-
Development (Cambridge, England) Dec 2023Development can proceed in 'fits and starts', with rapid transitions between cell states involving concerted transcriptome-wide changes in gene expression. However, it...
Development can proceed in 'fits and starts', with rapid transitions between cell states involving concerted transcriptome-wide changes in gene expression. However, it is not clear how these transitions are regulated in complex cell populations, in which cells receive multiple inputs. We address this issue using Dictyostelium cells undergoing development in their physiological niche. A continuous single cell transcriptomics time series identifies a sharp 'jump' in global gene expression marking functionally different cell states. By simultaneously imaging the physiological dynamics of transcription and signalling, we show the jump coincides with the onset of collective oscillations of cAMP. Optogenetic control of cAMP pulses shows that different jump genes respond to distinct dynamic features of signalling. Late jump gene expression changes are almost completely dependent on cAMP, whereas transcript changes at the onset of the jump require additional input. The coupling of collective signalling with gene expression is a potentially powerful strategy to drive robust cell state transitions in heterogeneous signalling environments. Based on the context of the jump, we also conclude that sharp gene expression transitions may not be sufficient for commitment.
Topics: Dictyostelium; Signal Transduction; Transcriptome; Gene Expression Profiling
PubMed: 37921687
DOI: 10.1242/dev.201946 -
Microbiology Spectrum Mar 2024species are clinically relevant free-living amoebae (FLA) ubiquitously found in soil and water bodies. Metabolically active trophozoites graze on diverse microbes via...
species are clinically relevant free-living amoebae (FLA) ubiquitously found in soil and water bodies. Metabolically active trophozoites graze on diverse microbes via phagocytosis. However, functional studies on Rab GTPases (Rabs), which are critical for controlling vesicle trafficking and maturation, are scarce for this FLA. This knowledge gap can be partly explained by the limited genetic tools available for cell biology. Here, we developed plasmids to generate fusions of strain Neff proteins to the N- or C-termini of mEGFP and mCherry2. Phylogenomic and structural analyses of the 11 Neff Rab7 paralogs found in the RefSeq assembly revealed that eight of them had non-canonical sequences. After correcting the gene annotation for the Rab7A ortholog, we generated a line stably expressing an mEGFP-Rab7A fusion, demonstrating its correct localization to acidified macropinocytic and phagocytic vacuoles using fluorescence microscopy live cell imaging (LCI). Direct labeling of live ESTM1D_MKCAZ16_6a (Sm18) cells with pHrodo Red, a pH-sensitive dye, demonstrated that they reside within acidified, Rab7A-positive vacuoles. We constructed new mini-Tn7 delivery plasmids and tagged Sm18 with constitutively expressed mScarlet-I. Co-culture experiments of Neff trophozoites with Sm18::mTn7TC1_Pc_mScarlet-I, coupled with LCI and microplate reader assays, demonstrated that Sm18 underwent multiple replication rounds before reaching the extracellular medium via non-lytic exocytosis. We conclude that belongs to the class of bacteria that can use amoeba as an intracellular replication niche within a -containing vacuole that interacts extensively with the endocytic pathway.IMPORTANCEDiverse lineages (genotypes) are of increasing clinical concern, mainly causing amoebic keratitis and granulomatous amebic encephalitis among other infections. ranks among the top 10 most prevalent multidrug-resistant opportunistic nosocomial pathogens and is a recurrent member of the microbiome hosted by and other free-living amoebae. However, little is known about the molecular strategies deployed by for an intracellular lifestyle in amoebae and other professional phagocytes such as macrophages, which allow the bacterium to evade the immune system and the action of antibiotics. Our plasmids and easy-to-use microtiter plate co-culture assays should facilitate investigations into the cellular microbiology of interactions with and other opportunistic pathogens, which may ultimately lead to the discovery of new molecular targets and antimicrobial therapies to combat difficult-to-treat infections caused by these ubiquitous microbes.
Topics: Acanthamoeba castellanii; Stenotrophomonas maltophilia; Vacuoles; Phylogeny; Bacteria
PubMed: 38319117
DOI: 10.1128/spectrum.02988-23 -
Dientamoeba fragilis associated with microbiome diversity changes in acute gastroenteritis patients.Parasitology International Dec 2023This study examined the correlation between intestinal protozoans and the bacterial microbiome in faecal samples collected from 463 patients in New Zealand who were...
