Carbohydrate analysis of Acanthamoeba castellanii

We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting “cyst wall” material was subsequently lyophilized and subjected to glycosyl composition analysis. Transmission electron microscopy confirmed the removal of intracystic material following enzymatic treatment. Our results showed that treated A. castellanii trophozoites, intact cysts and cyst walls contained various sugar moieties, of which a high percentage was galactose and glucose, in addition to small amounts of mannose, and xylose. Linkage analysis revealed several types of glycosidic linkages including the 1,4-linked glucosyl conformation, indicative of cellulose. Inhibitor studies suggested that, beside sugar synthesis, cytoskeletal re-arrangement and mitogen-activated protein kinase-mediated pathways are involved in A. castellanii encystment.     

Acanthamoeba spp.: In vitro effects of clinical isolates on murine macrophages, osteosarcoma and HeLa cells

Three different cell lines (murine macrophages, HeLa and osteosarcoma cells) were assayed in order to check for the manifestation of the cytopathic effects of three strains of Acanthamoeba recently isolated in our laboratory from contact lens cases: CLC-16, CLC-41.r and CLC-51-l. Adhesion and cytotoxicity assays were carried out with these strains and the type strain Acanthamoeba castellanii Neff as a control. Briefly, the ability of these amoebae to bind to the three cell lines was calculated and supernatants were examined for cytotoxicity by measuring lactate dehydrogenase released as an estimate of cytotoxicity using a commercial detection kit. The three strains showed high adhesion and cytotoxicity levels when tested in the three cell lines. This study demonstrates the ability of these amoebae to degrade any of the tested cell lines. To the best of our knowledge, this is the first report of the in vitro effects of acanthamoebae on osteosarcoma cells.     

Acanthamoeba castellanii Neff: In vitro activity against the trophozoite stage of a natural sesquiterpene and a synthetic cobalt(II)-lapachol complex

In this study, the in vitro activities of a natural sesquiterpene, α-cyperotundone, isolated from the root bark of Maytenus retusa and a cobalt(II)-complex of a natural occurring prenyl hydroxynaphthoquinone (lapachol) were evaluated against the trophozoite stage of Acanthamoeba castellanii Neff using a previously developed colorimetric 96-well microtiter plate assay, based on the oxido-reduction of Alamar Blue®. The obtained activities showed that these two compounds were able to inhibit the in vitro growth of the amoebae at relatively low concentrations. Further identification of the molecular targets of these products and their effects on acanthamoebae should be determined to evaluate their possible therapeutic use.     

Toxoplasma gondii: uptake and survival of oocysts in free-living amoebae

Waterborne transmission of the oocyst stage of Toxoplasma gondii can cause outbreaks of clinical toxoplasmosis in humans and infection of marine mammals. In water-related environments and soil, free-living amoebae are considered potential carriers of various pathogens, but knowledge on interactions with parasitic protozoa remains elusive. In the present study, we assessed whether the free-living Acanthamoebacastellanii, due to its phagocytic activity, can interact with T. gondii oocysts. We report that amoebae can internalize T. gondii oocysts by active uptake. Intracellular oocysts in amoebae rarely underwent phagocytic lysis, retained viability and established infection in mice. Interaction of T. gondii with amoebae did not reduce the infectivity and pathogenicity of oocysts even after prolonged co-cultivation. Our results show that uptake of oocysts by A. castellanii does not restrain the transmission of T. gondii in a murine infection model.     

alr0882 encoding a hypothetical protein of Anabaena PCC7120 protects Escherichia coli from nutrient starvation and abiotic stresses

This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (?uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform ?uspA E. coli strain. The IPTG induced expression of a 56.6 kDa GST fusion protein was visualized on SDS–PAGE and attested by immunoblotting. E. coli ?uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl₂, H₂O₂, UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl₂ demonstrated about 22.6–51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria.     

Resistance of Cysts of Amoebae to Microbial Decomposition

Cyst walls of Acanthamoeba rhysodes, A. palestinensis, A. castellanii, and one other strain of Acanthamoeba contain 36 to 45% protein and 20 to 34% carbohydrate. More than half of the protein in the walls of A. palestinensis, A. castellanii and Acanthamoeba sp. is accessible to and hydrolyzed by protease, and 67 to 69% of the carbohydrate of A. palestinensis and A. rhysodes walls is hydrolyzed by cellulase. The extent of hydrolysis of walls of the other amoebae by these enzymes is appreciably less, and chitinase and β-1,3-glucanase have no detectable effect. Protease solubilizes 10% or less of the weight of intact cysts, and no solubilization is observed with cellulase. Walls of A. palestinensis are extensively degraded in soil, the activity is less with A. rhysodes, and little attack on the other amoebae occurs. When added to soil, the protozoa excyst and grow for short periods, the trophozoites then die, and chiefly cysts persist thereafter.     

