Plasmodium falciparum gametocyte development 1 (Pfgdv1) and gametocytogenesis early gene identification and commitment to sexual development.

Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut. Although essential for the spread of malaria through the population, little is known about the initiation of gametocytogenesis in vitro or in vivo. Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparumgametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation. Transcriptional analysis of Pfgdv1 negative and positive parasite lines identified a set of gametocytogenesis early genes (Pfge) that were significantly down-regulated (>10 fold) in the absence of Pfgdv1 and expression was restored after Pfgdv1 complementation. Progressive accumulation of Pfge transcripts during successive rounds of asexual replication in synchronized cultures suggests that gametocytes are induced continuously during asexual growth. Comparison of Pfge gene transcriptional profiles in patient samples divided the genes into two groups differing in their expression in mature circulating gametocytes and providing candidates to evaluate gametocyte induction and maturation separately in vivo. The expression profile of one of the early gametocyte specific genes, Pfge1, correlated significantly with asexual parasitemia, which is consistent with the ongoing induction of gametocytogenesis during asexual growth observed in vitro and reinforces the need for sustained transmission-blocking strategies to eliminate malaria.

Taenia infestation in the appendix: a case report.

Taenia saginata is a zoonotic cestode causing taeniasis. Taeniasis refers to the intestinal infection with the adult stage of this tapeworm. An association between teaniasis and acute appendicitis is uncommon. We present the case of a 37 year old male who presented with abdominal pain for one day. He was diagnosed with having appendicitis and an appendectomy was performed. Pathology of the appendix showed Taenia saginata with eggs in the lumen. Histological analysis showed acute inflammation consistent with acute appendicitis caused by T. saginata.

Protein targeting to the parasitophorous vacuole membrane of Plasmodium falciparum.

Red blood cell (RBC) invasion and parasitophorous vacuole (PV) formation by Plasmodium falciparum are critical for the development and pathogenesis of malaria, a continuing global health problem. Expansion of the PV membrane (PVM) during growth is orchestrated by the parasite. This is particularly important in mature RBCs, which lack internal organelles and no longer actively synthesize membranes. Pfs16, a 16-kDa integral PVM protein expressed by gametocytes, was chosen as a model for studying the trafficking of material from the parasite across the PV space to the PVM. The locations of Pfs16-green fluorescent protein (GFP) reporter proteins containing distinct regions of Pfs16 were tracked from RBC invasion to emergence. Inclusion of the 53 C-terminal amino acids (aa) of Pfs16 to a GFP reporter construct already containing the N-terminal secretory signal sequence was sufficient for targeting to and retention on the PVM. An amino acid motif identified in this region was also found in seven other known PVM proteins. Removal of the 11 C-terminal aa did not affect PVM targeting, but membrane retention was decreased. Additionally, during emergence from the PVM and RBC, native Pfs16 and the full-length Pfs16-GFP reporter protein were found to concentrate on the ends of the gametocyte. Capping was not observed in constructs lacking the amino acids between the N-terminal secretory signal sequence and the transmembrane domain, suggesting that this region, which is not required for PVM targeting, is involved in capping. This is the first report to define the amino acid domains required for targeting to the P. falciparum PVM.

Functional analysis of the exported type IV HSP40 protein PfGECO in Plasmodium falciparum gametocytes.

During Plasmodium falciparum infection, host red blood cell (RBC) remodeling is required for the parasite's survival. Such modifications are mediated by the export of parasite proteins into the RBC that alter the architecture of the RBC membrane and enable cytoadherence. It is probable that some exported proteins also play a protective role against the host defense response. This may be of particular importance for the gametocyte stage of the life cycle that is responsible for malaria transmission, since the gametocyte remains in contact with blood as it proceeds through five morphological stages (I to V) during its 12-day maturation. Using microarray analysis, we identified several genes with encoded secretory or export sequences that were differentially expressed during early gametocytogenesis. One of these, PfGECO, encodes a predicted type IV heat shock protein 40 (HSP40) that we show is expressed in gametocyte stages I to IV and is exported to the RBC cytoplasm. HSPs are traditionally induced under stressful conditions to maintain homeostasis, but PfGECO expression was not increased upon heat shock, suggesting an alternate function. Targeted disruption of PfGECO indicated that the gene is not essential for gametocytogenesis in vitro, and quantitative reverse transcriptase PCR (RT-PCR) showed that there was no compensatory expression of the other type IV HSP40 genes. Although P. falciparum HSP40 members are implicated in the trafficking of proteins to the RBC surface, removal of PfGECO did not affect the targeting of other exported gametocyte proteins. This work has expanded the repertoire of known gametocyte-exported proteins to include a type IV HSP40, PfGECO.

