Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their ...biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood.
To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells.
LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO.
Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.
Polymicrobial biofilms are of large medical importance, but relatively little is known about the role of interspecies interactions for their physiology and virulence. Here, we studied two human ...pathogens co-occuring in the oral cavity, the opportunistic fungus Candida albicans and the caries-promoting bacterium Streptococcus mutans. Dual-species biofilms reached higher biomass and cell numbers than mono-species biofilms, and the production of extracellular polymeric substances (EPSs) by S. mutans was strongly suppressed, which was confirmed by scanning electron microscopy, gas chromatography-mass spectrometry and transcriptome analysis. To detect interkingdom communication, C. albicans was co-cultivated with a strain of S. mutans carrying a transcriptional fusion between a green fluorescent protein-encoding gene and the promoter for sigX, the alternative sigma factor of S. mutans, which is induced by quorum sensing signals. Strong induction of sigX was observed in dual-species biofilms, but not in single-species biofilms. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion of comS encoding the synthesis of the sigX-inducing peptide precursor abolished this activity, whereas deletion of comC encoding the competence-stimulating peptide precursor had no effect. Transcriptome analysis of S. mutans confirmed induction of comS, sigX, bacteriocins and the downstream late competence genes, including fratricins, in dual-species biofilms. We show here for the first time the stimulation of the complete quorum sensing system of S. mutans by a species from another kingdom, namely the fungus C. albicans, resulting in fundamentally changed virulence properties of the caries pathogen.
Streptococcus pyogenes (group A Streptococcus, GAS) is a human‐adapted pathogen responsible for a wide spectrum of disease. GAS can cause relatively mild illnesses, such as strep throat or impetigo, ...and less frequent but severe life‐threatening diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. GAS is an important public health problem causing significant morbidity and mortality worldwide. The main route of GAS transmission between humans is through close or direct physical contact, and particularly via respiratory droplets. The upper respiratory tract and skin are major reservoirs for GAS infections. The ability of GAS to establish an infection in the new host at these anatomical sites primarily results from two distinct physiological processes, namely bacterial adhesion and colonization. These fundamental aspects of pathogenesis rely upon a variety of GAS virulence factors, which are usually under strict transcriptional regulation. Considerable progress has been made in better understanding these initial infection steps. This review summarizes our current knowledge of the molecular mechanisms of GAS adhesion and colonization.
Bacteria of the phylum Planctomycetes have been previously reported to possess several features that are typical of eukaryotes, such as cytosolic compartmentalization and endocytosis-like ...macromolecule uptake. However, recent evidence points towards a Gram-negative cell plan for Planctomycetes, although in-depth experimental analysis has been hampered by insufficient genetic tools. Here we develop methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail. Super-resolution light microscopy of mutants, cryo-electron tomography, bioinformatic predictions and proteomic analyses support an altered Gram-negative cell plan for Planctomycetes, including a defined outer membrane, a periplasmic space that can be greatly enlarged and convoluted, and an energized cytoplasmic membrane. These conclusions are further supported by experiments performed with two other Planctomycetes, Gemmata obscuriglobus and Rhodopirellula baltica. We also provide experimental evidence that is inconsistent with endocytosis-like macromolecule uptake; instead, extracellular macromolecules can be taken up and accumulate in the periplasmic space through unclear mechanisms.
As a step towards better understanding of diversity and biology of phages and their hosts in haloalkaline Lake Elmenteita, phages were isolated from sediment samples and overlying water using ...indigenous bacteria as hosts. 17 seemingly different phages of diverse morphotypes with different dimensions and partly exhibiting remarkably unusual ultrastructures were revealed by transmission electron microscopy. 12 clonal phage isolates were further characterized. Infection capability of the phages was optimum at 30-35°C and in alkali condition with optimum at pH 10-12. Structural protein profiles and restriction fragment length polymorphism analyses patterns were distinct for each of the phage type. Complete nucleotide sequences of phages vB-VmeM-32, vB_EauS-123 and vB_BhaS-171 genomes varied in size from 30,926-199,912 bp and G + C content of between 36.25-47.73%. A range of 56-260 potential open reading frames were identified and annotated. The results showed that the 12 phages were distinct from each other and confirmed the presence and diversity of phages in extreme environment of haloalkaline Lake Elmenteita. The phages were deposited at the German Collection of Microorganisms and Cell Cultures and three of their genomes uploaded to NCBI GenBank.
The recently isolated strain L21-Fru-AB
represents moderately halophilic, obligately anaerobic and saccharolytic bacteria that thrive in the suboxic transition zones of hypersaline microbial mats. ...Phylogenetic analyses based on 16S rRNA genes, RpoB proteins and gene content indicated that strain L21-Fru-AB
represents a novel species and genus affiliated with a distinct phylum-level lineage originally designated Verrucomicrobia subdivision 5. A survey of environmental 16S rRNA gene sequences revealed that members of this newly recognized phylum are wide-spread and ecologically important in various anoxic environments ranging from hypersaline sediments to wastewater and the intestine of animals. Characteristic phenotypic traits of the novel strain included the formation of extracellular polymeric substances, a Gram-negative cell wall containing peptidoglycan and the absence of odd-numbered cellular fatty acids. Unusual metabolic features deduced from analysis of the genome sequence were the production of sucrose as osmoprotectant, an atypical glycolytic pathway lacking pyruvate kinase and the synthesis of isoprenoids via mevalonate. On the basis of the analyses of phenotypic, genomic and environmental data, it is proposed that strain L21-Fru-AB
and related bacteria are specifically adapted to the utilization of sulfated glycopolymers produced in microbial mats or biofilms.
