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Pharmaceutics Jul 2023produces several classes of antimicrobial substances, including bacteriocins, which are peptides or proteins with different structural composition and molecular mass:... (Review)
Review
produces several classes of antimicrobial substances, including bacteriocins, which are peptides or proteins with different structural composition and molecular mass: ribosomally synthesized by bacteria (1.4-20 kDa), non-ribosomally synthesized peptides and cyclic lipopeptides (0.8-42 kDa) and exopolysaccharides (>1000 kDa). Different bacteriocins act against Gram-positive or Gram-negative bacteria, fungal pathogens and amoeba cells. The main mechanisms of bacteriocin lytic activity include interaction of peptides with membranes of target cells resulting in structural alterations, pore-forming, and inhibition of cell wall biosynthesis. DNase and RNase activity for some bacteriocines are also postulated. Non-ribosomal peptides are synthesized by special non-ribosomal multimodular peptide synthetases and contain unnatural amino acids or fatty acids. Their harmful effect is due to their ability to form pores in biological membranes, destabilize lipid packaging, and disrupt the peptidoglycan layer. Lipopeptides, as biosurfactants, are able to destroy bacterial biofilms. Secreted polysaccharides are high molecular weight compounds, composed of repeated units of sugar moieties attached to a carrier lipid. Their antagonistic action was revealed in relation to bacteria, viruses, and fungi. Exopolysaccharides also inhibit the formation of biofilms by pathogenic bacteria and prevent their colonization on various surfaces. However, mechanism of the harmful effect for many secreted antibacterial substances remains unknown. The antimicrobial activity for most substances has been studied in vitro only, but some substances have been characterized in vivo and they have found practical applications in medicine and veterinary. The cyclic lipopeptides that have surfactant properties are used in some industries. In this review, special attention is paid to the antimycobacterials produced by as a possible approach to combat multidrug-resistant and latent tuberculosis. In particular, licheniformins and bacitracins have shown strong antimycobacterial activity. However, the medical application of some antibacterials with promising in vitro antimycobacterial activity has been limited by their toxicity to animals and humans. As such, similar to the enhancement in the antimycobacterial activity of natural bacteriocins achieved using genetic engineering, the reduction in toxicity using the same approach appears feasible. The unique capability of to synthesize and produce a range of different antibacterial compounds means that this organism can act as a natural universal vehicle for antibiotic substances in the form of probiotic cultures and strains to combat various types of pathogens, including mycobacteria.
PubMed: 37514078
DOI: 10.3390/pharmaceutics15071893 -
Microorganisms Apr 2024Some free-living amoebae can behave as opportunistic parasites, causing rare but dangerous diseases in humans and animals, primarily amoebic keratitis, with loss of...
Some free-living amoebae can behave as opportunistic parasites, causing rare but dangerous diseases in humans and animals, primarily amoebic keratitis, with loss of vision, and encephalitis, which is almost always fatal [...].
PubMed: 38792695
DOI: 10.3390/microorganisms12050865 -
Nature Protocols Jan 2024Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology,... (Review)
Review
Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology, in the spotlight for bioengineering advances. Their host, Acanthamoeba castellanii, is an accidental human pathogen that acts as a natural host and environmental reservoir of other human pathogens. Tools for genetic manipulation of viruses and host were lacking. Here, we provide a detailed method for genetic manipulation of A. castellanii and the GVs it plays host to by using CRISPR-Cas9 or homologous recombination. We detail the steps of vector preparation (4 d), transfection of amoeba cells (1 h), infection (1 h), selection (5 d for viruses, 2 weeks for amoebas) and cloning of recombinant viruses (4 d) or amoebas (2 weeks). This procedure takes ~3 weeks or 1 month for the generation of recombinant viruses or amoebas, respectively. This methodology allows the generation of stable gene modifications, which was not possible by using RNA silencing, the only previously available reverse genetic tool. We also include detailed sample-preparation steps for protein localization by immunofluorescence (4 h), western blotting (4 h), quantification of viral particles by optical density (15 min), calculation of viral lethal dose 50 (7 d) and quantification of DNA replication by quantitative PCR (4 h) to allow efficient broad phenotyping of recombinant organisms. This methodology allows the function of thousands of ORFan genes present in GVs, as well as the complex pathogen-host, pathogen-pathogen or pathogen-symbiont interactions in A. castellanii, to be studied in vivo.
