Acanthamoeba castellanii : growth on human cell layers reactivates attenuated properties after prolonged axenic culture

Abstract

The free-living, but potentially pathogenic, bacteriovorous amoebae of the genus Acanthamoeba can be easily grown axenically in a laboratory culture. This, however, often leads to considerable losses in virulence, and encystment capacity, and to changes in drug susceptibility. We evaluated potential options for a reactivation of a number of physiological properties, attenuated by prolonged axenic laboratory culture, including encystment potential, protease activity, heat resistance, growth rates and drug susceptibility against N-chlorotaurine (NCT). Toward this end, a strain that had been grown axenically for 10 years was repeatedly passaged on human HEp-2 cell monolayers or treated with 5′-azacytidine (AzaC), a methyltransferase inhibitor, and trichostatin A (TSA), a histone deacetylase inhibitor, in order to uplift epigenetic gene regulation. Culture on human cell monolayers resulted in significantly enhanced encystment potentials and protease activities, and higher susceptibility against NCT, whereas the resistance against heat shock was not altered. Treatment with AzaC/TSA resulted in increased encystment rates and protease activities, indicating the participation of epigenetic mechanisms. However, lowered resistances against heat shock indicate that possible stress responses to AzaC/TSA have to be taken into account. Repeated growth on human cell monolayers appears to be a potential method to reactivate attenuated characteristics in Acanthamoeba.

Major Role for Cysteine Proteases during the Early Phase of Acanthamoeba castellanii Encystment

“…However, E64d permeates membranes much more efficiently than E64 and is The prominent role of proteolytic activity during encystment, as described in this study, is in accordance with earlier reports on autophagic processes in encysting A. castellanii (2). This study also provides further support for a causal connection between protease activity and encystment capacity of A. castellanii, a connection which has been indicated by our previously published observation that both characteristics are less marked in aged and axenically grown cultures (11).…”

“…Later in the encystment process, however, the synthesis of encystment-specific proteins is required, including Csp21, serine proteases, glycogen phosphorylase, and cellulose synthase (5,6,14,20,21). It has to be emphasized, though, that defining "later in the encystment process" or "more advanced stage of encystment" in a strictly temporal sense can be misleading because the Acanthamoeba strains used by different research groups do not encyst equally fast (5,6,14,(20)(21)(22), probably due to physiological adaptations to prolonged axenic culture (10,11). Thus, the time points given in this study apply only to strain PAT06 after approximately 2 years of axenic culture.…”

Acanthamoeba strains show reduced temperature tolerance after long-term axenic culture

“…Certain physiologic characteristics may indicate the pathogenic potential of a particular isolate, however, as shown in the current study, most of these are no permanent traits but alter over long-term culture; particularly, when cultured axenically at stable laboratory conditions. Traits that are not needed are lost or at least down-regulated as has recently also been shown by Koehsler et al (2008Koehsler et al ( , 2009 concerning encystment and protease activities.…”

“…Long-term adaptions might be achieved by epigenetic regulation (Koehsler et al 2008(Koehsler et al , 2009). However, multiple animal passages or human HEp-2 cell monolayer passages can restore altered physiological characteristics in Acanthamoeba (Mazur and Hadas 1994;Koehsler et al 2009). Growth rate per se also is a physiological characteristic corresponding to pathogenic potential (Walochnik et al 2000b).…”

Acanthamoeba Activates Macrophages Predominantly through Toll-Like Receptor 4- and MyD88-Dependent Mechanisms To Induce Interleukin-12 (IL-12) and IL-6

“…However, this can lead to the loss of virulence factors, an encystment capability, and reduced susceptibility to drugs (29). Therefore, we compared the ability of the A. castellanii Neff strain, a classical laboratory strain, to that of an A. castellanii strain isolated from a case of .…”