Elastase secretion in Acanthamoeba polyphaga

Acanthamoeba species are frequently isolated from soil and water collections. In the environment, the organisms multiply as phagotrophic trophozoites and encyst under adverse conditions. Several species are known to infect man, causing keratitis and opportunistic diseases. The mechanisms underlying tissue damage and invasion by the amoebae are being elucidated and the involvement of secreted peptidases, particularly serine peptidases, has been demonstrated. Here, elastase activity was examined in Acanthamoeba-conditioned medium (ACM), making use of elastin-Congo red (ECR) and synthetic peptide p-nitroanilide substrates. ACM hydrolysed ECR over a broad pH range and optimally at a pH of 7.5 and above. Indicating the activity of serine and metallopeptidases, Congo red release was potently inhibited by PMSF, antipain, chymostatin and 1,10-phenanthroline, partially reduced by elastatinal and EDTA, and unaffected by 1,7-phenanthroline and E-64. Screening with synthetic substrates mainly showed the activity of serine peptidases. ACM efficiently hydrolysed Suc-Ala(2)-Pro-Leu-pNA and Suc-Ala(2)-Pro-Phe-pNA over a broad pH range (7.0-9.5) and was weakly active against Suc-Ala(3)-pNA, a substrate found to be optimally hydrolysed at a pH around 7.0. Following ammonium sulfate precipitation of ACM proteins and FPLC analysis, the majority of the ECR-splitting activity, characterised as serine peptidases, bound to CM-sepharose and co-eluted with part of the Suc-Ala(2)-Pro-Phe-pNA-hyd to lysing activity in a gradient of 0-0.6 M NaCl. In the corresponding FPLC fractions, serine peptidases resolving in the region of 70-130 kDa were detected in gelatin gels. Overall, the results demonstrate that trophozoites secrete elastases, and additionally suggest the high molecular weight serine peptidases as possible elastase candidates. (C) 2009 Elsevier B.V. All rights reserved.

Serine-like proteolytic enzymes correlated with differential pathogenicity in patients with acute Acanthamoeba keratitis

P>Acute ocular infection due to free-living amoebae of the genus Acanthamoeba is characterized by severe pain, loss of corneal transparency and, eventually, blindness. Proteolytic enzymes secreted by trophozoites of virulent Acanthamoeba strains have an essential role in the mechanisms of pathogenesis, including adhesion, invasion and destruction of the corneal stroma. In this study, we analysed the relationship between the extracellular proteases secreted by clinical isolates of Acanthamoeba and the clinical manifestations and severity of disease that they caused. Clinical isolates were obtained from patients who showed typical symptoms of Acanthamoeba keratitis. Trophozoites were cultivated axenically, and extracellular proteins were collected from cell culture supernatants. Secreted enzymes were partially characterized by gelatin and collagen zymography. Acanthamoeba trophozoites secreted proteases with different molecular masses, proteolysis rates and substrate specificities, mostly serine-like proteases. Different enzymatic patterns of collagenases were observed, varying between single and multiple collagenolytic activities. Low molecular weight serine proteases were secreted by trophozoites associated with worse clinical manifestations. Consequently, proteolytic enzymes of some Acanthamoeba trophozoites could be related to the degree of their virulence and clinical manifestations of disease in the human cornea.

Growth, encystment and survival of Acanthamoeba castellanii grazing on different bacteria

Free-living amoebae of the genus Acanthamoeba are widely distributed in soil and water collections, where trophozoites (vegetative, multiplicative stages) feed mainly by phagocytosis and thus control bacterial populations in the environment. Here, we examined the growth, encystment and survival of Acanthamoeba castellanii receiving different bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Bacillus subtilis, Bacillus megaterium, Micrococcus luteus, and Staphylococcus aureus) in nonnutrient saline. All bacteria assayed induced a dose-dependent proliferative response, in most cases maximized with a bacterial dose of 1 x 10(9) mL(-1); except for M. luteus, trophozoites grew better with viable than with heat-killed bacteria. In addition, Acanthamoeba growth was improved by adding bacteria on alternate days. Single-dose experiments indicated a temporal association between the growth of trophozoite and bacterial consumption, and higher consumption of M. luteus, E. coli and P. aeruginosa, bacterial species that allowed the highest trophozoite yields. Long-term Acanthamoeba-bacteria incubation revealed that encystment was significantly delayed by almost all the bacteria assayed (including S. aureus, which elicited a poor growth response) and that the presence of bacteria markedly increased cyst yield; final cyst recovery clearly depended on both the dose and the type of the bacterium given, being much higher with E. coli, M. luteus and P. aeruginosa.

