-
Parasitology Research Dec 2023Several antimicrobial agents are commonly included in contact lens disinfectant solutions including chlorhexidine diacetate (CHX), polyhexamethylene biguanide (PHMB) or...
Several antimicrobial agents are commonly included in contact lens disinfectant solutions including chlorhexidine diacetate (CHX), polyhexamethylene biguanide (PHMB) or myristamidopropyl dimethylamine (MAPD); however, their mode of action, i.e. necrosis versus apoptosis is incompletely understood. Here, we determined whether a mechanism of cell death resembling that of apoptosis was present in Acanthamoeba castellanii of the T4 genotype (NEFF) following exposure to the aforementioned antimicrobials using the anticoagulant annexin V that undergoes rapid high affinity binding to phosphatidylserine in the presence of calcium, making it a sensitive probe for phosphatidylserine exposure. The results revealed that under the conditions employed in this study, an apoptotic pathway of cell death in this organism at the tested conditions does not occur. Our findings suggest that necrosis is the likely mode of action; however, future mechanistic studies should be accomplished in additional experimental conditions to further comprehend the molecular mechanisms of cell death in Acanthamoeba.
Topics: Humans; Acanthamoeba castellanii; Contact Lens Solutions; Phosphatidylserines; Acanthamoeba Keratitis; Apoptosis; Necrosis; Contact Lenses
PubMed: 38060008
DOI: 10.1007/s00436-023-08061-5 -
Frontiers in Physiology 2023Having characterized actin from (Weihing and Korn, Biochemistry, 1971, 10, 590-600) and knowing that myosin had been isolated from the slime mold (Hatano and Tazawa,... (Review)
Review
Having characterized actin from (Weihing and Korn, Biochemistry, 1971, 10, 590-600) and knowing that myosin had been isolated from the slime mold (Hatano and Tazawa, Biochim. Biophys. Acta, 1968, 154, 507-519; Adelman and Taylor, Biochemistry, 1969, 8, 4976-4988), we set out in 1969 to find myosin in . We used K-EDTA-ATPase activity to assay myosin, because it is a unique feature of muscle myosins. After slightly less than 3 years, we purified a K-EDTA ATPase that interacted with actin. Actin filaments stimulated the Mg-ATPase activity of the crude enzyme, but this was lost with further purification. Recombining fractions from the column where this activity was lost revealed a "cofactor" that allowed actin filaments to stimulate the Mg-ATPase of the purified enzyme. The small size of the heavy chain and physical properties of the purified myosin were unprecedented, so many were skeptical, assuming that our myosin was a proteolytic fragment of a larger myosin similar to muscle or myosin. Subsequently our laboratories confirmed that myosin-I is a novel unconventional myosin that interacts with membrane lipids (Adams and Pollard, Nature, 1989, 340 (6234), 565-568) and that the cofactor is a myosin heavy chain kinase (Maruta and Korn, J. Biol. Chem., 1977, 252, 8329-8332). Phylogenetic analysis (Odronitz and Kollmar, Genome Biology, 2007, 8, R196) later established that class I myosin was the first myosin to appear during the evolution of eukaryotes.
PubMed: 38046947
DOI: 10.3389/fphys.2023.1324623 -
Parasites, Hosts and Diseases Nov 2023Free-living amoebae (FLA) rarely cause human infections but can invoke fatal infections in the central nervous system (CNS). No consensus treatment has been established...
Free-living amoebae (FLA) rarely cause human infections but can invoke fatal infections in the central nervous system (CNS). No consensus treatment has been established for FLA infections of the CNS, emphasizing the urgent need to discover or develop safe and effective drugs. Flavonoids, natural compounds from plants and plant-derived products, are known to have antiprotozoan activities against several pathogenic protozoa parasites. The anti-FLA activity of flavonoids has also been proposed, while their antiamoebic activity for FLA needs to be emperically determined. We herein evaluated the antiamoebic activities of 18 flavonoids against Naegleria fowleri and Acanthamoeba species which included A. castellanii and A. polyphaga. These flavonoids showed different profiles of antiamoebic activity against N. fowleri and Acanthamoeba species. Demethoxycurcumin, kaempferol, resveratrol, and silybin (A+B) showed in vitro antiamoebic activity against both N. fowleri and Acanthamoeba species. Apigenin, costunolide, (‒)-epicatechin, (‒)-epigallocatechin, rosmarinic acid, and (‒)-trans-caryophyllene showed selective antiamoebic activity for Acanthamoeba species. Luteolin was more effective for N. fowleri. However, afzelin, berberine, (±)-catechin, chelerythrine, genistein, (+)-pinostrobin, and quercetin did not exhibit antiamoebic activity against the amoeba species. They neither showed selective antiamoebic activity with significant cytotoxicity to C6 glial cells. Our results provide a basis for the anti-FLA activity of flavonoids, which can be applied to develope alternative or supplemental therapeutic agents for FLA infections of the CNS.
