Iron economy in Naegleria gruberi reflects its metabolic flexibility

Naegleria gruberi is a free-living amoeba, closely related to the human pathogen Naegleria fowleri, the causative agent of the deadly human disease primary amoebic meningoencephalitis. Herein, we investigated the effect of iron limitation on different aspects of N. gruberi metabolism. Iron metabolism is among the most conserved pathways found in all eukaryotes. It includes the delivery, storage and utilisation of iron in many cell processes. Nevertheless, most of the iron metabolism pathways of N. gruberi are still not characterised, even though iron balance within the cell is crucial. We found a single homolog of ferritin in the N. gruberi genome and showed its localisation in the mitochondrion. Using comparative mass spectrometry, we identified 229 upregulated and 184 down-regulated proteins under iron-limited conditions. The most down-regulated protein under iron-limited conditions was hemerythrin, and a similar effect on the expression of hemerythrin was found in N. fowleri. Among the other down-regulated proteins were [FeFe]-hydrogenase and its maturase HydG and several heme-containing proteins. The activities of [FeFe]-hydrogenase, as well as alcohol dehydrogenase, were also decreased by iron deficiency. Our results indicate that N. gruberi is able to rearrange its metabolism according to iron availability, prioritising mitochondrial pathways. We hypothesise that the mitochondrion is the center for iron homeostasis in N. gruberi, with mitochondrially localised ferritin as a potential key component of this process.     

Naegleria fowleri: Light and electron microscopy study of mitosis

DAPI and Feulgen stains were used as specific DNA markers for studying the mitosis process in Naegleria fowleri. Both DAPI and Feulgen stains reacted with DNA in the nuclei of the amoebae. Representative figures of N. fowleri mitotic nuclei with a defined arrangement according to the phase of the cell cycle were observed. A notable characteristic is that the nucleolus is present throughout the stages of mitosis. During metaphase, several deeply stained DNA condensations following an elongated pattern were observed, corresponding almost certainly to tightly grouped chromosomes. Ultrastructural observations demonstrated that the nucleus divides by cryptomitosis, a process in which the nuclear membrane does not disappear during the mitosis. Centrioles were not found, and a spindle of microtubules was observed running the length of the nucleus from pole to pole however, they did not come to a focal point.     

Naegleria lovaniensis new species: Isolation and identification of six thermophilic strains of a new species found in association with Naegleria fowleri

Six Naegleria strains, isolated previously in association with N. fowleri from thermal waters, were studied to further determine their antigenic relationship to N. fowleri and other Naegleria species. Results of immunofluorescent antibody and immunoelectrophoretic studies clearly established the antigenic divergence of the variants from N. fowleri, N. gruberi, and N. jadini. The variants were further distinguished from known Naegleria species by several ultrastructural characteristics, which included the complete enclosure of their nuclei with one or several layers of rough endoplasmic reticulum (RER) and extrusion of their nuclear material. Extruded nuclear material was observed in the cytoplasm completely sequestered by cisternae of the RER. The variants were also shown to be sensitive to agglutination induced by concanavalin A but not by wheat germ agglutinin. Based on the differences in the antigenicity, morphology, and lectin sensitivity between these six variants and established Naegleria species, we proposed that they should be established as a new species.     

A real-time PCR diagnostic method for detection of Naegleria fowleri

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence.Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.     

Nested PCR assay for the rapid detection of Naegleria fowleri from swimming pools in Egypt

