Random encounters and amoeba locomotion drive the predation of Listeria monocytogenes by Acanthamoeba castellanii.

Predatory protozoa play an essential role in shaping microbial populations. Among these protozoa, Acanthamoeba are ubiquitous in the soil and aqueous environments inhabited by Listeria monocytogenes. Observations of predator-prey interactions between these two microorganisms revealed a predation strategy in which Acanthamoeba castellanii assemble L. monocytogenes in aggregates, termed backpacks, on their posterior. The rapid formation and specific location of backpacks led to the assumption that A. castellanii may recruit L. monocytogenes by releasing an attractant. However, this hypothesis has not been validated, and the mechanisms driving this process remained unknown. Here, we combined video microscopy, microfluidics, single-cell image analyses, and theoretical modeling to characterize predator-prey interactions of A. castellanii and L. monocytogenes and determined whether bacterial chemotaxis contributes to the backpack formation. Our results indicate that L. monocytogenes captures are not driven by chemotaxis. Instead, random encounters of bacteria with amoebae initialize bacterial capture and aggregation. This is supported by the strong correlation between experimentally derived capture rates and theoretical encounter models at the single-cell level. Observations of the spatial rearrangement of L. monocytogenes trapped by A. castellanii revealed that bacterial aggregation into backpacks is mainly driven by amoeboid locomotion. Overall, we show that two nonspecific, independent mechanisms, namely random encounters enhanced by bacterial motility and predator surface-bound locomotion, drive backpack formation, resulting in a bacterial aggregate on the amoeba ready for phagocytosis. Due to the prevalence of these two processes in the environment, we expect this strategy to be widespread among amoebae, contributing to their effectiveness as predators.

Homeostasis of cellular amino acids in Acanthamoeba castellanii exposed to different media under amoeba-bacteria coculture conditions.

BACKGROUND: Acanthamoeba castellanii is a free-living protist that feeds on diverse bacteria. A. castellanii has frequently been utilized in studies on microbial interactions. Grazing bacteria also exhibit diverse effects on the physiological characteristics of amoebae, such as their growth, encystation, and cytotoxicity. Since the composition of amoebae amino acids is closely related to cellular activities, it can indicate the overall responses of A. castellanii to various stimuli. METHOD: A. castellanii was exposed to different culture conditions in low-nutrient medium with heat-killed DH5α to clarify their effects. A targeted metabolomic technique was utilized to evaluate the concentration of cellular amino acids. The amino acid composition and pathways were analyzed by two web-based tools: MetaboAnalyst and Pathview. Then, long-term exposure to A. castellanii was investigated through in silico and in vitro methods to elucidate the homeostasis of amino acids and the growth of A. castellanii. RESULTS: Under short-term exposure, all kinds of amino acids were enriched in all exposed groups. In contrast to the presence of heat-killed bacteria, the medium exhibited obvious effects on the amino acid composition of A. castellanii. After long-term exposure, the amino acid composition was more similar to that of the control group. A. castellanii may achieve amino acid homeostasis through pathways related to alanine, aspartate, citrulline, and serine. DISCUSSION: Under short-term exposure, compared to the presence of bacteria, the type of medium exerted a more powerful effect on the amino acid composition of the amoeba. Previous studies focused on the interaction of the amoeba and bacteria with effective secretion systems and effectors. This may have caused the effects of low-nutrient environments to be overlooked. CONCLUSION: When A. castellanii was stimulated in the coculture system through various methods, such as the presence of bacteria and a low-nutrient environment, it accumulated intracellular amino acids within a short period. However, different stimulations correspond to different amino acid compositions. After long-term exposure, A. castellanii achieved an amino acid equilibrium by downregulating the biosynthesis of several amino acids.

Potential role of Acanthamoeba Rab7.

Acanthamoeba castellanii is a free-living protozoan that causes several severe human parasitic diseases such as Acanthamoeba keratitis and granulomatous encephalitis. A. castellanii feeds on bacteria, yeasts, and other organic particles as food sources, but the mechanisms of digestion in acanthamoebal cells are unclear. Rab GTPases participate in endosomal delivery in eukaryotes after phagocytosis. This study aimed to determine the potential functions of A. castellanii Rab7 (AcRab7), which is involved in phagocytosis, and the relationship between AcRab7 and further cellular physiological phenomena. In this study, the inhibitor CID1067700 (CID) was used to specifically inhibit the binding of nucleotides to confirm the potential functions of AcRab7. Cellular proliferation and ATP assays were also used to detect underlying cellular physiological functions after blocking the phagocytosis pathway. We found that AcRab7 expression increased as the co-culture time with Escherichia coli increased. Immunofluorescence staining showed that AcRab7 colocalized with lysosomes in its GTP-activating form. In addition, AcRab7 inhibition resulted in a reduction in cell proliferation and ATP levels. Our results suggest that AcRab7 participates in endosomal delivery and dominates energy production and cell growth.