Topics: Humans; Amoeba; Acanthamoeba; Naegleria fowleri; Flavonoids; Amebiasis
PubMed: 38043540
DOI: 10.3347/PHD.23078 -
Parasites, Hosts and Diseases Nov 2023Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae....
Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.
Topics: Acanthamoeba castellanii; Escherichia coli; Phagocytosis; Legionella pneumophila; Phagosomes
PubMed: 38043535
DOI: 10.3347/PHD.23088 -
International Microbiology : the... Nov 2023Acanthamoeba are free living amoebae that are the causative agent of keratitis and granulomatous amoebic encephalitis. Alpha-Mangostin (AMS) is a significant xanthone;...
Acanthamoeba are free living amoebae that are the causative agent of keratitis and granulomatous amoebic encephalitis. Alpha-Mangostin (AMS) is a significant xanthone; that demonstrates a wide range of biological activities. Here, the anti-amoebic activity of α-Mangostin and its silver nano conjugates (AMS-AgNPs) were evaluated against pathogenic A. castellanii trophozoites and cysts in vitro. Amoebicidal assays showed that both AMS and AMS-AgNPs inhibited the viability of A. castellanii dose-dependently, with an IC of 88.5 ± 2.04 and 20.2 ± 2.17 μM, respectively. Both formulations inhibited A. castellanii-mediated human keratinocyte cell cytopathogenicity. Functional assays showed that both samples caused apoptosis through the mitochondrial pathway and reduced mitochondrial membrane potential and ATP production, while increasing reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome-c reductase in the cytosol. Whole transcriptome sequencing of A. castellanii showed the expression of 826 genes, with 447 genes being up-regulated and 379 genes being down-regulated post treatment. The Kyoto Encyclopedia of Genes and Genomes analysis showed that the majority of genes were linked to apoptosis, autophagy, RAP1, AGE-RAGE and oxytocin signalling pathways. Seven genes (PTEN, H3, ARIH1, SDR16C5, PFN, glnA GLUL, and SRX1) were identified as the most significant (Log2 (FC) value 4) for molecular mode of action in vitro. Future in vivo studies with AMS and nanoconjugates are needed to realize the clinical potential of this work.
PubMed: 38015290
DOI: 10.1007/s10123-023-00450-1 -
Encystation and Stress Responses under the Control of Ubiquitin-like Proteins in Pathogenic Amoebae.Microorganisms Oct 2023Amoebae found in aquatic and terrestrial environments encompass various pathogenic species, including the parasite and the free-living . Both microorganisms pose... (Review)
Review
Amoebae found in aquatic and terrestrial environments encompass various pathogenic species, including the parasite and the free-living . Both microorganisms pose significant threats to public health, capable of inducing life-threatening effects on humans. These amoebae exist in two cellular forms: trophozoites and cysts. The trophozoite stage is the form used for growth and reproduction while the cyst stage is the resistant and disseminating form. Cysts occur after cellular metabolism slowdown due to nutritional deprivation or the appearance of environmental conditions unfavourable to the amoebae's growth and division. The initiation of encystation is accompanied by the activation of stress responses, and scarce data indicate that encystation shares factors and mechanisms identified in stress responses occurring in trophozoites exposed to toxic compounds derived from human immune defence. Although some "omics" analyses have explored how amoebae respond to diverse stresses, these studies remain limited and rarely report post-translational modifications that would provide knowledge on the molecular mechanisms underlying amoebae-specific stress responses. In this review, we discuss ubiquitin-like proteins associated with encystation and cell survival during oxidative damage. We aim to shed light on the signalling pathways involved in amoebic defence mechanisms, with a focus on their potential clinical implications against pathogenic amoebae, addressing the pressing need for effective therapies.
PubMed: 38004682
DOI: 10.3390/microorganisms11112670 -
Nature Protocols Jan 2024Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology,... (Review)
Review
Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology, in the spotlight for bioengineering advances. Their host, Acanthamoeba castellanii, is an accidental human pathogen that acts as a natural host and environmental reservoir of other human pathogens. Tools for genetic manipulation of viruses and host were lacking. Here, we provide a detailed method for genetic manipulation of A. castellanii and the GVs it plays host to by using CRISPR-Cas9 or homologous recombination. We detail the steps of vector preparation (4 d), transfection of amoeba cells (1 h), infection (1 h), selection (5 d for viruses, 2 weeks for amoebas) and cloning of recombinant viruses (4 d) or amoebas (2 weeks). This procedure takes ~3 weeks or 1 month for the generation of recombinant viruses or amoebas, respectively. This methodology allows the generation of stable gene modifications, which was not possible by using RNA silencing, the only previously available reverse genetic tool. We also include detailed sample-preparation steps for protein localization by immunofluorescence (4 h), western blotting (4 h), quantification of viral particles by optical density (15 min), calculation of viral lethal dose 50 (7 d) and quantification of DNA replication by quantitative PCR (4 h) to allow efficient broad phenotyping of recombinant organisms. This methodology allows the function of thousands of ORFan genes present in GVs, as well as the complex pathogen-host, pathogen-pathogen or pathogen-symbiont interactions in A. castellanii, to be studied in vivo.