Variação sazonal da comunidade protozooplanctônica em um ambiente oligotrófico tropical (reservatório de Ilha Solteira, Brasil)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Revisiting the Acanthamoeba species that form star-shaped cysts (genotypes T7, T8, T9, and T17): characterization of seven new Brazilian environmental isolates and phylogenetic inferences

Free-living amoebae of the genus Acanthamoeba are the agents of both opportunistic and non-opportunistic infections and are frequently isolated from the environment. Of the 17 genotypes (T1-T17) identified thus far, 4 (T7, T8, T9, and T17) accommodate the rarely investigated species of morphological group I, those that form large, star-shaped cysts. We report the isolation and characterization of 7 new Brazilian environmental Acanthamoeba isolates, all assigned to group I. Phylogenetic analyses based on partial (similar to 1200 bp) SSU rRNA gene sequences placed the new isolates in the robustly supported clade composed of the species of morphological group I. One of the Brazilian isolates is closely related to A. comandoni (genotype T9), while the other 6, together with 2 isolates recently assigned to genotype T17, form a homogeneous, well-supported group (2-0% sequence divergence) that likely represents a new Acanthamoeba species. Thermotolerance, osmotolerance, and cytophatic effects, features often associated with pathogenic potential, were also examined. The results indicated that all 7 Brazilian isolates grow at temperatures up to 40 degrees C, and resist under hvperosmotic conditions. Additionally, media conditioned by each of the new Acanthamoeba isolates induced the disruption of SIRC and HeLa cell monolayers.

Simkania negevensis: clinical and laboratory studies in the population of hemodialized and kidney transplanted patients.

Simkania negevensis is a bacterium belonging to the order Chlamydiales but with certain biological characteristics different from those of chlamydia, according to which it was classified in the family Simkaniaceae. It is widespread in the environment, due to its ability to survive in amoebae also in phase cystic, for which it was hypothesized a possible transmission after contact with water in which they are present amoebae. So far it is known its role in diseases of the lower respiratory tract, such as childhood bronchiolitis and pneumonia in adults of the community, following its transmission through infected aerosols. A recent American study showed, by PCR, a high prevalence of S. negevensis in patients with lung transplant than other transplant recipients, assuming an association between the presence of the bacterium in these patients, and transplant rejection, were more frequent in lung transplant recipients infected compared to uninfected. There are no data so far analyzed in Italy relative to the population of dialysis and kidney transplant recipients relative to simkania negevensis why this study was undertaken in order to start a specific location and evaluate the scientific implications. Because its ability to assume persistent forms of infection, which may lead to a prolonged inflammatory response, Simkania negevensis, similar to other persistent bacteria or viruses, may be ivolved in pathologic complication. Sn may be a factor in graft rejection in mmunesuppressed lung transplant recipients, and further studies are planned to explore the posible association of Sn infections with various in vivo pathologies.

Alternative host model to evaluate Aeromonas virulence

Bacterial virulence can only be assessed by confronting bacteria with a host. Here, we present a new simple assay to evaluate Aeromonas virulence, making use of Dictyostelium amoebae as an alternative host model. This assay can be modulated to assess virulence of very different Aeromonas species.

