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European Journal of Protistology Jun 2024Acanthamoeba castellanii, a free-living amoeba, can be pathogenic to humans causing a corneal infection named Acanthamoeba keratitis (AK). The mannose-binding protein...
Acanthamoeba castellanii, a free-living amoeba, can be pathogenic to humans causing a corneal infection named Acanthamoeba keratitis (AK). The mannose-binding protein (MBP) is well established as the major factor related to Acanthamoeba pathogenesis. However, additional factors that participate in the adhesion process and protect trophozoites from cytolytic effects caused by host immune responses remain unknown. Ectonucleotidases, including 3'-nucleotidase/nuclease (3'-NT/NU), a bifunctional enzyme that was recently reported in A. castellanii, are frequently related to the establishment of parasitic infections. We verified that trophozoites can hydrolyze 3'-AMP, and this activity is similar to that observed in other protists. The addition of 3'-AMP increases the adhesion of trophozoites to LLC-MK2 epithelial cells, and this stimulation is completely reversed by DTT, an inhibitor of ecto-3'-nucleotidase activity. Lesions in corneal cells caused by AK infection may elevate the extracellular level of 3'-AMP. We believe that ecto-3'-nucleotidase activity can modulate the host immune response, thus facilitating the establishment of parasitic infection. This activity results from the generation of extracellular adenosine, which can bind to purinergic receptors present in host immune cells. Positive feedback may occur in this cascade of events once the ecto-3'-nucleotidase activity of trophozoites is increased by the adhesion of trophozoites to LLC-MK2 cells.
Topics: Acanthamoeba castellanii; Trophozoites; Adenosine; Cell Adhesion; Cell Line; Animals; Nucleotidases; Epithelial Cells
PubMed: 38688045
DOI: 10.1016/j.ejop.2024.126086 -
Journal of Medicinal Chemistry May 2024are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The...
are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from . The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC 0.45 μM), VT1598 (0.25 μM), and VT1161 (0.20 μM). The crystal structures of CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.
Topics: Sterol 14-Demethylase; 14-alpha Demethylase Inhibitors; Structure-Activity Relationship; Acanthamoeba; Acanthamoeba castellanii; Crystallography, X-Ray; Antiprotozoal Agents; Models, Molecular; Molecular Structure
PubMed: 38683753
DOI: 10.1021/acs.jmedchem.4c00303 -
MBio Jun 2024The causative agent of Legionnaires' disease, , is an environmental bacterium, that replicates in macrophages, parasitizes amoeba, and forms biofilms. employs the...
UNLABELLED
The causative agent of Legionnaires' disease, , is an environmental bacterium, that replicates in macrophages, parasitizes amoeba, and forms biofilms. employs the quorum sensing (Lqs) system and the transcription factor LvbR to control various bacterial traits, including virulence and biofilm architecture. LvbR negatively regulates the nitric oxide (NO) receptor Hnox1, linking quorum sensing to NO signaling. Here, we assessed the response of to NO and investigated bacterial receptors underlying this process. Chemical NO donors, such as dipropylenetriamine (DPTA) NONOate and sodium nitroprusside (SNP), delayed and reduced the expression of the promoters for flagellin (P) and the 6S small regulatory RNA (P). Marker-less mutant strains lacking individual (Hnox1, Hnox2, or NosP) or all three NO receptors (triple knockout, TKO) grew like the parental strain in media. However, in the TKO strain, the reduction of P expression by DPTA NONOate was less pronounced, suggesting that the NO receptors are implicated in NO signaling. In the Δ mutant, the promoter was upregulated, indicating that NosP negatively regulates LvbR. The single and triple NO receptor mutant strains were impaired for growth in phagocytes, and phenotypic heterogeneity of non-growing/growing bacteria in amoebae was regulated by the NO receptors. The single NO receptor and TKO mutant strains showed altered biofilm architecture and lack of response of biofilms to NO. In summary, we provide evidence that regulates virulence, intracellular phenotypic heterogeneity, and biofilm formation through NO and three functionally non-redundant NO receptors, Hnox1, Hnox2, and NosP.
IMPORTANCE
The highly reactive diatomic gas molecule nitric oxide (NO) is produced by eukaryotes and bacteria to promote short-range and transient signaling within and between neighboring cells. Despite its importance as an inter-kingdom and intra-bacterial signaling molecule, the bacterial response and the underlying components of the signaling pathways are poorly characterized. The environmental bacterium forms biofilms and replicates in protozoan and mammalian phagocytes. harbors three putative NO receptors, one of which crosstalks with the quorum sensing (Lqs)-LvbR network to regulate various bacterial traits, including virulence and biofilm architecture. In this study, we used pharmacological, genetic, and cell biological approaches to assess the response of to NO and to demonstrate that the putative NO receptors are implicated in NO detection, bacterial replication in phagocytes, intracellular phenotypic heterogeneity, and biofilm formation.