Balamuthia and Acanthamoeba-binding antibodies in West African human sera

Little is known about the prevalence of Balamuthia mandrillaris amoebae and Balamuthia amoebic encephalitis in Africa. As an approach, relative concentrations of amoebae-binding serum antibodies (Ab) were assessed by flow cytometry using formaldehyde-fixed B. mandrillaris, Acanthamoeba lenticulata 72-2 and Acanthamoeba castellanii 1BU amoebae for specific Ab capture (B.m.-Ab, A.l.-Ab, A.c.-Ab). One hundred and ninety-two sera from West African (Côte d’Ivoire) donors aged 11-95 years (mean 38 a; 51% males), and living in villages surrounded by rainforest near the Liberian border, were tested and related to reference sera from Berlin. While B.m.-Ab tended to increase with donor age, A.l.-Ab and A.c.-Ab did not. Accordingly, B.m.-Ab correlated only weakly with A.l.-Ab or A.c.-Ab. Of the nine individuals with the highest B.m.-Ab concentrations, most were elderly (mean 58 a), male (78%), and professed intensive outdoor activity (hunting, farming). Only three of these sera also showed elevated A.l.-Ab, and none elevated A.c.-Ab.     

Acanthamoeba polyphaga is a possible host for Vibrio cholerae in aquatic environments

Acanthamoeba is a genus of free-living amoebae found to be able to host many bacterial species living in the environment. Acanthamoebae and Vibrio cholerae are found in the aquatic environments of cholera endemic areas. Previously it has been shown that V. cholerae O1 and O139 can survive and grow in Acanthamoeba castellanii. The aim of this study was to examine the ability of Acanthamoeba polyphaga to host V. cholerae O1 and O139. The interaction between A. polyphaga and V. cholerae strains was studied by means of viable amoeba cell counts and viable count of the bacteria in the absence and presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Electron microscopy was used to determine the localization of V. cholerae inside A. polyphaga. The results showed that A. polyphaga enhanced growth and survival of V. cholerae, which grew and survived inside the amoeba cells for 2 weeks. The electron microscopy showed that A. polyphaga hosted intracellular V. cholerae localized in the vacuoles of amoeba cell. Neither the presence of V. cholerae together with A. polyphaga nor the intracellular localization of the bacteria inhibited growth and survival of A. polyphaga. The outcome of the interaction between these microorganisms may support strongly the role of A. polyphaga as host for V. cholerae O1 and O139.     

Effects of Mannose on Pathogenesis of Acanthamoeba castellanii

Acanthamoeba spp. are single-celled protozoan organisms that are widely distributed in the environment. In this study, to understand functional roles of a mannose-binding protein (MBP), Acanthamoeba castellanii was treated with methyl-alpha-D-mannopyranoside (mannose), and adhesion and cytotoxicity of the amoeba were analyzed. In addition, to understand the association of MBP for amoeba phagocytosis, phagocytosis assay was analyzed using non-pathogenic bacterium, Escherichia coli K12. Amoebae treated with mannose for 20 cycles exhibited larger vacuoles occupying the most area of the amoebic cytoplasm in comparison with the control group amoebae and glucose-treated amoebae. Mannose-selected amoebae exhibited lower levels of binding to Chinese hamster ovary (CHO) cells. Exogenous mannose inhibited >50% inhibition of amoebae (control group) binding to CHO cells. Moreover, exogenous mannose inhibited amoebae (i.e., man-treated) binding to CHO cells by <15%. Mannose-selected amoebae exhibited significantly decreased cytotoxicity to CHO cells compared with the control group amoebae, 25.1% vs 92.1%. In phagocytic assay, mannose-selected amoebae exhibited significant decreases in bacterial uptake in comparison with the control group, 0.019% vs 0.03% (P<0.05). Taken together, it is suggested that mannose-selected A. castellanii trophozoites should be severely damaged and do not well interact with a target cell via a lectin of MBP.     

Identification of Protein Arginine Methyltransferase 5 as a Regulator for Encystation of Acanthamoeba

Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa.     

Acanthamoeba castellanii: identification and distribution of actin cytoskeleton

The presence of the cytoskeleton of Acanthamoeba castellanii was observed by means of cryo-electronmicroscopy and immunofluorescence techniques. This structure is formed largely by fibers and networks of actin located mainly in cytoplasmic locomotion structures as lamellipodia and as well as in various endocytic structures. In addition, the comparision between total actin content in whole extracts among different amoebae was made. The molecular weight of actin in A. castellanii was 44 kDa, and 45 kDa for Naegleria fowleri and Entamoeba histolytica.     