This study examined the correlation between intestinal protozoans and the bacterial microbiome in faecal samples collected from 463 patients in New Zealand who were diagnosed with gastroenteritis. In comparison to traditional microscopic diagnosis methods, Multiplexed-tandem PCR proved to be more effective in detecting intestinal parasites. Among the identified protozoans, Blastocystis sp. and Dientamoeba fragilis were the most prevalent. Notably, D. fragilis was significantly associated with an increase in the alpha-diversity of host prokaryotic microbes. Although the exact role of Blastocystis sp. and D. fragilis as the primary cause of gastroenteritis remains debatable, our data indicates a substantial correlation between these protozoans and the prokaryote microbiome of their hosts, particularly when compared to other protists or patients with gastroenteritis but no detectable parasitic cause. These findings underscore the significance of comprehending the contributions of intestinal protozoans, specifically D. fragilis, to the development of gastroenteritis and their potential implications for disease management.
Topics: Animals; Humans; Dientamoeba; Intestinal Diseases, Parasitic; Blastocystis; Gastroenteritis; Parasites; Feces
PubMed: 37482266
DOI: 10.1016/j.parint.2023.102788 -
Microorganisms Oct 2023The northeastern edge of the Tibetan Plateau exhibits diverse climate and landform variations, and has experienced substantial recent environmental changes, which may...
The northeastern edge of the Tibetan Plateau exhibits diverse climate and landform variations, and has experienced substantial recent environmental changes, which may significantly impact local agricultural practices. Understanding the microeukaryote community structure within agricultural soils is crucial for finding out the biological responses to such changes and may guide future agricultural practices. In this study, we employed high-throughput amplicon sequencing to examine 29 agricultural soil samples from seven research areas around the northeastern edge of the Tibetan Plateau. The findings revealed that the predominant biological communities in these soils were characterized by a high abundance of Alveolata, Amoebozoa, and Rhizaria. Ascomycota displayed the highest relative abundance among fungal communities. Moreover, notable distinctions in microeukaryote community composition were observed among the study sites. Co-occurrence network analysis highlighted interactions between the biological communities. Furthermore, our results elucidated that deterministic and stochastic processes exerted diverse influences on the distribution of protozoan and fungal communities. This study provides valuable insight into the microeukaryote structure in the agricultural soils of the northeastern edge of the Tibetan Plateau, shedding light on the intricate relationships between environmental factors, microeukaryote communities, and agricultural productivity.
PubMed: 37894168
DOI: 10.3390/microorganisms11102510 -
Molecular Biology and Evolution Dec 2023The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine...
Functional Prokaryotic-Like Deoxycytidine Triphosphate Deaminases and Thymidylate Synthase in Eukaryotic Social Amoebae: Vertical, Endosymbiotic, or Horizontal Gene Transfer?
The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine triphosphate into deoxyuridine triphosphate, whereas eukaryotes and gram-positive bacteria instead use deoxycytidine monophosphate deaminase to transform deoxycytidine monophosphate to deoxyuridine monophosphate. It is then unusual that in addition to deoxycytidine monophosphate deaminases, the eukaryote Dictyostelium discoideum has 2 deoxycytidine triphosphate deaminases (Dcd1Dicty and Dcd2Dicty). Expression of either DcdDicty can fully rescue the slow growth of an Escherichia coli dcd knockout. Both DcdDicty mitigate the hydroxyurea sensitivity of a Schizosaccharomyces pombe deoxycytidine monophosphate deaminase knockout. Phylogenies show that Dcd1Dicty homologs may have entered the common ancestor of the eukaryotic groups of Amoebozoa, Obazoa, Metamonada, and Discoba through an ancient horizontal gene transfer from a prokaryote or an ancient endosymbiotic gene transfer from a mitochondrion, followed by horizontal gene transfer from Amoebozoa to several other unrelated groups of eukaryotes. In contrast, the Dcd2Dicty homologs were a separate horizontal gene transfer from a prokaryote or a virus into either Amoebozoa or Rhizaria, followed by a horizontal gene transfer between them. ThyXDicty, the D. discoideum thymidylate synthase, another enzyme of the deoxythymidine triphosphate biosynthesis pathway, was suggested previously to be acquired from the ancestral mitochondria or by horizontal gene transfer from alpha-proteobacteria. ThyXDicty can fully rescue the E. coli thymidylate synthase knockout, and we establish that it was obtained by the common ancestor of social amoebae not from mitochondria but from a bacterium. We propose horizontal gene transfer and endosymbiotic gene transfer contributed to the enzyme diversity of the deoxythymidine triphosphate synthesis pathway in most social amoebae, many Amoebozoa, and other eukaryotes.
