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Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK), was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMKΔ932). Expression of the carboxy-truncation of PATMKΔ932 also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

Clathrin-mediated endocytosis (CME) is the major pathway for internalization of membrane proteins from the cell surface. A half-century of studies have uncovered tremendous insights into how a clathrin-coated vesicle is formed. More recently, the advent of live-cell imaging has provided a dynamic view of this process. Since CME is highly conserved from yeast to man, budding yeast provides an evolutionary template for this process, and has been a valuable system for dissecting the underlying molecular mechanisms. In this review we trace the formation of a clathrin-coated vesicle from initiation to uncoating, focusing on key findings from the yeast system.

We identified in the Entamoeba histolytica genome a family of over 80 putative transmembrane kinases (TMKs). The TMK extracellular domains had significant similarity to the intermediate subunit (Igl) of the parasite Gal/GalNAc lectin. The closest homolog to the E. histolytica TMK kinase domain was a cytoplasmic dual-specificity kinase, SplA, from Dictyostelium discoideum. Sequence analysis of the TMK family demonstrated similarities to both serine/threonine and tyrosine kinases. TMK genes from each of six phylogenetic groups were expressed as mRNA in trophozoites, as assessed by spotted oligoarray and real-time PCR assays, suggesting nonredundant functions of the TMK groups for sensing and responding to extracellular stimuli. Additionally, we observed changes in the expression profile of the TMKs in continuous culture. Antisera produced against the conserved kinase domain identified proteins of the expected molecular masses of the expressed TMKs. Confocal microscopy with anti-TMK kinase antibodies revealed a focal distribution of the TMKs on the cytoplasmic face of the trophozoite plasma membrane. We conclude that E. histolytica expresses members of each subgroup of TMKs. The presence of multiple receptor kinases in the plasma membrane offers for the first time a potential explanation of the ability of the parasite to respond to the changing environment of the host.The Gal/GalNAc lectin of Entamoeba histolytica mediates parasite adherence to the host and signals the initiation of cytolysis (41,44,45,49). It is a heterotrimer consisting of covalently linked heavy (Hgl) and light (Lgl) subunits with a noncovalently linked intermediate (Igl) subunit (9,36,37,43,46). The Igl subunit of the Gal/GalNAc lectin has two known family members, Igl1 and Igl2. The Igl subunit has sequence similarity to the variant surface protein (VSP) of Giardia. We have previously identified a large number of proteins in the genome of E. histolytica containing CXXC motifs similar to those of Igl (8). Here we show that these CXXC-rich proteins form a large family of E. histolytica transmembrane kinases (TMKs) with highly variable extracellular domains homologous to Igl and VSPs of Giardia and with cytoplasmic kinase domains.Amebic trophozoites have been demonstrated to persist in humans for longer than 6 months (21,22). This prolonged period of infection suggests that the amebae evade the immune system. Other protozoan parasites, such as Plasmodium, Giardia, and Trypanosoma brucei, are also able to infect the host for long periods in spite of inducing robust immune responses. The mechanism(s) of persistence of these organisms is thought in part to be due to the variation of surface proteins. Plasmodium falciparum has three families of var genes that are independently expressed (29). The highest variation rate of these families is 2% per generation (52). Giardia encodes a family of 100 to 150 VSPs whose surface expression changes at a rate of one variation every 5 to 13 generations (38). T. brucei has a family of over 1,000 variant surface g...

Aspergillus fumigatusCgrA is the ortholog of a yeast nucleolar protein that functions in ribosome synthesis. To determine how CgrA contributes to the virulence of A. fumigatus, a ⌬cgrA mutant was constructed by targeted gene disruption, and the mutant was reconstituted to wild type by homologous introduction of a functional cgrA gene. The ⌬cgrA mutant had the same growth rate as the wild type at room temperature. However, when the cultures were incubated at 37°C, a condition that increased the growth rate of the wild-type and reconstituted strains approximately threefold, the ⌬cgrA mutant was unable to increase its growth rate. The absence of cgrA function caused a delay in both the onset and rate of germination at 37°C but had little effect on germination at room temperature. The ⌬cgrA mutant was significantly less virulent than the wild-type or reconstituted strain in immunosuppressed mice and was associated with smaller fungal colonies in lung tissue. However, this difference was less pronounced in a Drosophila infection model at 25°C, which correlated with the comparable growth rates of the two strains at this temperature. To determine the intracellular localization of CgrA, the protein was tagged at the C terminus with green fluorescent protein, and costaining with propidium iodide revealed a predominantly nucleolar localization of the fusion protein in living hyphae. Together, these findings establish the intracellular localization of CgrA in A. fumigatus and demonstrate that cgrA is required for thermotolerant growth and wild-type virulence of the organism.Aspergillus fumigatus is a saprophytic filamentous fungus that inhabits soil, water, and organic debris, where it has an essential role in the recycling of carbon and nitrogen (37). The organism propagates itself by the release into the air of high concentrations of asexual spores (conidia), which are unavoidably inhaled on a daily basis (26,46). Since the conidia are efficiently cleared by normal defenses, their inhalation is of minor consequence to healthy individuals. However, in the absence of adequate host immunity, the conidia germinate into highly invasive hyphae that cause severe lung damage and eventually disseminate to other organs. Patients with depressed immunity are at increased risk for infection with A. fumigatus, and the prognosis for invasive disease is very poor in these individuals (40). Of particular concern is the rising incidence of aspergillosis, a situation that has arisen as a consequence of aggressive cancer treatments and the widespread use of potent immunosuppressive regimens that support organ transplantation (33,44,54,58).Since A. fumigatus conidia are no more prevalent in the environment than the spores of some nonpathogenic molds (34), it is generally assumed that the organism has unique features that allow it to survive in humans, and thermotolerance has long been suspected to play a role (37). As a major component of the biomass in a self-heating compost pile, A. fumigatus has evolved mechanisms that allow it to grow we...

