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Applied and Environmental Microbiology Feb 2024Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate...
Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in , a -related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as β-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, TA modules may be involved in survival to stress. Moreover, as can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which has to share its replicative niche.IMPORTANCEThe response to adverse conditions, such as exposure to antibiotics, nutrient starvation and competition with other microorganisms, is essential for the survival of a bacterial population. TA systems are modules composed of two elements, a toxic protein and an antitoxin (protein or RNA) that counteracts the toxin. Although many aspects of TA biological functions still await to be elucidated, TAs have often been implicated in bacterial response to stress, including the response to nutrient starvation, antibiotic treatment and bacteriophage infection. TAs are ubiquitous in free-living bacteria but rare in obligate intracellular species such as chlamydiae. We identified functional TA systems in , a chlamydial species with a strikingly broad host range compared to other chlamydiae. Our work contributes to understand how obligate intracellular bacteria react to adverse conditions that might arise from competition with other viruses/bacteria for the same replicative niche and would threaten their ability to replicate.
Topics: Animals; Toxin-Antitoxin Systems; Chlamydia; Toxins, Biological; Antitoxins; Anti-Bacterial Agents; RNA; Mammals; Chlamydiales
PubMed: 38214519
DOI: 10.1128/aem.00681-23 -
Frontiers in Immunology 2023Primary Amoebic Meningoencephalitis (PAM), a severe lethal brain disease, is caused by a parasite, , also known as the "brain-eating amoeba". The chances of a patient's...
Primary Amoebic Meningoencephalitis (PAM), a severe lethal brain disease, is caused by a parasite, , also known as the "brain-eating amoeba". The chances of a patient's recovery after being affected by this parasite are very low. Only 5% of people are known to survive this life-threatening infection. Despite the fact that causes a severe, fatal infection, there is no proper treatment available to prevent or cure it. In this context, it is necessary to formulate a potential vaccine that could be able to combat infection. The current study aimed at developing a multi-epitope subunit vaccine against by utilizing immunoinformatics techniques and reverse vaccinology approaches. The T- and B-cell epitopes were predicted by various tools. In order to choose epitopes with the ability to trigger both T- and B-cell-mediated immune responses, the epitopes were put through a screening pipeline including toxicity, antigenicity, cytokine-inductivity, and allergenicity analysis. Three vaccine constructs were designed from the generated epitopes linked with linkers and adjuvants. The modeled vaccines were docked with the immune receptors, where vaccine-1 showed the highest binding affinity. Binding affinity and stability of the docked complex were confirmed through normal mode analysis and molecular dynamic simulations. Immune simulations developed the immune profile, and cloning affirmed the expression probability of the vaccine construct in ( strain K12. This study demonstrates an innovative preventative strategy for the brain-eating amoeba by developing a potential vaccine through immunoinformatics and reverse vaccinology approaches. This study has great preventive potential for Primary Amoebic Meningoencephalitis, and further research is required to assess the efficacy of the designed vaccine.
Topics: Humans; Naegleria fowleri; Escherichia coli; Central Nervous System Protozoal Infections; Brain; Epitopes, B-Lymphocyte; Informatics; Vaccines, Subunit
PubMed: 37965306
DOI: 10.3389/fimmu.2023.1284621 -
ACS Omega Mar 2024Pathogenic () are opportunistic free-living amoebae and are the causative agents of a very rare but severe brain infection called primary amoebic meningoencephalitis...
