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Biology Nov 2022Arginine-rich peptides can have broad-spectrum anti-bacterial and anti-fungal activities. Polyhomoarginine consists of highly cationic residues which can act on the...
Arginine-rich peptides can have broad-spectrum anti-bacterial and anti-fungal activities. Polyhomoarginine consists of highly cationic residues which can act on the negatively charged microbial cell membranes. Acanthamoeba is a free-living protozoan known to cause a rare corneal infection which is difficult to diagnose and treat. This study evaluated the activity of the polyhomoarginines against Acanthamoeba castellanii. Acanthamoeba amoebicidal, amoebistatic, encystation and excystment assays were performed using protocols described in the literature. The activity of polyhomoarginines (PHAs) of different lengths (10 to 400 residues) was measured against the trophozoites and cysts of Acanthamoeba castellanii ATCC30868 in concentrations ranging from 0.93 μM to 15 μM. Data were represented as mean ± SE and analysed using one-way ANOVA. Overall, PHAs demonstrated good anti-acanthamoeba activity against both trophozoites and cysts. PHA 30 reduced the number of viable trophozoites by 99%, inhibited the formation of cysts by 96% and the emergence of trophozoites from cysts by 67% at 3.75 μM. PHA 10 was similarly active, but at a slightly higher concentration of 15 μM, reducing the numbers of viable trophozoites by 98%, inhibiting cyst formation by 84% and preventing the emergence of trophozoites from cysts by 99%. At their greatest anti-amoeba concentrations, PHA 10 gave only 8% haemolysis at 15 μM while PHA 30 gave <40 % haemolysis at 3.75 μM. Polyhomoarginine 10 showed excellent anti-amoebic activity against both forms of Acanthamoeba castellanii and was non-toxic at its most active concentrations. This implies that polyhomoarginines can be developed into a potential therapeutic agent for Acanthamoeba keratitis. However, there is a need to carry out further pre-clinical and then in vivo experiments in the AK animal model.
PubMed: 36552236
DOI: 10.3390/biology11121726 -
Investigative Ophthalmology & Visual... Jan 2022To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii.
PURPOSE
To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii.
METHODS
A. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0-2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration.
RESULTS
The toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL.
CONCLUSIONS
pɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.
Topics: Acanthamoeba Keratitis; Acanthamoeba castellanii; Amebicides; Animals; Cells, Cultured; Contact Lens Solutions; Cornea; Disease Models, Animal; Epithelium, Corneal; Eye Infections, Parasitic; Humans; Hydrogels; Microscopy, Fluorescence; Polylysine; Swine; Trophozoites
PubMed: 34994769
DOI: 10.1167/iovs.63.1.11 -
Microorganisms Aug 2022Although the prevalence of keratitis (AK) is rare, its incidence in contact lens wearers has increased. infections can lead to the loss of vision if the diagnosis and...
Although the prevalence of keratitis (AK) is rare, its incidence in contact lens wearers has increased. infections can lead to the loss of vision if the diagnosis and treatment are delayed. In this study, we investigated the diagnostic potential of two antibodies raised against the adenylyl cyclase-associated protein (ACAP) and periplasmic binding protein (PBP) of in the AK mouse model. The specificity of ACAP and PBP antibodies to was confirmed by immunocytochemistry. AK mouse models were produced by corneal infections with trophozoites for 7 days and 21 days. Enzyme-linked immunosorbent assay results revealed that both ACAP and PBP antibodies successfully detected antigens in the tears and eyeball lysates of the AK mouse model. The detection levels of antigens were similar at both infection time points. Anti- IgG, IgA, and IgM antibodies were evaluated from the sera of the AK mouse model. Notably, IgM and IgA antibody responses were highest and lowest at both time points, respectively. Our findings revealed that both ACAP and PBP antibodies could detect antigens in the tears and eyeball lysates of the AK mouse model. These results provide important information for understanding infections and developing a new diagnostic tool for AK.
PubMed: 36144313
DOI: 10.3390/microorganisms10091711 -
Parasites & Vectors Nov 2020Acanthamoeba spp. are free-living amoeba that are ubiquitously distributed in the environment. This study examines pathogenic Acanthamoeba cysteine proteases (AcCPs)...
BACKGROUND
Acanthamoeba spp. are free-living amoeba that are ubiquitously distributed in the environment. This study examines pathogenic Acanthamoeba cysteine proteases (AcCPs) belonging to the cathepsin L-family and explores the mechanism of AcCP3 interaction with host cells.
METHODS
Six AcCP genes were amplified by polymerase chain reaction (PCR). Quantitative real-time PCR was used to analyse the relative mRNA expression of AcCPs during the encystation process and between pre- and post-reactivated trophozoites. To further verify the role of AcCP3 in these processes, AcCP3 recombinant proteins were expressed in Escherichia coli, and the hydrolytic activity of AcCP3 was determined. The influence of the AcCP3 on the hydrolytic activity of trophozoites and the toxicity of trophozoites to human corneal epithelial cells (HCECs) was examined by inhibiting AcCP3 expression using siRNA. Furthermore, the levels of p-Raf and p-Erk were examined in HCECs following coculture with AcCP3 gene knockdown trophozoites by Western blotting.