The DEAD-box RNA helicase Vad1 regulates multiple virulence-associated genes in Cryptococcus neoformans.

The study of fungal regulatory networks is essential to the understanding of how these pathogens respond to host environmental signals with effective virulence-associated traits. In this study, a virulence-associated DEAD-box RNA helicase-encoding gene (VAD1) was isolated from a mutant defective in the virulence factor laccase. A Deltavad1 mutant exhibited a profound reduction in virulence in a mouse model that was restored after reconstitution with WT VAD1. Loss of VAD1 resulted in upregulation of NOT1, a gene encoding a global repressor of transcription. NOT1 was found to act as an intermediary transcriptional repressor of laccase. Vad1 was located within macromolecular complexes that formed cytoplasmic granular bodies in mature cells and during infection of mouse brain. In addition, VAD1 was shown by in situ hybridization to be expressed in the brain of an AIDS patient coinfected with C. neoformans. To understand the role of VAD1 in virulence, a functional genomics approach was used to identify 3 additional virulence determinants dependent on VAD1: PCK1, TUF1, and MPF3, involved in gluconeogenesis, mitochondrial protein synthesis, and cell wall integrity, respectively. These data show that fungal virulence-associated genes are coordinately regulated and that an analysis of such transcriptomes allows for the identification of important new genes involved in the normal growth and virulence of fungal pathogens.

Sex- and stage-specific reporter gene expression in Plasmodium falciparum.

For malaria transmission, Plasmodium parasites must successfully complete gametocytogenesis in the vertebrate host. Differentiation into mature male or female Plasmodium falciparum gametocytes takes 9-12 days as the parasites pass through five distinct morphologic stages (I-V). To evaluate the signals controlling the initiation of stage- and/or sex-specific expression, reporter constructs containing the 5'-flanking regions (FR) of seven genes with distinct expression patterns through gametogenesis were developed. The regulatory information present in the 5'-FR of each selected gene was found to be sufficient to drive appropriate sex- and stage-specific reporter gene expression. The transformed parasite lines also provide in vivo markers to identify gametocytes at specific stages, including a subpopulation of schizonts that express early gametocyte markers.

Pfs47, paralog of the male fertility factor Pfs48/45, is a female specific surface protein in Plasmodium falciparum.

The genome of Plasmodium falciparum contains a small gene family that expresses proteins characterized by the presence of 6-cysteine domains. Most of these proteins are expressed on the surface of the parasite and some are known to play a role in cell-cell interactions. Two members of this family, Pfs48/45 and Pfs230, form a complex localized on the surface of gametes and are recognized as important targets for transmission-blocking vaccines. In this study we report the analysis of an additional member of this family, Pfs47 the closest paralog of Pfs48/45. We demonstrate that Pfs47 is expressed only in female gametocytes and is located on the surface of female gametes following emergence from red blood cells. In contrast to the critical function of P48/45 for male fertility, Pfs47 does not appear crucial for female fertility. Parasites lacking Pfs47 through targeted gene disruption, produce normal numbers of oocysts when included in the blood meal of the mosquito vector. In addition, three monoclonal antibodies against Pfs47 were unable to inhibit oocyst development when present in a blood meal containing wild type parasites. These results show redundancy in protein function for Pfs47 and reduce the support for candidacy of Pfs47 as a transmission-blocking vaccine target.

PaCaHa inhibits proliferation of human cancer cells in vitro.

