ModularImageAnalysis (MIA): Assembly of modularised image and object analysis workflows in ImageJ.

ModularImageAnalysis (MIA) is an ImageJ plugin providing a code-free graphical environment in which complex automated analysis workflows can be constructed and distributed. The broad range of included modules cover all stages of a typical analysis workflow, from image loading through image processing, object detection, extraction of measurements, measurement-based filtering, visualisation and data exporting. MIA provides out-of-the-box compatibility with many advanced image processing plugins for ImageJ including Bio-Formats, DeepImageJ, MorphoLibJ and TrackMate, allowing these tools and their outputs to be directly incorporated into analysis workflows. By default, modules support spatially calibrated 5D images, meaning measurements can be acquired in both pixel and calibrated units. A hierarchical object relationship model allows for both parent-child (one-to-many) and partner (many-to-many) relationships to be established. These relationships underpin MIA's ability to track objects through time, represent complex spatial relationships (e.g. topological skeletons) and measure object distributions (e.g. count puncta per cell). MIA features dual graphical interfaces: the 'editing view' offers access to the full list of modules and parameters in the workflow, while the simplified 'processing view' can be configured to display only a focused subset of controls. All workflows are batch-enabled by default, with image files within a specified folder being processed automatically and exported to a single spreadsheet. Beyond the included modules, functionality can be extended both internally, through integration with the ImageJ scripting interface, and externally, by developing third-party Java modules that extend the core MIA framework. Here we describe the design and functionality of MIA in the context of a series of real-world example analyses.

Correction: Back et al. A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge. Mar. Drugs 2021, 19, 105.

After publication of this article [...].

Quantification of Extracellular DNA Network Abundance and Architecture within Streptococcus gordonii Biofilms Reveals Modulatory Factors.

Extracellular DNA (eDNA) is an important component of biofilm matrix that serves to maintain biofilm structural integrity, promotes genetic exchange within the biofilm, and provides protection against antimicrobial compounds. Advances in microscopy techniques have provided evidence of the cobweb- or lattice-like structures of eDNA within biofilms from a range of environmental niches. However, methods to reliably assess the abundance and architecture of eDNA remain lacking. This study aimed to address this gap by development of a novel, high-throughput image acquisition and analysis platform for assessment of eDNA networks in situ within biofilms. Utilizing Streptococcus gordonii as the model, the capacity for this imaging system to reliably detect eDNA networks and monitor changes in abundance and architecture (e.g., strand length and branch number) was verified. Evidence was provided of a synergy between glucans and eDNA matrices, while it was revealed that surface-bound nuclease SsnA could modify these eDNA structures under conditions permissive for enzymatic activity. Moreover, cross talk between the competence and hexaheptapeptide permease systems was shown to regulate eDNA release by S. gordonii. This novel imaging system can be applied across the wider field of biofilm research, with potential to significantly advance interrogation of the mechanisms by which the eDNA network architecture develops, how it can influence biofilm properties, and how it may be targeted for therapeutic benefit. IMPORTANCE Extracellular DNA (eDNA) is critical for maintaining the structural integrity of many microbial biofilms, making it an attractive target for the management of biofilms. However, our knowledge and targeting of eDNA are currently hindered by a lack of tools for the quantitative assessment of eDNA networks within biofilms. Here, we demonstrate use of a novel image acquisition and analysis platform with the capacity to reliably monitor the abundance and architecture of eDNA networks. Application of this tool to Streptococcus gordonii biofilms has provided new insights into how eDNA networks are stabilized within the biofilm and the pathways that can regulate eDNA release. This highlights how exploitation of this novel imaging system across the wider field of biofilm research has potential to significantly advance interrogation of the mechanisms by which the eDNA network architecture develops, how it can influence biofilm properties, and how it may be targeted for therapeutic benefit.

PDIA3/ERp57 promotes a matrix-rich secretome that stimulates fibroblast adhesion through CCN2.