RESULTS
During encystation, five out of six AcCPs exhibited decreased expression, and only AcCP6 was substantially up-regulated at the mRNA level, indicating that most AcCPs were not directly correlated to encystation. Furthermore, six AcCPs exhibited increased expression level following trophozoite reactivation with HEp-2 cells, particularly AcCP3, indicating that these AcCPs might be virulent factors. After refolding of recombinant AcCP3 protein, the 27 kDa mature protein from the 34 kDa pro-protein hydrolysed host haemoglobin, collagen and albumin and showed high activity in an acidic environment. After AcCP3 knockdown, the hydrolytic activity of trophozoite crude protein against gelatin was decreased, suggesting that these trophozoites had decreased toxicity. Compared with untreated trophozoites or negative control siRNA-treated trophozoites, AcCP3-knockdown trophozoites were less able to penetrate and damage monolayers of HCECs. Western blot analysis showed that the activation levels of the Ras/Raf/Erk/p53 signalling pathways in HCECs decreased after inhibiting the expression of trophozoite AcCP3.
CONCLUSIONS
AcCP6 was correlated to encystation. Furthermore, AcCP3 was a virulent factor in trophozoites and participated in the activation of the Ras/Raf/Erk/p53 signalling pathways of host cells.
Topics: Acanthamoeba castellanii; Cathepsin L; Cysteine Proteases; Gene Expression; HeLa Cells; Host-Parasite Interactions; Humans; Parasite Encystment; Protozoan Proteins; Recombinant Proteins; Sequence Alignment; Trophozoites
PubMed: 33228764
DOI: 10.1186/s13071-020-04474-8 -
Antimicrobial Agents and Chemotherapy Jun 2022Infection with pathogenic free-living amoebae, including Naegleria fowleri, spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because...
Infection with pathogenic free-living amoebae, including Naegleria fowleri, spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase (Glck) as a potential target for therapeutic intervention, as glucose metabolism is critical for viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii (Glck) and (Glck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, Glck and Glck have apparent values of 472.5 and 41.0 μM, while Homo sapiens Glck (Glck) has a value of 310 μM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent values of 45.9 μM (Glck) and 124 μM (Glck) compared to ~8 mM for Glck. Additionally, Glck and Glck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.
Topics: Acanthamoeba; Amebiasis; Amoeba; Balamuthia mandrillaris; Glucokinase; Humans; Naegleria fowleri
PubMed: 35604214
DOI: 10.1128/aac.02373-21 -
Antibiotics (Basel, Switzerland) Nov 2022is a ubiquitous free-living amoeba capable of instigating keratitis and granulomatous amoebic encephalitis in humans. Treatment remains limited and inconsistent....
is a ubiquitous free-living amoeba capable of instigating keratitis and granulomatous amoebic encephalitis in humans. Treatment remains limited and inconsistent. Accordingly, there is a pressing need for novel compounds. Nanotechnology has been gaining attention for enhancing drug delivery and reducing toxicity. Previous work has shown that various antibiotic classes displayed antiamoebic activity. Herein, we employed two antibiotics: ampicillin and ceftriaxone, conjugated with the nanocarrier zinc oxide and β-cyclodextrin, and tested them against via amoebicidal, amoebistatic, encystment, excystment, cytopathogenicity, and cytotoxicity assays at a concentration of 100 μg/mL. Notably, zinc oxide β-cyclodextrin ceftriaxone significantly inhibited growth and cytopathogenicity. Additionally, both zinc oxide β-cyclodextrin ceftriaxone and ceftriaxone markedly inhibited encystment. Furthermore, all the tested compounds displayed negligible cytotoxicity. However, minimal anti-excystment or amoebicidal effects were observed for the compounds. Accordingly, this novel nanoconjugation should be employed in further studies in hope of discovering novel anti- compounds.
PubMed: 36551378
DOI: 10.3390/antibiotics11121721 -
Frontiers in Cell and Developmental... 2022is a widespread Free-Living Amoeba (FLA) that can cause severe ocular or cerebral infections in immunocompetent and immunocompromised patients, respectively, besides...
