-
Nature Communications Jul 2022Encystment is a common stress response of most protists, including free-living amoebae. Cyst formation protects the amoebae from eradication and can increase virulence...
Encystment is a common stress response of most protists, including free-living amoebae. Cyst formation protects the amoebae from eradication and can increase virulence of the bacteria they harbor. Here, we mapped the global molecular changes that occur in the facultatively pathogenic amoeba Acanthamoeba castellanii during the early steps of the poorly understood process of encystment. By performing transcriptomic, proteomic, and phosphoproteomic experiments during encystment, we identified more than 150,000 previously undescribed transcripts and thousands of protein sequences absent from the reference genome. These results provide molecular details to the regulation of expected biological processes, such as cell proliferation shutdown, and reveal new insights such as a rapid phospho-regulation of sites involved in cytoskeleton remodeling and translation regulation. This work constitutes the first time-resolved molecular atlas of an encysting organism and a useful resource for further investigation of amoebae encystment to allow for a better control of pathogenic amoebae.
Topics: Acanthamoeba castellanii; Amoeba; Bacteria; Proteomics; Virulence
PubMed: 35835784
DOI: 10.1038/s41467-022-31832-0 -
Scientific Reports Sep 2018Gemmata spp. bacteria thrive in the same aquatic environments as free-living amoebae. DNA-based detection of Gemmata spp. sequences in the microbiota of the human...
Gemmata spp. bacteria thrive in the same aquatic environments as free-living amoebae. DNA-based detection of Gemmata spp. sequences in the microbiota of the human digestive tract and blood further questioned the susceptibility of Gemmata spp. to phagocytes. Here, Gemmata obscuriglobus and Gemmata massiliana were co-cultured with the amoebae Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba griffini and THP-1 macrophage-like phagocytes. All experiments were performed in five independant replicates. The ratio amoeba/bacteria was 1:20 and the ratio THP-1/bacteria was 1:10. After a 2-hour co-culture, extracellular bacteria were killed by kanamycin or amikacin and eliminated. The intracellular location of Gemmata bacteria was specified by confocal microscopy. Microscopic enumerations and culture-based enumerations of colony-forming units were performed at T = 0, 1, 2, 3, 4, 8, 16, 24, 48 and 72 hours post-infection. Then, Gemmata bacteria were engulfed into the phagocytes' cytoplasmic vacuoles, more than (98 ± 2)% of Gemmata bacteria, compared to controls, were destroyed by phagocytic cells after a 48-h co-culture according to microscopy and culture results, and no positive culture was observed at T = 72-hours. Under our co-culture conditions, Gemmata bacteria were therefore susceptible to the environmental and host phagocytes here investigated. These data suggest that these Acanthamoeba species and THP-1 cells cannot be used to isolate G. massiliana and G. obscuriglobus under the co-culture conditions applied in this study. Although the THP-1 response can point towards potential responses that might occur in vivo, these responses should first bevalidated by in vivo studies to draw definite conclusions.
Topics: Acanthamoeba; Coculture Techniques; Humans; Macrophages; Planctomycetales; THP-1 Cells
PubMed: 30190504
DOI: 10.1038/s41598-018-31667-0 -
PloS One 2018The detection and identification of two endocytobiotic bacterial strains, one affiliated to the "Candidatus Caedibacter acanthamoebae"/"Ca. Paracaedimonas acanthamoeba",...
The detection and identification of two endocytobiotic bacterial strains, one affiliated to the "Candidatus Caedibacter acanthamoebae"/"Ca. Paracaedimonas acanthamoeba", and another to the endosymbiont of Acanthamoeba UWC8 and "Ca. Jidaibacter acanthamoeba" are described. For endocytobiont screening, we developed a PCR method with a set of broad-range bacterial 16S rRNA primers to substitute the commonly used but technically demanding fluorescent in situ hybridization technique. Our PCR test alone without sequencing failed to discriminate the endocytobiont-containing and endocytobiont-free Acanthamoeba sp. due to the presence of mismatched primers to host mitochondrial DNA. We highlighted the need to perform bacterial primer checking against the Acanthamoeba genome to avoid false positive detection in PCR. Although the genetic aspect of "Ca. Caedibacter acanthamoebae"/"Ca. Paracaedimonas acanthamoeba" and the endosymbiont of Acanthamoeba UWC8/"Ca. Jidaibacter acanthamoeba" are well studied, knowledge pertaining to their morphologies are quite vague. Hence, we used transmission electron microscopy to examine our endocytobionts which are affiliated to previously described intracellular bacteria of Acanthamoeba sp. We used good-quality TEM images for the localization and the fate of the current endocytobionts inside different life stages of the hosts. Furthermore, to the best of our knowledge, our TEM findings are the first to provide morphological evidence for the clearance of defective Acanthamoeba endocytobionts via an autophagic-like process.
