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Microbiology Spectrum Mar 2023The respiratory pathogenic bacterium Bordetella bronchiseptica can persistently survive in terrestrial and aquatic environments, providing a source of infection....
The respiratory pathogenic bacterium Bordetella bronchiseptica can persistently survive in terrestrial and aquatic environments, providing a source of infection. However, the environmental lifestyle of the bacterium is poorly understood. In this study, expecting repeated encounters of the bacteria with environmental protists, we explored the interaction between B. bronchiseptica and a representative environmental amoeba, Acanthamoeba castellanii, and found that the bacteria resisted amoeba digestion and entered contractile vacuoles (CVs), which are intracellular compartments involved in osmoregulation, to escape amoeba cells. In prolonged coculture, A. castellanii supported the proliferation of B. bronchiseptica. The avirulent Bvg phase, but not the virulent Bvg phase, of the bacteria was advantageous for survival in the amoebae. We further demonstrate that two Bvg phase-specific virulence factors, filamentous hemagglutinin and fimbriae, were targeted for predation by A. castellanii. These results are evidence that the BvgAS two-component system, the master regulator for Bvg phase conversion, plays an indispensable role in the survival of B. bronchiseptica in amoebae. The pathogenic bacterium Bordetella bronchiseptica, which causes respiratory diseases in various mammals, exhibits distinct Bvg and Bvg phenotypes. The former represents the virulent phase, in which the bacteria express a set of virulence factors, while the role of the latter in the bacterial life cycle remains to be understood. In this study, we demonstrate that B. bronchiseptica in the Bvg phase, but not the Bvg phase, survives and proliferates in coculture with Acanthamoeba castellanii, an environmental amoeba. Two Bvg phase-specific virulence factors, filamentous hemagglutinin and fimbriae, were targeted by A. castellanii predation. B. bronchiseptica turns into the Bvg phase at temperatures in which the bacteria normally encounter these amoebae. These findings demonstrate that the Bvg phase of B. bronchiseptica is advantageous for survival outside mammalian hosts and that the bacteria can utilize protists as transient hosts in natural environments.
PubMed: 36971600
DOI: 10.1128/spectrum.00487-23 -
Acta Tropica Jun 2024Soluble factors in the secretome of Acanthamoeba castellanii play crucial roles in the pathogenesis of Acanthamoeba keratitis (AK). Investigating the pathological...
Soluble factors in the secretome of Acanthamoeba castellanii play crucial roles in the pathogenesis of Acanthamoeba keratitis (AK). Investigating the pathological effects of A. castellanii-derived conditioned medium (ACCM) on ocular cells can provide insights into the damage inflicted during AK. This study examined ACCM-induced cytotoxicity in primary human corneal stromal cells (CSCs) and a human SV40 immortalized corneal epithelial cell line (ihCECs) at varying ACCM concentrations (25 %, 50 %, 75 %, and 100 %). MTT, AlamarBlue, Sulforhodamine B, lactate dehydrogenase, and Caspase-3/7 activation assays were used to assess the impact of ACCM on the cell viability, proliferation and apoptosis. Additionally, fluorescent staining was used to reveal actin cytoskeleton changes. ACCM exposure significantly decreased cell viability, increased apoptosis, and disrupted the actin cytoskeleton, particularly at higher concentrations and longer exposures. Proteases were found to mediate these cytopathogenic effects, highlighting the need for characterization of A. castellanii proteases as key virulence factors in AK pathogenesis.
PubMed: 38901524
DOI: 10.1016/j.actatropica.2024.107288 -
Parasites & Vectors Jul 2020The encystation of Acanthamoeba leads to the development of resilient cysts from vegetative trophozoites. This process is essential for the survival of parasites under...
BACKGROUND
The encystation of Acanthamoeba leads to the development of resilient cysts from vegetative trophozoites. This process is essential for the survival of parasites under unfavorable conditions. Previous studies have reported that, during the encystation of A. castellanii, the expression levels of encystation-related factors are upregulated. However, the regulatory mechanisms for their expression during the encystation process remains unknown. Proteins in the sirtuin family, which consists of nicotinamide adenine dinucleotide-dependent deacetylases, are known to play an important role in various cellular functions. In the present study, we identified the Acanthamoeba silent-information regulator 2-like protein (AcSir2) and examined its role in the growth and encystation of Acanthamoeba.