The matricellular glycoprotein thrombospondin-1 (TSP1) has complex roles in the extracellular matrix (ECM) and at cell surfaces, but relatively little is known about its intracellular associations prior to secretion. To search for novel intracellular interactions of TSP1 in situ, we carried out a biotin ligase-based TSP1 interactome screen and identified protein disulfide isomerase A3 (PDIA3/ERp57) as a novel candidate binding protein. In validation, TSP1 and PDIA3 were established to bind in vitro and to colocalize in the endoplasmic reticulum of human dermal fibroblasts (HDF). Loss of PDIA3 function, either by pharmacological inhibition in HDF or in Pdia3-/- mouse embryo fibroblasts (Pdia3-/- MEFs), led to alterations in the composition of cell-derived extracellular matrix, involving changed abundance of fibronectin and TSP1, was correlated with reduced cell spreading, altered organization of F-actin, and reduced focal adhesions. These cellular phenotypes of Pdia3-/- MEFs were normalized by exposure to conditioned medium (WTCM) or extracellular matrix (WTECM) from wild-type (WT)-MEFs. Rescue depended on PDIA3 activity in WT-MEFs and was not prevented by immunodepletion of fibronectin. Heparin-binding proteins in WTCM were found to be necessary for rescue. Comparative quantitative tandem-mass-tag proteomics and functional assays on the heparin-binding secretomes of WT-MEFs and Pdia3-/- MEFs identified multiple ECM and growth factor proteins to be downregulated in the CM of Pdia3-/- MEFs. Of these, cell communication network 2 (CCN2) was identified to be necessary for the adhesion-promoting activity of WTCM on Pdia3-/- MEFs and to bind TSP1. Thus, PDIA3 coordinates fibroblast production of an ECM-rich, proadhesive microenvironment, with implications for PDIA3 as a translational target.

A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge.

To tackle the growing problem of antibiotic resistance, it is essential to identify new bioactive compounds that are effective against resistant microbes and safe to use. Natural products and their derivatives are, and will continue to be, an important source of these molecules. Sea sponges harbour a diverse microbiome that co-exists with the sponge, and these bacterial communities produce a rich array of bioactive metabolites for protection and resource competition. For these reasons, the sponge microbiota constitutes a potential source of clinically relevant natural products. To date, efforts in bioprospecting for these compounds have focused predominantly on sponge specimens isolated from shallow water, with much still to be learned about samples from the deep sea. Here we report the isolation of a new Micromonospora strain, designated 28ISP2-46T, recovered from the microbiome of a mid-Atlantic deep-sea sponge. Whole-genome sequencing reveals the capacity of this bacterium to produce a diverse array of natural products, including kosinostatin and isoquinocycline B, which exhibit both antibiotic and antitumour properties. Both compounds were isolated from 28ISP2-46T fermentation broths and were found to be effective against a plethora of multidrug-resistant clinical isolates. This study suggests that the marine production of isoquinocyclines may be more widespread than previously supposed and demonstrates the value of targeting the deep-sea sponge microbiome as a source of novel microbial life with exploitable biosynthetic potential.

Understanding the Matrix: The Role of Extracellular DNA in Oral Biofilms.

Dental plaque is the key etiological agent in caries formation and the development of the prevalent chronic oral inflammatory disease, periodontitis. The dental plaque biofilm comprises a diverse range of microbial species encased within a rich extracellular matrix, of which extracellular DNA (eDNA) has been identified as an important component. The molecular mechanisms of eDNA release and the structure of eDNA have yet to be fully characterized. Nonetheless, key functions that have been proposed for eDNA include maintaining biofilm structural integrity, initiating adhesion to dental surfaces, acting as a nutrient source, and facilitating horizontal gene transfer. Thus, eDNA is a potential therapeutic target for the management of oral disease-associated biofilm. This review aims to summarize advances in the understanding of the mechanisms of eDNA release from oral microorganisms and in the methods of eDNA detection and quantification within oral biofilms.

Effects of food-borne nanomaterials on gastrointestinal tissues and microbiota.

