Pathogenesis and Cell Biology
A malaria parasite formin regulates actin polymerisation and localises to the parasite-erythrocyte moving junction during invasion
J Baum, CJ Tonkin M Rug, D Richard, AF Cowman in collaboration with BJ Smith (Bioinformatics), SB Gould (School of Botany, University of Melbourne),
TD Pollard, A Paul (Yale University, New Haven, CT USA) Pub ref: 7
Malaria parasites are obligate intracellular parasites, requiring development within the human erythrocyte to complete their lifecycle. Invasion of the erythrocyte is driven by an actomyosin motor that requires turnover of short, dynamic actin filaments (F-actin); however, the cell factors (or nucleators) that regulate where and when F-actin forms are unknown. Previously, we showed through bioinformatic investigation that the malaria parasites and other parasites in phylum Apicomplexa lack Arp2/3 complex, a key actin nucleator found across most eukaryotes. In its absence, the most likely nucleator would be a formin, a recently described family of proteins that catalyse the formation of linear actin filaments. All Apicomplexa have at least two formins, prompting us to investigate their role in invasion by the most virulent human malaria Plasmodium falciparum. By immunofluorescence microscopy PfFormin1 localizes at the apical pole of invading parasites and follows the moving junction complex that forms between host and parasite cell during invasion. This distinct localisation is predicted to be the precise site of the activated molecular motor responsible for driving invasion, which directly implicates formins in actin regulation of malaria parasite motility. Furthermore, in vitro, the actin-binding domain of PfFormin1 behaves like a true nucleator. It is a potent catalyst initiating growth of new actin filaments. PfFormin1 is the first demonstrated regulator of actin polymerisation described for malaria parasites. Our findings highlight its potential as a key controller of cell invasion and, as such, an important determinant of malaria parasite pathogenicity, opening the way to investigating actin regulation as a target for new anti-malarial drugs.
(A) On the left, phase contrast micrograph of free blood stage malaria parasites (merozoites, ~1uM in length) awaiting entry into a human red blood cell. On the right, indirect immunofluorescent microscopy of the same merozoites labelled with antibodies raised against Formin1 (red) localising it to the extreme apical pole of the parasite. Merozoites are co-stained with a marker of the parasite pellicle (green) and the nucleus (blue). (B) A TIRF microscopy image of fluorescently labelled actin filaments polymerizing in vitro in the absence (left) or presence (right) of the, recombinantly expressed, Formin1 actin binding domain, demonstrating Formin1’s potency as an actin filament nucleator.
Plasmodium falciparum erythrocyte membrane protein-1 specifically suppresses early production of host interferon-γ
MC D’Ombrain, TS Voss, AG Maier, JA Pearce, DS Hansen, AF Cowman, L Schofield. Pub ref: 30
Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a variable antigen expressed by P. falciparum, the malarial parasite. PfEMP-1, present on the surface of infected host erythrocytes, mediates erythrocyte binding to vascular endothelium, enabling the parasite to avoid splenic clearance. In addition, PfEMP-1 is proposed to regulate host immune responses via interactions with the CD36 receptor on antigen presenting cells. We investigated the immunoregulatory function of PfEMP-1 by comparing host cell responses to erythrocytes infected with either wild-type parasites or transgenic parasites lacking PfEMP-1, on two distinct parasite genetic backgrounds. We showed that PfEMP-1 suppresses the production of the cytokine interferon-γ (IFNγ )by human peripheral blood mononuclear cells (PBMCs) early after exposure to P. falciparum. Suppression of this rapid pro-inflammatory response was CD36-independent and specific to IFNγ production by γδ-T, NK and αβ-T cells. No evidence for PfEMP-1-mediated polyclonal activation of these cell populations was found. The data suggest the parasite may be interacting directly with these effector cells, rather than indirectly through antigen-presenting cells. PfEMP-1 had no effect on early TNF-α, IL-6, IL-10, IL-2 or IL-4 responses of PBMCs to P. falciparum. Furthermore, we found no evidence for a role for PfEMP-1 as target of cytotoxicity by NK cells or cytotoxic T cell populations. These data demonstrate a parasite strategy for specifically downregulating the pro-inflammatory IFNγ response and further establish transgenic parasites lacking PfEMP-1 as powerful tools for elucidating the functional and immunological roles of this important virulence protein in human malaria.
Vaccination with live non-persistent phosphomannomutase-deficient Leishmania major parasites protects via early IL-10 suppression and increased magnitude of T cell responses
L Kedzierski, JM Curtis, E Handman in collaboration with PC Doherty, K Kedzierska (Department of Microbiology and Immunology, University of Melbourne)
Since the importance of persistent infection for maintaining an effective long-lasting protection is controversial, we asked whether immunisation with non-persistent parasites leads to protection against Leishmania and recruitment of T cells of a specific phenotype. We have demonstrated that vaccination of susceptible BALB/c mice with non-persistent ∆PMM parasites induces protection against virulent challenge and increases precursor frequency of memory T cells, which are rapidly recruited to draining lymph nodes. Vaccination with ∆PMM parasites also enables mice to suppress early IL-10 production following infection, which results in high IFN-γ/IL-10 ratio, thus affecting the outcome of the disease in favour of the host.
The role of osmiophilic bodies and Pfg377 expression in female gametocyte emergence and mosquito infectivity in the human malaria parasite Plasmodium falciparum
TF de Koning-Ward, J Healer, BS Crabb in collaboration with T Papenfuss (Bioinformatics), A Olivieri, L Bertuccini, F Silvestrini, G Camardia, L Baldassarri, P Alano (Instituto Superiore di Sanita, Rome, Italy), A Hood, K Charvalias, PB Diaz, LC Ranford-Cartwright (University of Glasgow, UK), TF McElwain (Washington State University, Pullman, WA USA) Pub ref: 37
Osmiophilic bodies are membrane-bound vesicles found predominantly in Plasmodium females and have been postulated to aid in the escape of gametocytes from the erythrocyte after ingestion by the mosquito. By targeted gene disruption, we show that Pfg377, which is currently the only protein known to be associated with osmiophilic bodies in Plasmodium falciparum, plays a fundamental role in the formation of these organelles. Furthermore, female gametocytes lacking the full complement of osmiophilic bodies are significantly less efficient both in vitro and in vivo in their emergence from the erythrocytes upon induction of gametogenesis. This explains the significant defect in oocyst formation in mosquitoes fed blood meals containing Pfg377- negative gametocytes, resulting in an almost complete blockade of infection.