Acanthamoeba castellanii is a facultative pathogen that has a two-stage life cycle comprising the vegetatively growing trophozoite stage and the dormant cyst stage. Cysts are formed when the cell encounters unfavorable conditions, such as environmental stress or food deprivation. Due to their rigid double-layered wall, Acanthamoeba cysts are highly resistant to antiamoebic drugs. This is problematic as cysts can survive initially successful chemotherapeutic treatment and cause relapse of the disease. We studied the Acanthamoeba encystment process by using two-dimensional gel electrophoresis (2DE) and found that most changes in the protein content occur early in the process. Truncated actin isoforms were found to abound in the encysting cell, and the levels of translation elongation factor 2 (EF2) were sharply decreased, indicating that the rate of protein synthesis must be low at this stage. In the advanced stage of encystment, however, EF2 levels and the trophozoite proteome were partly restored. The protease inhibitors PMSF (phenylmethylsulfonyl fluoride) and E64d [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester] inhibited the onset of encystment, whereas the protein synthesis inhibitor cycloheximide was ineffective. Changes in the protein profile, similar to those of encysting cells, could be observed with trophozoite homogenates incubated at room temperature for several hours. Interestingly, these changes could be inhibited significantly by cysteine protease inhibitors but not by inhibitors against other proteases. Taken together, we conclude that the encystment process in A. castellanii is of a bipartite nature consisting of an initial phase of autolysis and protein degradation and an advanced stage of restoration accompanied by the expression of encystment-specific genes.

The free-living but facultatively pathogenic amoebae of the genus Acanthamoeba are frequently infected with bacterial endosymbionts that can have a profound influence on the physiology and viability of their host. Parachlamydia acanthamoebae, a chlamydial endosymbiont in acanthamoebae, is known to be either symbiotic or lytic to its host, depending on the ambient conditions, for example, temperature. Moreover, parachlamydiae can also inhibit the encystment process in Acanthamoeba, an essential survival strategy of their host for the evasion of chemotherapeutic agents, heat, desiccation and radiation. To obtain a more detailed picture of the intracellular interactions of parachlamydiae and acanthamoebae, we studied parachlamydial infection in several Acanthamoeba isolates at the proteomic level by means of two-dimensional gel electrophoresis (2DE) and mass spectrometry. We observed that P. acanthamoebae can infect all three morphological subtypes of the genus Acanthamoeba and that the proteome pattern of released P. acanthamoebae elementary bodies was always practically identical regardless of the Acanthamoeba strain infected. Moreover, by comparing proteome patterns of encysting cells from infected and uninfected Acanthamoeba cultures, it was shown that encystment is blocked by P. acanthamoebae at a very early stage. Finally, on 2D-gels of purified P. acanthamoebae from culture supernatants, a subunit of the NADH-ubiquinone oxidoreductase complex, that is, an enzyme that has been described as an indicator for bacterial virulence was identified by a mass spectrometric and bioinformatic approach.

Abstract. Building communities that are resilient and adaptive to climate change requires the development of education strategies that train community members in higher order thinking skills that can be used to solve complex environmental problems. This study provides an empirical test of hypotheses within social-ecological systems resilience theory that have suggested metacognitive learning strategies could increase resilience thinking skills such as scenarios thinking, systems thinking, and the ability to interpret and apply ecological data in complex problem solving. During a 6-week long ecology unit with 108 seventh-grade students, we taught half the students using standard inquiry teaching methods and the other half using the same method, with the addition of a daily metacognitive learning intervention. We investigated the short-term (after six weeks of intervention) and long-term (1 yr after the intervention ended) effects of the intervention on student's metacognitive ability and resilience thinking skills. Over the long term, we found a modest increase in the metacognitive ability of students who received the daily metacognitive journaling exercise. Interview data suggest that the structured metacognitive practice did most to improve the resilience thinking level of students who had low resilience thinking ability prior to the intervention period. However, the interaction between pre-treatment ability level and the treatment group was not detected in the written assessment data. These data suggest that the metacognitive learning intervention we used can benefit metacognitive ability over the long term, but has limited transferability to resilience thinking skills for most students. We suggest additional instructional practices for implementing metacognitive teaching approaches that could enhance the generalizability of their benefits across resilience thinking skills and student's ability levels.