Entamoeba histolytica: Oxygen resistance and virulence

Entamoeba histolytica virulence has been attributed to several amoebic molecules such as adhesins, amoebapores and cysteine proteinases, but supporting evidence is either partial or indirect. In this work we compared several in vitro and in vivo features of both virulent E. histolytica (vEh) and non-virulent E. histolytica (nvEh) axenic HM-1 IMSS strains, such as complement resistance, proteinase activity, haemolytic, phagocytic and cytotoxic capacities, survival in mice caecum, and susceptibility to O₂. The only difference observed was a higher in vitro susceptibility of nvEh to O₂. The molecular mechanism of that difference was analyzed in both groups of amoebae after high O₂ exposure. vEh O₂ resistance correlated with: (i) higher O₂ reduction ( and H₂O₂ production); (ii) increased H₂O₂ resistance and thiol peroxidase activity, and (iii) reversible pyruvate: ferredoxin oxidoreductase (PFOR) inhibition. Despite the high level of carbonylated proteins in nvEh after O₂ exposure, membrane oxidation by reactive oxygen species was not observed. These results suggest that the virulent phenotype of E. histolytica is related to the greater ability to reduce O₂ and H₂O₂ as well as PFOR reactivation, whereas nvEh undergoes irreversible PFOR inhibition resulting in metabolic failure and amoebic death.     

Inheritance of resistance to Bacillus thuringiensis Cry1Ab protein in the sugarcane borer (Lepidoptera: Crambidae)

Inheritance traits of a Cry1Ab-resistant strain of the sugarcane borer, Diatraea saccharalis (F.) were analyzed using various genetic crosses. Reciprocal parental crosses between Cry1Ab-susceptible and Cry1Ab-resistant populations, F₁ by F₁ crosses, and backcrosses of F₁ with the Cry1Ab-resistant population were successfully completed. Larval mortality of the parental and cross-populations were assayed on Cry1Ab diet and Bacillus thuringiensis (Bt)-corn leaf tissue. Maternal effects and sex linkage were examined by comparing the larval mortality between the two F₁ populations. Dominance levels of resistance were measured by comparing the larval mortality of the Cry1Ab-resistant, -susceptible, and -heterozygous populations. Number of genes associated with the resistance was evaluated by fitting the observed mortality of F₂ and backcross populations with a Mendelian monogenic inheritance model. Cry1Ab resistance in D. saccharalis was likely inherited as a single or a few tightly linked autosomal genes. The resistance was incompletely recessive on Bt corn leaf tissue, while the effective dominance levels (D_ML) of resistance increased as Cry1Ab concentrations decreased with Cry1Ab-treated diet. D_ML estimated based on larval mortality on intact Bt corn plants reported in a previous study ranged from 0.08 to 0.26. This variability in D_ML levels of Cry1Ab resistance in D. saccharalis suggests that Bt corn hybrids must express a sufficient dose of Bt proteins to make the resistance genes functionally recessive. Thus, Bt resistant heterozygous individuals can be killed as desired in the “high/dose refuge” resistance management strategy for Bt corn.     

Starvation and Encystment of a Soil Amoeba Hartmannella castellanii*

SYNOPSIS. The behavior of the amoeba H. castellanii was investigated in various carbon and nitrogen deficient media with a view to developing a satisfactory replacement medium for the study of encystment and excystment. Media which had been devised for other soil amoebae did not cause H. castellanii to encyst. In these media there was an efflux of material from the cells which was independent of osmolarity but which was minimized by the addition of magnesium. Maximal encystment occurred in a medium containing magnesium chloride alone. The cysts produced in the magnesium chloride replacement medium are viable and readily excyst when resuspended in the growth medium. The cysts contain cellulose, which is not present in the vegetative amoebae, and differ from the amoebae in their greater resistance to induced lysis and mechanical injury.     

Survival and Growth of Francisella tularensis in Acanthamoeba castellanii

Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.     

斑膜合垫盲蝽若虫在国槐上的空间分布型及抽样技术

斑膜合垫盲蝽Orthotylus(O.)sophorae Josifov是近年来临夏地区国槐Sophora japonica Linnaeus上严重发生的害虫之一。应用6种分布型指数法分析判定了斑膜合垫盲蝽若虫在国槐上的空间分布型,利用Taylor幂法则和Iwao回归方程分析聚集原因,结果表明,斑膜合垫盲蝽若虫在国槐上呈聚集分布,公共k c值为0.6169,且符合负二项分布;其种群聚集是由某些环境作用引起的。在此基础上,采用Iwao的方法确定了斑膜合垫盲蝽若虫的田间最适抽样数和序贯抽样表;并根据Gerrard的零频率模型建立了估计该种群平均密度的零频率公式:x=1.7457(-lnP0)1.1119。     