Background Actin polymerization by Arp2/3 complex must be tightly regulated to promote endocytosis. While many Arp2/3 complex activators have been identified, mechanisms for its negative regulation have remained more elusive. We analyzed the yeast arp2-7 allele, which is biochemically unique in causing unregulated actin assembly in vitro in the absence of Arp2/3 activators. Results We examined endocytosis in arp2-7 mutants by live cell imaging of Sla1-GFP, a coat marker, and Abp1-RFP, which marks the later actin phase of endocytosis. Sla1-GFP and Abp1-RFP lifetimes were accelerated in arp2-7 mutants, which is opposite to actin nucleation-impaired arp2 alleles or deletions of Arp2/3 activators. We performed a screen for multi-copy suppressors of arp2-7 and identified SYP1, a FCHO1 homologue, which contains F-BAR and AP-2μ homology domains. Over-expression of SYP1 in arp2-7 cells slowed Sla1-GFP lifetimes closer to wild type cells. Further, purified Syp1 directly inhibited Las17/WASp stimulation of Arp2/3 complex-mediated actin assembly. This activity was mapped to a fragment of Syp1 located between its F-BAR and AP-2μ homology domains, and depends on non-VCA sequences in Las17/WASp. Conclusions Together, these data identify Syp1 as a novel negative regulator of WASp-Arp2/3 complex that helps choreograph the precise timing of actin assembly during endocytosis.

The clathrin light-chain (LC) N-terminal region interacts with the Sla2/Hip1/Hip1R family of ANTH/talin–like proteins. In vivo evidence shows that LC–Sla2 binding is important for releasing Sla2 attachments to actin in the endocytic coat. Loss of this regulation can suppress major actin defects during endocytosis.

Amebic erythrophagocytosis is characteristic of invasive amebiasis, and mutants deficient in erythrocyte ingestion are avirulent. We sought to understand the molecular mechanisms underlying erythrocyte phagocytosis by Entamoeba histolytica. Following adherence to amebae, erythrocytes became round and crenulated, and phosphatidylserine (PS) was exposed on their outer membrane leaflets. These changes were similar to the effects of calcium treatment on erythrocytes, which we utilized to separate ameba-induced exposure of erythrocyte PS from the process of phagocytosis. The adherence and phagocytosis of calcium-treated erythrocytes were less inhibited by galactose than were those of healthy erythrocytes, suggesting the existence of an amebic coreceptor specific for PS. To test whether PS was recognized by amebae, calcium-treated cells were incubated with annexin V prior to adherence to or ingestion by E. histolytica. Annexin V blocked both adherence (50% ؎ 12% inhibition; P < 0.05) and phagocytosis (65% ؎ 10%; P < 0.05), providing evidence that at least one galactose-independent coreceptor was involved in the adherence and ingestion of red blood cells. The coreceptor was inhibited by phospho-L-serine and to a lesser extent by phospho-D-serine but not by phospho-Lthreonine, which is consistent with the coreceptor functioning in the adherence and ingestion of erythrocytes via recognition of PS. We expanded our investigations to the highly related but noninvasive parasite Entamoeba dispar and demonstrated that it was deficient in red-blood-cell adherence, induction of PS exposure, and phagocytosis. These findings establish phosphatidylserine involvement in erythrophagocytosis by amebae and suggest the existence of a PS receptor on the surfaces of both E. histolytica and E. dispar.Entamoeba histolytica is the causative agent of amebiasis. This disease is most prevalent in the poorest areas of the developing world, causing the second-highest rates of morbidity and mortality due to protozoan parasites (57). Clinical manifestations of amebiasis include asymptomatic colonization, colitis, and liver or brain abscesses (24). In patients with invasive amebiasis, E. histolytica causes vast tissue damage (11,23) that is believed to be a direct result of its ability to induce host cell apoptosis (26, 27, 37, 47) and necrosis (3). The antiinflammatory nature of cell death via apoptosis may explain the paucity of inflammatory changes with invasive amebiasis (28, 50).Invasive infection by E. histolytica is thought to involve a stepwise process of adherence and cytolysis followed by phagocytosis. Adherence via the Gal/GalNAc lectin is required for the latter steps to occur (26,32,38,39). Cytolysis occurs in a contact-dependent manner and has been associated with increased intracellular calcium (40), activation of caspase 3 (27), and exposure of phosphatidylserine (PS) on the outer leaflet of the host cell membrane (26). These changes are consistent with the induction of apoptosis, but there is also evidence of necrosis (3) occurring ...