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Bacterial EVs have been related to inter-kingdom communication between probiotic/pathogenic bacteria and their hosts. Our aim was to investigate the transcytosis process of B. subtilis EVs using an in vitro intestinal epithelial cell model. In this study, using Confocal Laser Scanning Microscopy, we report that uptake and internalization of CFSE-labeled B. subtilis EVs (115 nm ± 27 nm) by Caco-2 cells are time-dependent. To study the transcytosis process we used a transwell system and EVs were quantified in the lower chamber by Fluorescence and Nanoparticle Tracking Analysis measurements. Intact EVs are transported across a polarized cell monolayer at 60–120 min and increased after 240 min with an estimated average uptake efficiency of 30% and this process is dose-dependent. EVs movement into intestinal epithelial cells was mainly through Z axis and scarcely on X and Y axis. This work demonstrates that EVs could be transported across the gastrointestinal epithelium. We speculate this mechanism could be the first step allowing EVs to reach the bloodstream for further delivery up to extraintestinal tissues and organs. The expression and further encapsulation of bioactive molecules into natural nanoparticles produced by probiotic bacteria could have practical implications in food, nutraceuticals and clinical therapies.

Bacterial viruses are widespread and abundant across natural and engineered habitats. They influence ecosystem functioning through interactions with their hosts. Laboratory studies of phage–host pairs have advanced our understanding of phenotypic and genetic diversification in bacteria and phages. However, the dynamics of phage–host interactions have been seldom recorded in complex natural environments. We conducted an observational metagenomic study of the dynamics of interaction between Gordonia and their phages using a three-year data series of samples collected from a full-scale wastewater treatment plant. The aim was to obtain a comprehensive picture of the coevolution dynamics in naturally evolving populations at relatively high time resolution. Coevolution was followed by monitoring changes over time in the CRISPR loci of Gordonia metagenome-assembled genome, and reciprocal changes in the viral genome. Genome-wide analysis indicated low strain variability of Gordonia , and almost clonal conservation of the trailer end of the CRISPR loci. Incorporation of newer spacers gave rise to multiple coexisting bacterial populations. The host population carrying a shorter CRISPR locus that contain only ancestral spacers, which has not acquired newer spacers against the coexisting phages, accounted for more than half of the total host abundance in the majority of samples. Phages genome co-evolved by introducing directional changes, with no preference for mutations within the protospacer and PAM regions. Metagenomic reconstruction of time-resolved variants of host and viral genomes revealed how the complexity at the population level has important consequences for bacteria-phage coexistence.

Advances in DNA sequencing technology have facilitated the determination of hundreds of complete genome sequences both for bacteria and their bacteriophages. Some of these bacteria have well-developed and facile genetic systems for constructing mutants to determine gene function, and recombineering is a particularly effective tool. However, generally applicable methods for constructing defined mutants of bacteriophages are poorly developed, in part because of the inability to use selectable markers such as drug resistance genes during viral lytic growth. Here we describe a method for simple and effective directed mutagenesis of bacteriophage genomes using Bacteriophage Recombineering of Electroporated DNA (BRED), in which a highly efficient recombineering system is utilized directly on electroporated phage DNA; no selection is required and mutants can be readily detected by PCR. We describe the use of BRED to construct unmarked gene deletions, in-frame internal deletions, base substitutions, precise gene replacements, and the addition of gene tags.

Mycobacterium tuberculosis , the bacterium responsible for tuberculosis, remains a global health concern. Unlike most well-studied model bacilli, mycobacteria possess a distinctive and complex cell envelope, as well as an asymmetric polar growth mode. However, the proteins and mechanisms that drive cell asymmetric elongation in these bacteria are still not well understood. This study sheds light on the role of the protein FhaA in this process. Our findings demonstrate that FhaA localizes at the septum and asymmetrically to the poles, with a preference for the fast-growing pole. Furthermore, we showed that FhaA is essential for population heterogeneity and asymmetric polar elongation and plays a role in the precise subcellular localization of the cell wall biosynthesis machinery. Mycobacterial asymmetric elongation results in a physiologically heterogeneous bacterial population which is important for pathogenicity and response to antibiotics, stressing the relevance of identifying new factors involved in these still poorly characterized processes.

Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively-drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections.

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.

Fil: Rodríguez Taño, Azalia. Universidad de la República. Facultad de Química. Programa de Posgrado; Uruguay

Five newly isolated mycobacteriophages--Angelica, CrimD, Adephagia, Anaya, and Pixie--have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them--with the exception of TM4--form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species.

Background: Bacillus subtilis 168 is a regular resident of the mammalian gastrointestinal (GI) microbiome and has been used in food fermentations, being awarded the status of \"Generally Recognized As Safe\" (GRAS). Extracellular vesicles (EVs) have been proposed to be involved in signalling between probiotic bacteria and their mammalian hosts. B. subtilis 168 produces EVs which were on the nanometric size range (50-300 nm). EVs carried cytoplasmic components, such as specific proteins, which suggest a role for the EVs in the bacteria-GI cells interface. We hypothesize that transcytosis of EVs across intestinal epithelial cells is a crucial step in the host-probiotic communication. To test this, the ability of EVs produced by the probiotic strain B. subtilis 168 to cross intestinal epithelial cellbarrierwas investigated in an in vitro model of human Caco-2 cells. Methods: B. subtilis 168 was grown in BHI medium at 37°C under agitation for 18 h. Cells were removed from the culture by centrifugation. Supernatant was then concentrated using a 100-kDa filter membrane. The concentrated supernatant was spun at 110000 g for 2 h to pellet EVs. Isolated EVs were stained with carboxyfluorescein succinimidyl ester. Human colon carcinoma Caco-2 cells were differentiated for 14 days (100% confluence). EVs' uptake was analysed as the number of EVs labelled inside the cell by confocal laser scanning microscopy. Transcytosis was studied as the fluorescence measured in the collected medium from the transwell lower chamber and EVs were also observed. The cytotoxicity of the EVs was evaluated using MTT assay. Results: Intact EVs uptake in Caco-2 cells was linear for up to 30 min: y = 1.02 x -1.25 and R2 = 0.97 (p < 0.05). In transcytosis studies, fluorescence was recorded after 120 min elapsed and increased 50% at 240 min (n = 3). We also found intact EVs in the collected medium from the lower chamber of the transwell. EVs did not significantly reduce cell viability (p > 0.05). Summary/Conclusion: EVs produced by the probiotic strain B. subtilis 168 crossed intestinal epithelial cell barrier of human Caco-2 cells. This evidence suggests that EVs could play a key role in signalling between GI bacteria and mammalian hosts. The expression and further encapsulation of proteins into EVs of GRAS bacteria could represent a scientific novelty, with applications in food and clinical therapies.