Topics: Humans; Acanthamoeba castellanii; Giant Viruses; Viruses
PubMed: 37964008
DOI: 10.1038/s41596-023-00910-y -
PLoS Neglected Tropical Diseases Mar 2024Acanthamoeba keratitis (AK) is a corneal sight-threatening infection caused by the free-living amoebae of the genus Acanthamoeba. Early and appropriate treatment...
BACKGROUND
Acanthamoeba keratitis (AK) is a corneal sight-threatening infection caused by the free-living amoebae of the genus Acanthamoeba. Early and appropriate treatment significantly impacts visual outcomes. Mucoadhesive polymers such as chitosan are a potential strategy to prolong the residence time and bioavailability of the encapsulated drugs in the cornea. Regarding the recent administration of miltefosine (MF) for treating resistant AK, in the present study, we synthesized miltefosine-loaded chitosan nanoparticles (MF-CS-NPs) and evaluated them against Acanthamoeba.
METHODOLOGY/PRINCIPAL FINDINGS
Chitosan nanoparticles (CNPs) were prepared using the ionic gelation method with negatively charged tripolyphosphate (TPP). The zeta-potential (ZP) and the particle size of MF-CS-NPs were 21.8±3.2 mV and 46.61±18.16 nm, respectively. The release profile of MF-CS-NPs indicated linearity with sustained drug release. The cytotoxicity of MF-CS-NPs on the Vero cell line was 2.67 and 1.64 times lower than free MF at 24 and 48 hours. This formulation exhibited no hemolytic activity in vitro and ocular irritation in rabbit eyes. The IC50 of MF-CS-NPs showed a significant reduction by 2.06 and 1.69-fold in trophozoites at 24 and 48 hours compared to free MF. Also, the MF-CS-NPs IC50 in the cysts form was slightly decreased by 1.26 and 1.21-fold at 24 and 48 hours compared to free MF.
CONCLUSIONS
The MF-CS-NPs were more effective against the trophozoites and cysts than free MF. The nano-chitosan formulation was more effective on trophozoites than the cysts form. MF-CS-NPs reduced toxicity and improved the amoebicidal effect of MF. Nano-chitosan could be an ideal carrier that decreases the cytotoxicity of miltefosine. Further analysis in animal settings is needed to evaluate this nano-formulation for clinical ocular drug delivery.
Topics: Animals; Rabbits; Drug Carriers; Chitosan; Acanthamoeba; Nanoparticles; Phosphorylcholine
PubMed: 38527059
DOI: 10.1371/journal.pntd.0011976 -
Food and Waterborne Parasitology Sep 2023In this study, the presence of spp., sp., spp., sp., and was assessed in organic leafy green vegetables (lettuce, spinach, cabbage) and fruits (strawberry), which...
In this study, the presence of spp., sp., spp., sp., and was assessed in organic leafy green vegetables (lettuce, spinach, cabbage) and fruits (strawberry), which are usually consumed raw. A total of 110 organic samples were collected in Valencia (Spain). Protozoa were concentrated before detection by immunofluorescence ( spp. and sp.) or real-time qPCR ( spp., sp., , , and ). The most abundant protozoa in organic vegetables and berry fruits were (65.5%), followed by (37.2%), (17.3%), (12.7%), spp. (6.8%), sp. (1.8%) and sp. (1.7%). was not found in any of the organic samples. Thus, results showed that consumers can be exposed to protozoan parasites by consuming organic vegetables and berry fruits. This is the first report in Spain describing the presence of the protozoan pathogens spp., sp., , and , spp. and sp. in organic fresh produce. The results of this research will help determine the risk of foodborne protozoan parasites on organic leafy greens and strawberries that are available at local markets.