The free-living amoeboflagellate Naegleria fowleri is the only species infects humans world widely distributed. N. fowleri is the causative agent of very rare but severe brain infection called primary amoebic meningo-encephalitis (PAM), a rapidly fatal disease of the central nervous system mainly in immuno-compromised individuals. N. fowleri infects human through the entry of the nose, and it happens when human swimming or diving in warm freshwater, such as lakes, rivers and swimming pools. The disease is acute, and patients often die within 5–10 days and before the infectious agent can be diagnosed. Limited information is available about the existence of pathogenic N. fowleri, in Egypt, so the present of N. fowleri is an important public health. In the present study, we examined hundred water, dust and swap samples collected from 5 swimming pools in Cairo, Egypt. Based on morphological characteristics of trophozoite and cyst, flagellation test 56% of thermo-tolerant Naegleria like amoeba was detected. The incidence of thermo-tolerant free-living amoebae reached 84, 80and 70% from water, cotton swap and dust samples, respectively at cultivation temperature of 45 °C. The highest occurrence of thermo-tolerant amoebae were recorded in summer (100 & 87.5%) while the lowest one were recorded in winter (58 & 37.5%) in both water and dust samples, respectively. In swap samples, the highest occurrence of thermo-tolerant free-living amoeba was recorded in both summer and spring (100%), while the lowest one was recorded in winter (40%). N. fowleri was performed on 24 samples from a total of 56 (42.2%) samples which are positive by culture. Nested PCR using Mp2Cl5 gene primers that is unique to N. fowleri was carried out. The N. fowleri specific primer showed band at 166 bp against 24 of 56 (42.2%) samples. The majority of positive samples unique to N. fowleri was detected in water samples followed by swap samples and finally dust samples 14 of 24 (58%), 7 of 24 (29%), 3 of 24 (13%), respectively. In conclusion, swimming pools water may be the source of Naegleria invasion. The use of molecular methods to identify free-living amoebae N. fowleri could provide a more rapid means to diagnose infections caused by those amoebae.     

Ultrastructure of mitosis inAmoeba proteus

Summary The fine structure ofAmoeba proteus nuclei has been studied during interphase and mitosis. The interphase nucleus is discoidal, the nuclear envelope is provided with a honeycomb layer on the inside. There are numerous nucleoli at the periphery and many chromatin filaments and nuclear helices in the central part of nucleus.In prophase the nucleus becomes spherical, the numerous chromosomes are condensed, and the number of nucleoli decreases. The mitotic apparatus forms inside the nucleus in form of an acentric spindle. In metaphase the nuclear envelope loses its pore complexes and transforms into a system of rough endoplasmic reticulum cisternae (ERC) which separates the mitotic apparatus from the surrounding cytoplasm; the nucleoli and the honeycomb layer disappear completely. In anaphase the half-spindles become conical, and the system of ERC around the mitotic spindle persists. Electron dense material (possibly microtubule organizing centers—MTOCs) appears at the spindle pole regions during this stage. The spindle includes kinetochore microtubules attached to the chromosomes, and non-kinetochore ones which pierce the anaphase plate. In telophase the spindle disappears, the chromosomes decondense, and the nuclear envelope becomes reconstructed from the ERC. At this stage, nucleoli can already be revealed with the light microscope by silver staining; they are visible in ultrathin sections as numerous electron dense bodies at the periphery of the nucleus.The mitotic chromosomes consist of 10 nm fibers and have threelayered kinetochores. Single nuclear helices still occur at early stages of mitosis in the spindle region.     

Ultrastructure of mitosis in the aphid-pathogenic fungusErynia neoaphidis

Summary An account of mitosis in the aphid-pathogenic, entomophthoraceous fungusErynia neoaphidis is presented. The mitotic apparatus is characterized by a closed, intranuclear, polarized spindle. Chromosomes are permanently attached by kinetochore microtubules (kcMTs) to the poles during mitosis. The spindle develops as the spindle pole bodies migrate and separate. At metaphase the eccentric spindle contains only kcMTs and is located in a relatively chromatinfree zone. Paired sister kinetochores are arranged in a broad metaphase plate. During anaphase kcMTs shorten, astral and nonchromosomal microtubules develop and elongate and the interpolar distance increases.     

Ultrastructure of the Z organ in Xiphinema ifacolum

Bieve-Zacheo T., Zacheo G. and Lamberti F. 1985. Ultrastructure of the Z organ in Xiphinema ifacolum. International Journal for Parasitology15: 453–461. The uterus in Xiphinema ifacolum can be divided into uterus proper, a 77 μm long tube and a lemon-shaped Z organ, about 28 μm long and 18 μm wide, placed between the oviduct and the uterus proper. The Z organ consists of a thick outer muscular layer of 120 cells, arranged in 20 rings of six cells each and a thin inner epithelium layer, lining the lumen. The epithelial cell walls, lining the lumen of the Z organ are thicker than those lining the uterus proper and are strongly folded. The crests of some of these folds carry six large apophyses, all about the same size and shape, these occupy the full length of the organ, becoming thicker towards the center of the lumen. There are many tubules near the surfaces of the apophyses, the contents of which can be dissolved by treatment with pepsin and pronase, indicating that they are proteins. This material probably consists of secretions which are squeezed out of the apophyses by a passing egg and may function in the formation or hardening of the egg shell.     