Topics: Acanthamoeba; Alphaproteobacteria; DNA, Bacterial; DNA, Mitochondrial; Genome, Bacterial; Host-Pathogen Interactions; Microscopy, Electron, Transmission; RNA, Ribosomal, 16S
PubMed: 30356282
DOI: 10.1371/journal.pone.0204732 -
Eye (London, England) Feb 2023The true disease status of a population with suspected microbial keratitis (MK) cannot be verified. There is not an accurate (gold) reference standard to confirm...
PURPOSE
The true disease status of a population with suspected microbial keratitis (MK) cannot be verified. There is not an accurate (gold) reference standard to confirm infection and inter-test comparisons of sensitivity and specificity therefore lead to bias with questionable estimates of test utility. We present an alternative method to report results.
METHODS
We used a decision to treat as the definition for MK. We retrospectively compared the results of corneal culture and polymerase chain reaction (PCR) as these are objective tests available for the three principal groups of pathogens. We then estimated the potential contribution of positive results, either alone or in combination, to support the working diagnosis.
RESULTS
We included 2021 (77.4%) eyes with suspected bacterial keratitis, 365 (14.0%) with suspected acanthamoeba keratitis, and 226 (8.6%) with suspected fungal keratitis, all treated between July 2013 and December 2019. In these groups, there were 51.6% positive culture and 6.5% positive PCR results for bacteria, 19.0% and 40.5% for acanthamoeba, and 28.3% and 15.0% for fungi. Between groups the differences in the proportions of positive results from culture and PCR was statistically significant (P < 0.001). The added benefit of PCR to the result of culture in identifying a potential pathogen was 1.4% for bacteria (P = 0.6292), 24.4% for acanthamoeba (P = 0.0001), and 5.8% for fungi (P = 0.3853).
CONCLUSIONS
For suspected MK a comparison of the test positivity rate is an easily comprehensible outcome measure of test utility.
Topics: Humans; Retrospective Studies; Acanthamoeba Keratitis; Corneal Ulcer; Acanthamoeba; Cornea; Fungi; Bacteria
PubMed: 35102245
DOI: 10.1038/s41433-022-01952-4 -
PloS One 2020To determine whether Acanthamoeba keratitis (AK) patients have higher rates of Acanthamoeba and free-living amoeba (FLA) colonising domestic sinks than control contact...
PURPOSE
To determine whether Acanthamoeba keratitis (AK) patients have higher rates of Acanthamoeba and free-living amoeba (FLA) colonising domestic sinks than control contact lens (CL) wearers, and whether these isolates are genetically similar to the corneal isolates from their CL associated AK.
METHODS
129 AK patients from Moorefield Eye Hospital, London and 64 control CL wearers from the Institute of Optometry were included in this study. The participants self-collected home kitchen and bathroom samples from tap-spouts, overflows and drains using an instructional kit. The samples were cultured by inoculating onto a non-nutrient agar plate seeded with Escherichia coli, incubated at 32°C and examined for amoebae by microscopy for up to 2 weeks. Partial sequences of mitochondrial cytochrome oxidase genes (coxA) of Acanthamoeba isolates from four AK patients were compared to Acanthamoeba isolated from the patient's home. The association between sampling sites was analysed with the chi-square test.
RESULTS
A total of 513 samples from AK patients and 189 from CL controls were collected. The yield of FLA was significantly greater in patients' bathrooms (72.1%) than CL controls' bathrooms (53.4%) (p<0.05). Spouts (kitchen 6.7%, bathroom 11%) had the lowest rate of Acanthamoeba isolation compared to drains (kitchen 18.2%, bathroom 27.9%) and overflow (kitchen 39.1%, bathroom 25.9%) either in kitchens or bathrooms (p<0.05). There was no statistically significant difference between the average prevalence of Acanthamoeba in all three sample sites in kitchens (16.9%) compared to all three sample sites in bathrooms (21.5%) and no association for Acanthamoeba prevalence between AK patients and CL controls. All four corneal isolates had the same coxA sequence as at least one domestic water isolate from the patients' sink of the kitchen and the bathroom.
CONCLUSION
The prevalence of Acanthamoeba and FLA was high in UK homes. FLA colonisation was higher in AK patients compared to controls but the prevalence of Acanthamoeba between AK patients and CL controls domestic sinks was similar. This study confirms that domestic water isolates are probably the source of AK infection. Advice about avoiding water contact when using CL's should be mandatory.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Amoebozoa; Case-Control Studies; Contact Lenses; Disease Susceptibility; Environment; Housing; Humans; London; Risk Factors; Sanitary Engineering; Water
PubMed: 32160218
DOI: 10.1371/journal.pone.0229681 -
Parasite Immunology Feb 2019Acanthamoeba infections are characterized by an intense localized innate immune response associated with an influx of macrophages. Acanthamoeba protease production is...