METHODS
We obtained the full-length sequence for AcSir2 using reverse-transcription polymerase chain reaction. In Acanthamoeba transfectants that constitutively overexpress AcSir2 protein, SIRT deacetylase activity was measured, and the intracellular localization of AcSir2 and the effects on the growth and encystation of trophozoites were examined. In addition, the sirtuin inhibitor salermide was used to determine whether these effects were caused by AcSir2 overexpression RESULTS: AcSir2 was classified as a class-IV sirtuin. AcSir2 exhibited functional SIRT deacetylase activity, localized mainly in the nucleus, and its transcription was upregulated during encystation. In trophozoites, AcSir2 overexpression led to greater cell growth, and this growth was inhibited by treatment with salermide, a sirtuin inhibitor. When AcSir2 was overexpressed in the cysts, the encystation rate was significantly higher; this was also reversed with salermide treatment. In AcSir2-overexpressing encysting cells, the transcription of cellulose synthase was highly upregulated compared with that of control cells, and this upregulation was abolished with salermide treatment. Transmission electron microscope-based ultrastructural analysis of salermide-treated encysting cells showed that the structure of the exocyst wall and intercyst space was impaired and that the endocyst wall had not formed.
CONCLUSIONS
These results indicate that AcSir2 is a SIRT deacetylase that plays an essential role as a regulator of a variety of cellular processes and that the regulation of AcSir2 expression is important for the growth and encystation of A. castellanii.
Topics: Acanthamoeba castellanii; Amebiasis; Animals; Genes, Protozoan; Glucosyltransferases; Histone Deacetylases; Humans; Naphthols; Parasite Encystment; Phenylpropionates; Phylogeny; Protozoan Proteins; Sirtuins; Transfection; Trophozoites
PubMed: 32698828
DOI: 10.1186/s13071-020-04237-5 -
Translational Vision Science &... Sep 2023To investigate the combined anti-Acanthamoeba effects of nitric oxide (NO) donors and hypochlorite to maximize amoebicidal outcomes while minimizing damage to human...
PURPOSE
To investigate the combined anti-Acanthamoeba effects of nitric oxide (NO) donors and hypochlorite to maximize amoebicidal outcomes while minimizing damage to human corneal epithelial cells (HCECs).
METHODS
Acanthamoeba castellanii and primary cultured HCECs and keratocytes were treated with sodium hypochlorite (NaOCl), NO donors (sodium nitroprusside [SNP] and sodium nitrite [NaNO2]), or a combination of hypochlorite and NO donors. The viability of A. castellanii, HCECs, and keratocytes was assessed. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration of NaOCl and NO donors were determined. The activation of mammalian targets of rapamycin (mTOR) and ERK and the expression of nitrite reductase and Nrf2 were assessed in HCECs using Western blot analysis. The cysticidal effects of combined NaOCl and NO donors were also evaluated.
RESULTS
A dose-dependent toxicity was observed in A. castellanii, HCECs, and keratocytes when treated with NaOCl and SNP. The range of tested NaNO2 concentrations showed no significant toxicity to HCECs; however, dose-dependent toxicity to A. castellanii was observed. The MIC of NaOCl against HCECs and A. castellanii was 8.0 mg/mL. The MIC of NaNO2 and SNP was 500 mM and 10 mM in both HCECs and A. castellanii, respectively. Weak attenuation of the mTOR and ERK phosphorylation was observed and Nrf2 expression decreased slightly after exposure of HCECs to 2.0 mg/mL NaOCl. For the combination treatment, NaOCl (0.125 mg/mL) was selected based on the safety of HCECs and the toxicity of A. castellanii. A more potent anti-Acanthamoeba effect and HCEC toxicity were observed when NaOCl was combined with SNP rather than NaNO2.