Potentially human pathogenic Acanthamoeba isolated from a heated indoor swimming pool in Switzerland

Some free-living amoebae, including some species of the genus Acanthamoeba, can cause infections in humans and animals. These organisms are known to cause granulomatous amebic encephalitis (GAE) in predominantly immune-deficient persons. In the present study, we isolated a potentially human pathogenic Acanthamoeba isolate originating from a public heated indoor swimming pool in Switzerland. The amoebae, thermophilically preselected by culture at 37 degrees C, subsequently displayed a high thermotolerance, being able to grow at 42 degrees C, and a marked cytotoxicity, based on a co-culture system using the murine cell line L929. Intranasal infection of Rag2-immunodeficient mice resulted in the death of all animals within 24 days. Histopathology of brains and lungs revealed marked tissue necrosis and hemorrhagic lesions going along with massive proliferation of amoebae. PCR and sequence analysis, based on 18S rDNA, identified the agent as Acanthamoeba lenticulata. In summary, the present study reports on an Acanthamoeba isolate from a heated swimming pool suggestive of being potentially pathogenic to immunocompromised persons.

Palaeohydrology, fires and vegetation succession in the southern Baltic during the last 7500 years reconstructed from a raised bog based on multi-proxy data

We present the first 7500 yr long multi-proxy record from a raised bog located at the southern Baltic coast, Poland. Testate amoebae, plant macrofossils, pollen and microscopic charcoal were used to reconstruct environmental changes in Pomerania (northern Poland, Kaszuby Lakeland) from a 7-m thick peat archive of Stążki bog dated 5500 BC–AD 1250. We obtained a record of proxies representing different spatial scales: regional vegetation changed simultaneously with local vegetation, and testate amoebae showed a pattern of change similar to that of pollen and plant macrofossils. On the basis of the combined proxies, we distinguished three hydroclimatic stages: moist conditions 5500–3450 BC, drier conditions with regionally increased fires up to 600 BC, and again moist conditions from 600 BC onward. During the drier interval, a first climatic shift to wetter conditions at 1700 BC is indicated by regional pollen as the replacement of Corylus by Carpinus, and locally by, e.g., the increase of Hyalosphenia elegans and mire plants such as Sphagnum sec. Cuspidata. Furthermore, we observed a correlation since 600 BC among the re-expansion of Carpinus (after a sudden decline ca. 950 BC), increased peat accumulation, increase of Hyalosphenia species, and fewer fires, suggesting lower evapotranspiration and a stable high water table in the bog. Fagus started to expand after AD 810 gradually replacing Carpinus, which was possibly due to a gradually more oceanic climate, though we cannot exclude human impact on the forests. Peat accumulation, determined by radiocarbon dating, varied with bog surface wetness. The hydroclimatic phases found in Stążki peatland are similar to moisture changes recorded in other sites from Poland and Europe. This is the first detailed record of hydroclimatic change during the Holocene in the southern Baltic region, so it forms a reference site for further studies on other southern Baltic bogs that are in progress.

Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri

Species in the genus Naegleria are free-living amoebae of the soil and warm fresh water. Although around 30 species have been recognized, Naegleria fowleri is the only one that causes primary amoebic meningoencephalitis (PAM) in humans. PAM is an acute and fast progressing disease affecting the central nervous system. Most of the patients die within 1-2 weeks of exposure to the infectious water source. The fact that N. fowleri causes such fast progressing and highly lethal infections has opened many questions regarding the relevant pathogenicity factors of the amoeba. In order to investigate the pathogenesis of N. fowleri under defined experimental conditions, we developed a novel high- versus low-pathogenicity model for this pathogen. We showed that the composition of the axenic growth media influenced growth behaviour and morphology, as well as in vitro cytotoxicity and in vivo pathogenicity of N. fowleri. Trophozoites maintained in Nelson's medium were highly pathogenic for mice, demonstrated rapid in vitro proliferation, characteristic expression of surface membrane vesicles and a small cell diameter, and killed target mouse fibroblasts by both contact-dependent and -independent destruction. In contrast, N. fowleri cultured in PYNFH medium exhibited a low pathogenicity, slower growth, increased cell size and contact-dependent target cell destruction. However, cultivation of the amoeba in PYNFH medium supplemented with liver hydrolysate (LH) resulted in trophozoites that were highly pathogenic in mice, and demonstrated an intermediate proliferation rate in vitro, diminished cell diameter and contact-dependent target cell destruction. Thus, in this model, the presence of LH resulted in increased proliferation of trophozoites in vitro and enhanced pathogenicity of N. fowleri in mice. However, neither in vitro cytotoxicity mechanisms nor the presence of membrane vesicles on the surface correlated with the pathologic potential of the amoeba. This indicated that the pathogenicity of N. fowleri remains a complex interaction between as-yet-unidentified cellular mechanisms.