Pathogenic () are opportunistic free-living amoebae and are the causative agents of a very rare but severe brain infection called primary amoebic meningoencephalitis (PAM). The fatality rate of PAM in reported cases is more than 95%. Most of the drugs used against infections are repurposed drugs. Therefore, a large number of compounds have been tested against in vitro, but most of the tested compounds showed high toxicity and an inability to cross the blood-brain barrier. Andrographolide, forskolin, and borneol are important natural compounds that have shown various valuable biological properties. In the present study, the nanoconjugates (AND-AgNPs, BOR-AgNPs, and FOR-AgNPs) of these compounds were synthesized and assessed against both stages (trophozoite and cyst) of for their antiamoebic and cysticidal potential in vitro. In addition, cytotoxicity and host cell pathogenicity were also evaluated in vitro. FOR-AgNPs were the most potent nanoconjugate and showed potent antiamoebic activity againstwith an IC of 26.35 μM. Nanoconjugates FOR-AgNPs, BOR-AgNPs, and AND-AgNPs also significantly inhibit the viability of cysts. Cytotoxicity assessment showed that these nanoconjugates caused minimum damage to human keratinocyte cells (HaCaT cells) at 100 μg/mL, while also effectively reducing the cytopathogenicity of trophozoites to the HaCaT cells. The outcomes of our experiments have unveiled substantial potential for AND-AgNPs, BOR-AgNPs, and FOR-AgNPs in the realm of developing innovative alternative therapeutic agents to combat infections caused by . This study represents a significant step forward in the pursuit of advanced strategies for managing such amoebic infections, laying the foundation for the development of novel and more effective therapeutic modalities in the fight against free-living amoebae.
PubMed: 38497026
DOI: 10.1021/acsomega.3c08844 -
Frontiers in Cellular and Infection... 2023Entamoeba is an anaerobic protozoan that is strongly associated with inflamed periodontal pockets. It is able to invade the mucosal epithelium of the human host, where...
BACKGROUND
Entamoeba is an anaerobic protozoan that is strongly associated with inflamed periodontal pockets. It is able to invade the mucosal epithelium of the human host, where it can feed on epithelial cells and elicit a severe innate immune response. Unlike other species, it is considered that cannot form cysts, because it is a non-infectious protozoan. The lack of encystation capability would make it susceptible to periodontal treatment. However, it is not clear how the human host becomes infected with trophozoites. We investigated the ability of to encapsulate in response to an unfavorable environment .
METHODS
Different strains of , isolated from inflamed periodontal pocket samples, were cultured for 8 days in the presence or absence of the antimicrobials amoxycillin and metronidazole. To reveal cyst formation, we investigated the morphology and ultrastructure of the amoeba by light, fluorescence, transmission and scanning electron microscopy. We also used the fluorescent dye calcofluor white M2R to demonstrate chitin present in the cyst wall.
RESULTS
We observed exocysts and an intra-cystic space separating the encapsulated trophozoite from the environment. Remarkably, cysts showed a smooth surface, polygonal edges and smaller size compared to free-living trophozoites. In addition, encapsulated trophozoites that detached from the cyst wall had a dense cytoplasma without phagocytic vesicles. The cyst walls consisted of chitin as in other species. The encapsulated trophozoids were mononuclear after antibioticinduced encapsulation.
DISCUSSION
We conclude that cyst formation has significant implications for dissemination and infection and may explain why established treatment approaches often fail to halt periodontal tissue destruction during periodontitis and peri-implantitis.
Topics: Animals; Humans; Entamoeba; Trophozoites; Cysts; Anti-Bacterial Agents; Chitin
PubMed: 37469604
DOI: 10.3389/fcimb.2023.1201394 -
Frontiers in Microbiology 2023Free-living amoebae are described as potential reservoirs for pathogenic bacteria in the environment. It has been hypothesized that this might be the case for subsp. ,...
Free-living amoebae are described as potential reservoirs for pathogenic bacteria in the environment. It has been hypothesized that this might be the case for subsp. , the bacterium responsible for paratuberculosis. In a previous work, we isolated an amoeba from a water sample in the environment of infected cattle and showed that this amoeba was associated with subsp. . While a partial 18S rRNA gene has allowed us to suggest that this amoeba was -like, at that time we were not able to sub-cultivate it. In the present study, we succeeded in cultivating this strain at 20-25°C. This amoeba is among the smallest (5-7 μm) described. The sequencing of the whole genome allowed us to extract the full 18S rRNA gene and propose this strain as a new species of the genus, i.e., . Of note, the mitochondrial genome is particularly large (184,954 bp). Finally, we showed that this amoeba was able to phagocyte subsp. and that the bacterium was still observed within amoebae after at least 3 days. In conclusion, we characterized a new environmental amoeba species at the cellular and genome level that was able to interact with subsp. . As a result, is a potential candidate as environmental reservoir for subsp. but further experiments are needed to test this hypothesis.
