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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 -
Journal of Water and Health Sep 2023The present study aims to identify the Acanthamoeba genotypes and their pathogenic potential in three recreational lakes in Malaysia. Thirty water samples were collected...
The present study aims to identify the Acanthamoeba genotypes and their pathogenic potential in three recreational lakes in Malaysia. Thirty water samples were collected by purposive sampling between June and July 2022. Physical parameters of water quality were measured in situ while chemical and microbiological analyses were performed in the laboratory. The samples were vacuum filtered through nitrate filter, cultured onto non-nutrient agar and observed microscopically for amoebic growth. DNAs from positive samples were extracted and made to react with polymerase chain reaction using specific primers. Physiological tolerance tests were performed for all Acanthamoeba-positive samples. The presence of Acanthamoeba was found in 26 of 30 water samples by PCR. The highest rate in lake waters contaminated with amoeba was in Biru Lake (100%), followed by Titiwangsa Lake (80%) and Shah Alam Lake (80%). ORP, water temperature, pH and DO were found to be significantly correlated with the presence of Acanthamoeba. The most common genotype was T4. Temperature- and osmo-tolerance tests showed that 8 (30.8%) of the genotypes T4, T9 and T11 were highly pathogenic. The presence of genotype T4 in habitats related to human activities supports the relevance of this amoeba as a potential public health concern.
Topics: Humans; Acanthamoeba; Lakes; Malaysia; Virulence; Amoeba; Genotype
PubMed: 37756200
DOI: 10.2166/wh.2023.186 -
Cornea May 2024The purpose of this study was to identify conjunctival transcriptome differences in patients with Acanthamoeba keratitis compared with keratitis with no known associated...
PURPOSE
The purpose of this study was to identify conjunctival transcriptome differences in patients with Acanthamoeba keratitis compared with keratitis with no known associated pathogen.
METHODS
The host conjunctival transcriptome of 9 patients with Acanthamoeba keratitis (AK) is compared with the host conjunctival transcriptome of 13 patients with pathogen-free keratitis. Culture and/or confocal confirmed Acanthamoeba in 8 of 9 participants with AK who underwent metagenomic RNA sequencing as the likely pathogen. Cultures were negative in all 13 cases where metagenomic RNA sequencing did not identify a pathogen.
RESULTS
Transcriptome analysis identified 36 genes differently expressed between patients with AK and patients with presumed sterile, or pathogen-free, keratitis. Gene enrichment analysis revealed that some of these genes participate in several biologic pathways important for cellular signaling, ion transport and homeostasis, glucose transport, and mitochondrial metabolism. Notable relatively differentially expressed genes with potential relevance to Acanthamoeba infection included CPS1, SLC35B4, STEAP2, ATP2B2, NMNAT3, and AKAP12.
CONCLUSIONS
This research suggests that the local transcriptome in Acanthamoeba keratitis may be sufficiently robust to be detected in the conjunctiva and that corneas infected with Acanthamoeba may be distinguished from the inflamed cornea where no pathogen was identified. Given the low sensitivity for corneal cultures, identification of differentially expressed genes may serve as a suggestive transcriptional signature allowing for a complementary diagnostic technique to identify this blinding parasite. Knowledge of differentially expressed genes may also direct investigation of disease pathophysiology and suggest novel pathways for therapeutic targets.
PubMed: 38771726
DOI: 10.1097/ICO.0000000000003545 -
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 -
International Ophthalmology Aug 2023Acanthamoeba keratitis (AK) and fungal keratitis (FK) are two microbial keratitis that cause serious damage and, without early accurate diagnosis and treatment, may lead... (Meta-Analysis)
Meta-Analysis Review
PURPOSE
Acanthamoeba keratitis (AK) and fungal keratitis (FK) are two microbial keratitis that cause serious damage and, without early accurate diagnosis and treatment, may lead to blindness. In vivo corneal confocal scan, as an emerging ocular diagnostic method in comparison with microbiological smears and cultures as the gold standard, may assist in accelerating appropriate diagnosis.
OBJECTIVE
To determine the diagnostic accuracy of confocal scan for the diagnosis of AK and FK.
METHODS
Data were collected via a comprehensive literature search of PubMed, Web of Science, Cochrane Library, Embase and Scopus using keywords related to diagnostic accuracy of confocal scan in AK and FK up to October 2022. Pooled data underwent meta-analysis in terms of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall diagnostic odds ratio (DOR) of confocal scan for the diagnosis of AK and FK.
RESULTS
The final 14 relevant studies were identified, including 1950 eyes. Meta-analysis in AK group revealed 94% sensitivity, 87% specificity, 89% PPV, 92% NPV, and DOR of 143.32, and in FK group disclosed 88% sensitivity, 85% specificity, 85% PPV, 88% NPV, and DOR of 75.98.
CONCLUSION
The accuracy of confocal scan for the diagnosis of AK was significantly more than that for detecting FK; despite the limitations such as limited numbers of available retrospective studies for the detection of FK, confocal scan had an acceptable performance in detecting FK eyes. The overall performance of NCS was similar with that of HRT-RCM for the detection of both types of keratitis.
Topics: Humans; Acanthamoeba Keratitis; Retrospective Studies; Microscopy, Confocal; Corneal Ulcer; Cornea; Eye Infections, Fungal
PubMed: 36977847
DOI: 10.1007/s10792-023-02689-z -
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 -
Expert Opinion on Drug Metabolism &... Mar 2024Carbonic anhydrases (CAs, EC 4.2.1.1) have been established drug targets for decades, with their inhibitors and activators possessing relevant pharmacological activity... (Review)
Review
INTRODUCTION
Carbonic anhydrases (CAs, EC 4.2.1.1) have been established drug targets for decades, with their inhibitors and activators possessing relevant pharmacological activity and applications in various fields. At least 11 sulfonamides/sulfamates are clinically used as diuretics, antiglaucoma, antiepileptic, or antiobesity agents and one derivative, SLC-0111, is in clinical trials as antitumor/antimetastatic agent. The activators were less investigated with no clinically used agent.