BACKGROUND/AIM: The aim of this study was to investigate the antiproliferative and cytotoxic effects of a newly synthesized molecule named paracetamol acetohydroxamic acid (PaCaHa) on human neoplastic cell lines. MATERIALS AND METHODS: A549, CRL 2923, HeLa, and ARPE were treated with various concentrations of PaCaHa and DMSO (vehicle control). The cytotoxic/cytostatic effects of PaCaHa were determined after a 24-h incubation period and compared to the DMSO control. The cytotoxic and antiproliferative effects were determined by the trypan blue dye exclusion and MTT methods. RESULTS: A higher susceptibility to PaCaHA was found in CRL 2923 and HeLa cells, while A549 and ARPE cells were less responsive to PaCaHa. The percent of cytotoxicity resulting from 400 µg/mL of PaCaHa were >90 for CRL-2923 and HeLa, 68 for A549, and 64 for ARPE cells. The cytotoxic difference between CRL-2923/HeLa and ARPE/A549 cells was significant (P < 0.05). CONCLUSION: PaCaHa showed dose dependent cytotoxic and antiproliferative effects on three distinct human cancer cell lines. The differential effect of PaCaHa on different cancer cell lines suggests that PaCaHa could have a potential antitumor effect on specific cancer types. These results support further comprehensive studies on PaCaHa and its derivatives.

Identification of a subtelomeric gene family expressed during the asexual-sexual stage transition in Plasmodium falciparum.

For malaria transmission, the parasite must undergo sexual differentiation into mature gametocytes. However, the molecular basis for this critical transition in the parasites life cycle is unknown. Six previously uncharacterized genes, Pfg14.744, Pfg14.745, Pfg14.748, Pfg14.763, Pfg14.752 and Pfg6.6 that are members of a 36 gene Plasmodium falciparum-specific subtelomeric superfamily were found to be expressed in parasites that are committed to sexual development as suggested by co-expression of Pfs16 and Pfg27. Northern blots demonstrated that Pfg14.744 and Pfg14.748 were first expressed before the parasites differentiated into morphologically distinct gametocytes, transcription continued to increase until stage II gametocytes were formed and then rapidly decreased. Immunofluorescence assays indicated that both proteins were only produced in the subpopulation of ring stage parasites that are committed to gametocytogenesis and both localized to the parasitophorous vacuole (PV)b of the early ring stage parasites. As the parasites continued to develop Pfg14.748 remained within the parasitophorous vacuole, while Pfg14.744 was detected in the erythrocyte. The 5' flanking region of either gene alone was sufficient to drive early gametocyte specific expression of green fluorescent protein (GFP). In parasites transfected with a plasmid containing the Pfg14.748 5' flanking region immediately upstream of GFP, fluorescence was observed in a small number of schizonts the cycle before stage I gametocytes were observed. This expression pattern is consistent with commitment to sexual differentiation prior to merozoite release and erythrocyte invasion. Further investigation into the role of these genes in the transition from asexual to sexual differentiation could provide new strategies to block malaria transmission.

Male-specific expression of the paralog of malaria transmission-blocking target antigen Pfs230, PfB0400w.

Malaria transmission requires that Plasmodium parasites circulating in the vertebrate host develop into male and female gametocytes, which are then taken up by a mosquito to undergo fertilization and further development into infectious sporozoites. To understand the malaria specific events involved in this process, the gene products involved require identification and characterization. This work demonstrates that antibodies generated against the paralog of malaria transmission-blocking antigen Pfs230, PfB0400w, react only with stage V male gametocytes, not gametes or asexual parasites. In contrast, Pfs230 is expressed on the surface of all gametocytes and remains associated with emerged gametes as one of the primary surface antigens for several hours. Consistent with the localization findings, a high molecular weight band is recognized by anti-PfB0400w antibodies on western blots of extracts of late stage gametocytes, not asexual parasites, early (stage II/III) gametocytes, or gametes. PfB0400w mRNA is also not observed in asexual parasites. The transcript levels peak in stage III/IV gametocytes, then sharply decline in gametes. This work identifies a novel male-specific protein with an expression pattern that is distinctly different than its paralog.

Malaria parasites form filamentous cell-to-cell connections during reproduction in the mosquito midgut.

Physical contact is important for the interaction between animal cells, but it can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of > 100 µm and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive "nanotubes" in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut.

Malaria transmission-blocking antigen, Pfs230, mediates human red blood cell binding to exflagellating male parasites and oocyst production.