Ingestion of engineered nanomaterials is inevitable due to their addition to food and prevalence in food packaging and domestic products such as toothpaste and sun cream. In the absence of robust dosimetry and particokinetic data, it is currently challenging to accurately assess the potential toxicity of food-borne nanomaterials. Herein, we review current understanding of gastrointestinal uptake mechanisms, consider some data on the potential for toxicity of the most commonly encountered classes of food-borne nanomaterials (including TiO2 , SiO2, ZnO, and Ag nanoparticles), and discuss the potential impact of the luminal environment on nanoparticle properties and toxicity. Much of our current understanding of gastrointestinal nanotoxicology is derived from increasingly sophisticated epithelial models that augment in vivo studies. In addition to considering the direct effects of food-borne nanomaterials on gastrointestinal tissues, including the potential role of chronic nanoparticle exposure in development of inflammatory diseases, we also discuss the potential for food-borne nanomaterials to disturb the normal balance of microbiota within the gastrointestinal tract. The latter possibility warrants close attention given the increasing awareness of the critical role of microbiota in human health and the known impact of some food-borne nanomaterials on bacterial viability. WIREs Nanomed Nanobiotechnol 2018, 10:e1481. doi: 10.1002/wnan.1481 This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

Detection of a fluorescent-labeled avidin-nucleic acid nanoassembly by confocal laser endomicroscopy in the microvasculature of chronically inflamed intestinal mucosa.

Inflammatory bowel diseases are chronic gastrointestinal pathologies causing great discomfort in both children and adults. The pathogenesis of inflammatory bowel diseases is not yet fully understood and their diagnosis and treatment are often challenging. Nanoparticle-based strategies have been tested in local drug delivery to the inflamed colon. Here, we have investigated the use of the novel avidin-nucleic acid nanoassembly (ANANAS) platform as a potential diagnostic carrier in an experimental model of inflammatory bowel diseases. Fluorescent- labeled ANANAS nanoparticles were administered to mice with chemically induced chronic inflammation of the large intestine. Localization of mucosal nanoparticles was assessed in vivo by dual-band confocal laser endomicroscopy. This technique enables characterization of the mucosal microvasculature and crypt architecture at subcellular resolution. Intravascular nanoparticle distribution was observed in the inflamed mucosa but not in healthy controls, demonstrating the utility of the combination of ANANAS and confocal laser endomicroscopy for highlighting intestinal inflammatory conditions. The specific localization of ANANAS in inflamed tissues supports the potential of this platform as a targeted carrier for bioactive moieties in the treatment of inflammatory bowel disease.

Applications of microscopy in Salmonella research.

Salmonella enterica is a Gram-negative enteropathogen that can cause localized infections, typically resulting in gastroenteritis, or systemic infection, e.g., typhoid fever, in humans and many other animals. Understanding the mechanisms by which Salmonella induces disease has been the focus of intensive research. This has revealed that Salmonella invasion requires dynamic cross-talk between the microbe and host cells, in which bacterial adherence rapidly leads to a complex sequence of cellular responses initiated by proteins translocated into the host cell by a type 3 secretion system. Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second type 3 secretion system initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. Elucidation of the complex and highly dynamic pathogen-host interactions ultimately requires analysis at the level of single cells and single infection events. To achieve this goal, researchers have applied a diverse range of microscopy techniques to analyze Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and high-resolution light microscopy techniques such as confocal microscopy can reveal the precise location of Salmonella and its relationship to cellular components. Widefield light microscopy is a simpler approach with which to study the interaction of bacteria with host cells and often has advantages for live cell imaging, enabling detailed analysis of the dynamics of infection and cellular responses. Here we review the use of imaging techniques in Salmonella research and compare the capabilities of different classes of microscope to address specific types of research question. We also provide protocols and notes on some microscopy techniques used routinely in our own research.

Streptococcus gordonii comCDE (competence) operon modulates biofilm formation with Candida albicans.