AREAS COVERED
Drug interactions between CA inhibitors/activators and various other agents are reviewed in publications from the period March 2020 - January 2024.
EXPERT OPINION
Drug interactions involving these agents revealed several interesting findings. Acetazolamide plus loop diuretics is highy effective in acute decompensated heart failure, whereas ocular diseases such as X-linked retinoschisis and macular edema were treated by acetazolamide plus bevacizumab or topical NSAIDs. Potent anti-infective effects of acetazolamide and other CAIs, alone or in combination with other agents were demonstrated for the management of , vancomycin resistant enterococci, , and infections. Topiramate, in combination with phentermine is incresingly used for the management of obesity, whereas zonisamide plus levodopa is highly effective for Parkinson's disease. Acetazolamide, methazolamide, ethoxzolamide, and SLC-0111 showed synergistic antitumor/antimetastatic action in combination with many other antitumor drugs.
Topics: Humans; Carbonic Anhydrase Inhibitors; Acetazolamide; Sulfonamides; Drug Interactions; Antineoplastic Agents; Structure-Activity Relationship; Phenylurea Compounds
PubMed: 38450431
DOI: 10.1080/17425255.2024.2328152 -
Global prevalence of free-living amoebae in solid matrices - A systematic review with meta-analysis.Acta Tropica Nov 2023The ubiquitous free-living amoebae (FLA) are microorganisms of significant medical, sanitary, and ecological importance. However, their characterization within solid... (Meta-Analysis)
Meta-Analysis Review
The ubiquitous free-living amoebae (FLA) are microorganisms of significant medical, sanitary, and ecological importance. However, their characterization within solid matrices such as soil, dust, sediment, mud, sludge, and compost remain to be systematized. In this study, we conducted a systematic review with meta-analysis to explore the global distribution of FLA in solid matrices. From the analysis of 104 out of 4,414 scientific articles retrieved from different databases, it was found that the general global prevalence of FLA in solid matrices was of 55.13% (95% confidence interval (CI) 49.32-60.94). Specifically, FLA prevalence was high in soil (72.40%, 95% CI 69.08-75.73), sediment (57.91%, 95% CI 50.01-65.81), mud (52.90%, 95% CI 24.01-81.78), dust (48.60%, 95% CI 43.00-54.19), and sewage sludge (40.19%, 95% CI 30.68-49.70). In aerosols it was comparatively lower (17.21%, 95% CI 12.76-21.66). Acanthamoeba spp. (52.23%) and Hartmanella/Vermamoeba spp. (36.06%) were found to be more prevalent, whereas Naegleria spp. (34.98%) and Balamuthia spp. (27.32%) were less prevalent. The distribution of the highest global prevalence values for species of Acanthamoeba spp., considering different publication periods of the studies, is as follows: A. hatchetti (51.46%), A. rhysodes (47.49%), A. polyphaga (36.37%), A. culbertsoni (34.31%), A. castellanii (34.21%), and A. lenticulata (32.82%). For other FLA species, the distribution is: Hartmannella/Vermamoeba vermiformis (91.57%), Naegleria fowleri (42.32%), Naegleria gruberi (32.39%), and Balamuthia mandrillaris (25%). The most prevalent Acanthamoeba genotypes were T4 (33.38%) and T3 (23.94%). Overall, the global prevalence of FLA in solid matrices is as high as or greater than that reported in water by previous systematic reviews. Thus, actions aimed at reducing exposure to FLA or exploring their ecological dynamics should consider not only water but also the various solid matrices. The finding outlined here can provide valuable insights for such actions, e.g., informing on the level of exposure to FLA, or on the microbial biodiversity of specific environmental compartments.
Topics: Animals; Amoeba; Prevalence; Sewage; Acanthamoeba; Coleoptera; Dust; Soil; Water
PubMed: 37633571
DOI: 10.1016/j.actatropica.2023.107006 -
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 -
International Journal of Environmental... Dec 2023is a free-living amoeba (FLA) that is ubiquitous in nature and can cause serious pathologies in humans. This protozoan has been detected in several environmental...
is a free-living amoeba (FLA) that is ubiquitous in nature and can cause serious pathologies in humans. This protozoan has been detected in several environmental sources, such as soil, water, and swimming pools. The aim of this study was to evaluate the occurrence and molecular diversity of spp. in aquatic environments of the state of Sergipe, northeastern Brazil, and to determine the pathogenic potential of the isolated samples. A total of 138 samples were collected from 69 aquatic environments and, after cell culture, 74% of the samples were positive for FLA, 47% belonging to the genus . Genotypic analysis was performed using the primers JDP1 and JDP2, confirming distinct genotypes: 18 (75%) isolates belonging to genotype T4, two (8%) to T3, and one isolate (4%) to genotype T5. Tests carried out to analyze the pathogenic potential showed that 11 isolates could grow at 0.5 M mannitol concentration and seven isolates supported hyperosmolarity. In the thermotolerance test, two isolates grew at 37°C. These results confirm the presence and the pathogenic potential of FLA of the genus in aquatic environments of the municipalities of Sergipe.
Topics: Humans; Acanthamoeba; Brazil; Virulence; Water; Genotype
PubMed: 36041225
DOI: 10.1080/09603123.2022.2117280