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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 -
Diagnostic Microbiology and Infectious... Oct 2023Acanthamoeba keratitis is a devastating infectious disease of the cornea caused by an opportunistic amoeba, Acanthamoeba castellanii. It is poorly recognized, and...
Acanthamoeba keratitis is a devastating infectious disease of the cornea caused by an opportunistic amoeba, Acanthamoeba castellanii. It is poorly recognized, and diagnostic delays can lead to irreversible damage to the vision. The gold standard for diagnosis has been a sample culture that lasts approximately 2 weeks. Nevertheless, the essence of time has led to the need for an accurate and fast technique to detect A. castellanii from a sample. We developed both traditional and quantitative real-time-PCR-based methods to detect A. castellanii in less than 3 hours and with the sensitivity of one amoeba. Diagnostic laboratories can select the best-suited method for their purposes from 2 comparable methods. The correct treatment can be initiated from the emergency room when the diagnosis has been made quickly within a few hours, hence saving the patient from long-term complications.
Topics: Humans; Acanthamoeba castellanii; Rapid Diagnostic Tests; Acanthamoeba Keratitis; Cornea; Real-Time Polymerase Chain Reaction
PubMed: 37506594
DOI: 10.1016/j.diagmicrobio.2023.116014 -
Translational Vision Science &... Aug 2023To develop a feline model of acute Acanthamoeba keratitis using methods that replicate natural routes of infection transmission.
PURPOSE
To develop a feline model of acute Acanthamoeba keratitis using methods that replicate natural routes of infection transmission.
METHODS
Corneal Acanthamoeba castellanii inoculation was performed by three methods: topical inoculation with Acanthamoeba solution following corneal abrasion, placement of a contaminated contact lens for 7 days, and placement of a contaminated contact lens for 7 days following corneal abrasion. Sham inoculations with parasite-free medium and sterile contact lenses were also performed. Cats were monitored by ocular examination and in vivo corneal confocal microscopy for 21 days post-inoculation. Corneal samples were collected at intervals for microbiologic assessment, histopathology, and immunohistochemistry.
RESULTS
All cats in the corneal abrasion groups developed clinical keratitis. Clinical ocular disease was inconsistently detected in cats from the contaminated contact lens only group. Initial corneal lesions were characterized by multifocal epithelial leukocyte infiltrates. Ocular lesions progressed to corneal epithelial ulceration and diffuse stromal inflammation. After 14 days, corneal ulcerations resolved, and stromal inflammation consolidated into multifocal subepithelial and stromal infiltrates. Corneal amoebae were detected by culture, in vivo confocal microscopy, histopathology, and immunohistochemistry in cats with keratitis. Neutrophilic and lymphocytic keratoconjunctivitis with lymphoplasmacytic anterior uveitis were identified by histopathology. Coinfection with aerobic bacteria was detected in some, but not all, cats with keratitis. Ocular disease was not detected in the sham inoculation groups.
CONCLUSIONS
Feline Acanthamoeba keratitis is experimentally transmissible by contaminated contact lenses and topical inoculation following corneal epithelial trauma.
TRANSLATIONAL RELEVANCE
Experimentally induced acute Acanthamoeba keratitis in cats is clinically and histopathologically similar to its human counterpart.
Topics: Cats; Animals; Humans; Acanthamoeba Keratitis; Acanthamoeba castellanii; Cornea; Corneal Injuries; Inflammation
PubMed: 37566398
DOI: 10.1167/tvst.12.8.10 -
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 -
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 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 -
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 -
International Journal of Molecular... Aug 2023Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the...
Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the mitochondrial matrix, thereby uncoupling respiratory substrate oxidation from ATP synthesis. The aim of this study was to provide functional evidence that the putative gene of the free-living protozoan amoeba, , encodes the mitochondrial protein with uncoupling activity characteristic of UCPs and to investigate its role during oxidative stress. We report the sequencing and cloning of a complete coding sequence, its phylogenetic analysis, and the heterologous expression of AcUCP in the strain Measurements of mitochondrial respiratory activity and membrane potential indicate that the heterologous expression of AcUCP causes AcUCP-mediated uncoupling activity. In addition, in a model of oxidative stress with increased reactive oxygen species levels (superoxide dismutase 1 knockout yeasts), AcUCP expression strongly promotes cell survival and growth. The level of superoxide anion radicals is greatly reduced in the Δ strain expressing AcUCP. These results suggest that AcUCP targeted to yeast mitochondria causes uncoupling and may act as an antioxidant system. Phylogenetic analysis shows that the UCP diverges very early from other UCPs, but clearly locates within the UCP subfamily rather than among other mitochondrial anion carrier proteins.
PubMed: 37569876
DOI: 10.3390/ijms241512501 -
International Journal of Molecular... Sep 2023is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis...
is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the strain Heysham-1, lacking the -acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to , suggesting that the -acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose -acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection.
Topics: Humans; Legionella pneumophila; Lipopolysaccharides; Rhamnose; Serogroup; Bacterial Proteins; Legionnaires' Disease
PubMed: 37834049
DOI: 10.3390/ijms241914602 -
Translational Vision Science &... Sep 2023To investigate the combined anti-Acanthamoeba effects of nitric oxide (NO) donors and hypochlorite to maximize amoebicidal outcomes while minimizing damage to human...
PURPOSE
To investigate the combined anti-Acanthamoeba effects of nitric oxide (NO) donors and hypochlorite to maximize amoebicidal outcomes while minimizing damage to human corneal epithelial cells (HCECs).
METHODS
Acanthamoeba castellanii and primary cultured HCECs and keratocytes were treated with sodium hypochlorite (NaOCl), NO donors (sodium nitroprusside [SNP] and sodium nitrite [NaNO2]), or a combination of hypochlorite and NO donors. The viability of A. castellanii, HCECs, and keratocytes was assessed. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration of NaOCl and NO donors were determined. The activation of mammalian targets of rapamycin (mTOR) and ERK and the expression of nitrite reductase and Nrf2 were assessed in HCECs using Western blot analysis. The cysticidal effects of combined NaOCl and NO donors were also evaluated.
RESULTS
A dose-dependent toxicity was observed in A. castellanii, HCECs, and keratocytes when treated with NaOCl and SNP. The range of tested NaNO2 concentrations showed no significant toxicity to HCECs; however, dose-dependent toxicity to A. castellanii was observed. The MIC of NaOCl against HCECs and A. castellanii was 8.0 mg/mL. The MIC of NaNO2 and SNP was 500 mM and 10 mM in both HCECs and A. castellanii, respectively. Weak attenuation of the mTOR and ERK phosphorylation was observed and Nrf2 expression decreased slightly after exposure of HCECs to 2.0 mg/mL NaOCl. For the combination treatment, NaOCl (0.125 mg/mL) was selected based on the safety of HCECs and the toxicity of A. castellanii. A more potent anti-Acanthamoeba effect and HCEC toxicity were observed when NaOCl was combined with SNP rather than NaNO2.
CONCLUSIONS
Combined NaOCl and NO donors had a stronger anti-Acanthamoeba effect compared to either drug alone.
TRANSLATIONAL RELEVANCE
This study demonstrates that the combined use of various drugs for the treatment of Acanthamoeba infection can enhance the anti-Acanthamoeba effect while minimizing the toxicity of the individual drug.
Topics: Humans; Animals; Acanthamoeba castellanii; Nitric Oxide Donors; Hypochlorous Acid; NF-E2-Related Factor 2; TOR Serine-Threonine Kinases; Mammals
PubMed: 37768280
DOI: 10.1167/tvst.12.9.23