Topics: Humans; Acanthamoeba castellanii; Giant Viruses; Viruses
PubMed: 37964008
DOI: 10.1038/s41596-023-00910-y -
European Journal of Protistology Oct 2023Acanthamoeba castellanii is a free-living amoeba that acts as an opportunistic pathogen for humans and is the pathogenic agent of Acanthamoeba keratitis (AK). A....
Acanthamoeba castellanii is a free-living amoeba that acts as an opportunistic pathogen for humans and is the pathogenic agent of Acanthamoeba keratitis (AK). A. castellanii may present as proliferative and infective trophozoites or as resistant cysts during their life cycle. The immune response against AK is still poorly explored; however, it is well established that macrophages and neutrophils play essential roles in controlling corneal infection during the disease outcome. The release of NETs is one of the innate immune strategies to prevent parasite infection, especially when neutrophils interact with microorganisms that are too large to be phagocytosed, which is the case for amoeba species. The present work demonstrated that A. castellanii trophozoites can trigger NET formation upon in vitro interaction with neutrophils. Using DNase as a control, we observed increased parasite survival after coinciding with neutrophils, which may be correlated with NET degradation. Indeed, A. castellanii trophozoites degrade the NET DNA scaffold. Molecular analysis confirmed the occurrence of a 3'-nucleotidase/nuclease (3'-NT/NU) in the A. castellanii genome. We also demonstrated that trophozoites exhibit significantly higher 3'-NT/NU activity than cysts, which cannot trigger NET release. Considering that previous studies indicated the pathological role of 3'-NT-/NU in parasite infection, we suggest that this enzyme may act as the mechanism of escape of A. castellanii trophozoites from NETs.
Topics: Animals; Humans; Acanthamoeba castellanii; Trophozoites; Extracellular Traps; Acanthamoeba Keratitis
PubMed: 37948889
DOI: 10.1016/j.ejop.2023.126032 -
Archives of Microbiology Oct 2023Acanthamoeba castellanii is the causative agent of fatal encephalitis and blinding keratitis. Current therapies remain a challenge, hence there is a need to search for...
Acanthamoeba castellanii is the causative agent of fatal encephalitis and blinding keratitis. Current therapies remain a challenge, hence there is a need to search for new therapeutics. Here, we tested embelin (EMB) and silver nanoparticles doped with embelin (EMB-AgNPs) against A. castellanii. Using amoebicidal assays, the results revealed that both compounds inhibited the viability of Acanthamoeba, having an IC of 27.16 ± 0.63 and 13.63 ± 1.08 μM, respectively, while causing minimal cytotoxicity against HaCaT cells in vitro. The findings suggest that both samples induced apoptosis through the mitochondria-mediated pathway. Differentially expressed genes analysis showed that 652 genes were uniquely expressed in treated versus untreated cells, out of which 191 were significantly regulated in the negative control vs. conjugate. Combining the analysis, seven genes (ARIH1, RAP1, H3, SDR16C5, GST, SRX1, and PFN) were highlighted as the most significant (Log2 (FC) value ± 4) for the molecular mode of action in vitro. The KEGG analysis linked most of the genes to apoptosis, the oxidative stress signaling pathway, cytochrome P450, Rap1, and the oxytocin signaling pathways. In summary, this study provides a thorough framework for developing therapeutic agents against microbial infections using EMB and EMB-AgNPs.
Topics: Acanthamoeba castellanii; Metal Nanoparticles; Silver; Apoptosis
PubMed: 37898989
DOI: 10.1007/s00203-023-03698-3 -
Applied and Environmental Microbiology Nov 2023Persistence of in the aquatic environment contributes to the fatal diarrheal disease cholera, which remains a global health burden. In the environment, bacteria face...
Persistence of in the aquatic environment contributes to the fatal diarrheal disease cholera, which remains a global health burden. In the environment, bacteria face predation pressure by heterotrophic protists such as the free-living amoeba . This study explores how a mutant of adapts to acquire essential nutrients and survive predation. Here, we observed that up-regulation of iron acquisition genes and genes regulating resistance to oxidative stress enhances pathogen fitness. Our data show that can defend predation to overcome nutrient limitation and oxidative stress, resulting in an enhanced survival inside the protozoan hosts.
Topics: Animals; Vibrio cholerae; Amoeba; Predatory Behavior; Cholera; Iron
PubMed: 37882527
DOI: 10.1128/aem.01095-23