AIM
Acanthamoeba infections are characterized by an intense localized innate immune response associated with an influx of macrophages. Acanthamoeba protease production is known to affect virulence. Herein, the ability of Acanthamoeba trophozoite proteases, of either the laboratory Neff strain or a recently isolated clinical strain, to stimulate IL-12 and IL-6 and to activate protease-activated receptors, PAR and PAR expressed on murine macrophages, was investigated.
METHOD AND RESULTS
Using selected protease inhibitors, leupeptin and E64, we showed that Acanthamoeba proteases can stimulate IL-12 and IL-6 by murine macrophages. Subsequently, using specific antagonists to inhibit PAR , and bone marrow-derived macrophages from PAR gene-deficient mice, we demonstrate that PAR , but not PAR contributes to macrophage IL-12 production in response to Acanthamoeba. In contrast, Acanthamoeba-induced IL-6 production is PAR and PAR independent.
CONCLUSION
This study shows for the first time the involvement of PARs, expressed on macrophages, in the response to Acanthamoeba trophozoites and might provide useful insight into Acanthamoeba infections and their future treatments.
Topics: Acanthamoeba; Amebiasis; Animals; Cell Cycle Proteins; Immunity, Innate; Interleukin-12; Macrophage Activation; Macrophages; Male; Mice; Mice, Inbred BALB C; Peptide Hydrolases; Protein Serine-Threonine Kinases; Receptor, PAR-2; Signal Transduction
PubMed: 30578557
DOI: 10.1111/pim.12612 -
Parasites & Vectors Sep 2020Acanthamoeba spp. are cosmopolitan protozoans that cause infections in the brain, as well as extracerebral infections in the cornea, lungs and skin. Little is known...
BACKGROUND
Acanthamoeba spp. are cosmopolitan protozoans that cause infections in the brain, as well as extracerebral infections in the cornea, lungs and skin. Little is known about the mechanisms of the immunological response to these parasites in organs which are not their main biotope. Therefore, the purpose of this study was to determine the expression of TLR2 and TLR4 in the kidneys and heart of Acanthamoeba spp.-infected mice, with respect to the host's immunological status.
METHODS
The mice were grouped into four groups: immunocompetent control mice; immunosuppressed control mice; immunocompetent Acanthamoeba spp.-infected mice; and immunosuppressed Acanthamoeba spp. infected mice. In the study, we used the amoebae T16 genotype which was isolated from a patient. The TLRs expressions in the kidneys and heart of mice were assessed by quantitative real-time polymerase chain reaction. Moreover, we visualized TLR2 and TLR4 proteins in the organs by immunohistochemical staining.
RESULTS
In the kidneys, we observed a higher TLR2 expression in immunosuppressed mice at 24 days post-Acanthamoeba spp. infection (dpi) compared to the uninfected mice. There were no statistically significant differences in TLR4 expression in the kidneys between the immunocompetent and immunosuppressed mice, both of infected and uninfected mice. In the heart, we observed a difference in TLR2 expression in immunocompetent mice at 24 dpi compared to immunocompetent mice at 8 dpi. The immunocompetent Acanthamoeba spp.-infected mice had higher TLR4 expression at 8 dpi compared to the immunocompetent uninfected mice.
CONCLUSIONS
Our results indicate that TLR2 is involved in response to Acanthamoeba spp. infection in the kidneys, whereas in the heart, both studied TLRs are involved.
Topics: Acanthamoeba; Amebiasis; Animals; Humans; Immunocompromised Host; Kidney; Male; Mice; Mice, Inbred BALB C; Toll-Like Receptor 2; Toll-Like Receptor 4
PubMed: 32958053
DOI: 10.1186/s13071-020-04351-4 -
Clinical & Experimental Optometry Nov 2020This study examined the prevalence of free-living Acanthamoeba in domestic tap water in the greater Sydney region, Australia, and determined any seasonal variation in...
BACKGROUND
This study examined the prevalence of free-living Acanthamoeba in domestic tap water in the greater Sydney region, Australia, and determined any seasonal variation in prevalence.
METHODS
Fifty-four participants were included in this study following approval from an institutional human research ethics committee. Each participant self-collected two samples (one in summer and another in winter) from the surface of the drain of the bathroom sink using an instructional kit. The samples were cultured by inoculating onto a non-nutrient agar plate seeded with Escherichia coli and incubation at 32°C for two weeks. The plates were microscopically examined for the presence of free-living amoeba. DNA was isolated from 20 samples and a polymerase chain reaction (PCR) assay was performed for amplification of the partial sequence of the 18S ribosomal RNA gene. The PCR amplified products were sequenced using Sanger sequencing and genotyping was performed based on the variation in nucleotide sequences.