Ultrastructure of the attachment of Pyrsonympha to the hind-gut wall of Reticulitermes tibialis

Protozoa of the genus Pyrsonympha are found attached to the cuticular lining of the hind-gut wall of the termite Reticulitermes tibialis by a specialized anterior structure referred to here as the ‘attachment organelle’. The structure of the hind-gut, the attachment organelle, and the nature of the attachment are described using light and electron microscopy. The attachment organelle is a highly folded membranous structure containing large 20 to 26 nm filaments. Although no unambiguous functional significance can presently be assigned to this attachment, it may serve to prevent loss of the protozoa from the intestinal tract or it may be involved in the nutritional symbiosis between termite and protozoa.     

Ultrastructure of the nematode pathogen, Bacillus penetrans

The ultrastructure and development of Bacillus penetrans in root-knot nematodes, Meloidogyne spp., was studied with a transmission electron microscope. Host infection was by a germ tube from the cup-shaped sporangium containing the endospore. The prokaryotic vegetative cells contained septa formed by an ingrowth of the inner layer of the trilaminate cell wall and were associated with mesosomes. Structure of the endospore was similar to other bacteria with a spore protoplast enclosed within two cortical layers and three spore coats. An exosporium which may function in attachment and host specificity surrounded the endospore. Ultrastructural changes accompanying sporulation were similar to those reported for other endospore-forming bacteria but with some parasite specialization. The filamentous vegetative growth was characteristic of some Actinomycetales. Endospore development at the apices of dichotomously branched filaments of the thallus resembled the genus Actinobifida.     

Ultrastructure of the median neurosecretory cells of Manduca sexta in vivo and in vitro

Ultrastructural comparisons of median neurosecretory cells of Manduca sexta were made by using fresh and cultured specimens. Examination of the endomembrane system, the abundance and electron density of neurosecretory granules, and the condition and abundance of mitochondria showed that brains cultured for 4 days were similar to those taken directly from the insect. After 10 days, the neurosecretory cells had broken down but the neurosecretory granules were still present.     

Axenic cultivation of Naegleria gruberi : Requirement for methionine

A simplified axenic medium for Naegleria gruberi strain NEG-M contains -methionine, dextrose, yeast extract, a macromolecular fraction of fetal calf serum, and phosphate buffer. Amoebae cultured in suspension in this medium grow with doubling times of 8–10 h (at 32 °C) to yield 2–4 × 10⁶ cells/ml. Amoebae from growing or early stationary phase cultures, transferred to nonnutrient buffer, differentiate synchronously into flagellates. Differentiation occurs reproducibly 80 min after initiation (time for 50% flagellates at 25 °C) if amoebae are taken from a culture maintained at pH 6.6.     

拟目乌贼精子发生和精子的超微结构

应用扫描电镜和透射电镜观察了拟目乌贼(Sepia lycidas)精子的发生过程和超微结构。结果表明,精子发生经历了精原细胞、初级精母细胞、次级精母细胞、精细胞和成熟精子5个阶段,其中精细胞可以分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ5个时期,精细胞Ⅱ期又可分为前期和后期。细胞核经历了一个横向收缩、纵向拉长的过程,由圆形或椭圆形,变为不规则的纺锤形、稍弯曲的长柱形;核内染色质由絮状,变为絮块状、致密颗粒状、细纤维状、粗纤维状和片层状,直至高电子密度均质状;顶体由圆形,变为头盔形、圆锥形、倒"U"字形,直至子弹头形;线粒体由空泡状经过融合和迁移,变为内嵴丰富的椭球形,形成不完全包围鞭毛的线粒体距。成熟精子全长101.28μm,由头部和尾部组成,头部呈长辣椒状,长7.73μm,宽1.51μm,由顶体和细胞核组成;尾部细长,为93.18μm,为典型的"9+2"结构,由中段、主段和末段三部分组成。     

Ultrastructure of some developmental stages of Helicosporidium sp. in the navel orangeworm, Paramyelois transitella

The ultrastructure of the developmental stages of Helicosporidium sp. is described from the early spherical cell stage to the maturation and germination of the spore. The presence of well-defined Golgi bodies and mitotic division of the nucleus suggests that this pathogen is not an ascomycete, as had been previously reported, and indicates an affinity to the Protozoa.     

