and H pylori determination, and duodenal biopsies for exclusion of coeliac disease. Stool samples were collected for parasitology/microbiology. Thirteen children were excluded following parasitology and duodenal histopathology, and five due to impaired blood analysis. Results Ten children were hypochlorhydric (pH>4) and 33 were H pylori positive. In H pylori-positive children with pH>4 (n=6) serum iron and transferrin saturation levels % were significantly lower (p<0.01) than H pylori-positive children with pH≤4. No differences in ferritin, or total iron binding capacity, were observed. In H pylori-negative children with pH>4, iron and transferrin saturation were not significantly different from children with pH≤4. Conclusions Low serum iron and transferrin in childhood H pylori infection is associated with hypochlorhydria. In uninfected children, hypochlorhydria was not associated with altered serum iron parameters, indicating a combination of H pylori infection and/or inflammation, and hypochlorhydria has a role in the aetiology of ID. Although H pylori-associated hypochlorhydria is transient during acute gastritis, this alters iron homeostasis with clinical impact in developing countries with a high H pylori prevalence.

CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission

To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/- 0.0154 and 0.0171 +/- 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/- 0.0079 virions/image) than glans tissue (0.0167 +/- 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/- 0.0188 vs. 0.0151 +/- 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/- 3.908 vs. 12.466 +/- 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV transmission in uncircumcised men.