The effect of polyhydroxyalkanoates in Pseudomonas chlororaphis PA23 biofilm formation, stress endurance, and interaction with the protozoan predator Acanthamoeba castellanii.

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola against the fungal pathogen Sclerotinia sclerotiorum. In addition to producing antifungal compounds, this bacterium synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds. Because the role of PHA in PA23 physiology is currently unknown, we investigated the impact of this polymer on stress resistance, adherence to surfaces, and interaction with the protozoan predator Acanthamoeba castellanii. Three PHA biosynthesis mutants were created, PA23phaC1, PA23phaC1ZC2, and PA23phaC1ZC2D, which accumulated reduced PHA. Our phenotypic assays revealed that PA23phaC1ZC2D produced less phenazine (PHZ) compared with the wild type (WT) and the phaC1 and phaC1ZC2 mutants. All three mutants exhibited enhanced sensitivity to UV irradiation, starvation, heat stress, cold stress, and hydrogen peroxide. Moreover, motility, exopolysaccharide production, biofilm formation, and root attachment were increased in strains with reduced PHA levels. Interaction studies with the amoeba A. castellanii revealed that the WT and the phaC1 and phaC1ZC2 mutants were consumed less than the phaC1ZC2D mutant, likely due to decreased PHZ production by the latter. Collectively these findings indicate that PHA accumulation enhances PA23 resistance to a number of stresses in vitro, which could improve the environmental fitness of this bacterium in hostile environments.

Type 1 Fimbriae and Motility Play a Pivotal Role During Interactions of Salmonella typhimurium with Acanthamoeba castellanii (T4 Genotype).

Amoebic bacterial interactions are the most ancient form of host pathogen interactions. Here, we investigate the fate of Salmonella typhimurium and Acanthamoeba castellanii T4 genotype upon mutual interactions in a nutrition free environment. The role of type 1 fimbriae and motility of S. typhimurium during interactions with A. castellanii has also been investigated. Deletion of genes encoding the type 1 fimbriae subunit FimA, type 1 fimbriae tip protein FimH, chemotaxis regulatory proteins CheA and CheY and major flagella subunits FliC and FljB was performed through homologous recombination. In vitro association, invasion and survival assays of S. typhimurium wild-type and mutant strains were performed upon co-incubation of bacteria with A. castellanii trophozoites in a nutrition free environment. The deletion gene encoding type 1 fimbriae subunit FimA reduced, whereas the deletion of genes encoding flagella subunits FliC and FljB of flagella enhanced the association capability of S. typhimurium with A. castellanii. Invasion of A. castellanii by Salmonella was significantly reduced upon the loss of type 1 fimbriae subunit FimA and type 1 fimbriae tip protein FimH. Co-incubation of S. typhimurium with A. castellanii in phosphate buffered saline medium stimulated the growth of S. typhimurium wild-type and mutant strains. Viable A. castellanii trophozoites count became significantly reduced upon co-incubation with S. typhimurium within 48 h. Type 1 fimbriae play a pivotal role in the adherence of S. typhimurium to the A. castellanii cell surface. Subsequently, this interaction provides S. typhimurium an advantage in growth.

Effects of Acanthamoeba castellanii on the dissolved oxygen and the microbial community under the experimental aquatic model.