PubMed: 38188567
DOI: 10.3389/fmicb.2023.1324985 -
PloS One 2023Primary amoebic meningoencephalitis (PAM) is a rapidly progressing central nervous system (CNS) infection caused by Naegleria fowleri, a free-living amoeba found in warm...
Primary amoebic meningoencephalitis (PAM) is a rapidly progressing central nervous system (CNS) infection caused by Naegleria fowleri, a free-living amoeba found in warm freshwater. The disease progression is very rapid, and the outcome is nearly always fatal. We aim to describe the disease course in patients admitted with PAM in a tertiary care center in Karachi, Pakistan between the periods of 2010 to 2021. A total of 39 patients were included in the study, 33 males (84.6%). The median age of the patients was 34 years. The most frequent presenting complaint was fever, which was found in 37 patients (94.9%) followed by headache in 28 patients (71.8%), nausea and vomiting in 27 patients (69.2%), and seizures in 10 patients (25.6%). Overall, 39 patients underwent lumbar puncture, 27 patients (69.2%) had a positive motile trophozoites on CSF wet preparation microscopy, 18 patients (46.2%) had a positive culture, and 10 patients had a positive PCR. CSF analysis resembled bacterial meningitis with elevated white blood cell counts with predominantly neutrophils (median, 3000 [range, 1350-7500] cells/μL), low glucose levels median, 14 [range, 1-92] mg/dL), and elevated protein levels (median, 344 [range, 289-405] mg/dL). Imaging results were abnormal in approximately three-fourths of the patients which included cerebral edema (66.7%), hydrocephalus (25.6%), and cerebral infarctions (12.8%). Only one patient survived. PAM is a fatal illness with limited treatment success. Early diagnosis and prompt initiation of treatment can improve the survival of the patients and reduce mortality.
Topics: Male; Humans; Adult; Pakistan; Central Nervous System Protozoal Infections; Naegleria fowleri; Spinal Puncture; Amebiasis; Meningoencephalitis
PubMed: 37939056
DOI: 10.1371/journal.pone.0290394 -
BioRxiv : the Preprint Server For... Nov 2023Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a...
Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. and were particularly fascinating for their apparent redundant functions during human macrophage infection, while alone was essential for virulence in the amoeban host . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.
PubMed: 36945652
DOI: 10.1101/2023.02.03.527066 -
European Journal of Protistology Oct 2023Cercozoans and heterolobose amoebae are found across terrestrial habitats where they feed on other unicellular microbes, including bacteria, fungi and microalgae. They...
Cercozoans and heterolobose amoebae are found across terrestrial habitats where they feed on other unicellular microbes, including bacteria, fungi and microalgae. They constitute a significant fraction of soil ecosystems and are integral members of plant microbiota. Here, we present the results on the isolation of protozoans from the rhizosphere and phyllosphere of Browningia candelaris (Meyen) in the Andean Altiplano and Eulychnia taltalensis (F. Ritter) from the Coastal Cordillera of the Atacama Desert, both endemic to this ancient desert. We identified a new heterolobose amoeba species of the genus Allovahlkampfia isolated from cactus soil, three new species of the different glissomonad genera Allapsa, Neoheteromita, Neocercomonas and one new thecofilosean amoeba of the genus Rhogostoma isolated from the phyllosphere of one studied cactus. In addition, one bacterivorous flagellate was isolated from cactus spines and identified as a member of the non-scaled imbricatean family Spongomonadidae (Spongomonas). The isolation of protists from cactus spines extends the knowledge on the habitat ranges of taxa typically found on plant leaves or soils. The molecular data presented here is a prerequisite for further investigations on the ecology and diversity of protists including next-generation sequencing of microhabitats in plants and the rhizosphere, allowing for deeper taxonomic classification.