Determinants of Acidobacteria activity inferred from the relative abundances of 16S rRNA transcripts in German grassland and forest soils

16S rRNA genes and transcripts of Acidobacteria were investigated in 57 grassland and forest soils of three different geographic regions. Acidobacteria contributed 9-31% of bacterial 16S rRNA genes whereas the relative abundances of the respective transcripts were 4-16%. The specific cellular 16S rRNA content (determined as molar ratio of rRNA:rRNA genes) ranged between 3 and 80, indicating a low in situ growth rate. Correlations with flagellate numbers, vascular plant diversity and soil respiration suggest that biotic interactions are important determinants of Acidobacteria 16S rRNA transcript abundances in soils. While the phylogenetic composition of Acidobacteria differed significantly between grassland and forest soils, high throughput denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism fingerprinting detected 16S rRNA transcripts of most phylotypes in situ. Partial least squares regression suggested that chemical soil conditions such as pH, total nitrogen, C:N ratio, ammonia concentrations and total phosphorus affect the composition of this active fraction of Acidobacteria. Transcript abundance for individual Acidobacteria phylotypes was found to correlate with particular physicochemical (pH, temperature, nitrogen or phosphorus) and, most notably, biological parameters (respiration rates, abundances of ciliates or amoebae, vascular plant diversity), providing culture-independent evidence for a distinct niche specialization of different Acidobacteria even from the same subdivision.

A 13,600-year diatom oxygen isotope record from the South Carpathians (Romania): Reflection of winter conditions and possible links with North Atlantic circulation changes

This study provides a continuous lateglacial and Holocene record of diatom silica oxygen isotope changes (delta O-18(DIAT)) in a subalpine lake sediment sequence obtained from the Retezat Mts (Taul dintre Brazi, 1740 m a.s.l.). This through-flow, shallow, high-altitude lake with a surface area of only 0.4 ha has short water residence time and is predominantly fed by snowmelt and rainwater. Its delta O-18(DIAT) record principally reflects the oxygen isotope composition of the winter and spring precipitation, as diatom blooms occur mainly in the spring and early summer. Hence, changes in delta O-18(DIAT) are interpreted as seasonal scale changes: in the amount of winter precipitation. Low oxygen isotope values (27-28.5 parts per thousand) occurred during the lateglacial until 12,300 cal BP, followed by a sharp increase thereafter. In the Holocene delta O-18(DIAT) values ranged from 29 to 31 parts per thousand until 3200 cal BP, followed by generally lower values during the late Holocene (27-30 parts per thousand). Short-term decreases in the isotopic values were found between 10,140-9570, 9000-8500, 7800-7300, 6300-5800, 5500-5000 and at 8015, 4400, 4000 cal BP. After 3200 cal BP a decreasing trend was visible with the lowest values between 3100-2500 and after 2100 cal BP The general trend in the record suggests that contribution of winter precipitation was generally lower between 11,680 and 3200 cal BP, followed by increased contribution during the last millennia. The late Holocene decrease in delta O-18(DIAT) shows good agreement with the speleothem delta O-18, lake level and testate amoebae records from the Carpathian Mountains that also display gradual delta O-18 decrease and lake level/mire water table level rise after 3200 cal BR Strong positive correlation with North Atlantic circulation and solar activity proxies, such as the Austrian and Hungarian speleothem records, furthermore suggested that short-term increases in the isotopic ratios in the early and mid Holocene are likely connectable to high solar activity phases and high frequency of positive North Atlantic Oscillation indexes that may have resulted in decreased winter precipitation in this region.