CONCLUSIONS
Combined NaOCl and NO donors had a stronger anti-Acanthamoeba effect compared to either drug alone.
TRANSLATIONAL RELEVANCE
This study demonstrates that the combined use of various drugs for the treatment of Acanthamoeba infection can enhance the anti-Acanthamoeba effect while minimizing the toxicity of the individual drug.
Topics: Humans; Animals; Acanthamoeba castellanii; Nitric Oxide Donors; Hypochlorous Acid; NF-E2-Related Factor 2; TOR Serine-Threonine Kinases; Mammals
PubMed: 37768280
DOI: 10.1167/tvst.12.9.23 -
Applied and Environmental Microbiology Jul 2017Plague is a flea-borne rodent-associated zoonotic disease caused by The disease is characterized by epizootics with high rodent mortalities, punctuated by...
Plague is a flea-borne rodent-associated zoonotic disease caused by The disease is characterized by epizootics with high rodent mortalities, punctuated by interepizootic periods when the bacterium persists in an unknown reservoir. This study investigates the interaction between and the ubiquitous soil free-living amoeba (FLA) to assess if the bacterium can survive within soil amoebae and whether intracellular mechanisms are conserved between infection of mammalian macrophages and soil amoebae. The results demonstrate that during coculture with amoebae, representative strains of epidemic biovars Medievalis, Orientalis, and Antiqua are phagocytized and able to survive within amoebae for at least 5 days. Key determinants of the intracellular interaction of and phagocytic macrophages, PhoP and the type three secretion system (T3SS), were then tested for their roles in the -amoeba interaction. Consistent with a requirement for the PhoP transcriptional activator in the intracellular survival of in macrophages, a PhoP mutant is unable to survive when cocultured with amoebae. Additionally, induction of the T3SS blocks phagocytic uptake of by amoebae, similar to that which occurs during macrophage infection. Electron microscopy revealed that in , resides intact within spacious vacuoles which were characterized using lysosomal trackers as being separated from the lysosomal compartment. This evidence for prolonged survival and subversion of intracellular digestion of within FLA suggests that protozoa may serve as a protective soil reservoir for is a reemerging flea-borne zoonotic disease. Sylvatic plague cycles are characterized by an epizootic period during which the disease spreads rapidly, causing high rodent mortality, and an interepizootic period when the bacterium quiescently persists in an unknown reservoir. An understanding of the ecology of in the context of its persistence in the environment and its reactivation to initiate a new epizootic cycle is key to implementing novel surveillance strategies to more effectively predict and prevent new disease outbreaks. Here, we demonstrate prolonged survival and subversion of intracellular digestion of within a soil free-living amoeba. This suggests the potential role for protozoa as a protective soil reservoir for , which may help explain the recrudescence of plague epizootics.
Topics: Acanthamoeba castellanii; Bacterial Proteins; Humans; Microbial Viability; Plague; Type III Secretion Systems; Yersinia pestis
PubMed: 28455335
DOI: 10.1128/AEM.00593-17 -
MSystems Jun 2024We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two strains, Neff (environmental) and T4 (clinical). Morphological analysis via...
We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for -related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of -related diseases.
Topics: Acanthamoeba castellanii; Extracellular Vesicles; Proteomics; Humans; Lipid Metabolism; Protozoan Proteins; Proteome
PubMed: 38717186
DOI: 10.1128/msystems.01226-23 -
Cornea May 2023Acanthamoeba castellanii ( A. castellanii ) displays host specificity at the level of the ocular surface. This study determined the susceptibility of the intact and...
PURPOSE
Acanthamoeba castellanii ( A. castellanii ) displays host specificity at the level of the ocular surface. This study determined the susceptibility of the intact and traumatized feline cornea to A. castellanii binding and invasion relative to other host species with established susceptibility and resistance to Acanthamoeba binding.