is a widespread Free-Living Amoeba (FLA) that can cause severe ocular or cerebral infections in immunocompetent and immunocompromised patients, respectively, besides its capacity to transport diverse pathogens. During their life cycle, FLA can alternate between a vegetative form, called a trophozoite, and a latent and resistant form, called a cyst. This resistant form is characterized by the presence of a cell wall containing two layers, namely the ectocyst and the endocyst, mainly composed of cellulose and proteins. In the present work, we aimed to stimulate excystment by treating their cysts with a cellulolytic enzyme, i.e., cellulase, or two proteolytic enzymes, i.e., collagenase and pepsin. While 11 days were necessary to obtain total excystment in the control at 27°C, only 48 h were sufficient at the same temperature to obtain 100% trophozoites in the presence of 25 U/mL cellulase, 50 U/mL collagenase or 100 U/mL pepsin. Additionally, more than 96% amoebae have excysted after only 24 h with 7.5 U/mL cellulase at 30°C. Nevertheless, no effect of the three enzymes was observed on the excystment of and . Surprisingly, trophozoites excysted in the presence of cellulase displayed a markedly shorter doubling time at 7 h, in comparison to the control at 23 h. Likewise, trophozoites doubled their population in 9 h when both cellulose and cellulase were added to the medium, indicating that cyst wall degradation products promote their trophozoite proliferation. The analysis of cysts in epifluorescent microscopy using FITC-lectins and in electron microscopy revealed a disorganized endocyst and a reduction of the intercystic space area after cellulase treatment, implying that these cellular events are preliminary to trophozoite release during excystment. Further studies would be necessary to determine the signaling pathways involved during this amoebal differentiation process to identify new therapeutic targets for the development of anti-acanthamoebal drugs.
PubMed: 36172275
DOI: 10.3389/fcell.2022.982897 -
Frontiers in Microbiology 2020is a pathogenic and opportunistic free-living amoeba that causes keratitis (AK) and granulomatous amebic encephalitis (GAE) in immunocompromised individuals. The...
is a pathogenic and opportunistic free-living amoeba that causes keratitis (AK) and granulomatous amebic encephalitis (GAE) in immunocompromised individuals. The biological and pathogenic characterizations behind this opportunistic protozoan is not fully understood. This study aimed to determine the biological functions of heat shock protein (HSP)-20 of (-HSP20) involved in the maintenance of life cycle and the infectivity of . Immunoscreening cDNA library with infected rabbit sera identified three positive clones, one of them was a putative heat shock protein (-HSP20). The recombinant 23 kDa -HSP20 protein (r-HSP20) was successfully expressed in BL21 (DE3) and purified using metal affinity chromatography. The rabbits immunized with r-HSP20 produced high titer antibody (1:25,600). Immunolocalization with the antibody identified the expression of native -HSP20 on the surface of both trophozoites and cysts. Further, Western blot with antibody identified that the expression of native Ac-HSP20 was 7.5 times higher in cysts than in trophozoites. Blocking -HSP20 on the membrane of trophozoites with specific antibody or silencing gene transcription by siRNA inhibited their transformation into cysts at the early stage but returned to normal at the late stage by stimulating the transcription of . Incubation of trophozoites with anti--HSP20 IgG increased macrophage-involved phagocytosis to the protozoa and inhibited trophozoite infectivity on the cornea of rabbits compared with that without antibody. Our study provides that -HSP20 is a surface antigen involved in the encystation and infectivity of and thus an important target for vaccine and drug development.
PubMed: 33510719
DOI: 10.3389/fmicb.2020.595080 -
Communications Biology Mar 2023Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology, consisting of catalytic, tRNA-recognition, editing, and C-Ala domains. The...
Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology, consisting of catalytic, tRNA-recognition, editing, and C-Ala domains. The catalytic and tRNA-recognition domains catalyze aminoacylation, the editing domain hydrolyzes mischarged tRNA, and C-Ala-the major tRNA-binding module-targets the elbow of the L-shaped tRNA. Interestingly, a mini-AlaRS lacking the editing and C-Ala domains is recovered from the Tupanvirus of the amoeba Acanthamoeba castellanii. Here we show that Tupanvirus AlaRS (TuAlaRS) is phylogenetically related to its host's AlaRS. Despite lacking the conserved amino acid residues responsible for recognition of the identity element of tRNA (G3:U70), TuAlaRS still specifically recognized G3:U70-containing tRNA. In addition, despite lacking C-Ala, TuAlaRS robustly binds and charges micro (an RNA substrate corresponding to the acceptor stem of tRNA) as well as tRNA, indicating that TuAlaRS exclusively targets the acceptor stem. Moreover, this mini-AlaRS could functionally substitute for yeast AlaRS in vivo. This study suggests that TuAlaRS has developed a new tRNA-binding mode to compensate for the loss of C-Ala.
Topics: Alanine-tRNA Ligase; RNA, Transfer, Ala; Escherichia coli; RNA, Transfer
PubMed: 36959394
DOI: 10.1038/s42003-023-04699-0 -
PloS One 2022Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms...
Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Animals; Antibodies, Protozoan; Antibody Specificity; Carboxylesterase; Cell Line; Cells, Cultured; Contact Lenses; Culture Media, Conditioned; Early Diagnosis; Epithelial Cells; Epithelium, Corneal; Humans; Immunization; Male; Mice; Protozoan Proteins
PubMed: 34986189
DOI: 10.1371/journal.pone.0262223