PubMed: 37405064
DOI: 10.1016/j.fawpar.2023.e00200 -
International Journal of Molecular... Sep 2023Extracellular vesicles (EVs) of protozoan parasites have diverse biological functions that are essential for parasite survival and host-parasite interactions. In this...
Extracellular vesicles (EVs) of protozoan parasites have diverse biological functions that are essential for parasite survival and host-parasite interactions. In this study, we characterized the functional properties of EVs from , a pathogenic amoeba that causes a fatal brain infection called primary amoebic meningoencephalitis (PAM). EVs (NfEVs) have been shown to be internalized by host cells such as C6 glial cells and BV-2 microglial cells without causing direct cell death, indicating their potential roles in modulating host cell functions. NfEVs induced increased expression of proinflammatory cytokines and chemokines such as TNF-α, IL-1α, IL-1β, IL-6, IL-17, IFN-γ, MIP-1α, and MIP-2 in BV-2 microglial cells; these increases were initiated via MyD88-dependent TLR-2/TLR-4. The production levels of proinflammatory cytokines and chemokines in NfEVs-stimulated BV-2 microglial cells were effectively downregulated by inhibitors of MAPK, NF-κB, or JAK-STAT. Phosphorylation levels of JNK, p38, ERK, p65, JAK-1, and STAT3 were increased in NfEVs-stimulated BV-2 microglial cells but were effectively suppressed by each corresponding inhibitor. These results suggest that NfEVs could induce proinflammatory immune responses in BV-2 microglial cells via the NF-κB-dependent MAPK and JAK-STAT signaling pathways. Taken together, these findings suggest that NfEVs are pathogenic factors involved in the contact-independent pathogenic mechanisms of by inducing proinflammatory immune responses in BV-2 microglial cells, further contributing to deleterious inflammation in infected foci by activating subsequent inflammation cascades in other brain cells.
Topics: Microglia; NF-kappa B; Naegleria fowleri; Extracellular Vesicles; Cytokines; Blood Group Antigens; Immunity
PubMed: 37686429
DOI: 10.3390/ijms241713623 -
RNA (New York, N.Y.) Nov 2023The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune...
The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here, we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity toward RNA containing secondary structures and a free 5' end, and a broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.
Topics: Animals; Phylogeny; RNA, Messenger; Nucleotidyltransferases; RNA; Methyltransferases; Guanine; RNA Caps; Mammals
PubMed: 37625853
DOI: 10.1261/rna.079738.123 -
Translational Vision Science &... Aug 2023To develop a feline model of acute Acanthamoeba keratitis using methods that replicate natural routes of infection transmission.
PURPOSE
To develop a feline model of acute Acanthamoeba keratitis using methods that replicate natural routes of infection transmission.
METHODS
Corneal Acanthamoeba castellanii inoculation was performed by three methods: topical inoculation with Acanthamoeba solution following corneal abrasion, placement of a contaminated contact lens for 7 days, and placement of a contaminated contact lens for 7 days following corneal abrasion. Sham inoculations with parasite-free medium and sterile contact lenses were also performed. Cats were monitored by ocular examination and in vivo corneal confocal microscopy for 21 days post-inoculation. Corneal samples were collected at intervals for microbiologic assessment, histopathology, and immunohistochemistry.
RESULTS
All cats in the corneal abrasion groups developed clinical keratitis. Clinical ocular disease was inconsistently detected in cats from the contaminated contact lens only group. Initial corneal lesions were characterized by multifocal epithelial leukocyte infiltrates. Ocular lesions progressed to corneal epithelial ulceration and diffuse stromal inflammation. After 14 days, corneal ulcerations resolved, and stromal inflammation consolidated into multifocal subepithelial and stromal infiltrates. Corneal amoebae were detected by culture, in vivo confocal microscopy, histopathology, and immunohistochemistry in cats with keratitis. Neutrophilic and lymphocytic keratoconjunctivitis with lymphoplasmacytic anterior uveitis were identified by histopathology. Coinfection with aerobic bacteria was detected in some, but not all, cats with keratitis. Ocular disease was not detected in the sham inoculation groups.