Topics: DCMP Deaminase; Gene Transfer, Horizontal; Escherichia coli; Amoeba; Thymidylate Synthase; Dictyostelium; Deoxycytidine Monophosphate
PubMed: 38064674
DOI: 10.1093/molbev/msad268 -
Genes Feb 2024, the causative agent of amebiasis, is the third leading cause of death among parasitic diseases globally. Its life cycle includes encystation, which has been mostly...
, the causative agent of amebiasis, is the third leading cause of death among parasitic diseases globally. Its life cycle includes encystation, which has been mostly studied in , responsible for reptilian amebiasis. However, the molecular mechanisms underlying this process are not fully understood. Therefore, we focused on the identification and characterization of Myb proteins, which regulate the expression of encystation-related genes in various protozoan parasites. Through bioinformatic analysis, we identified 48 genes in encoding MYB-domain-containing proteins. These were classified into single-repeat 1R (20), 2R-MYB proteins (27), and one 4R-MYB protein. The in-silico analysis suggests that these proteins are multifunctional, participating in transcriptional regulation, chromatin remodeling, telomere maintenance, and . Transcriptomic data analysis revealed expression signatures of genes, suggesting a potential orchestration in the regulation of early and late encystation-excystation genes. Furthermore, we identified probable target genes associated with reproduction, the meiotic cell cycle, ubiquitin-dependent protein catabolism, and endosomal transport. In conclusion, our findings suggest that Myb proteins regulate stage-specific proteins and a wide array of cellular processes. This study provides a foundation for further exploration of the molecular mechanisms governing encystation and unveils potential targets for therapeutic intervention in amebiasis.
Topics: Humans; Entamoeba; Entamoeba histolytica; Gene Expression Profiling; Gene Expression Regulation; Amebiasis
PubMed: 38397191
DOI: 10.3390/genes15020201 -
International Journal of Molecular... Sep 2023species (sp.) is one of the less well-understood water- and foodborne protozoa of medical and veterinary importance linked to different gastrointestinal disorders....
species (sp.) is one of the less well-understood water- and foodborne protozoa of medical and veterinary importance linked to different gastrointestinal disorders. Soldiers participating in military missions are particularly vulnerable to infection with this protozoa. The present study used molecular methods to detect, identify, and subtype (ST) sp. in Polish soldiers stationed in the Republic of Kosovo. Fecal samples were collected from 192 soldiers on arrival and after four months of stay. After DNA extraction, the barcoding region of the small subunit ribosomal RNA (SSU-rRNA) gene was amplified and sequenced. The DNA of sp. was detected in six (3.13%) and thirty (15.16%) samples in the first and second batch, respectively. Sequencing analysis revealed infections with ST 2, 3, 4, and 7. There was no statistical association between sp. infection and the parasite's ST or the age or rank of soldiers. The results indicate that the visit to a new environment and prolonged stay in the area of military operation in Kosovo resulted in a significant increase in both sp. infections and ST diversity among surveyed soldiers. This shows the need to undertake appropriate countermeasures to reduce infections in the military environment abroad.
Topics: Blastocystis; Kosovo; Poland; Feces; Water
PubMed: 37762408
DOI: 10.3390/ijms241814100 -
The Science of the Total Environment Nov 2023Free-living amoebae (FLA) are ubiquitous protozoa mainly found in aquatic environments. They are well-known reservoirs and vectors for the transmission of...
Bacterial microbiota management in free-living amoebae (Heterolobosea lineage) isolated from water: The impact of amoebae identity, grazing conditions, and passage number.
Free-living amoebae (FLA) are ubiquitous protozoa mainly found in aquatic environments. They are well-known reservoirs and vectors for the transmission of amoeba-resistant bacteria (ARB), most of which are pathogenic to humans. Yet, the natural bacterial microbiota associated with FLA remains largely unknown. Herein, we characterized the natural bacterial microbiota of different FLA species isolated from recreational waters in Guadeloupe. Monoxenic cultures of Naegleria australiensis, Naegleria sp. WTP3, Paravahlkampfia ustiana and Vahlkampfia sp. AK-2007 (Heterolobosea lineage) were cultivated under different grazing conditions, during successive passages. The whole bacterial microbiota of the waters and the amoebal cysts was characterized using 16S rRNA gene metabarcoding. The culturable subset of ARB was analyzed by mass spectrometry (MALDI-TOF MS), conventional 16S PCR, and disk diffusion method (to assess bacterial antibiotic resistance). Transmission electron microscopy was used to locate the ARB inside the amoebae. According to alpha and beta-diversity analyses, FLA bacterial microbiota were significantly different from the ones of their habitat. While Vogesella and Aquabacterium genera were detected in water, the most common ARB belonged to Pseudomonas, Bosea, and Escherichia/Shigella genera. The different FLA species showed both temporary and permanent associations with differentially bacterial taxa, suggesting host specificity. These associations depend on the number of passages and grazing conditions. Additionally, Naegleria, Vahlkampfia and Paravahlkampfia cysts were shown to naturally harbor viable bacteria of the Acinetobacter, Escherichia, Enterobacter, Pseudomonas and Microbacterium genera, all being pathogenic to humans. To our knowledge, this is the first time Paravahlkampfia and Vahlkampfia have been demonstrated as hosts of pathogenic ARB in water. Globally, the persistence of these ARB inside resistant cysts represents a potential health risk. To ensure the continued safety of recreational waters, it is crucial to (i) regularly control both the amoebae and their ARB and (ii) improve knowledge on amoebae-bacteria interactions to establish better water management protocols.