Candida albicans is a pleiomorphic fungus that forms mixed species biofilms with Streptococcus gordonii, an early colonizer of oral cavity surfaces. Activation of quorum sensing (QS; intercellular signalling) promotes monospecies biofilm development by these micro-organisms, but the role of QS in mixed species communities is not understood. The comCDE genes in S. gordonii encode a sensor-regulator system (ComDE), which is activated by the comC gene product (CSP, competence stimulating peptide) and modulates expression of QS-regulated genes. Dual species biofilms of S. gordonii ΔcomCDE or ΔcomC mutants with C. albicans showed increased biomass compared to biofilms of S. gordonii DL1 wild-type with C. albicans. The ΔcomCDE mutant dual species biofilms in particular contained more extracellular DNA (eDNA), and could be dispersed with DNase I or protease treatment. Exogenous CSP complemented the S. gordonii ΔcomC transformation deficiency, as well as the ΔcomC-C. albicans biofilm phenotype. Purified CSP did not affect C. albicans hyphal filament formation but inhibited monospecies biofilm formation by C. albicans. The results suggest that the S. gordonii comCDE QS-system modulates the production of eDNA and the incorporation of C. albicans into dual species biofilms.

Axenic culture of a candidate division TM7 bacterium from the human oral cavity and biofilm interactions with other oral bacteria.

The diversity of bacterial species in the human oral cavity is well recognized, but a high proportion of them are presently uncultivable. Candidate division TM7 bacteria are almost always detected in metagenomic studies but have not yet been cultivated. In this paper, we identified candidate division TM7 bacterial phylotypes in mature plaque samples from around orthodontic bonds in subjects undergoing orthodontic treatment. Successive rounds of enrichment in laboratory media led to the isolation of a pure culture of one of these candidate division TM7 phylotypes. The bacteria formed filaments of 20 to 200 µm in length within agar plate colonies and in monospecies biofilms on salivary pellicle and exhibited some unusual morphological characteristics by transmission electron microscopy, including a trilaminated cell surface layer and dense cytoplasmic deposits. Proteomic analyses of cell wall protein extracts identified abundant polypeptides predicted from the TM7 partial genomic sequence. Pleiomorphic phenotypes were observed when the candidate division TM7 bacterium was grown in dual-species biofilms with representatives of six different oral bacterial genera. The TM7 bacterium formed long filaments in dual-species biofilm communities with Actinomyces oris or Fusobacterium nucleatum. However, the TM7 isolate grew as short rods or cocci in dual-species biofilms with Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, or Streptococcus gordonii, forming notably robust biofilms with the latter two species. The ability to cultivate TM7 axenically should majorly advance understanding of the physiology, genetics, and virulence properties of this novel candidate division oral bacterium.

O-mannosylation in Candida albicans enables development of interkingdom biofilm communities.

Candida albicans is a fungus that colonizes oral cavity surfaces, the gut, and the genital tract. Streptococcus gordonii is a ubiquitous oral bacterium that has been shown to form biofilm communities with C. albicans. Formation of dual-species S. gordonii-C. albicans biofilm communities involves interaction of the S. gordonii SspB protein with the Als3 protein on the hyphal filament surface of C. albicans. Mannoproteins comprise a major component of the C. albicans cell wall, and in this study we sought to determine if mannosylation in cell wall biogenesis of C. albicans was necessary for hyphal adhesin functions associated with interkingdom biofilm development. A C. albicans mnt1Δ mnt2Δ mutant, with deleted α-1,2-mannosyltransferase genes and thus defective in O-mannosylation, was abrogated in biofilm formation under various growth conditions and produced hyphal filaments that were not recognized by S. gordonii. Cell wall proteomes of hypha-forming mnt1Δ mnt2Δ mutant cells showed growth medium-dependent alterations, compared to findings for the wild type, in a range of protein components, including Als1, Als3, Rbt1, Scw1, and Sap9. Hyphal filaments formed by mnt1Δ mnt2Δ mutant cells, unlike wild-type hyphae, did not interact with C. albicans Als3 or Hwp1 partner cell wall proteins or with S. gordonii SspB partner adhesin, suggesting defective functionality of adhesins on the mnt1Δ mnt2Δ mutant. These observations imply that early stage O-mannosylation is critical for activation of hyphal adhesin functions required for biofilm formation, recognition by bacteria such as S. gordonii, and microbial community development. IMPORTANCE In the human mouth, microorganisms form communities known as biofilms that adhere to the surfaces present. Candida albicans is a fungus that is often found within these biofilms. We have focused on the mechanisms by which C. albicans becomes incorporated into communities containing bacteria, such as Streptococcus. We find that impairment of early stage addition of mannose sugars to C. albicans hyphal filament proteins deleteriously affects their subsequent performance in mediating formation of polymicrobial biofilms. Our analyses provide new understanding of the way that microbial communities develop, and of potential means to control C. albicans infections.