Topics: Rhizosphere; Cactaceae; Bacteria; Microbiota; Plants; Soil Microbiology; Soil
PubMed: 38006640
DOI: 10.1016/j.ejop.2023.126034 -
BioRxiv : the Preprint Server For... Dec 2023This study investigates the genomic characteristics of , a small-sized amoeba within the Tubulinea clade of the Amoebozoa supergroup. Despite Tubulinea's significance...
This study investigates the genomic characteristics of , a small-sized amoeba within the Tubulinea clade of the Amoebozoa supergroup. Despite Tubulinea's significance in various fields, genomic data for this clade have been scarce presents the smallest free-living amoeba genome within Tubulinea and Amoebozoa to date. Comparative analysis reveals intriguing parallels with parasitic lineages in terms of genome size and predicted gene numbers, emphasizing the need to understand the consequences of reduced genomes in free-living amoebae. Functional categorization of predicted genes in shows similar percentages of ortholog groups to other amoebae in various categories, but a distinctive feature is the extensive gene contraction in orphan (ORFan) genes and those involved in biological processes. Notably, among the few genes that underwent expansion, none are related to cellular components, suggesting adaptive processes that streamline biological processes and cellular components for efficiency and energy conservation. The investigation delves into genomic structural evidence, including gene content and repetitive elements, illuminating the distinctive genomic traits of and providing reinforcement for its compact genome size. Overall, this research underscores the diversity within Tubulinea, highlights knowledge gaps in Amoebozoa genomics, and positions as a valuable addition to genomic datasets, prompting further exploration of complexities in Amoebozoa diversity and genome evolution.
PubMed: 38106205
DOI: 10.1101/2023.12.07.570689 -
Microbiome Jul 2023Legionella are parasites of freshwater protozoa, responsible for Legionellosis. Legionella can be found in a variety of aquatic environments, including rivers, lakes,...
BACKGROUND
Legionella are parasites of freshwater protozoa, responsible for Legionellosis. Legionella can be found in a variety of aquatic environments, including rivers, lakes, and springs, as well as in engineered water systems where they can potentially lead to human disease outbarks. Legionella are considered to be predominantly freshwater organisms with a limited ability to proliferate in saline environments. Exposure of Legionella to high sodium concentrations inhibits growth and virulence of laboratory strains, particularly under elevated temperatures. Nonetheless, Legionella have been identified in some saline environments where they likely interact with various protozoan hosts. In this work, we examine how these selection pressures, sodium and grazing, help shape Legionella ecology within natural environments. Utilizing Legionella-specific primers targeting a variable region of the Legionella 16S rRNA gene, we characterized Legionella abundance, diversity, and community composition in natural spring clusters of varying sodium concentrations, focusing on high sodium concentrations and elevated temperatures.
RESULTS
We observed the highest abundance of Legionella in spring clusters of high salinity, particularly in combination with elevated temperatures. Legionella abundance was strongly related to sodium concentrations. The Legionella community structure in saline environments was characterized by relatively low diversity, compared to spring clusters of lower salinity. The community composition in high salinity was characterized by few dominant Legionella genotypes, not related to previously described species. Protozoan microbial community structure and composition patterns resembled those of Legionella, suggesting a common response to similar selection pressures. We examined Legionella co-occurrence with potential protozoan hosts and found associations with Ciliophora and Amoebozoa representatives.
CONCLUSIONS
Our results indicate that selection forces in saline environments favor a small yet dominant group of Legionella species that are not closely related to known species. These novel environmental genotypes interact with various protozoan hosts, under environmental conditions of high salinity. Our findings suggest that alternative survival mechanisms are utilized by these species, representing mechanisms distinct from those of well-studied laboratory strains. Our study demonstrate how salinity can shape communities of opportunistic pathogens and their hosts, in natural environments, shedding light on evolutionary forces acting within these complex environments. Video Abstract.
Topics: Humans; Legionella; RNA, Ribosomal, 16S; Ecology; Water Microbiology; Fresh Water
PubMed: 37518067
DOI: 10.1186/s40168-023-01611-0