Most bacteria contain a peptidoglycan (PG) cell wall, which is critical for maintenance of shape and important for cell division. In contrast, Planctomycetes have been proposed to produce a ...proteinaceous cell wall devoid of PG. The apparent absence of PG has been used as an argument for the putative planctomycetal ancestry of all bacterial lineages. Here we show, employing multiple bioinformatic methods, that planctomycetal genomes encode proteins required for PG synthesis. Furthermore, we biochemically demonstrate the presence of the sugar and the peptide components of PG in Planctomycetes. In addition, light and electron microscopic experiments reveal planctomycetal PG sacculi that are susceptible to lysozyme treatment. Finally, cryo-electron tomography demonstrates that Planctomycetes possess a typical PG cell wall and that their cellular architecture is thus more similar to that of other Gram-negative bacteria. Our findings shed new light on the cellular architecture and cell division of the maverick Planctomycetes.
Streptococcus suis is an economically important pathogen of pigs as well as a zoonotic cause of human disease. Serotyping is used for further characterization of isolates; some serotypes seem to be ...more virulent and more widely spread than others. This study characterizes a collection of German field isolates of Streptococcus suis from pigs dating from 1996 to 2016 with respect to capsular genes (cps) specific for individual serotypes and pathotype by multiplex PCR and relates results to the clinical background of these isolates. The most prominent finding was the reduction in prevalence of serotype-2/serotype-1/2 among invasive isolates during this sampling period, which might be attributed to widely implemented autogenous vaccination programs in swine against serotype 2 in Germany. In diseased pigs (systemically ill; respiratory disease) isolates of serotype-1/serotype-14, serotype-2/serotype-1/2, serotype 3 to 5 and 7 to 9 were most frequent while in carrier isolates a greater variety of cps types was found. Serotype-1/serotype-14 seemed to be preferentially located in joints, serotype 4 and serotype 3 in the central nervous system, respectively. The virulence associated extracellular protein factor was almost exclusively associated with invasive serotype-1/serotype-14 and serotype-2/serotype-1/2 isolates. In contrast, lung isolates of serotype-2/serotype-1/2 mainly harbored the gene for muramidase-released protein. Serotype 4 and serotype 9 isolates from clinically diseased pigs most frequently carried the muramidase-released protein gene and the suilysin gene. When examined by transmission electron microscopy all but one of the isolates which were non-typable by molecular and serological methods showed various amounts of capsular material indicating potentially new serotypes among these isolates. Given the variety of cps types/serotypes detected in pigs, not only veterinarians but also medical doctors should consider other serotypes than just serotype 2 when investigating potential human cases of Streptococcus suis infection.
Acidobacteria represent abundant members of soil microbial communities but only few representatives could be isolated and validly described so far. Currently, eighteen species of subdivision 1, one ...species of subdivision 3, three species of subdivision 8, and one species of subdivision 10 are recognized. In contrast, Acidobacteria of subdivision 4 have largely escaped cultivation although they belong to the most abundant and diverse acidobacterial groups in soils. A member of subdivision 4, strain A2-16T, was isolated from a semiarid savanna soil. Cells were motile spheres to rods with a tendency to form chains and larger aggregates. Cultures were orange to pink colored, neutrophilic mesophiles, and showed aerobic chemoorganoheterotrophic growth on very few complex substrates and protocatechuate, and weak growth on chitin, cellulose and starch. While protein substrates such as casamino acids or peptone were utilized, individual amino acids did not promote growth. Also, growth on alternative electron acceptors or fermentative growth could not be observed. Major fatty acids were summed features 1 (15:1 iso H/13:0 3-OH) and 3 (16:1ω7c/15:0 iso 2-OH). The major quinone was MK-8. The DNA G+C content was 46.5mol%. Phylogenetic analysis placed A2-16T amidst uncultured members of Acidobacteria subdivision 4. The most closely related environmental 16S rRNA gene sequences (96–97% nucleotide identity) were several clone sequences from terrestrial environments. Based on these characteristics, the isolated strain is proposed as a new species of a novel genus, Blastocatella fastidiosa gen. nov., sp. nov. The type strain of B. fastidiosa is A2-16T (=DSM 25172T=LMG26944T).
These days it has been increasingly recognized that mast cells (MCs) are critical components of host defense against pathogens. In this study, we have provided the first evidence that MCs can kill ...bacteria by entrapping them in extracellular structures similar to the extracellular traps described for neutrophils (NETs). We took advantage of the ability of MCs to kill the human pathogen Streptococcus pyogenes by a phagocytosis-independent mechanism in order to characterize the extracellular antimicrobial activity of MCs. Close contact of bacteria and MCs was required for full antimicrobial activity. Immunofluorescence and electron microscopy revealed that S pyogenes was entrapped by extracellular structures produced by MCs (MCETs), which are composed of DNA, histones, tryptase, and the antimicrobial peptide LL-37. Disruption of MCETs significantly reduced the antimicrobial effect of MCs, suggesting that intact extracellular webs are critical for effective inhibition of bacterial growth. Similar to NETs, production of MCETs was mediated by a reactive oxygen species (ROS)–dependent cell death mechanism accompanied by disruption of the nuclear envelope, which can be induced after stimulation of MCs with phorbol-12-myristate-13-acetate (PMA), H2O2, or bacterial pathogens. Our study provides the first experimental evidence of antimicrobial extracellular traps formation by an immune cell population other than neutrophils.
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