Fate of resting cysts of Alexandrium spp. ingested by Perinereis nuntia (Polychaeta) and Theola fragilis (Mollusca)

The ingestion of resting cysts of Alexandrium spp. by Perinereis nuntia (Polychaeta) and Theola fragilis (Mollusca) was experimentally examined in the laboratory. P. nuntia and T. fragilis were cultured in bottom sediment containing a high density of Alexandrium cysts under dark conditions. Moreover, to evaluate the degree and consequence of being ingested, the density of cysts in the control sediment (no macrobenthic organisms) and the germination capability of the cysts in the faecal pellets of the two species of macrobenthos were examined.Cysts in the culture sediment were found to be ingested by both P. nuntia and T. fragilis. No difference in the density of cysts between the sediments cultured with and without P. nuntia was observed. However, the density of cysts in the sediments with T. fragilis decreased by 24% compared to the density in the control sediment. It is possible that most of the cysts ingested were digested by T. fragilis. The rate of Alexandrium cyst digestion by this species is estimated 594 cysts/individual/day. It is estimated that 91% of the cysts ingested by T. fragilis were partially or totally digested and only 9% were excreted in a viable state during the experiment. Thus, T. fragilis has a stronger affect on the abundance of Alexandrium cysts compared with P. nuntia.No significant difference was observed between the germination success of the cysts from faecal pellets of P. nuntia and T. fragilis compared to the cysts in the control sediment. If, however, the necessary light for the cysts to germinate is cut off by being enclosed within the faecal pellet, the germination rate of cysts from the faecal pellets may be suppressed.     

Spin label studies of microsomal membranes from Acanthamoeba castellanii in different states of differentiation

Two fatty acid spin labels—[I(1,14)], stearic acid bearing a paramagnetic nitroxide group on carbon 16, and [I(12,3)], stearic acid bearing a paramagnetic nitroxide group on carbon 5—have been used to compare the physical properties of lipid in rough and smooth microsomal membranes from trophozoites and cysts of Acanthamoeba castellanii. Arrhenius plots of rotational correlation times (τ_c) calculated from the spectra for I(1,14) showed an abrupt discontinuity in slope for membranes from both trophozoites and cysts. This occurred at temperatures ranging from ?3 to 1 °C for smooth microsomes and from 8 to 11 °C for rough microsomes for both cysts and amoebae. The value of τ_c at 29 °C, the culturing temperature, in effect scores fluidity of the membrane matrix, and did not show any significant difference for either rough or smooth microsomes during the transition from exponential to stationary phase growth. However, smooth microsomes from cysts showed a 14% increase in fluidity relative to trophozoites, and the fluidity of rough microsomes from cysts tended to be lower. An order parameter (S) calculated from spectra for I(12,3) did not change as a function of encystment for the smooth membranes and increased only slightly for rough microsomes. The activation energy (E_a) for Arrhenius plots of τ_c above the inflection temperature increased as a result of encystment, indicating a greater degree of molecular interaction within the cyst membranes. Moreover, the τ_c plots for both rough and smooth microsomal membranes from trophozoites tended to converge at 29 °C, the growth temperature, whereas plots for cyst membranes were virtually parallel, bracketing those for the trophozoite membranes. This suggests that the trophozoite is able to regulate its membrane fluidity and that cysts, which are resting cells, have lost this regulatory capacity.     

Specific Detection of Acanthamoeba species using Polyclonal Peptide Antibody Targeting the Periplasmic Binding Protein of A. castellanii

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.     

The role of a 2-on-2 haemoglobin in oxidative and nitrosative stress resistance of Antarctic Pseudoalteromonas haloplanktis TAC125

The 2-on-2 haemoglobins, previously named truncated, are monomeric, low-molecular weight oxygen-binding proteins that share the overall topology with vertebrate haemoglobins. Although several studies on 2-on-2 haemoglobins have been reported, their physiological and biochemical functions are not yet well defined, and various roles have been suggested. The genome of the psychrophilic Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is endowed with three genes encoding 2-on-2 haemoglobins. To investigate the function played by one of the three trHbs, PhHbO, a PhTAC125 genomic mutant strain was constructed, in which the encoding gene was knocked-out. The mutant strain was grown under controlled conditions and several aspects of bacterium physiology were compared with those of wild-type cells when dissolved oxygen pressure in solution and growth temperature were changed. Interestingly, inactivation of the PhHbO encoding gene makes the mutant bacterial strain sensitive to high solution oxygen pressure, to H₂O₂, and to a nitrosating agent, suggesting the involvement of PhHbO in oxidative and nitrosative stress resistance.