Acanthamoeba castellanii is a protist that has a high predation efficiency for bacteria in a number of monoxenic culture experiments. However, the role of A. castellanii in the microbial community is still unknown because of the lack of studies on multiple-species interactions. The aim of this study was to investigate the change of bacterial composition after A. castellanii emerges in a water environment. We added A. castellanii to an environmental water sample and incubated it for two days. Then, we performed 16S ribosomal RNA sequencing techniques to analyze the changes in bacterial composition. In this study, A. castellanii slightly increased the relative abundance of a few opportunistic pathogens, such as Legionella, Roseomonas, and Haemophilus. This result may be related to the training ground hypothesis. On the other hand, the growth of some bacteria was inhibited, such as Cyanobacteria and Firmicutes. Although A. castellanii did not drastically change the whole bacterial community, we surprisingly found the dissolved oxygen concentration was increased after incubation with A. castellanii. We applied environmental water at the laboratory scale to investigate the interactions among A. castellanii, complex microbial communities and the environment. We identified the bacteria that are sensitive to A. castellanii and further found the novel relationship between dissolved oxygen and microbial interaction. Our results helped to clarify the role of A. castellanii in microbial communities.

The interaction between Sporothrix schenckii sensu stricto and Sporothrix brasiliensis with Acanthamoeba castellanii.

BACKGROUND: Sporotrichosis is a group of zoonotic subcutaneous mycoses, found worldwide and caused by fungi belonging to the genus Sporothrix. Protozoans of the genus Acanthamoeba are widely distributed, and some species may be pathogenic and/or opportunistic. These organisms coexist in the same environment and may interact. OBJECTIVES: This study determined the profile of interactions of S schenckii sensu stricto and S brasiliensis with A castellanii, using an in vitro co-culture model to evaluate the intrinsic characteristics of the two Sporothrix species and A castellanii. METHODS: We compared the rate of phagocytosis of S schenckii sensu stricto and S brasiliensis by A castellanii; the viability of S schenckii sensu stricto and S brasiliensis after contact with A castellanii; the viability of the amoeba after contact with a fungal species; and the influence of S schenckii sensu stricto and S brasiliensis on the encystment process of A castellanii. RESULTS: The analyses indicated that A castellanii phagocytised both S schenckii and S brasiliensis, with significantly more S schenckii than S brasiliensis in the first two hours of contact. Our results showed a significant increase in conidia and hyphae count after 72 hours of co-culture of A castellanii with S brasiliensis, and the amoebae lysed after they ingested the fungi, indicating that the fungi probably used the amoebae as a source of nutrition. CONCLUSIONS: Our results were obtained in vitro and these organisms may not behave similarly in vivo; in vivo studies of co-infections are necessary in order to gain a thorough understanding of this relationship.

Acanthamoeba castellanii as an alternative interaction model for the dermatophyte Trichophyton rubrum.

BACKGROUND: Trichophyton rubrum (Tr) is the main aetiological agent of human dermatophytosis, being isolated from the environment and keratinised tissues. In the environment, Tr can interact with other organisms, such as free-living amoebas (FLA), which can act as an alternative host system to study the interaction between microbes and phagocytic cells. OBJECTIVES: To characterise the Acanthamoeba castellanii (ALX)-Tr interaction. METHODS: Interaction was characterised in three conditions: trophozoites (PYG), late (PYG/NES) and early (NES) encystation stimulus, evaluating encystation kinetics, phagocytosis, exocytosis and fungicidal activity dynamics. RESULTS: Tr was able to induce ALX encystation and be internalised by ALX. The number of internalised conidia was high at 1 hour, and ALX presented fungicidal activity with increased intracellular ROS production and exocytosis. In PYG/NES, phagocytosis and ROS production were reduced, with decreased ALX's fungicidal activity. However, in NES there was an increased fungal engulfment, and a reduced ROS production and higher fungal burden. Furthermore, exogenous mannose decreased phagocytosis of Tr conidia, and divalent cations induced ROS production and increased ALX's fungicidal activity. Interestingly, phagocytosis was reduced in the presence of cytoskeleton inhibitor, but exocytosis was increased, suggesting that Tr conidia may have alternative pathways to escape ALX's cells. CONCLUSION: A castellanii is a proper model for studying Tr-FLA interaction, since ALX can engulf, produce ROS and kill Tr, and all these parameters are influenced by an encystation stimulus and divalent cations. Moreover, this interaction is likely to occur in the environment implicating in the adaptation to environmental stressful conditions in both organisms.

Transient superdiffusion of polydisperse vacuoles in highly motile amoeboid cells.

We perform a detailed statistical analysis of diffusive trajectories of membrane-enclosed vesicles (vacuoles) in the supercrowded cytoplasm of living Acanthamoeba castellanii cells. From the vacuole traces recorded in the center-of-area frame of moving amoebae, we examine the statistics of the time-averaged mean-squared displacements of vacuoles, their generalized diffusion coefficients and anomalous scaling exponents, the ergodicity breaking parameter, the non-Gaussian features of displacement distributions of vacuoles, the displacement autocorrelation function, as well as the distributions of speeds and positions of vacuoles inside the amoeba cells. Our findings deliver novel insights into the internal dynamics of cellular structures in these infectious pathogens.