Palaeoenvironmental changes in Central Europe (NE Poland) during the last 6200 years reconstructed from a high-resolution multi-proxy peat archive

Peat deposits from an ombrotrophic bog (north-eastern Poland) were analysed to reconstruct peatland development and environmental changes. This paper presents reconstructions of hydrological changes and plant succession over the last 6000 years. The methods included the high-resolution analysis of plant macrofossils, pollen and testate amoebae, supported by radiocarbon dating. Three main phases were identified in the history of the bog and surrounding woodland vegetation: 4000–400 BC, 400 BC–AD 1700 and AD 1700–2011. Except for terrestrialisation and the fen-to-bog transition phase, the development of bog vegetation was mainly dependent on the climate until approximately AD 1700. The dominant taxon in Gązwa bog was Sphagnum fuscum/rubellum. Woodland development was significantly affected by human activity at several time periods. The most visible human activity, manifested by the decline of deciduous species, occurred ca. 350 BC, ca. AD 250, ca. AD 1350 and after AD 1700. These events correspond to phases of human settlement in the area. During 400–300 BC, the decline of deciduous trees, primarily Carpinus, coincided with an increase in indicators of human activity and fire frequency. At ca. AD 200, Carpinus and Tilia abundance decreased, corresponding to an increased importance of cereals (Secale and Triticum). Since ca. AD 1350, the impact of Teutonic settlement is apparent, and after AD 1700, deciduous forests largely disappeared.

Epitheliocystis and the importance of Chlamydia-related bacteria on fish health

This book offers unique coverage of all presently known amoeba-resistant microorganisms and their significance in the study of infectious diseases. It highlights the role of free-living amoebae as a widespread evolutionary crib for the development of virulence traits in resistant microbes, including the ability of intracellular bacteria to survive to other phagocytic cells such as human macrophages. The emphasis is on public health risks associated with the presence in drinking water of intra-amoebal bacteria as well as the ecology and pathogenic role of amoebae-resisting bacteria as new emerging human pathogens

Long-term hydrological dynamics and fire history over the last 2000 years in CE Europe reconstructed from a high-resolution peat archive

Sphagnum peatlands in the oceanic-continental transition zone of Poland are currently influenced by climatic and anthropogenic factors that lead to peat desiccation and susceptibility to fire. Little is known about the response of Sphagnum peatland testate amoebae (TA) to the combined effects of drought and fire. To understand the relationships between hydrology and fire dynamics, we used high-resolution multi-proxy palaeoecological data to reconstruct 2000 years of mire history in northern Poland. We employed a new approach for Polish peatlands – joint TA-based water table depth and charcoal-inferred fire activity reconstructions. In addition, the response of most abundant TA hydrological indicators to charcoal-inferred fire activity was assessed. The results show four hydrological stages of peatland development: moderately wet (from ∼35 BC to 800 AD), wet (from ∼800 to 1390 AD), dry (from ∼1390 to 1700 AD) and with an instable water table (from ∼1700 to 2012 AD). Fire activity has increased in the last millennium after constant human presence in the mire surroundings. Higher fire activity caused a rise in the water table, but later an abrupt drought appeared at the onset of the Little Ice Age. This dry phase is characterized by high ash contents and high charcoal-inferred fire activity. Fires preceded hydrological change and the response of TA to fire was indirect. Peatland drying and hydrological instability was connected with TA community changes from wet (dominance of Archerella flavum, Hyalosphenia papilio, Amphitrema wrightianum) to dry (dominance of Cryptodifflugia oviformis, Euglypha rotunda); however, no clear fire indicator species was found. Anthropogenic activities can increase peat fires and cause substantial hydrology changes. Our data suggest that increased human fire activity was one of the main factors that influenced peatland hydrology, though the mire response through hydrological changes towards drier conditions was delayed in relation to the surrounding vegetation changes.