METHODS
Full-thickness buttons of fresh feline, porcine, and canine corneas were prepared. The corneal epithelium was confirmed intact by fluorescein staining or lightly scarified with a 25-G needle to simulate corneal trauma. Acanthamoeba castellanii was axenically cultivated. Corneal buttons were incubated with the parasite suspension or parasite-free medium for 18 hours at 35°C. Corneal buttons were rinsed, fixed, and processed for histopathology and immunohistochemistry using immunoperoxidase and immunofluorescence methods of amoeba detection.
RESULTS
Numerous amoebae were bound to feline and porcine corneas incubated with parasites. In both intact and traumatized corneas, amoebae were detected at all levels in the corneal epithelium and within the anterior stroma. In traumatized corneal sections, amoebae were frequently present in regions of epithelial damage. Corneal architecture was well-preserved in sections incubated with parasite-free medium; however, epithelial cell sloughing, separation of epithelial layers, and epithelial detachment from the stroma were observed in corneas incubated with amoebae. Intact and traumatized canine corneas were relatively free of adherent amoebae, and corneal architecture was indistinguishable between sections incubated with the parasite suspension and parasite-free medium.
CONCLUSIONS
The feline cornea is highly susceptible to in vitro binding and invasion by A. castellanii . Acanthamoeba binding to the feline cornea does not require a previous epithelial defect.
Topics: Animals; Cats; Dogs; Swine; Acanthamoeba castellanii; Cornea; Epithelium, Corneal; Acanthamoeba Keratitis; Corneal Injuries
PubMed: 36518074
DOI: 10.1097/ICO.0000000000003220 -
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 -
Virulence Dec 2018Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad...
Extracellular vesicles and vesicle-free secretome of the protozoa Acanthamoeba castellanii under homeostasis and nutritional stress and their damaging potential to host cells.
Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad range of clinical presentations to humans. Although several molecules have been described to play a role in Ac pathogenesis, including parasite host-tissue invasion and escaping of host-defense, little information is available on their mechanisms of secretion. Herein, we describe the molecular components secreted by Ac, under different protein availability conditions to simulate host niches. Ac extracellular vesicles (EVs) were morphologically and biochemically characterized. Dynamic light scattering analysis of Ac EVs identified polydisperse populations, which correlated to electron microscopy measurements. High-performance thin liquid chromatography of Ac EVs identified phospholipids, steryl-esters, sterol and free-fatty acid, the last two also characterized by GC-MS. Secretome composition (EVs and EVs-free supernatants) was also determined and proteins biological functions classified. In peptone-yeast-glucose (PYG) medium, a total of 179 proteins were identified (21 common proteins, 89 exclusive of EVs and 69 in EVs-free supernatant). In glucose alone, 205 proteins were identified (134 in EVs, 14 common and 57 proteins in EVs-free supernatant). From those, stress response, oxidative and protein and amino acid metabolism proteins prevailed. Qualitative differences were observed on carbohydrate metabolism enzymes from Krebs cycle and pentose phosphate shunt. Serine proteases and metalloproteinases predominated. Analysis of the cytotoxicity of Ac EVs (upon uptake) and EVs-free supernatant to epithelial and glioblastoma cells revealed a dose-dependent effect. Therefore, the Ac secretome differs depending on nutrient conditions, and is also likely to vary during infection.
Topics: Acanthamoeba castellanii; Amebiasis; Animals; Cell Line; Extracellular Vesicles; Homeostasis; Humans; Protein Transport; Proteome; Proteomics; Protozoan Proteins; Secretory Pathway
PubMed: 29560793
DOI: 10.1080/21505594.2018.1451184 -
Parasites & Vectors May 2024Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing...
Characterization of novel extracellular proteases produced by Acanthamoeba castellanii after contact with human corneal epithelial cells and their relevance to pathogenesis.
BACKGROUND
Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs).
METHODS
We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography.
RESULTS
We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba.
CONCLUSIONS
This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.
Topics: Humans; Acanthamoeba castellanii; Epithelial Cells; Coculture Techniques; Epithelium, Corneal; Peptide Hydrolases; Acanthamoeba Keratitis; Serine Proteases; Protozoan Proteins; Virulence
PubMed: 38812022
DOI: 10.1186/s13071-024-06304-7