Cytopathic Change and Inflammatory Response of Human Corneal Epithelial Cells Induced by Acanthamoeba castellanii Trophozoites and Cysts.

Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.

Yersinia pestis Resists Predation by Acanthamoeba castellanii and Exhibits Prolonged Intracellular Survival.

Plague is a flea-borne rodent-associated zoonotic disease caused by Yersinia pestis The disease is characterized by epizootics with high rodent mortalities, punctuated by interepizootic periods when the bacterium persists in an unknown reservoir. This study investigates the interaction between Y. pestis and the ubiquitous soil free-living amoeba (FLA) Acanthamoeba castellanii to assess if the bacterium can survive within soil amoebae and whether intracellular mechanisms are conserved between infection of mammalian macrophages and soil amoebae. The results demonstrate that during coculture with amoebae, representative Y. pestis strains of epidemic biovars Medievalis, Orientalis, and Antiqua are phagocytized and able to survive within amoebae for at least 5 days. Key Y. pestis determinants of the intracellular interaction of Y. pestis and phagocytic macrophages, PhoP and the type three secretion system (T3SS), were then tested for their roles in the Y. pestis-amoeba interaction. Consistent with a requirement for the PhoP transcriptional activator in the intracellular survival of Y. pestis in macrophages, a PhoP mutant is unable to survive when cocultured with amoebae. Additionally, induction of the T3SS blocks phagocytic uptake of Y. pestis by amoebae, similar to that which occurs during macrophage infection. Electron microscopy revealed that in A. castellanii, Y. pestis resides intact within spacious vacuoles which were characterized using lysosomal trackers as being separated from the lysosomal compartment. This evidence for prolonged survival and subversion of intracellular digestion of Y. pestis within FLA suggests that protozoa may serve as a protective soil reservoir for Y. pestisIMPORTANCEYersinia pestis is a reemerging flea-borne zoonotic disease. Sylvatic plague cycles are characterized by an epizootic period during which the disease spreads rapidly, causing high rodent mortality, and an interepizootic period when the bacterium quiescently persists in an unknown reservoir. An understanding of the ecology of Y. pestis in the context of its persistence in the environment and its reactivation to initiate a new epizootic cycle is key to implementing novel surveillance strategies to more effectively predict and prevent new disease outbreaks. Here, we demonstrate prolonged survival and subversion of intracellular digestion of Y. pestis within a soil free-living amoeba. This suggests the potential role for protozoa as a protective soil reservoir for Y. pestis, which may help explain the recrudescence of plague epizootics.

Legionella pneumophila decreases velocity of Acanthamoeba castellanii.

Acanthamoeba castellanii is a free-living amoeba commonly found in aquatic environment. It feeds on bacteria even if some bacteria resist amoebal digestion. Thus, A. castellanii is described as a Trojan horse able to harbor pathogenic bacteria. L. pneumophila is one of the amoeba-resisting bacteria able to avoid host degradation by phagocytosis and to multiply inside the amoeba. When infecting its host, L. pneumophila injects hundreds of effectors via a type IV secretion system that change physiology of the amoeba to its profit. In this study, we assess mobility of A. castellanii upon infection with L. pneumophila. Electron-microscopy analysis of amoebae revealed a reduction of acanthopodia on cells infected with L. pneumophila. Analysis of velocity showed that migration of A. castellanii infected with L. pneumophila was significantly impaired compare to uninfected cells. Taken together, infection with L. pneumophila could prevent formation of cytoplasmic extensions such as acanthopodia with consequences on the shape, adherence and mobility of A. castellanii.

Acanthamoeba castellanii interactions with Streptococcus pneumoniae and Streptococcus pyogenes.

