-
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 -
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 -
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 -
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 -
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 -
Frontiers in Cellular and Infection... 2018Environmental bacteria of the genus naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow... (Review)
Review
Environmental bacteria of the genus naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the -containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of "effector" proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for spp., but also useful cellular models for eukaryotic phagocytes. In particular, and emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors.
Topics: Acanthamoeba; Acanthamoeba castellanii; Amoeba; Animals; Autophagy; Bacterial Proteins; Dictyostelium; Disease Models, Animal; Drug Evaluation, Preclinical; Evolution, Molecular; GTP Phosphohydrolases; Host-Pathogen Interactions; Legionella; Legionella pneumophila; Legionnaires' Disease; Macrophages; Phosphatidylinositols; Proteomics; Type IV Secretion Systems; Vacuoles
PubMed: 29552544
DOI: 10.3389/fcimb.2018.00061 -
Microorganisms Mar 2023genotype T4 is a clinically significant free-living amoeba that causes granulomatous amoebic encephalitis and amoebic keratitis in human beings. During the initial...
genotype T4 is a clinically significant free-living amoeba that causes granulomatous amoebic encephalitis and amoebic keratitis in human beings. During the initial stages of infection, trophozoites interact with various host immune responses, such as lactoferrin (Lf), in the corneal epithelium, nasal mucosa, and blood. Lf plays an important role in the elimination of pathogenic microorganisms, and evasion of the innate immune response is crucial in the colonization process. In this study, we describe the resistance of to the microbicidal effect of bovine apo-lactoferrin (apo-bLf) at different concentrations (25, 50, 100, and 500 µM). trophozoites incubated with apo-bLf at 500 µM for 12 h maintained 98% viability. Interestingly, despite this lack of effect on viability, our results showed that the apo-bLf inhibited the cytopathic effect of in MDCK cells culture, and analysis of amoebic proteases by zymography showed significant inhibition of cysteine and serine proteases by interaction with the apo-bLf. From these results, we conclude that bovine apo-Lf influences the activity of secretion proteases, which in turn decreases amoebic cytopathic activity.
PubMed: 36985284
DOI: 10.3390/microorganisms11030708 -
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 -
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