Topics: Biofilms; Legionella pneumophila; Nitric Oxide; Signal Transduction; Virulence; Gene Expression Regulation, Bacterial; Bacterial Proteins; Phenotype; Macrophages; Quorum Sensing
PubMed: 38682908
DOI: 10.1128/mbio.00710-24 -
Microorganisms Apr 2024Acanthamoeba keratitis (AK) is a rare but potentially sight-threatening corneal infection caused by the Acanthamoeba parasite. This microorganism is found ubiquitously... (Review)
Review
Acanthamoeba keratitis (AK) is a rare but potentially sight-threatening corneal infection caused by the Acanthamoeba parasite. This microorganism is found ubiquitously in the environment, often in freshwater, soil, and other sources of moisture. Despite its low incidence, AK presents significant challenges due to delayed diagnosis and the complex nature of therapeutic management. Early recognition is crucial to prevent severe ocular complications, including corneal ulceration and vision loss. Diagnostic modalities and treatment strategies may vary greatly depending on the clinical manifestation and the available tools. With the growing reported cases of Acanthamoeba keratitis, it is essential for the ophthalmic community to thoroughly understand this condition for its effective management and improved outcomes. This review provides a comprehensive overview of AK, encompassing its epidemiology, risk factors, pathophysiology, clinical manifestations, diagnosis, and treatment.
PubMed: 38674702
DOI: 10.3390/microorganisms12040758 -
BMJ Open Ophthalmology Apr 2024Microbial keratitis (MK) is a significant cause of blindness in sub-Saharan Africa. We investigated the feasibility of using a novel corneal impression membrane (CIM)...
OBJECTIVE
Microbial keratitis (MK) is a significant cause of blindness in sub-Saharan Africa. We investigated the feasibility of using a novel corneal impression membrane (CIM) for obtaining and processing samples by culture, PCR and whole-genome sequencing (WGS) in patients presenting with suspected MK in Malawi.
METHODS AND ANALYSIS
Samples were collected from patients presenting with suspected MK using a 12 mm diameter polytetrafluoroethylene CIM disc. Samples were processed using culture and PCR for , herpes simplex virus type 1 (HSV-1) and the bacterial 16S rRNA gene. Minimum inhibitory concentrations of isolates to eight antimicrobials were measured using susceptibility strips. WGS was used to characterise isolates.
RESULTS
71 eyes of 71 patients were included. The overall CIM isolation rate was 81.7% (58 positive samples from 71 participants). 69 (81.2%) of isolates were Gram-positive cocci. Coagulase-negative 31.8% and species 14.1% were the most isolated bacteria. Seven (9.9%) participants were positive for HSV-1. Fungi and were not detected. Moxifloxacin and chloramphenicol offered the best coverage for both Gram-positive and Gram-negative isolates when susceptibility was determined using known antimicrobial first quartile concentrations and European Committee on Antimicrobial Susceptibility Testing breakpoints, respectively. WGS identified known virulence genes associated with keratitis.
CONCLUSIONS
In a resource-poor setting, a CIM can be used to safely sample the cornea in patients presenting with suspected MK, enabling identification of causative microorganisms by culture and PCR. Although the microbiological spectrum found was limited to the dry season, these preliminary results could be used to guide empirical treatment.
Topics: Humans; Pilot Projects; Malawi; Male; Female; Adult; Middle Aged; Eye Infections, Bacterial; Young Adult; Bacteria; Microbial Sensitivity Tests; Cornea; Keratitis; Anti-Bacterial Agents; Aged; Polymerase Chain Reaction; Adolescent; Acanthamoeba; RNA, Ribosomal, 16S
PubMed: 38653537
DOI: 10.1136/bmjophth-2024-001682 -
Klinische Monatsblatter Fur... Apr 2024
Topics: Humans; Acanthamoeba Keratitis; Administration, Topical; Antifungal Agents; Antiprotozoal Agents; Drug Therapy, Combination; Phosphorylcholine; Treatment Outcome; Voriconazole; Middle Aged
PubMed: 38653269
DOI: 10.1055/a-2196-6011 -
Parasitology Research Apr 2024The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming...
The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO gas. ClO gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.