Regulatory function of trefoil peptides (TFF) on intestinal cell junctional complexes.

Abstract Trefoil peptides (TFF) are constitutively expressed in the gastrointestinal tract and are involved in gastrointestinal defence and repair by promoting epithelial restitution. Although there is a general consensus regarding the pro-motogenic activity of trefoil peptides, the cellular mechanisms through which they mediate these processes are not completely understood. Pertubation of the E-cadherin/catenin complex at intercellular junctions appears to be a functional pathway through which TFF2 and TFF3 promote cell migration. Tight junction complexes seal the paracellular spaces between cells and contribute to epithelial barrier function. TFF3 peptide stimulation stabilises these junctions through upregulation of the tightening protein claudin-1 and redistribution of ZO-1 from the cytoplasm to the intercellular membrane with an increase in binding to occludin. Modulation of the functional activity and subcellular localisation of epithelial junctional adhesion molecules represent important mechanisms by which trefoil peptides may promote migration of intestinal epithelial cells in vitro and healing of mucosal damage in vivo.

Characterization of cerium oxide nanoparticles-part 1: size measurements.

The present study gives an overview of some of the major aspects for consideration in the characterization of nanomaterials (NMs). Part 1 focuses on the measurement of particle size and size-related parameters using several analytical techniques such as transmission electron microscopy, atomic force microscopy, dynamic light scattering, X-ray diffraction, and Brunauer, Emmett, and Teller surface area measurements as applied to commercially available cerium oxide nanoparticles (NPs) and microparticles (MPs). Part 2 (see companion paper) considers nonsize-related characterization and analysis. The results are discussed in relation to the nature of the sample and preparation, and the analytical principles, limitations, and advantages of each technique. Accurate information on the particle size of the different fractions of a sample can be obtained by using a combination of different types of microscopy, spectroscopy, separation, and other techniques; this should inform ecotoxicological and environmental studies. The good agreement between the measured primary particle size of the NPs (~15 nm) by atomic force microscopy, transmission electron microscopy, X-ray diffraction, and Brunauer, Emmett, and Teller suggests that the primary particles are formed of semispherical single crystals. For MPs, all measurements agree that they are large particles in the range above the NPs (100 nm), with some difference between the measured sizes, possibly as a result of polydispersity effects. Additionally, our findings suggest that atomic force microscopy and transmission electron microscopy prepared by centrifugation methods provide consistent data at low concentrations when dynamic light scattering fails.

Characterization of cerium oxide nanoparticles-part 2: nonsize measurements.