Malaria transmission requires that the parasites differentiate into gametocytes prior to ingestion by a mosquito during a blood meal. Once in the mosquito midgut the gametocytes emerge from red blood cells (RBCs), fertilize, develop into ookinetes and finally infectious sporozoites. Gamete surface antigen, Pfs230, is an important malaria transmission-blocking vaccine candidate, but its function has remained unclear. Two clones with distinct Pfs230 gene disruptions (Delta1.356 and Delta2.560) and a clone with a disruption of Pfs48/45 were used to evaluate the role of Pfs230 in the mosquito midgut. Pfs230 disruptants successfully emerge from RBCs and male gametes exflagellate producing microgametes. However, exflagellating Pfs230-minus males, in the presence or absence of Pfs48/45, are unable to interact with RBCs and form exflagellation centres. Oocyst production and mosquito infectivity is also significantly reduced, 96-92% and 76-71% respectively. In contrast, in the Pfs230 disruptants the expression and localization of other known sexual stage-specific antigens, including Pfs48/45, Pfs47, the Pfs230 paralogue (PfsMR5), Pfs16 or Pfs25, were not altered and the Pfs230-minus gametes retained resistance to the alternative pathway of human complement. These results suggest that Pfs230 is the surface molecule on males that mediates RBC binding and plays an important role in oocyst development, a critical step in malaria transmission.

Targeted disruption of Plasmodium falciparum cysteine protease, falcipain 1, reduces oocyst production, not erythrocytic stage growth.

Cysteine proteases are currently targets for drug development in a number of parasitic diseases, including malaria. In Plasmodium falciparum, the parasite responsible for the most virulent form of human malaria, there are four members of the cathepsin L-like family of cysteine proteases. Three of these (falcipains 2A, 2B and 3) are thought to be primarily involved in haemoglobin digestion, whereas falcipain 1 has recently been linked to erythrocyte invasion. Neither their expression nor their role in P. falciparum gametocytogenesis, which is required for malaria transmission, has been evaluated. In this study, RNA transcripts for the falcipain family members were identified as the parasite developed through all five stages of gametocytogenesis. Falcipain 1 transcript was upregulated in gametocytes, while levels of falcipain 2A/2B decreased in late-stage gametocytes and gametes. To evaluate the function of falcipain 1, the gene was disrupted, and clones from independent transformations were isolated. The asexual growth of the falcipain 1 minus clones was not overtly affected, and they produced morphologically normal gametocytes and gametes. However, when falcipain 1 minus parasites were fed to a mosquito, oocyst production was reduced by 70-90%, suggesting an important role for falcipain 1 during parasite development in the mosquito midgut.

Targeting and sequestration of truncated Pfs230 in an intraerythrocytic compartment during Plasmodium falciparum gametocytogenesis.

For malaria to be transmitted, the Plasmodium falciparum parasite must invade an erythrocyte and undergo gametocytogenesis. When mature intraerythrocytic gametocytes are taken up in a blood meal by a mosquito they emerge as gametes and, once fertilized, continue to differentiate into infectious sporozoites. One of the major proteins associated with the surface of the parasite during gamete differentiation is Pfs230, a 360 kDa member of a family of P. falciparum proteins that contains a repeated cysteine motif domain. To characterize the role of different regions of Pfs230, the gene was disrupted by targeted integration and clones isolated that expressed distinct sections of Pfs230. Independent clones D1.356 a and b express the first 452 amino acids (aa) of Pfs230 and do not contain a cysteine motif domain, whereas clones D2.850 a and b express the first 950 aa, including the first cysteine motif domain. Although both sets of clones undergo gametogenesis and produce morphologically normal gametes, neither truncated Pfs230 is located on the surface of the gamete. In clones D1.356 a and b, the 452 aa Pfs230 is secreted into the parasitophorous vacuole and released as a soluble protein when the parasite emerges from the erythrocyte as a gamete. In marked contrast, the 950 aa form of Pfs230 expressed by clones D2.850 a and b is sequestered in a novel tubular compartment in the erythrocyte cytoplasm. This sexual-stage tubular intraerythrocytic compartment (STIC) is not recognized by antibodies specific for proteins associated with the parasitophorous vacuole membrane (Pfs16 or Exp-1) or Maurer's clefts (Pfsbp 1 or mAb LWL1) or intraerythrocytic asexual parasite proteins (PfEMP2 or HRP II).