RESULTS
A total of 97 samples were collected over the two collection periods, with 28.6 per cent of samples morphologically classified as Acanthamoeba. The summer period yielded 16 of 54 (29.6 per cent) samples classified as Acanthamoeba, while the winter period yielded 12 of 43 (27.9 per cent) samples classified as Acanthamoeba. There was no statistically significant difference (p = 0.85) between the prevalence of free-living Acanthamoeba in summer compared to winter. Phylogenetic analysis showed that 15 of 20 (75 per cent) isolates belonged to genotype T4, the most frequent genotype isolated in Acanthamoeba keratitis.
CONCLUSION
The prevalence of free-living Acanthamoeba characterised morphologically in domestic tap water of the greater Sydney region was higher than expected, especially considering the low incidence of Acanthamoeba keratitis in Australia. However, this study did not find variation between seasons. As the T4 genotype was most common, Sydney-based practitioners must always consider Acanthamoeba as a possible causative organism in cases of microbial keratitis, regardless of the season.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Humans; Phylogeny; Prevalence; Seasons; Water
PubMed: 32227362
DOI: 10.1111/cxo.13065 -
Parasites, Hosts and Diseases May 2024Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based...
Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based solely on their presence. In this study, we determined the presence of FLA in river water by filtering water samples collected from various sites and culturing the resulting filtrates. FLA were detected in all the water samples with varying quality grades (Grades Ι-V). The significant increase in the size of the amoebae population with the deterioration in the water quality. Monoxenic cultures of the amoebae were performed, and genomic DNAs were isolated, among which 18S rDNAs were sequenced to identify the amoeba species. Of the 12 species identified, 10 belonged to the Acanthamoeba genus; of the remaining 2 species, one was identified as Vannella croatica and the other as a species of Vermamoeba. Acanthamoeba was detected in samples with Grades Ι to VI quality, whereas the Vermamoeba species was present only in Grade Ι water. V. croatica was found exclusively in water with Grade ΙΙ quality. Following morphological observations, genomic DNA was sequenced using 16S rDNA to determine whether the species of Acanthamoeba harbored endosymbionts. Most of the isolated Acanthamoeba contained endosymbionts, among which 4 species of endogenous bacteria were identified and examined using transmission electron microscopy. This study provides evidence that the distribution of amoebae other than Acanthamoeba may be associated with water quality. However, further confirmation will be required based on accurate water quality ratings and assessments using a more diverse range of FLA.
Topics: Amoeba; Water Quality; Phylogeny; Rivers; DNA, Protozoan; Acanthamoeba; RNA, Ribosomal, 18S; DNA, Ribosomal; Biodiversity; Sequence Analysis, DNA; RNA, Ribosomal, 16S
PubMed: 38835259
DOI: 10.3347/PHD.24020 -
Microbiology Spectrum Dec 2021Acanthamoeba castellanii is a free-living, pathogenic ameba found in the soil and water. It invades the body through ulcerated skin, the nasal passages, and eyes and can...
Acanthamoeba castellanii is a free-living, pathogenic ameba found in the soil and water. It invades the body through ulcerated skin, the nasal passages, and eyes and can cause blinding keratitis and granulomatous encephalitis. However, the mechanisms underlying the opportunistic pathogenesis of A. castellanii remain unclear. In this study, we observed that commensal bacteria significantly reduced the cytotoxicity of the ameba on mammalian cells. This effect occurred in the presence of both Gram-positive and Gram-negative commensals. Additionally, commensals mitigated the disruption of cell junctions. experiments on mouse eyeballs further showed that the commensals protected the corneal epithelial layer. Together, these findings indicate that A. castellanii is pathogenic to individuals with a dysbiosis of the microbiota at infection sites, further highlighting the role of commensals as a natural barrier during parasite invasion. Acanthamoeba castellanii, an opportunistic protozoan widely present in the environment, can cause keratitis and encephalitis in humans. However, only a few reports describe how the ameba acts as an opportunistic pathogen. Our study showed that the normal microbiota interfered with the cytotoxicity of , persevered during invasion, and reduced corneal epithelium peeling in the mouse eyeball model. This suggests that commensals may act as a natural barrier against invasion. In future, individuals who suffer from keratitis should be examined for microbiota absence or dysbiosis to reduce the incidence of infection in clinical settings.
Topics: Acanthamoeba Keratitis; Acanthamoeba castellanii; Animals; Cornea; Epithelium; Female; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Symbiosis
PubMed: 34935418
DOI: 10.1128/Spectrum.00512-21