Ultrastructure of mitosis in the cowpea rust fungus Uromyces phaseoli var. Vignae

Aspects of the ultrastructure of mitotic nuclei of the fungus Uromyces phaseoli var. vignae are described from both intercellular hyphae in the cowpea host and infection structures induced to differentiate in vitro. The interphase nucleus-associated organelle (NAO) consists of two trilamellar acircular disks connceted by an osmiophilic bar. The intranuclear spindle develops between these disks when they separate. The spindle contains pole to pole, interdigitating, chromosomal, and fragmentary microtubules arranged to form a central bundle along the surface of which lie the metaphase chromosomes. No metaphase plate is found. There are up to three microtubules per kinetochore and approximately 14 chromosomes on the haploid spindle. Telophase elongation appears to involve extension of pole to pole microtubules with no evidence for the remaining presence of interdigitating microtubules. Concomitantly, numerous cytoplasmic microtubules develop from each NAO disk where few or none are present in other phases. Reformation of the interphase NAO involves the formation of a sausage- shaped intermediate at late telophase. The nuclear envelope remains intact and the nucleolus persists throughtout division. Various aspects of the spindle and NAOs appear to be evolutionary intermediates between Ascomycetes and higher Basidiomycetes, thus supporting the theory of Basidiomycete evolution from the former group and demonstrating an encouraging correlation between mitotic characteristics and other phylogenetic markers.     

Plasmodium simium: Ultrastructure of erythrocytic phase

An electron microscopic study of Plasmodium simium infections in the squirrel monkey has supplied information on the ultrastructure of erythrocytic trophozoites, schizonts, merozoites, and gametocytes, in addition to an unusual form of host cell pathology. In general, the structural features, as well as certain specialized functions, e.g., hemoglobin ingestion and utilization, nuclear and cytoplasmic division, were found to be similar to those described for other malarial parasites. Some striking features were noted, however. A highly asynchronous mode of merozoite production was observed within single segmenting parasites in spite of the overall developmental synchrony displayed by the population as a whole. Secondly, during parasite segmentation, newly formed merozoites are connected to one another, as well as to the parasitophorous membrane, by periodic surface strands. It is speculated that these interparasite bridges serve as structural support to the segmenting parasite. When merozoites are matured fully, these interconnections break, leaving a uniform array of short surface bristles. In addition, a number of different pathological changes in host cell structure have been noted. Localized surface discontinuities appear in region of infected cells where apical regions of developing or fully mature merozoites are abutted against the plasma membrane. These profiles suggest that these specialized apical regions of the merozoite function in release as well as in host cell penetration. More generalized surface pathology occurs within parasitized erythrocytes in the form of surface blebs, surface clefts, and associated cytoplasmic microvesicles. The severity of this pathology increases as the intraerythrocytic parasite matures. Topographically these altered cells have a “berry-like” surface texture which makes them quite distinctive when viewed by scanning electron microscopy.     

旋毛虫肌幼虫细胞传代培养及超微结构观察

消化、分离观察旋毛虫(Trichinella spiralis)肌幼虫,获得肌幼虫细胞,用含10%胎牛血清的RPMI-1640培养液培养原代细胞,胰酶(含0.02?TA)消化法进行传代,透射电镜观察培养细胞超微结构,用多重PCR鉴定培养细胞。结果表明,在培养24～72h原代细胞开始贴壁,7～8d形成单层细胞,细胞间融合现象不明显,10～12d传一代。透射电镜显示旋毛虫细胞核为椭圆形,核膜、核仁清晰,核内染色质较丰富,胞浆含丰富的线粒体。细胞主要有两种类型:椭圆形和多角形,以椭圆形为主。多重PCR扩增培养细胞DNA,可见1条与旋毛虫肌幼虫DNA扩增产物相同的条带(173bp)。结果表明,旋毛虫肌幼虫细胞可在含10%胎牛血清的RPMI-1640培养液中传代培养。