Among the genus Streptococcus, S. pyogenes and S. pneumoniae are the major causes of pharyngitis, impetigo, pneumonia and meningitis in humans. Streptococcus spp. are facultative anaerobes that are nutritionally fastidious, yet survive in the environment and target the predisposed population. Antibacterial disinfectants have been partially effective only, indicating the need for novel preventative measures and to understand mechanisms of bacterial resistance. Acanthamoeba is a free-living protist that is known to harbour microbial pathogens, provide shelter, and assist in their transmission to susceptible population. The overall aim of this study was to determine whether S. pyogenes and S. pneumoniae can interact with A. castellanii by associating, invading, and surviving inside trophozoites and cysts. It was observed that both S. pyogenes and S. pneumoniae were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. Notably, S. pyogenes and S. pneumoniae survived the encystation process, avoided phagocytosis, multiplied, and exhibited higher recovery from the mature cysts, compared with the trophozoite stage (approximately 2 bacteria per amoebae ratio for cyst stage versus 0.02 bacteria per amoeba ration for trophozoite stage). As Acanthamoeba cysts are resilient and can disperse through the air, A. castellanii can act as a vector in providing shelter, facilitating growth and possibly genetic exchanges. In addition, these interactions may contribute to S. pyogenes and S. pneumoniae survival in harsh environments, and transmission to susceptible population and possibly affecting their virulence. Future studies will determine the molecular mechanisms associated with Acanthamoeba interactions with Streptococcus and the evolution of pathogenic bacteria and in turn expedite the discovery of novel therapeutic and/or preventative measures.

In vitro interactions of Acanthamoeba castellanii Neff and Vibrio harveyi.

Free-living amoebae (FLA) are opportunistic protozoa widely distributed in the environment. They are frequently found in water and soil samples, but they have also been reported to be associated with bacterial human pathogens such as Legionella spp. Campylobacter spp or Vibrio cholerae among others. Including within Vibrio spp. V. harveyi (Johnson and Shunk, 1936) is a bioluminescent marine bacteria which has been found swimming freely in tropical marine waters, being part of the stomach and intestine microflora of marine animals, and as both a primary and opportunistic pathogen of marine animals. Our aim was to study the interactions between Vibrio harveyi and Acanthamoeba castellanii Neff. Firstly, in order to analyze changes in it cultivability, V. harveyi was coincubated with A. castellanii Neff axenic culture and with Acanthamoeba Conditioned Medium (ACM) at different temperatures in aerobic conditions. Interestingly, at 4 °C and 18-20 °C bacteria were still cultivable in marine agar, at 28 °C, in aerobic conditions, but there weren't significant differences comparing with the controls. We also noted an enhanced migration of Acanthamoeba toward V. harveyi on non-nutrient agar plates compared to controls with no bacteria.

Variation in Campylobacter jejuni culturability in presence of Acanthamoeba castellanii Neff.

Free-living amoebae (FLA) are protozoa that are widely distributed in the environment mainly in water and soil related habitats. These amoebae have also been reported to be associated with some bacterial pathogens for humans such as Campylobacter spp. The species C. jejuni is the causative agent of about 90% of human campylobacteriosis cases worldwide and this disease may even end up in severe autoimmune sequelae as Guillain-Barré syndrome (GBS). In this study, the interactions between the strain Acanthamoeba castellanii Neff and Campylobacter jejuni was investigated. Campylobacter jejuni was coincubated with Acanthamoeba castellanii Neff trophozoites at different temperatures, in order to evaluate the C. jejuni ability to grow in presence A. castellanii culture and Acanthamoeba Conditioned Medium (ACM). C. jejuni was coincubated with A. castellanii axenic culture at different temperatures in aerobic conditions. Our results revealed that bacteria were still cultivable (Blood Agar medium, at 37 °C, in microaerophilic atmosphere) after a 14 days C. jejuni - A. castellanii coculture, comparing with C. jejuni alone, which was only cultivable for 24 h.

Status of the effectiveness of contact lens disinfectants in Malaysia against keratitis-causing pathogens.