CONCLUSIONS
Feline Acanthamoeba keratitis is experimentally transmissible by contaminated contact lenses and topical inoculation following corneal epithelial trauma.
TRANSLATIONAL RELEVANCE
Experimentally induced acute Acanthamoeba keratitis in cats is clinically and histopathologically similar to its human counterpart.
Topics: Cats; Animals; Humans; Acanthamoeba Keratitis; Acanthamoeba castellanii; Cornea; Corneal Injuries; Inflammation
PubMed: 37566398
DOI: 10.1167/tvst.12.8.10 -
Journal of Fish Diseases Sep 2023Nodular gill disease (NGD) is an infectious condition characterized by proliferative gill lesions leading to respiratory problems, oxygen deficiency and mortality in...
Nodular gill disease (NGD) is an infectious condition characterized by proliferative gill lesions leading to respiratory problems, oxygen deficiency and mortality in fish. Globally, NGD primarily impacts freshwater salmonids in intensive aquaculture systems. In recent years, numerous outbreaks of severe gill disease have affected more than half of the larger rainbow trout (Oncorhynchus mykiss) farms in Switzerland, mainly during spring and early summer. Mortality has reached up to 50% in cases where no treatment was administered. Freshwater amoeba are the presumed aetiologic agent of NGD. The gross gill score (GS) categorising severity of gill pathology is a valuable first-line diagnostic tool aiding fish farmers in identifying and quantifying amoebic gill disease (AGD) in farmed marine salmonids. In this study, the GS was adapted to the NGD outbreak in farmed trout in Switzerland. In addition to scoring disease severity, gill swabs from NGD-affected rainbow trout were sampled and amoeba were cultured from these swabs. Morphologic and molecular methods identified six amoeba strains: Cochliopodium sp., Naegleria sp., Vannella sp., Ripella sp., Saccamoeba sp. and Mycamoeba sp. However, the importance of the different amoeba species for the onset and progression of NGD still has to be evaluated. This paper presents the first description of NGD with associated amoeba infection in farmed rainbow trout in Switzerland.
Topics: Animals; Amoeba; Oncorhynchus mykiss; Gills; Switzerland; Fish Diseases; Aquaculture
PubMed: 37294659
DOI: 10.1111/jfd.13819 -
PLoS Pathogens Nov 2023The "Amoeboid Predator-Fungal Animal Virulence Hypothesis" posits that interactions with environmental phagocytes shape the evolution of virulence traits in fungal...
The "Amoeboid Predator-Fungal Animal Virulence Hypothesis" posits that interactions with environmental phagocytes shape the evolution of virulence traits in fungal pathogens. In this hypothesis, selection to avoid predation by amoeba inadvertently selects for traits that contribute to fungal escape from phagocytic immune cells. Here, we investigate this hypothesis in the human fungal pathogens Cryptococcus neoformans and Cryptococcus deneoformans. Applying quantitative trait locus (QTL) mapping and comparative genomics, we discovered a cross-species QTL region that is responsible for variation in resistance to amoeba predation. In C. neoformans, this same QTL was found to have pleiotropic effects on melanization, an established virulence factor. Through fine mapping and population genomic comparisons, we identified the gene encoding the transcription factor Bzp4 that underlies this pleiotropic QTL and we show that decreased expression of this gene reduces melanization and increases susceptibility to amoeba predation. Despite the joint effects of BZP4 on amoeba resistance and melanin production, we find no relationship between BZP4 genotype and escape from macrophages or virulence in murine models of disease. Our findings provide new perspectives on how microbial ecology shapes the genetic architecture of fungal virulence, and suggests the need for more nuanced models for the evolution of pathogenesis that account for the complexities of both microbe-microbe and microbe-host interactions.
Topics: Animals; Humans; Mice; Amoeba; Metagenomics; Predatory Behavior; Cryptococcus neoformans; Cryptococcosis
PubMed: 37956179
DOI: 10.1371/journal.ppat.1011763