Topics: Humans; Water; RNA, Ribosomal, 16S; Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Amoeba; Bacteria; Microbiota
PubMed: 37506913
DOI: 10.1016/j.scitotenv.2023.165816 -
Parasites, Hosts and Diseases Nov 2023Free-living amoebae (FLA) rarely cause human infections but can invoke fatal infections in the central nervous system (CNS). No consensus treatment has been established...
Free-living amoebae (FLA) rarely cause human infections but can invoke fatal infections in the central nervous system (CNS). No consensus treatment has been established for FLA infections of the CNS, emphasizing the urgent need to discover or develop safe and effective drugs. Flavonoids, natural compounds from plants and plant-derived products, are known to have antiprotozoan activities against several pathogenic protozoa parasites. The anti-FLA activity of flavonoids has also been proposed, while their antiamoebic activity for FLA needs to be emperically determined. We herein evaluated the antiamoebic activities of 18 flavonoids against Naegleria fowleri and Acanthamoeba species which included A. castellanii and A. polyphaga. These flavonoids showed different profiles of antiamoebic activity against N. fowleri and Acanthamoeba species. Demethoxycurcumin, kaempferol, resveratrol, and silybin (A+B) showed in vitro antiamoebic activity against both N. fowleri and Acanthamoeba species. Apigenin, costunolide, (‒)-epicatechin, (‒)-epigallocatechin, rosmarinic acid, and (‒)-trans-caryophyllene showed selective antiamoebic activity for Acanthamoeba species. Luteolin was more effective for N. fowleri. However, afzelin, berberine, (±)-catechin, chelerythrine, genistein, (+)-pinostrobin, and quercetin did not exhibit antiamoebic activity against the amoeba species. They neither showed selective antiamoebic activity with significant cytotoxicity to C6 glial cells. Our results provide a basis for the anti-FLA activity of flavonoids, which can be applied to develope alternative or supplemental therapeutic agents for FLA infections of the CNS.
Topics: Humans; Amoeba; Acanthamoeba; Naegleria fowleri; Flavonoids; Amebiasis
PubMed: 38043540
DOI: 10.3347/PHD.23078 -
Microbiology (Reading, England) Aug 2023The predation and engulfment of bacteria by facilitates intimate interactions between host and prey. This process plays an important and underestimated role in the...
The predation and engulfment of bacteria by facilitates intimate interactions between host and prey. This process plays an important and underestimated role in the physiology, ecology and evolution of pathogenic bacteria. species can be reservoirs for many important human pathogens including is the leading cause of bacterial foodborne enteritis worldwide, despite being a microaerophile that is incapable of withstanding atmospheric levels of oxygen long-term. The persistence and transmission of this major pathogen in the natural environment outside its avian and mammalian hosts is not fully understood. Recent evidence has provided insight into the relationship of and spp. where are a transient host for this pathogen. Mutations to the flagella components were shown to affect interactions. Here, we show that the motility function of flagella is not a prerequisite for interactions and that specific -linked glycan modifications of the major flagellin, FlaA, are important for the recognition, interaction and phagocytosis by . Substitution of the -linked glycosylated serine 415 and threonine 477 with alanine within FlaA abolished interactions with and these mutants were indistinguishable from a Δ mutant. By contrast, mutation to serine 405 did not affect 11168H and interactions. Given the abundance of flagella glycosylation among clinically important pathogens, our observations may have a wider implication for understanding host-pathogen interactions.
Topics: Humans; Animals; Acanthamoeba castellanii; Campylobacter jejuni; Flagellin; Alanine; Polysaccharides; Mammals
PubMed: 37610804
DOI: 10.1099/mic.0.001386