Part 1 (see companion paper) of the present study discussed the application of a multimethod approach in characterizing the size of cerium oxide nanoparticles (NPs). However, other properties less routinely investigated, such as shape and morphology, structure, chemical composition, and surface properties, are likely to play an important role in determining the behavior, reactivity, and potential toxicity of these NPs. The present study describes the measurement of the aforementioned physicochemical properties of NPs (applied also to nanomaterials [NMs]) compared with micrometer particles (MPs). The authors use a wide range of techniques, including high resolution-transmission electron microscopy, scanning electron microscopy, atomic force microscopy, X-ray diffraction, X-ray energy dispersive spectroscopy, electron energy loss spectroscopy, X-ray photoelectron spectroscopy, and electrophoresis, and compare these techniques, their advantages, and their limitations, along with recommendations about how best to approach NM characterization, using an application to commercial cerium oxide NPs and MPs. Results show that both cerium oxide NPs and MPs are formed of single polyhedron or truncated polyhedron crystals. Cerium oxide NPs contain a mixture of Ce(3+) and Ce(4+) cations, whereas the MPs contain mainly Ce(4+) , which is potentially important in understanding the toxicity of cerium oxide NPs. The isoelectric point of cerium oxide NPs was approximately pH 8, which explains their propensity to aggregate in aqueous media (see companion paper).

Interspecies comparisons on the uptake and toxicity of silver and cerium dioxide nanoparticles.

An increasing number and quantity of manufactured nanoparticles are entering the environment as the diversity of their applications increases, and this will lead to the exposure of both humans and wildlife. However, little is known regarding their potential health effects. We compared the potential biological effects of silver (Ag; nominally 35 and 600-1,600 nm) and cerium dioxide (CeO(2;) nominally <25 nm and 1-5 µm) particles in a range of cell (human hepatocyte and intestinal and fish hepatocyte) and animal (Daphnia magna, Cyprinus carpio) models to assess possible commonalities in toxicity across taxa. A variety of analytical techniques were employed to characterize the particles and investigate their biological uptake. Silver particles were more toxic than CeO(2) in all test systems, and an equivalent mass dose of Ag nanoparticles was more toxic than larger micro-sized material. Cellular uptake of all materials tested was shown in C3A hepatocytes and Caco-2 intestinal cells, and for Ag, into the intestine, liver, gallbladder, and gills of carp exposed via the water. The commonalities in toxicity of these particle types across diverse biological systems suggest that cross-species extrapolations may be possible for metal nanoparticle test development in the future. Our findings also suggest transport of particles through the gastrointestinal barrier, which is likely to be an important uptake route when assessing particle risk.

Comparative analysis of EspF variants in inhibition of Escherichia coli phagocytosis by macrophages and inhibition of E. coli translocation through human- and bovine-derived M cells.

The EspF protein is secreted by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). EspF sequences differ between EHEC O157:H7, EHEC O26:H11, and EPEC O127:H6 in terms of the number of SH3-binding polyproline-rich repeats and specific residues in these regions, as well as residues in the amino domain involved in cellular localization. EspF(O127) is important for the inhibition of phagocytosis by EPEC and also limits EPEC translocation through antigen-sampling cells (M cells). EspF(O127) has been shown to have effects on cellular organelle function and interacts with several host proteins, including N-WASP and sorting nexin 9 (SNX9). In this study, we compared the capacities of different espF alleles to inhibit (i) bacterial phagocytosis by macrophages, (ii) translocation through an M-cell coculture system, and (iii) uptake by and translocation through cultured bovine epithelial cells. The espF gene from E. coli serotype O157 (espF(O157)) allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell coculture system in comparison to espF(O127) and espF(O26). In contrast, espF(O157) was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonization site of EHEC O157 in cattle and a site containing M-like cells. Although LUMIER binding assays demonstrated differences in the interactions of the EspF variants with SNX9 and N-WASP, we propose that other, as-yet-uncharacterized interactions contribute to the host-based variation in EspF activity demonstrated here.

Streptococcus pyogenes antigen I/II-family polypeptide AspA shows differential ligand-binding properties and mediates biofilm formation.