Topics: Chlorine Compounds; Naegleria fowleri; Acanthamoeba; Oxides; Disinfectants; Time Factors; Survival Analysis; Amebicides
PubMed: 38652173
DOI: 10.1007/s00436-024-08215-z -
Biometals : An International Journal on... Apr 2024Acanthamoeba spp. emerged as a clinically important pathogen related to amoebic keratitis. It is among the main causes of corneal transplantation and vision loss in...
Acanthamoeba spp. emerged as a clinically important pathogen related to amoebic keratitis. It is among the main causes of corneal transplantation and vision loss in ophthalmology. The treatment protocols have a low cure rate, high toxicity, and need for drug combination. Transition metal compounds have shown promising antiprotozoal effects. This study evaluates the amoebicidal activity of copper(II) coordination compounds in combination with chlorhexidine and the cytotoxicity to topical ocular application. These copper(II) coordination compounds were screened against Acanthamoeba castellanii trophozoites (ATCC 50492). The cytotoxicity on rabbit corneal cell line (ATCC-CCL 60) was performed. The compounds showed high amoebicidal potential, with inhibition of trophozoite viability above 80%. The Cp12 and Cp13 compounds showed Minimal Inhibitory Amoebicidal Concentration (MIAC) at 200 µM and mean inhibitory concentration (IC) values lower than 10 µM. Against the cysts, Cp12 showed a reduction in viability (48%) in the longest incubation period. A synergistic effect for Cp12 with chlorhexidine was observed. The compounds have a dose-dependent effect against rabbit corneal cells. Compound Cp12 has potential for future application in developing ophthalmic formulations against Acanthamoeba keratitis and its use in multipurpose solutions is highlighted.
PubMed: 38647983
DOI: 10.1007/s10534-024-00602-4 -
Revista Argentina de Microbiologia Apr 2024Free-living amoebae (FLA) of the genus Acanthamoeba are ubiquitous and amphizoic protozoa that colonize aquatic and terrestrial habitats and can serve as reservoirs for...
Free-living amoebae (FLA) of the genus Acanthamoeba are ubiquitous and amphizoic protozoa that colonize aquatic and terrestrial habitats and can serve as reservoirs for other microorganisms. They are considered econoses that can cause severe and rare pathologies. Due to limited epidemiological data available, the objective of this study was to investigate the presence of Acanthamoeba in coastal wetlands of the southeast of Buenos Aires province and evaluate their association with bacteriological and environmental variables. From February 2021 to July 2022, 22 seawater samples were collected at different points along the coast of the city of Mar del Plata (Buenos Aires, Argentina). Environmental parameters were determined and physicochemical and bacteriological studies, morphological identification, cultures and molecular typification were conducted. Regardless of the environmental and bacteriological variables, the presence of Acanthamoeba spp. was molecularly confirmed in 54.54% of the samples, being the first report of these protozoa in seawater in Argentina.
PubMed: 38632019
DOI: 10.1016/j.ram.2024.02.006 -
Omics : a Journal of Integrative Biology Apr 2024With their unusually large genome and particle sizes, giant viruses (GVs) defy the conventional definition of viruses. Although most GVs isolated infect unicellular...
With their unusually large genome and particle sizes, giant viruses (GVs) defy the conventional definition of viruses. Although most GVs isolated infect unicellular protozoans, such as amoeba, studies in the last decade have established their much wider prevalence infecting most eukaryotic supergroups and some giant viral families with the potential to be human pathogens. Their complexity, almost autonomous life cycle, and enigmatic evolution necessitate the study of GVs. The accurate assessment of GV proteome is a veritable challenge. We have compared the coverage of global protein identification using different methods for GVs isolated in Mumbai, Mimivirus Bombay (MVB), Powai Lake Megavirus (PLMV), and Kurlavirus (KV), along with two previously studied GVs, Acanthamoeba polyphaga Mimivirus (APMV) and Marseillevirus (MV). Our study shows that the simultaneous use of in-gel and in-solution digestion methods can significantly increase the coverage of protein identification in the global proteome analysis of purified GV particles. Combining the two methods of analyses, we identified an additional 72 proteins in APMV and 114 in MV compared with what have been previously reported. Similarly, proteomes of MVB, PLMV, and KV were analyzed, and a total of 242 proteins in MVB, 287 proteins in PLMV, and 174 proteins in KV were identified. Our results suggest that a combined methodology of in-gel and in-solution methods is more efficient and opens up new avenues for innovation in global proteome analysis of GVs. Future planetary health research on GVs can benefit from consideration of a broader range of proteomics methodologies as illustrated by the present study.
Topics: Proteomics; Giant Viruses; Proteome; Viral Proteins
PubMed: 38621149
DOI: 10.1089/omi.2024.0012