The aim of this study was (i) to assess the antimicrobial effects of contact lens disinfecting solutions marketed in Malaysia against common bacterial eye pathogens and as well as eye parasite, Acanthamoeba castellanii, and (ii) to determine whether targeting cyst wall would improve the efficacy of contact lens disinfectants. Using ISO 14729 Stand-Alone Test for disinfecting solutions, bactericidal and amoebicidal assays of six different contact lens solutions including Oxysept®, AO SEPT PLUS, OPTI-FREE® pure moist®, Renu® fresh™, FreshKon® CLEAR and COMPLETE RevitaLens™ were performed using Manufacturers Minimum recommended disinfection time (MRDT). The efficacy of contact lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, and Acanthamoeba castellanii. In addition, using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, we determined whether combination of both agents can enhance efficacy of marketed contact lens disinfectants against A. castellanii trophozoites and cysts, in vitro. The results revealed that all contact lens disinfectants tested showed potent bactericidal effects exhibiting 100% kill against all bacterial species tested. In contrast, none of the contact lens disinfectants had potent effects against Acanthamoeba cysts viability. When tested against trophozoites, two disinfectants, Oxysept Multipurpose and AO-sept Multipurpose showed partial amoebicidal effects. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents in contact lens disinfectants abolished viability of A. castellanii cysts and trophozoites. Given the inefficacy of contact lens disinfectants tested in this study, these findings present a significant concern to public health. These findings revealed that targeting cyst wall by using cyst wall degrading molecules in contact lens disinfecting solutions will enhance their efficacy against this devastating eye infection.

Apoptosis in Acanthamoeba castellanii belonging to the T4 genotype.

Here we describe features of apoptosis in unicellular Acanthamoeba castellanii belonging to the T4 genotype. When exposed to apoptosis-inducing compounds such as doxorubicin, A. castellanii trophozoites exhibited cell shrinkage and membrane blebbing as observed microscopically, DNA fragmentation using agarose gel electrophoresis, and phosphatidylserine (PS) externalization using annexin V immunostaining. Overall, these findings suggest the existence of apoptosis in A. castellanii possibly mediated by intrinsic apoptotic cascade. Further research in this field could provide avenues to selectively induce apoptosis in A. castellanii by triggering intrinsic apoptotic cascade.

Efficient Liquid Media for Encystation of Pathogenic Free-Living Amoebae.

Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page's amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.

The effect of peptidic and non-peptidic proteasome inhibitors on the biological properties of Acanthamoeba castellanii belonging to the T4 genotype.

The treatment of Acanthamoeba infections remains problematic, suggesting that new targets and/or chemotherapeutic agents are needed. Bioassay-guided screening of drugs that are clinically-approved for non-communicable diseases against opportunistic eukaryotic pathogens is a viable strategy. With known targets and mode of action, such drugs can advance to clinical trials at a faster pace. Recently Bortezomib (proteasome inhibitor) has been approved by FDA in the treatment of multiple myeloma. As proteasomal pathways are well known regulators of a variety of eukaryotic cellular functions, the overall aim of the present study was to study the effects of peptidic and non-peptidic proteasome inhibitors on the biology and pathogenesis of Acanthamoeba castellanii of the T4 genotype, in vitro. Zymographic assays revealed that inhibition of proteasome had detrimental effects on the extracellular proteolytic activities of A. castellanii. Proteasome inhibition affected A. castellanii growth (using amoebistatic assays), but not viability of A. castellanii. Importantly, proteasome inhibitors affected encystation as determined by trophozoite transformation into the cyst form, as well as excystation, as determined by cyst transformation into the trophozoite form. The ability of proteasome inhibitor to block Acanthamoeba differentiation is significant, as it presents a major challenge in the successful treatment of Acanthamoeba infection. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic infections.

Effect of non-steroidal anti-inflammatory drugs on biological properties of Acanthamoeba castellanii belonging to the T4 genotype.

Non-steroidal anti-inflammatory drug, Diclofenac, targeting COX have shown promise in the treatment of Acanthamoeba keratitis, but the underlying mechanisms remain unknown. Using various NSAIDs, Diclofenac sodium, Indomethacin, and Acetaminophen, here we determined the effects of NSAIDs on the biological properties of Acanthamoeba castellanii belonging to the T4 genotype. Using amoebicidal assays, the results revealed that Diclofenac sodium, and Indomethacin affected growth of A. castellanii. In contrast, none of the compounds tested had any effect on the viability of A. castellanii. Importantly, all NSAIDs tested abolished A. castellanii encystation. This is a significant finding as the ability of amoebae to transform into the dormant cyst form presents a significant challenge in the successful treatment of infection. The NSAIDs inhibit production of cyclo-oxegenase, which regulates the synthesis of prostaglandins suggesting that cyclooxygenases (COX-1 and COX-2) and prostaglandins play significant role(s) in Acanthamoeba biology. As NSAIDs are routinely used in the clinical practice, these findings may help design improved preventative strategies and/or of therapeutic value to improve prognosis, when used in combination with other anti-amoebic drugs.