The streptococcal antigen I/II (AgI/II)-family polypeptides are cell wall-anchored adhesins expressed by most indigenous oral streptococci. Proteins sharing 30-40% overall amino acid sequence similarities with AgI/II-family proteins are also expressed by Streptococcus pyogenes. The S. pyogenes M28_Spy1325 polypeptide (designated AspA) displays an AgI/II primary structure, with alanine-rich (A) and proline-rich (P) repeats flanking a V region that is projected distal from the cell. In this study it is shown that AspA from serotype M28 S. pyogenes, when expressed on surrogate host Lactococcus lactis, confers binding to immobilized salivary agglutinin gp-340. This binding was blocked by antibodies to the AspA-VP region. In contrast, the N-terminal region of AspA was deficient in binding fluid-phase gp-340, and L. lactis cells expressing AspA were not agglutinated by gp-340. Deletion of the aspA gene from two different M28 strains of S. pyogenes abrogated their abilities to form biofilms on saliva-coated surfaces. In each mutant strain, biofilm formation was restored by trans complementation of the aspA deletion. In addition, expression of AspA protein on the surface of L. lactis conferred biofilm-forming ability. Taken collectively, the results provide evidence that AspA is a biofilm-associated adhesin that may function in host colonization by S. pyogenes.

Differences in Salmonella enterica serovar Typhimurium strain invasiveness are associated with heterogeneity in SPI-1 gene expression.

Most studies on Salmonella enterica serovar Typhimurium infection focus on strains ATCC SL1344 or NTCC 12023 (ATCC 14028). We have compared the abilities of these strains to induce membrane ruffles and invade epithelial cells. S. Typhimurium strain 12023 is less invasive and induces smaller membrane ruffles on MDCK cells compared with SL1344. Since the SPI-1 effector SopE is present in SL1344 and absent from 12023, and SL1344 sopE mutants have reduced invasiveness, we investigated whether 12023 is less invasive due to the absence of SopE. However, comparison of SopE(+) and SopE(-) S. Typhimurium strains, sopE deletion mutants and 12023 expressing a sopE plasmid revealed no consistent relationship between SopE status and relative invasiveness. Nevertheless, absence of SopE was closely correlated with reduced size of membrane ruffles. A PprgH-gfp reporter revealed that relatively few of the 12023 population (and that of the equivalent strain ATCC 14028) express SPI-1 compared to other S. Typhimurium strains. Expression of a PhilA-gfp reporter mirrored that of PprgH-gfp in 12023 and SL1344, implicating reduced signalling via the transcription factor HilA in the heterogeneous SPI-1 expression of these strains. The previously unrecognized strain heterogeneity in SPI-1 expression and invasiveness has important implications for studies of Salmonella infection.

Effects of silver and cerium dioxide micro- and nano-sized particles on Daphnia magna.

Acute (96 h) and chronic (21 d) exposures of Daphnia magna neonates were carried out with nano- and micro-sized Ag and CeO(2) particles to assess the influence of both material and size of particles on mortality and moulting. Mortality rates for silver in the acute exposures were: AgNP, 56.7 ± 23.3% at 0.1 mg L(-1) and 100 ± 20% at 1 mg L(-1), and micro-Ag, 13.3 ± 6.7% at 0.1 mg L(-1) and 80 ± 20% at 1 mg L(-1). CeO(2) was not acutely toxic at concentrations up to 10 mg L(-1). Mortality for Ag over 21d at concentrations of up to 0.05 mg L(-1) was low, while mortality of 30% was observed for 0.001 mg L(-1) of nano-Ag. CeO(2), with the exception of the 10 mg L(-1) of nano-CeO(2) (100% mortality by day 7), was non-toxic. Inhibition of moulting and growth in the acute study occurred at toxic concentrations (Ag particles), and at 10 mg L(-1) of nano-CeO(2). The chronic study revealed reduced moulting at 0.001 mg L(-1) of nano-Ag and 0.01 and 0.05 mg L(-1) of both sizes of Ag, but there was no impact on D. magna size, and no effects of CeO(2). The toxicity of nano-CeO(2) may be attributed to reduced feeding and physical interference with the daphnids' carapace, resulting in reduced swimming ability. Our results suggest that Ag NPs in particular have the potential to be harmful to aquatic invertebrates after release into the environment, whereas CeO(2) particles appear to cause little adverse effects, and only at environmentally irrelevant concentrations.